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Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by rational engineering of pKa values
Accepted Manuscript Title: Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by rational engineering of pKa values Authors: Cheng-Hua Wang, Xiao-Ling Liu, Ri-Bo Huang, Bing-Fang He, Mou-Ming Zhao PII: DOI: Reference:
S1369-703X(18)30308-5 https://doi.org/10.1016/j.bej.2018.08.015 BEJ 7026
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
11-4-2018 25-8-2018 27-8-2018
Please cite this article as: Wang C-Hua, Liu X-Ling, Huang R-Bo, He B-Fang, Zhao MMing, Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by rational engineering of pKa values, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by
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rational engineering of pKa values
Cheng-Hua Wang a, *, Xiao-Ling Liu a, Ri-Bo Huang b, *, Bing-Fang He c, Mou-Ming Zhao a a College
of Light Industry and Food Engineering, Guangxi University, Nanning 530004,
People’s Republic of China.
Key Laboratory of Non-Food Biomass and Enzyme Technology, National
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b State
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Engineering Research Center for Non-food Biorefinery, Guangxi Key Laboratory of
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of
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Biorefinery, Guangxi Academy of Sciences, Nanning 530007, People’s Republic of China.
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Technology, Nanjing 211816, People’s Republic of China.
Cheng-Hua Wang, E-mail: [email protected]
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Xiao-Ling Liu, E-mail: [email protected]
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Ri-Bo Huang, E-mail: [email protected] Bing-Fang He, E-mail: [email protected]
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Mou-Ming Zhao, E-mail: [email protected]
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*Corresponding
authors.
Cheng-Hua Wang, Address: College of Light Industry and Food Engineering, 100 Daxue
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East Road, Nanning 530004, People’s Republic of China. Tel: +86-771-323-2874, Fax: +86-771-323-2874, E-mail: [email protected];
Ri-Bo Huang, Address: Guangxi Academy of Sciences, 98 Daling Road, Nanning 530007,
People’s Republic of China. Tel: +86-771-250-3902, Fax: +86-771-250-3902, E-mail:
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[email protected]
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Graphical abstract
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Highlights
Three mutations decreasing pKa values of catalytic residues of Amy7C were
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designed. Three single and two double mutants were constructed and characterized.
All mutants shifted pH optima and pH-activity profiles toward more acidic values.
N271H decreased pH optimum by 2 units without comprising the catalytic efficiency.
A270K/N271H increased catalytic efficiency by 3.94-fold at optimum pH of 4.5.
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Abstract
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The aim of this study was to rationally engineer the acidic adaptation of B. subtilis
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Ca-independent alpha-amylase (Amy7C) by decreasing the pKa values of catalytic residues
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through mutations at active site. Within 4.5 Å of three catalytic residues of Amy7C, three mutations R172K, A270K and N271H were identified by computational homology
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modeling and pKa prediction analyses. Five single and double mutants consisting of these
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three mutations were constructed and characterized. Compared to the wild-type, all mutants shifted the pH optima and pH-activity profiles toward lower pH values without comprising the thermostablity. Double mutants showed simultaneous accumulation of advantageous mutations. The best mutant, A270K/N271H showed 2 units decrease in optimum pH and
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about 3.94-fold increase of catalytic efficiency. Structural analysis suggested that the improved acidic adaptation could be attributed to the decreased pKa values of catalytic
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nucleophile and proton donor residues. Protein engineering of α-amylase for acidic adaptation here provides a successful example of the extent to which mutations near active site and computational models can be used for industrial enzyme improvements.
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Keywords α-amylase, acidic adaptation, pKa, protein engineering, rational design
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1. Introduction The most widely used commercial alpha-amylases (EC 3.2.1.1) in starch processing,
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represented by Bacillus licheniformis α-amylase (BLA), usually operate optimally at high temperature (95 oC for BLA) and neutral pH (about pH 6.0 for BLA) conditions, and require addition of calcium ions to sustain stability and/or activity [1-3]. To make the best
use of these α-amylases, starch slurry must be firstly adjusted from native pH 3.2-4.5 to pH
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5.8-6.2 for liquefaction and then back to pH 4.2-4.5 for the next saccharification step.
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Meanwhile, calcium ions must be added for liquefaction and then to remove salts from
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downstream products on a large scale [3-5]. Consequently, there is great interest in
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changing the pH-performance profiles toward acidity and reducing the dependence on Ca2+
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of α-amylases in terms of economics and feasibility [6-8]. However, because it has proved
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difficult to eliminate calcium dependence of conventional Ca-dependent α-amylases, it is superior to make such novel Ca-independent and acidic α-amylase by engineering the
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acidic adaptation of existing Ca-independent α-amylases [9, 10].
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Previous protein engineering endeavors have been directed to pH-activity profiles of α-amylases [11]. These include improving the activity and thermostability of BLA at strong
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acid pH values by directed evolution and rational design methods [6, 7, 12, 13], exploring the determinants of pH-activity profile of Bacillus subtilis Ba2 by rational design [14], and enhancing pH-performance and higher specific activity of B. subtilis BAA by directed evolution [15]. It was indicated that the determinants of pH-activity profiles of α-amylases 5
could be attributed to electrostatics due to charged groups, dynamic structural changes of active site and hydrophobic effects [6, 14, 16, 17]. To the best of our knowledge, all the
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protein engineering of pH-activity profiles were carried out on Ca2+-dependent α-amylases including BLA, Ba2 and BAA, while no acidic engineering of Ca2+-independent α-amylases has ever been reported [16].
As a typical glycoside hydrolase, α-amylase obeys the double-displacement retaining
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mechanism [18, 19]. Based on the following three facts of α-amylase: 1) the nucleophile
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and proton donor must remain deprotonated and protonated respectively for optimal
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catalysis [20]; 2) the titrations of nucleophile and proton donor determine the acidic and
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basic limbs of the pH-activity profile [21], and 3) the catalysis is expected to be limited at
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the low pH by the protonation of the nucleophile and at high pH by deprotonation of the
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hydrogen donor [22], it can be concluded that the pKa values of nuclophile and proton donor residues determine the catalysis process and pH-activity profile. It is a promising
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way to engineer the acidic adaptation of α-amylase by rationally decreasing the pKa values
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of catalytic general acid/base. Bacillus subtilis CN7 α-amylase (Amy7C) is a Ca-independent and acid-resistant
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liquefying α-amylase, which has a optimum pH of 6.5 and keeps more than 75% activity within the range of pH 4.5-7.5 [23]. The elimination of calcium addition and pH adjustment of Amy7C excels in specific applications such as simultaneous saccharification and fermentation (SSF) process and production of high-fructose corn syrup (HFCS) [24]. 6
However, the desire for a better enzyme with improved acidic pH adaptation and catalytic activity still remains. In this study, Amy7C was engineered toward enhanced acidic
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adaptation by rationally decreasing pKa values of catalytic residues through mutations near the active site.
2. Material and methods
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2.1. Materials
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Luria-Bertani (LB) media were purchased from Difco Laboratories (Detroit, USA). The
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recombinant plasmid pSA7C, which was constructed previously by cloning the truncated
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α-amylase gene (GenBank ID: JN980090) from B. subtilis CN7 (CCTCC M 2012061) into
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the plasmid pSE380 [23], was used as template for site-directed mutagenesis. The PCR
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primers were listed in Table S1. The host strains E. coli XL1-Blue (Agilent Technologies Inc., Shanghai, China) and JM109 (Promega Corporation, Shanghai, China) were employed
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for cloning and expression of the recombinant α-amylases, respectively. All other
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chemicals and reagents used in this study were acquired commercially and were of reagent grade unless otherwise stated.
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2.2 Rational design As previously described [23], 3-D structure of wild-type Amy7C was constructed by
homology modeling via the SWISS-MODEL server (http://swissmodel.expasy.org/) by using an experimentally determined X-ray structure of α-amylase from Bacillus subtilis complexed with maltopentaose (PDB ID: 1BAG) as template, which shares sequence 7
identity of 91% with Amy7C and has the most conservation of sequence match in the vicinity of the active site, ensuring sufficient accuracy to generate reliable three-dimensional protein structure models [25, 26]. The enzyme-ligand complex structure
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was built by replacing the apoenzyme coordinates of 1BAG by that of the Amy7C after the superimposition between 1BAG and Amy7C while retaining all the coordinates of ligands
in 1BAG, and then further optimized by docking and energy minimization in MOE software with the default settings (Molecular Operation Environment, version2008.10). The energy minimization process was confined to the atoms within 4.5 Å neighboring maltopentaose in the holo-form structure. The enzyme-substrate interactions of the
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optimized holo-form structure with maltopentaose were analyzed by using PyMOL
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software (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.).
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The pKa values of catalytic residues were predicted by using the PROPKA 3.1 server
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(http://propka.ki.ku.dk/). The mutations were designed by five steps: 1) select the amino acid residues within the range of 4.5 Å of three catalytic triad residues, Asp174, Glu206
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and Asp276, and the substrate maltopentaose in the wild-type Amy7C; 2) mutate the
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selected residues to the other 19 amino acid residues; 3) construct predicted structure for each single mutant by SWISS-MODEL and MOE; 4) calculate the pKa values of the
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catalytic residues and mutations in the wild-type and mutants; 5) choose mutants predicted by PROPKA (see below) to significantly (> 3 units) decrease the pKa of the catalytic
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residues. All the mutants were modeled and further utilized to calculate the pKa values of catalytic residues and relevant mutations in the same way as that of the wild-type Amy7C.
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All the molecular docking and structural analysis of the enzyme-substrate interactions were performed with MOE and PyMOL software. 2.3. Site-directed mutagenesis
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The recombinant plasmids to produce the mutant α-amylases were constructed using QuikChange® II XL site-directed mutagenesis kit (Agilent Technologies Inc., Shanghai,
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China). As shown in Table A1, three pairs of primers R172K-S and R172K-A, A270K-S and A270K-A, N271H-S and N271H-A were used for single mutants R172K, A270K and N271H, respectively. The primers A270KN271H-S and A270KN271H-A were used to
construct double mutant A270K/N271H. The double mutant R172K/N271H was
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constructed using the recombinant plasmid of single mutant N271H as template by the
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primer pairs of R172K-S and R172K-A. The PCR conditions were: 95 oC for 3 min,
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followed by 30 cycles of 98 °C 10 s, 68 oC 5 min 30 s, and finally 72 °C for 10 min. PCR
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products were digested with 10 U Dpn I enzyme to remove the template plasmid and were
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then transformed into the competent E. coli XL1-Blue cells. Transformed cells were
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selected on the basis of their ampicillin resistance and the recombinant plasmids were confirmed by DNA sequencing.
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2.4. Determination of kinetic parameters of purified α-amylases
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All the recombinant wild-type and mutant α-amylases were expressed, purified and analyzed as described in our previous work [23]. Alpha-amylase activity was quantitatively
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assayed by the dinitrosalicylic acid method (DNS) [27]. The activities of the α-amylases were assayed with 1 μg α-amylase per milliliter at their optimal temperatures and pH values using 0.5% (w/v) soluble starch (Sigma) in 50 mM phosphate-citrate buffer for 5 min. One unit of α-amylase activity was defined as the amount of enzyme required to release 1 μmol 9
reducing sugars per minute under test conditions. The kinetic parameters were calculated from the Lineweaver-Burk plots.
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2.5. Characterization of purified α-amylases Aliquots (50 μL) of diluted enzymes were mixed with 450 μL 1% (w/v) soluble starch
(Sigma company, Shanghai, China) at different pH and incubated for 5 min at 65 oC. The buffer systems were 50 mM phosphate citrate (pH 2.5-6.0) and 50 mM sodium phosphate
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(pH 6.0-8.0) with pH intervals of 0.5 pH units. All the buffer solutions were corrected for
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the temperature dependence on pH to ensure the expected pH values for measuring at the
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assay temperatures. The relative activities of α-amylases against the pH values were used to
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characterize the pH-activity profile. For the temperature-activity profiles, 50 μL diluted
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enzymes were mixed with 450 μL of 1% (w/v) soluble starch in 50 mM phosphate citrate
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buffer (pH 6.5), and then the mixtures were incubated at temperatures from 50 oC to 80 oC for 5 min. The temperatures were created by a gradient thermocycler (T100™ Thermal
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Cycler, Bio-Rad, USA), consisting of 50oC, 50.7oC, 52.8oC, 56.1oC, 59.6oC, 63.2oC, 66.8oC,
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70.3oC, 73.9oC, 77.2oC, 79.3oC and 80oC.
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3. Results and Discussion 3.1. Rational design based on decreasing pKa values of catalytic residues As shown in Fig.1, the catalytic triad residues for general acid/base hydrolysis in the predicted structure of Amy7C, Asp174, Glu206 and Asp276 serving as the respective 10
nucleophile, proton donor and transition state stabilizer residues, were completely superimposed with that of 1BAG, and so did the hydrogen bonds formed with the
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glucosidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the non-reducing end glucose residue of the substrate, maltopentaose). Within the range of 4.5 Å of the side chain of Asp174, there were 38 atoms belonging to the substrate maltopentaose and side chains of Tyr60, Val97, His100, Arg172 and Ala175. As for Glu206, there were 35 atoms
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being of maltopentaose and Arg172, Ala175 and Leu208. As for Asp276, there were 32
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atoms pertaining to maltopentaose and Ser265, His266 and Asn271. Among the above nine
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neighboring amino acid residues, only Arg172, Ala270 and Asn271 stabilize the structural
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integrity of catalytic triad and substrate by direct side-chain hydrogen bonding (Fig. 2). So,
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theoretical structures of all the 57 possible single mutants at Arg172, Ala270 and Asn271
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sites (19 per site) and wild-type Amy7C were constructed and further utilized to calculate
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the pKa values of catalytic residues and relevant mutations. (Fig. 1), (Fig. 2)
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As shown in Table 1, three single mutants R172K, A270K and N271H showed obvious lowered pKa value for at least one catalytic residue (See Tables A2-A7 for detailed
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environmental perturbations causing the variations in pKa values based on PROPKA analysis). N271H decreased the pKa value of Asp174, both A270K and R172K reduced the pKa value of Glu206. What is worth noting, pKa of the nucleophile Asp174 (3.68) was lower than that of the acid/base catalyst Glu206 (7.68) in N271H, while it is completely 11
opposite in the wild-type, in which the pKa value of nucleophile Asp174 (8.05) was higher than that of acid/base catalyst Glu206 (6.14). This situation is similar to the naturally
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occurring GH 57 family α-amylases, represented by the Thermococcus litoralis 4-α-Glucanotransferase (TLGT) [28]. The Glu123 and Asp214 residues of TLGT act as respective catalytic nucleophile and acid/base catalyst, whose pKa values are 8.80 and
12.15 when calculated using the same PROPKA method here based on the acarbose bound
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structure, PDB ID: 1k1y [29]. These observations may indicate a plasticity of catalytic roles
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of Asp and Glu in the amylases, however more study is still required to make clear the
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relationship between their pKa shifts and catalytic role.
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(Table 1)
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In considering the cumulative effect of point mutations [30], two double mutants,
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A270K/N271H and R172K/N271H were also computationally modeled and evaluated. As shown in Table 1, the pKa values of three catalytic residues in A270K/N271H were nearly
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the same as that of N271H, while R172K/N271H showed very similar pKa values of
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catalytic residues to that of R172K. The predicted pKa values of mutated residues in double mutants were the same as that in single mutants.
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3.2. Characterization of purified recombinant α-amylases The recombinant wild-type and mutant α-amylases were expressed using E. coli JM109 as host strains and were further purified to homogeneity (>95% purity) by Ni-NTA chromatography [23]. There was no significant difference between the macroscopic 12
properties of the wild-type Amy7C and its mutants during the purification and characterization processes. All the mutants showed similar typical but narrower bell-shaped
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pH-activity profiles and shifted the pH-activity profiles toward acidity with decreased optimum pH values, in comparison to the wild type (Figs. 3&A1). The wild type showed
the maximum enzymatic activity at pH 6.5 and kept more than 75% of the maximum activity between pH 4.5 and pH 7.5. N271H reduced the optimum pH by 1.5 units and
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shifted the pH-activity range (>75% maximum activity) to pH 4.0-6.5. The combination of
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A270K and N271H further decreased the optimum pH to 4.5 and shifted the pH range (>75%
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maximum activity) at the acidic limb to pH 3.5. However, the pH optimum and pH range
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(>75%) of R172K/N271H fell in between that of their deconvolved single mutants, R172K
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and N271H.
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Mutations of R172K, A270K and N271H were sufficient to markedly shift pH-activity profiles and to increase the relative activity at acidic pH values (Table 2), while these
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rationally designed mutations here distributed within the range of 4.5 Å to the active site
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residues (Figs. 1&2b). These substitutions and their distances from the catalytic residues seemed to be different from the experimental results of Neilson et al. [6, 14] and Liu et al.
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[7], but were partially in line with the structural investigation of Sajedi et al. [17] Neilson et al. changed the pH-activity profiles of Bacillus α-amylase Ba2 by neutral → neutral and neutral → charged mutations at positions within 7-18 Å of the active site residues [14]. Liu et al. [7] reported two acid-stabilizing mutations L134R and S320A that located on the 13
surface and far away from the active site. Sajedi et al. performed a comparative structural investigation between a homology model of Ca2+-independent α-amylase from Bacillus sp.
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KR-8104 (KRA) with similar acidic pH-profile to the wild-type Amy7C and the crystal structure of Bacillus licheniformis BLA (PDB ID: 1BLI), then observed that substitutions with altered charge and decreased hydrophobic effects around the catalytic nucleophile of
KRA correspond thoroughly with the acidic pH-profile, and further speculated that these
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substitutions compared to the BLA might affect the pKa values of catalytic residues and
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consequently shift its pH profile [17]. However, all the observed 26 amino acid
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substitutions located 7.4-25.6 Å from the catalytic nucleophile (Asp231 numbering in
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KRA), which are much longer than the distance of less than 4.5 Å in this study, and neither
(Fig. 3)
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of the substitutions was the same as the three mutations of Amy7C reported here.
All the mutants except A270K performed the best activities at the same experimental
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temperature of 63.2oC as the wild-type with very similar temperature-activity profiles (Figs.
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4&A2). A270K enhanced the optimum temperature by 7.1oC, while the double mutant A270K/N271H did not inherit this thermostability improvement. To the best of our
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knowledge, this is the first report of the simultaneous improvement of optimum temperature (7.1oC increase) and optimum pH (1 unit reduction) achieved by only one single point mutation. The optimum temperature increase of 7.1oC is also one of the best ever accessed by introducing up to two point mutations into the α-amylases [23]. 14
(Fig. 4) 3.3. Comparison of kinetic parameters of purified α-amylases
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All the wild-type and mutant α-amylases displayed classic Michaelis-Menten kinetics using soluble starch as substrate under respective pH optima and temperature conditions. As shown in Table 2, compared to the wild-type, all three single mutants decreased the
turnover numbers (kcat) and catalytic efficiency (kcat/Km) values, while N271H reduced both
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the kcat and Km to nearly the same extent, bringing out a moderately changed kcat/Km value
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of 93% of the wild-type (see Fig. A3 for Lineweaver-Burk plots). A270K decreased the
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substrate affinity (1.35 fold Km), while both N271H and R172K increased the affinity for
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soluble starch (lower Km values). However, the double mutant A270K/N271H decreased
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the Km by 58% and increased the kcat by 84%, resulting in about 3.94-fold increase of
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catalytic efficiency (Table A8, see Supplementary materials). Up to date, the best acidic engineering result of α-amylase was achieved by a double
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mutant BLA (L134R/S320A), which decreased the optimum pH of BLA by 2 units and
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shifted the stable range pH from pH 5.5-7.0 to pH 4.0-6.5 [13]. The engineering of Ca-independent α-amylase first reported here has also successfully decreased the optimum
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pH by 2 unit and shifted stable range pH from 4.5-7.5 to pH 3.5-6.5. Compared to the previously reported Bacillus sp. KR-8104 acidic and Ca-independent α-amylase [31], which was optimally active at pH 4.0-6.0 and has kcat of 54 s-1 and kcat/ Km of 146 g-1.L.s-1, A270K/N271H showed better performance with optimum pH of 4.5, kcat of 2319.41±66.51 15
s-1 and kcat/ Km of 1878.83 g-1.L.s-1, which are among the best parameters ever reported [32]. (Table 2)
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3.4. Structural insight into acidic adaptation of mutant α-amylases Asn271 follows the α-helix harboring the transition state stabilizer residue, Asp267. As shown in Figs. 1c&5a the predicted active sites of wild-type and N271H mutant, the side
chain of His271 adopted a rotamer pointing away from the active site cleft and more
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solvent accessible than the Asn271, and eliminated the hydrogen bond Asn271↔ Glc2 of
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maltopentaose (Glc2 represents the second glucose unit in the substrate of maltopentose
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from the reducing end, the same below). Meanwhile, the N271H retained the hydrogen
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bonding network mediated by the substrate: Asp174 ↔ Glc3 of maltopentaose ↔ Glu206,
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and the hydrogen bond formed by main chain of His271 with catalytic Asp267 (Figs. 1b,
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1c&2a, Tables A2&A3). The local hydrogen bonding network interactions influenced dramatically the pKa values of catalytic residues [33]. With the substitution, the pKa values
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of Asp174, Glu206 and Asp267 were shifted to 3.68, 7.28 and 4.57 from 8.05, 6.14 and
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2.70, respectively (Tables 1, A2, A3&A4). The pKa values of proton donor (Asp 174) and nucleophile (Glu 206) in N271H were far lower than that of wild-type Amy7C. This would
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be the right reason for the shift of pH-activity profile toward acidity (Fig. 2&A2). In addition, the elimination of the acid-labile side chain amide group of Asn and formation of a stronger α-helix capping by the imidazole group of His may also benefit the acidic adaptation of N271H. 16
(Fig. 5) Ala270 is the last residue of the α-helix harboring the transition state stabilizer residue
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Asp267 in the predicted models of Amy7C and its mutants. The positively charged side chain amino group of Lys270 was deeply buried in a “π-cavity”, that was surrounded by five amino acid residues, Trp15, Tyr269, Phe302, Pro305 and Phe315 in A270K (Fig. 5b). The distance of less than 4.5 Å between the cationic moiety (side chain) of Lys270 and the
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faces of aromatic rings of “π-cavity” ensures considerable cation-π interactions [34], which
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would compensate the impairment by the small and hydrophobic (Ala) to the bulky and
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hydrophilic (Lys) substitution. Meanwhile, the location in random coils of Trp15, Pro305
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and Phe317 and the fulfillment of the cavity surrounding the A270 in the wild-type Amy7C
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by the bulky and hydrophilic side-chain of Lys270 may also contribute to the tolerance of
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the substitution. A structure-based multiple sequence alignment analysis showed that the naturally occurring residues at A270 position of α-amylases include arginine,
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proline, aspartic acid, threonine (not shown). For example, there is a hydrophobic and
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bulky amino acid, proline, in the counterpart of Lys270 of mutant A270K in naturally occurring thermostable α-amylases from the Bacillus licheniformis BLA (PDB ID: 1BLI)
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and Bacillus amyloliquefaciens BAA (PDB ID: 3BH4). Especially, another positively charged side chain amino group of Arg is also deeply buried in a “π-cavity” in the Alteromonas haloplanctis alpha-amylase AHA (PDB ID: 1AQH), Aspergillus oryzae alpha-amylase TAKA (PDB ID: 2TAA) and pig pancreatic alpha-amylase PPA (PDB ID: 17
3L2L). These observations may explain why the optimum temperature of A270K was increased by 7.1oC and the temperature-activity profile was shifted to higher temperatures
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(Figs. 4&A2). Because the electrostatic interactions between charged and polar amino acids can be felt at a significant distance, the positively charged Lys270 affected the pKa values
of active site residues in the positively charged environment [14]. The predicted pKa value of Asp174 was decreased by 0.03, while Glu206 and Asp267 were increased by respective
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0.04 and 2.01 in comparison to that of the wild-type (Table 1). Among the pKa changes, all
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the 0.03 decrease of Asp174, 0.04 increase of Glu206 and 2.61 increase of Asp267 came
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from coulombic interactions (Tables A2, A3&A4). These pKa shifts probably explain the
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one unit decrease of the optimum pH, the acidic shift of the acidic and basic limbs of
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pH-activity profile, and the alterations of kcat, Km values of A270K (Table 2).
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Arg172 is the third residue of fourth β stand of TIM barrel in the interior domain A, and is the antepenultimate residue before the nucleophile residue Asp174. Arg172 is the most
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conserved residue other than the three catalytic residues in α-amylase family, to which the
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only naturally occurring substitution is Lysine [35, 36]. We unexpectedly identified this mutation by only structure-based pKa analysis. The side chain of Arg 172 is involved in a
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hydrogen bonding network: Asp174↔Arg172↔Glc3 of maltopentaose (Figs. 1&5c). However, the side chain of Lys172 does not participate in any hydrogen bonding. The loss of hydrogen bonds by Lys172 made Asp174 more solvent accessible and higher pKa value (Table 1). The loss of hydrogen bond between Glc3 and Lys172 would strengthen the 18
hydrogen bond between Glu206 and Glc3, which decreased the pKa value of Glu206 (Tables 1, A3&A5). These were in good accordance with the environmental perturbations
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changing the pKa value of D174 site in R172K based on PROPKA analysis results, which divide the pKa increase of 3.72 (11.77 minus 8.05) into 0.16 desolvation effects, 0.09
unfavorable electrostatic reorganization energies, 1.52 side-chain hydrogen-bond interactions, and 2.03 Coulombic interactions (Table A2). The pKa decrease of 2.38 of
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Glu206 consists of 0.63 desolvation effects, 0.65 unfavorable electrostatic reorganization
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energies, -1.84 side-chain hydrogen-bond interactions, -0.51 backbone hydrogen-bond
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interactions, and -1.34 Coulombic interactions (Table A3). Such pKa value reduction of
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proton donor (Glu 206) and increase of nucleophile (Asp 174) would shift the acidic limb
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to more acidic values and the basic limb to more alkaline values for mutant R172K.
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Experimental results showed that R172K decreased the optimum pH by 0.5 unit and shifted the basic limb toward more acidity, but almost did not shift the acidic limb (Fig. 3&Table
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2). Meanwhile, mutant R172K decreased the kcat (4%), Km (38%) and kcat/Km (11%) values
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(Table 2). These results are consistent with previous mutational study on Bacillus stearothermophilus α-amylase [35], of which the counterpart R232K mutation resulted in
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lowered specific activity (around 12% of parental enzyme) but much broader optimum pH for activity. Numerous studies have shown strong non-additive effects when combining stabilizing mutations, especially likely for mutations in close proximity and mutations that would force 19
backbone movement, while some observed that stabilizing mutations could contribute independently to the overall stability of proteins [37, 38]. This study showed a cumulative
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effect between mutations shifting the pH-activity profiles in double mutant A270K/N271H (Table 2 and Fig. 3), suggesting relative independence between Lys 270 and His 271 (Figs.
5a, 5b&6a). However, A270K/N271H increased the catalytic activity greatly without
inheriting the thermostability of A270K substitution (Table 2). These results suggested an
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intricate reorientation of active site residues, substrate and a more optimized catalysis
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process of A270K/N271H, as evidenced by the alteration of kcat, Km values and substrate
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hydrolysis pattern (Table 2&Fig. A4).
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Double mutant R172K/N271H displayed moderate changes of pH-activity profile,
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optimum pH and enzymatic parameters in comparison to parental single mutations (Fig. 3
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&Table 2), despite the pKa values of D174, E206, D267, K172 and H271 of R172K/N271H were nearly the same as that in respective single mutants (Table 1), as indicated by their
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similar interactions around the mutations (Figs. 1c, 1e, 1g, 5a, 5c&6b, Tables A2-A7). In
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contrast to A270K/N271H, the additional incorporation of R172K mutation neither improved the acidic adaption property, nor increased the catalytic properties, compared to
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N271H. In addition, the simultaneous combination of three mutations offset the impact of each other, yielding a worse triple mutant (<5% activity, optimum pH=4.5) than R172K/N271H (data not shown). Obviously, the underlying structural basis for obviation of “cumulative effect” of R172K/N271H and triple mutant requires further investigation in the 20
future. (Fig. 6)
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4. Conclusions The acidic adaptation of B. subtilis Ca-independent α-amylase was for the first time
successfully engineered by rationally decreasing the pKa values of catalytic residues through mutations located within 4.5 Å of active site. All mutants enhanced acidic
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adaptation with decreased pH optima and shifted pH-activity profiles toward lower pH
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values. A270K/N271H showed 2 pH units decrease in optimum pH and about 3.94-fold
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increase of catalytic efficiency, yielding the best previously unreported Ca-independent and
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acidic α-amylase. Protein engineering of α-amylase for acidic adaptation here provides a
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successful example of the extent to which mutations near active site and computational
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models can be used for industrial enzyme improvements.
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Conflict of Interest
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The authors have no conflicts of interest.
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Acknowledgements This work was supported by grants from the Major Program of Natural Science Foundation of Guangxi [grant number 2016GXNSFEA380003], the Guangxi BaGui Scholars of Guangxi Zhuang Autonomous Region of China, the Chinese National Basic Research 21
Program (“973”)[grant 2009CB724703] and National Science and Technology Support
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Program [grant 2007BAD75B05].
Appendix A. Supplementary data
Additional experimental material and results, including olignonucleotide primers used for site-directed mutagenesis, normalized enzymatic parameters, normalized pH-activity
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profiles, normalized temperature-activity profiles and HPLC analysis of hydrolysis
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products of soluble starch by purified recombinant wild-type and mutant α-amylases.
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References [1] M. Vihinen, P. Mantsala, Microbial amylolytic enzymes, Crit Rev Biochem Mol Biol, 24 (1989) 329-418. [2] M. Violet, J.C. Meunier, Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase, Biochem J, 263 (1989) 665-670.
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[3] S. Sivaramakrishnan, D. Gangadharan, K.M. Nampoothiri, C.R. Soccol, A. Pandey, α-amylases from microbial sources- An overview on recent development, Food Technol Biotechnol, 44 (2006) 173-184.
[4] W.D. Crabb, J.K. Shetty, Commodity scale production of sugars from starches, Curr Opin Microbiol, 2 (1999) 252-256.
[5] A. Pandey, P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh, R. Mohan, Advances in microbial amylases, Biotechnol Appl Biochem, 31 ( Pt 2) (2000) 135-152.
[6] J.E. Nielsen, L. Beier, D. Otzen, T.V. Borchert, H.B. Frantzen, K.V. Andersen, A. Svendsen, Electrostatics in the active site of an alpha-amylase, Eur J Biochem, 264 (1999) 816-824.
[7] Y.H. Liu, F.P. Lu, Y. Li, J.L. Wang, C. Gao, Acid stabilization of Bacillus licheniformis alpha amylase through
U
introduction of mutations, Appl Microbiol Biotechnol, 80 (2008) 795-803.
N
[8] Q.G. Zhang, Y. Han, H.Z. Xiao, Microbial alpha-amylase: A biomolecular overview, Process Biochemistry, 53 (2017) 88-101.
A
[9] R. Priyadharshini, P. Gunasekaran, Site-directed mutagenesis of the calcium-binding site of alpha-amylase of Bacillus licheniformis, Biotechnol Lett, 29 (2007) 1493-1499.
M
[10] M. Ghollasi, K. Khajeh, H. Naderi-Manesh, A. Ghasemi, Engineering of a Bacillus alpha-amylase with improved thermostability and calcium independency, Appl Biochem Biotechnol, 162 (2010) 444-459. [11] T.B. Dey, A. Kumar, R. Banerjee, P. Chandna, R.C. Kuhad, Improvement of microbial alpha-amylase
D
stability: Strategic approaches, Process Biochemistry, 51 (2016) 1380-1390. [12] A. Shaw, R. Bott, A.G. Day, Protein engineering of alpha-amylase for low pH performance, Curr Opin
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Biotechnol, 10 (1999) 349-352.
[13] Y.H. Liu, F.P. Lu, Y. Li, X.B. Yin, Y. Wang, C. Gao, Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600, Appl Microbiol Biotechnol, 78 (2008) 85-94.
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[14] J.E. Nielsen, T.V. Borchert, G. Vriend, The determinants of alpha-amylase pH-activity profiles, Protein Eng, 14 (2001) 505-512.
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[15] C. Bessler, J. Schmitt, K.H. Maurer, R.D. Schmid, Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity, Protein Sci, 12 (2003) 2141-2149. [16] A. Sharma, T. Satyanarayana, Microbial acid-stable α-amylases: Characteristics, genetic engineering and applications, Process Biochemistry, 48 (2013) 201-211.
A
[17] R.H. Sajedi, M. Taghdir, H. Naderi-Manesh, K. Khajeh, B. Ranjbar, Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+-independent alpha-amylase with acidic pH-profile, J Biochem Mol Biol, 40 (2007) 315-324. [18] G. Davies, B. Henrissat, Structures and mechanisms of glycosyl hydrolases, Structure, 3 (1995) 853-859. [19] Z. Fujimoto, K. Takase, N. Doui, M. Momma, T. Matsumoto, H. Mizuno, Crystal structure of a catalytic-site mutant alpha-amylase from Bacillus subtilis complexed with maltopentaose, J Mol Biol, 277
23
(1998) 393-407. [20] Y. Matsuura, M. Kusunoki, W. Harada, M. Kakudo, Structure and possible catalytic residues of Taka-amylase A, J Biochem, 95 (1984) 697-702. [21] J.E. Nielsen, T.V. Borchert, Protein engineering of bacterial alpha-amylases, Biochim Biophys Acta, 1543 (2000) 253-274.
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[22] J. Kyte, Mechanism in protein chemistry, Garland, New York, 1995. [23] C. Wang, R. Huang, B. He, Q. Du, Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis, BMC Bioinformatics, 13 (2012) 263.
[24] C. Wang, R. Huang, Q. Wang, N. Shen, D. Chen, Z. Li, Z. Huang, Alpha-amylase truncated body and application thereof. CN Patent 201210079326 [P]. 2012-03-23.
[25] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-MODEL: homology modelling of protein structures and complexes, Nucleic Acids Res, 46 (2018) W296-W303.
[26] T. Schwede, J. Kopp, N. Guex, M.C. Peitsch, SWISS-MODEL: An automated protein homology-modeling
U
server, Nucleic Acids Res, 31 (2003) 3381-3385.
N
[27] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal Chem, 31 (1959) 426-428.
A
[28] H. Imamura, S. Fushinobu, M. Yamamoto, T. Kumasaka, B.S. Jeon, T. Wakagi, H. Matsuzawa, Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor, J Biol
M
Chem, 278 (2003) 19378-19386.
[29] D.C. Bas, D.M. Rogers, J.H. Jensen, Very fast prediction and rationalization of pKa values for protein-ligand complexes, Proteins, 73 (2008) 765-783.
D
[30] B.W. Matthews, Structural and genetic analysis of protein stability, Annu Rev Biochem, 62 (1993) 139-160.
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[31] A. Salimi, F. Yousefi, M. Ghollasi, S. Daneshjou, H. Tavoli, S. Ghobadi, K. Khajeh, Investigations on possible roles of C-terminal propeptide of a Ca-independent alpha-amylase from bacillus, J Microbiol Biotechnol, 22 (2012) 1077-1083.
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[32] I. Schomburg, A. Chang, D. Schomburg, BRENDA, enzyme data and metabolic information, Nucleic Acids Res, 30 (2002) 47-49.
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[33] H. Li, A.D. Robertson, J.H. Jensen, Very fast empirical prediction and rationalization of protein pKa values, Proteins, 61 (2005) 704-721. [34] J.P. Gallivan, D.A. Dougherty, Cation-pi interactions in structural biology, Proc Natl Acad Sci U S A, 96 (1999) 9459-9464.
A
[35] M. Vihinen, P. Ollikka, J. Niskanen, P. Meyer, I. Suominen, M. Karp, L. Holm, J. Knowles, P. Mantsala, Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues, J Biochem, 107 (1990) 267-272. [36] S. Janecek, How many conserved sequence regions are there in the α-amylase family?, Biologia, 57 (2002) 29-41. [37] M. Matsumura, S. Yasumura, S. Aiba, Cumulative effect of intragenic amino-acid replacements on the
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thermostability of a protein, Nature, 323 (1986) 356-358. [38] L. Serrano, A.G. Day, A.R. Fersht, Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability, J Mol Biol,
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233 (1993) 305-312.
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Figure captions
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Fig.1 2D representation of the alpha-amylase-maltopentaose complexes: (a) Bacillus subtilis alpha-amylase BSUA (PDB ID: 1BAG); (b) Amy7C; (c) N271H; (d) A270K; (e) R172K; (f) A270KN271H; (g) R172KN271H. The dotted outline surrounding the ligand denotes the proximity contour, meaning distance to the active site interior. The blue
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smudges that are drawn behind some of the ligand atoms denote ligand solvent exposure,
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indicating the extent of solvent exposure. The turquoise discs that are drawn behind some
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of the residues denote receptor solvent exposure, indicating the difference in solvent
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exposure due to the presence of the ligand. The catalytic residues, Asp176, Glu208 and
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Asp269 acting as respective nucleophile, general acid/base and transition state stabilizer,
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and the hydrogen bonds formed with the glucosidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the non-reducing end glucose residue of the substrate) in 1BAG (a)
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are well superimposed in Amy7C (b) and its mutants (c-g), indicating a similar catalytic
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mechanism. The 2D molecular graphics were generated by MOE software under the default
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settings (Molecular Operation Environment, version2008.10).
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Fig. 2 Mutation positions in Amy7C. Domains A, B and C of Amy7C are shown in light
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grey, cyan and yellow respectively. The maltopentaose substrate is shown in purple sticks.
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The active site amino acid residues, Asp174, Glu206 and Asp267, which serve as respective nucleophile, catalytic acid/base and transition state stabilizer, are shown in red ball and stick form. The chosen mutation sites of Arg172, Ala270 and Asn271 are indicated in green ball and stick form. The dot lines represent hydrogen bonds. (a) overall distribution of
27
mutations in cartoon form; (b) close-up view of mutations in molecular surface form, the
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hydrophobic and hydrophilic residues are colored in green and blue respectively.
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Fig.3 pH-activity profiles for the wild-type Amy7C and its mutants. The activities of
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α-amylases were evaluated at protein concentrations of approximately 1 μg.mL−1 except
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R172K (10 μg.mL−1) and R172K/N271H (5 μg.mL−1) with 5 g/L soluble starch at the
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indicated pH under their optimum temperatures for 5 min. The buffer solutions were 50
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mM phosphate citrate (pH 2.5-6.0) and 50 mM sodium phosphate (pH 6.0-8.0). All the
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α-amylases were assayed at 63.2oC except A270K at 70.3oC. One activity unit (U) was defined as the amount of enzyme required to release 1 μmol dextrose equivalents per
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minute under test conditions. All the assays were implemented in triplicate and represented
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by mean ± standard deviations.
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Fig.4 Temperature-activity profiles for the wild-type Amy7C and its mutants. The activities
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of α-amylases were evaluated at protein concentrations of approximately 1 μg.mL−1 except
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R172K (10 μg.mL−1) and R172K/N271H (5 μg.mL−1) with 5 g/L soluble starch at the indicated temperatures under their optimum pH values for 5 min. Amy7C, R172K, A270K
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and R172K/N271H, N271H and A270K/N271H were assayed in pH 6.5, pH 6.0, pH 5.5,
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pH 5.0 and pH 4.5, respectively. One activity unit (U) was defined as the amount of
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enzyme required to release 1 μmol dextrose equivalents per minute under test conditions. All the assays were implemented in triplicate and represented by mean ± standard
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deviations.
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Fig. 5 Superimpositions of wild-type Amy7C and single mutants N271H (a), A270K (b)
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and R172K (c). The wild-type residues are shown in grey (C atom), blue (N atom) and red
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(C atom) spheres and sticks. The mutations are shown in green spheres. The maltopentaose substrate is shown in purple sticks. Hydrogen bonds existing only in wild-type and in both
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wild-type and mutants are shown in blue and black dashed lines, respectively. The
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orange sticks.
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“π-cavity” amino acid residues Trp15, Tyr269, Phe302, Pro305 and Phe315 are shown in
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Fig. 6 Superimpositions of wild-type Amy7C and double mutants A270K/N271H (a) and R172K/N271H (b). The wild-type residues are shown in grey (C atom), blue (N atom) and
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red (C atom) spheres and sticks. The mutations are shown in green spheres. The
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maltopentaose substrate is shown in purple sticks. Hydrogen bonds existing only in wild-type and in both wild-type and mutants are shown in blue and black dashed lines,
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respectively. The “π-cavity” amino acid residues Trp15, Tyr269, Phe302, Pro305 and Phe315 are shown in orange sticks.
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Table 1. Predicted pKa values of catalytic residues and mutations of wild-type Amy7C and its mutants pKa a
Enzyme
Wild-type
8.05
6.14
2.70
-
18.24 -
-
N271H
3.68
7.28
4.57
-
18.04 -
6.62
A270K
8.02
6.18
4.71
-
18.22 3.81
-
R172K
11.77
3.76
2.86
14.31 -
A270K/N271H 3.68
7.28
4.59
-
R172K/N271H 11.23
3.58
2.70
14.24 -
-
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18.02 3.82 -
-
6.62 6.63
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pKa values were calculated by the PROPKA sever (http://propka.ki.ku.dk/) using the
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a
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D174 b E206 b D267 b K172 R172 K270 H271
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theoretical PDB files of the wild-type Amy7C and its mutants in protein-ligand complex format. b D174, E206 and D267 were the catalytic residues playing the roles as nucleophile,
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catalytic acid/base, and transition state stabilizer, respectively. “-” indicates no value.
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Table 2. Enzymatic parameters of wild-type and mutant α-amylases pHopt a
Enzyme
Topt(oC) a
pHpred b
Tpred b
pHopt b
Topt (oC) b
Molecular
Vmax d
Km d
Activity e
kcat d
kcat/Km d
(95% CI)
weight c
(U/mg)
(g/L)
(U/mg)
(s-1)
(L.g-1.s-1)
63.36-65.29
47575
1589.76±88.56
3.31±0.0.5
956.54±27.65
1260.55±70.22
380.33
4.99-5.32
60.74-64.32
47598
1016.96±33.97
2.28±0.03
698.09±16.42
806.75±26.95
353.71
A
(95% CI) 6.5
63.2
6.32
64.33
N271H
5.0
63.2
5.16
62.53
A270K
5.5
70.3
5.39
70.53
5.28-5.50
69.42-71.64
47632
905.08±70.01
4.47±0.07
477.94±9.49
718.51±55.57
159.74
R172K
6.0
63.2
5.55
64.11
5.45-5.65
63.01-65.21
47547
63.63±2.78
1.26±0.04
50.84±2.88
50.42±2.20
41.84
A270K/N271H
4.5
63.2
5.06
61.81
4.88-5.24
61.17-62.46
47655
2920.25±83.74
1.36±0.03
2296.84±122.65
2319.41±66.51
1878.83
R172K/N271H
5.5
63.2
5.73
62.61
5.48-5.98
61.63-63.60
47570
254.39±10.55
2.61±0.04
167.03±4.88
201.69±8.36
76.07
ED
PT
CC E a pH
opt
b pH
6.14-6.50
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Wild-type
and Topt were shown as the experimental pH and temperature optima, which were used to determine the kinetic parameters in this study.
pred,
Tpred, pHopt (95% CI) and Topt (95% CI) indicted the predicted mean pH optima, mean temperature optima, the 95% confidence intervals
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when the pH-activity and temperature-activity relationship data were fitted as Gaussian functions. c
Molecular weight were deduced from the protein sequence.
d
Vmax, Km, kcat and kcat/Km values were assayed with different concentrations of soluble starch (0.4 g/L-9 g/L) in 50 mM phosphate citrate (pH
2.5-6.0) and 50 mM sodium phosphate (pH 6.0-8.0) for 5 min under the optimum pH and temperature. The Vmax, Km, kcat values were averaged from
34
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A
CC E
PT
ED
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A
three independent experiments and shown as mean ± standard deviations. The kcat/Km values were calculated by dividing the means of kcat and
35
Km values. One activity unit (U) was defined as the amount of enzyme required to release 1 μmol dextrose equivalents per minute under test conditions, as described in “Material and methods”. e
Activity values were assayed at 5 g /L soluble starch in 50 mM phosphate citrate at optimum pH and
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A
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temperature values. All the experiments were carried out in triplicate.
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S1369-703X(18)30308-5 https://doi.org/10.1016/j.bej.2018.08.015 BEJ 7026
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
11-4-2018 25-8-2018 27-8-2018
Please cite this article as: Wang C-Hua, Liu X-Ling, Huang R-Bo, He B-Fang, Zhao MMing, Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by rational engineering of pKa values, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced acidic adaptation of Bacillus subtilis Ca-independent alpha-amylase by
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rational engineering of pKa values
Cheng-Hua Wang a, *, Xiao-Ling Liu a, Ri-Bo Huang b, *, Bing-Fang He c, Mou-Ming Zhao a a College
of Light Industry and Food Engineering, Guangxi University, Nanning 530004,
People’s Republic of China.
Key Laboratory of Non-Food Biomass and Enzyme Technology, National
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b State
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Engineering Research Center for Non-food Biorefinery, Guangxi Key Laboratory of
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of
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c
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Biorefinery, Guangxi Academy of Sciences, Nanning 530007, People’s Republic of China.
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Technology, Nanjing 211816, People’s Republic of China.
Cheng-Hua Wang, E-mail: [email protected]
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Xiao-Ling Liu, E-mail: [email protected]
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Ri-Bo Huang, E-mail: [email protected] Bing-Fang He, E-mail: [email protected]
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Mou-Ming Zhao, E-mail: [email protected]
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*Corresponding
authors.
Cheng-Hua Wang, Address: College of Light Industry and Food Engineering, 100 Daxue
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East Road, Nanning 530004, People’s Republic of China. Tel: +86-771-323-2874, Fax: +86-771-323-2874, E-mail: [email protected];
Ri-Bo Huang, Address: Guangxi Academy of Sciences, 98 Daling Road, Nanning 530007,
People’s Republic of China. Tel: +86-771-250-3902, Fax: +86-771-250-3902, E-mail:
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[email protected]
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Graphical abstract
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Highlights
Three mutations decreasing pKa values of catalytic residues of Amy7C were
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designed. Three single and two double mutants were constructed and characterized.
All mutants shifted pH optima and pH-activity profiles toward more acidic values.
N271H decreased pH optimum by 2 units without comprising the catalytic efficiency.
A270K/N271H increased catalytic efficiency by 3.94-fold at optimum pH of 4.5.
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Abstract
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The aim of this study was to rationally engineer the acidic adaptation of B. subtilis
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Ca-independent alpha-amylase (Amy7C) by decreasing the pKa values of catalytic residues
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through mutations at active site. Within 4.5 Å of three catalytic residues of Amy7C, three mutations R172K, A270K and N271H were identified by computational homology
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modeling and pKa prediction analyses. Five single and double mutants consisting of these
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three mutations were constructed and characterized. Compared to the wild-type, all mutants shifted the pH optima and pH-activity profiles toward lower pH values without comprising the thermostablity. Double mutants showed simultaneous accumulation of advantageous mutations. The best mutant, A270K/N271H showed 2 units decrease in optimum pH and
3
about 3.94-fold increase of catalytic efficiency. Structural analysis suggested that the improved acidic adaptation could be attributed to the decreased pKa values of catalytic
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nucleophile and proton donor residues. Protein engineering of α-amylase for acidic adaptation here provides a successful example of the extent to which mutations near active site and computational models can be used for industrial enzyme improvements.
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Keywords α-amylase, acidic adaptation, pKa, protein engineering, rational design
4
1. Introduction The most widely used commercial alpha-amylases (EC 3.2.1.1) in starch processing,
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represented by Bacillus licheniformis α-amylase (BLA), usually operate optimally at high temperature (95 oC for BLA) and neutral pH (about pH 6.0 for BLA) conditions, and require addition of calcium ions to sustain stability and/or activity [1-3]. To make the best
use of these α-amylases, starch slurry must be firstly adjusted from native pH 3.2-4.5 to pH
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5.8-6.2 for liquefaction and then back to pH 4.2-4.5 for the next saccharification step.
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Meanwhile, calcium ions must be added for liquefaction and then to remove salts from
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downstream products on a large scale [3-5]. Consequently, there is great interest in
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changing the pH-performance profiles toward acidity and reducing the dependence on Ca2+
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of α-amylases in terms of economics and feasibility [6-8]. However, because it has proved
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difficult to eliminate calcium dependence of conventional Ca-dependent α-amylases, it is superior to make such novel Ca-independent and acidic α-amylase by engineering the
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acidic adaptation of existing Ca-independent α-amylases [9, 10].
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Previous protein engineering endeavors have been directed to pH-activity profiles of α-amylases [11]. These include improving the activity and thermostability of BLA at strong
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acid pH values by directed evolution and rational design methods [6, 7, 12, 13], exploring the determinants of pH-activity profile of Bacillus subtilis Ba2 by rational design [14], and enhancing pH-performance and higher specific activity of B. subtilis BAA by directed evolution [15]. It was indicated that the determinants of pH-activity profiles of α-amylases 5
could be attributed to electrostatics due to charged groups, dynamic structural changes of active site and hydrophobic effects [6, 14, 16, 17]. To the best of our knowledge, all the
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protein engineering of pH-activity profiles were carried out on Ca2+-dependent α-amylases including BLA, Ba2 and BAA, while no acidic engineering of Ca2+-independent α-amylases has ever been reported [16].
As a typical glycoside hydrolase, α-amylase obeys the double-displacement retaining
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mechanism [18, 19]. Based on the following three facts of α-amylase: 1) the nucleophile
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and proton donor must remain deprotonated and protonated respectively for optimal
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catalysis [20]; 2) the titrations of nucleophile and proton donor determine the acidic and
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basic limbs of the pH-activity profile [21], and 3) the catalysis is expected to be limited at
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the low pH by the protonation of the nucleophile and at high pH by deprotonation of the
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hydrogen donor [22], it can be concluded that the pKa values of nuclophile and proton donor residues determine the catalysis process and pH-activity profile. It is a promising
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way to engineer the acidic adaptation of α-amylase by rationally decreasing the pKa values
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of catalytic general acid/base. Bacillus subtilis CN7 α-amylase (Amy7C) is a Ca-independent and acid-resistant
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liquefying α-amylase, which has a optimum pH of 6.5 and keeps more than 75% activity within the range of pH 4.5-7.5 [23]. The elimination of calcium addition and pH adjustment of Amy7C excels in specific applications such as simultaneous saccharification and fermentation (SSF) process and production of high-fructose corn syrup (HFCS) [24]. 6
However, the desire for a better enzyme with improved acidic pH adaptation and catalytic activity still remains. In this study, Amy7C was engineered toward enhanced acidic
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adaptation by rationally decreasing pKa values of catalytic residues through mutations near the active site.
2. Material and methods
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2.1. Materials
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Luria-Bertani (LB) media were purchased from Difco Laboratories (Detroit, USA). The
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recombinant plasmid pSA7C, which was constructed previously by cloning the truncated
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α-amylase gene (GenBank ID: JN980090) from B. subtilis CN7 (CCTCC M 2012061) into
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the plasmid pSE380 [23], was used as template for site-directed mutagenesis. The PCR
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primers were listed in Table S1. The host strains E. coli XL1-Blue (Agilent Technologies Inc., Shanghai, China) and JM109 (Promega Corporation, Shanghai, China) were employed
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for cloning and expression of the recombinant α-amylases, respectively. All other
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chemicals and reagents used in this study were acquired commercially and were of reagent grade unless otherwise stated.
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2.2 Rational design As previously described [23], 3-D structure of wild-type Amy7C was constructed by
homology modeling via the SWISS-MODEL server (http://swissmodel.expasy.org/) by using an experimentally determined X-ray structure of α-amylase from Bacillus subtilis complexed with maltopentaose (PDB ID: 1BAG) as template, which shares sequence 7
identity of 91% with Amy7C and has the most conservation of sequence match in the vicinity of the active site, ensuring sufficient accuracy to generate reliable three-dimensional protein structure models [25, 26]. The enzyme-ligand complex structure
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was built by replacing the apoenzyme coordinates of 1BAG by that of the Amy7C after the superimposition between 1BAG and Amy7C while retaining all the coordinates of ligands
in 1BAG, and then further optimized by docking and energy minimization in MOE software with the default settings (Molecular Operation Environment, version2008.10). The energy minimization process was confined to the atoms within 4.5 Å neighboring maltopentaose in the holo-form structure. The enzyme-substrate interactions of the
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optimized holo-form structure with maltopentaose were analyzed by using PyMOL
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software (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.).
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The pKa values of catalytic residues were predicted by using the PROPKA 3.1 server
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(http://propka.ki.ku.dk/). The mutations were designed by five steps: 1) select the amino acid residues within the range of 4.5 Å of three catalytic triad residues, Asp174, Glu206
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and Asp276, and the substrate maltopentaose in the wild-type Amy7C; 2) mutate the
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selected residues to the other 19 amino acid residues; 3) construct predicted structure for each single mutant by SWISS-MODEL and MOE; 4) calculate the pKa values of the
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catalytic residues and mutations in the wild-type and mutants; 5) choose mutants predicted by PROPKA (see below) to significantly (> 3 units) decrease the pKa of the catalytic
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residues. All the mutants were modeled and further utilized to calculate the pKa values of catalytic residues and relevant mutations in the same way as that of the wild-type Amy7C.
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All the molecular docking and structural analysis of the enzyme-substrate interactions were performed with MOE and PyMOL software. 2.3. Site-directed mutagenesis
8
The recombinant plasmids to produce the mutant α-amylases were constructed using QuikChange® II XL site-directed mutagenesis kit (Agilent Technologies Inc., Shanghai,
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China). As shown in Table A1, three pairs of primers R172K-S and R172K-A, A270K-S and A270K-A, N271H-S and N271H-A were used for single mutants R172K, A270K and N271H, respectively. The primers A270KN271H-S and A270KN271H-A were used to
construct double mutant A270K/N271H. The double mutant R172K/N271H was
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constructed using the recombinant plasmid of single mutant N271H as template by the
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primer pairs of R172K-S and R172K-A. The PCR conditions were: 95 oC for 3 min,
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followed by 30 cycles of 98 °C 10 s, 68 oC 5 min 30 s, and finally 72 °C for 10 min. PCR
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products were digested with 10 U Dpn I enzyme to remove the template plasmid and were
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then transformed into the competent E. coli XL1-Blue cells. Transformed cells were
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selected on the basis of their ampicillin resistance and the recombinant plasmids were confirmed by DNA sequencing.
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2.4. Determination of kinetic parameters of purified α-amylases
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All the recombinant wild-type and mutant α-amylases were expressed, purified and analyzed as described in our previous work [23]. Alpha-amylase activity was quantitatively
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assayed by the dinitrosalicylic acid method (DNS) [27]. The activities of the α-amylases were assayed with 1 μg α-amylase per milliliter at their optimal temperatures and pH values using 0.5% (w/v) soluble starch (Sigma) in 50 mM phosphate-citrate buffer for 5 min. One unit of α-amylase activity was defined as the amount of enzyme required to release 1 μmol 9
reducing sugars per minute under test conditions. The kinetic parameters were calculated from the Lineweaver-Burk plots.
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2.5. Characterization of purified α-amylases Aliquots (50 μL) of diluted enzymes were mixed with 450 μL 1% (w/v) soluble starch
(Sigma company, Shanghai, China) at different pH and incubated for 5 min at 65 oC. The buffer systems were 50 mM phosphate citrate (pH 2.5-6.0) and 50 mM sodium phosphate
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(pH 6.0-8.0) with pH intervals of 0.5 pH units. All the buffer solutions were corrected for
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the temperature dependence on pH to ensure the expected pH values for measuring at the
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assay temperatures. The relative activities of α-amylases against the pH values were used to
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characterize the pH-activity profile. For the temperature-activity profiles, 50 μL diluted
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enzymes were mixed with 450 μL of 1% (w/v) soluble starch in 50 mM phosphate citrate
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buffer (pH 6.5), and then the mixtures were incubated at temperatures from 50 oC to 80 oC for 5 min. The temperatures were created by a gradient thermocycler (T100™ Thermal
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Cycler, Bio-Rad, USA), consisting of 50oC, 50.7oC, 52.8oC, 56.1oC, 59.6oC, 63.2oC, 66.8oC,
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70.3oC, 73.9oC, 77.2oC, 79.3oC and 80oC.
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3. Results and Discussion 3.1. Rational design based on decreasing pKa values of catalytic residues As shown in Fig.1, the catalytic triad residues for general acid/base hydrolysis in the predicted structure of Amy7C, Asp174, Glu206 and Asp276 serving as the respective 10
nucleophile, proton donor and transition state stabilizer residues, were completely superimposed with that of 1BAG, and so did the hydrogen bonds formed with the
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glucosidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the non-reducing end glucose residue of the substrate, maltopentaose). Within the range of 4.5 Å of the side chain of Asp174, there were 38 atoms belonging to the substrate maltopentaose and side chains of Tyr60, Val97, His100, Arg172 and Ala175. As for Glu206, there were 35 atoms
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being of maltopentaose and Arg172, Ala175 and Leu208. As for Asp276, there were 32
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atoms pertaining to maltopentaose and Ser265, His266 and Asn271. Among the above nine
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neighboring amino acid residues, only Arg172, Ala270 and Asn271 stabilize the structural
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integrity of catalytic triad and substrate by direct side-chain hydrogen bonding (Fig. 2). So,
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theoretical structures of all the 57 possible single mutants at Arg172, Ala270 and Asn271
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sites (19 per site) and wild-type Amy7C were constructed and further utilized to calculate
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the pKa values of catalytic residues and relevant mutations. (Fig. 1), (Fig. 2)
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As shown in Table 1, three single mutants R172K, A270K and N271H showed obvious lowered pKa value for at least one catalytic residue (See Tables A2-A7 for detailed
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environmental perturbations causing the variations in pKa values based on PROPKA analysis). N271H decreased the pKa value of Asp174, both A270K and R172K reduced the pKa value of Glu206. What is worth noting, pKa of the nucleophile Asp174 (3.68) was lower than that of the acid/base catalyst Glu206 (7.68) in N271H, while it is completely 11
opposite in the wild-type, in which the pKa value of nucleophile Asp174 (8.05) was higher than that of acid/base catalyst Glu206 (6.14). This situation is similar to the naturally
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occurring GH 57 family α-amylases, represented by the Thermococcus litoralis 4-α-Glucanotransferase (TLGT) [28]. The Glu123 and Asp214 residues of TLGT act as respective catalytic nucleophile and acid/base catalyst, whose pKa values are 8.80 and
12.15 when calculated using the same PROPKA method here based on the acarbose bound
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structure, PDB ID: 1k1y [29]. These observations may indicate a plasticity of catalytic roles
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of Asp and Glu in the amylases, however more study is still required to make clear the
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relationship between their pKa shifts and catalytic role.
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(Table 1)
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In considering the cumulative effect of point mutations [30], two double mutants,
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A270K/N271H and R172K/N271H were also computationally modeled and evaluated. As shown in Table 1, the pKa values of three catalytic residues in A270K/N271H were nearly
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the same as that of N271H, while R172K/N271H showed very similar pKa values of
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catalytic residues to that of R172K. The predicted pKa values of mutated residues in double mutants were the same as that in single mutants.
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3.2. Characterization of purified recombinant α-amylases The recombinant wild-type and mutant α-amylases were expressed using E. coli JM109 as host strains and were further purified to homogeneity (>95% purity) by Ni-NTA chromatography [23]. There was no significant difference between the macroscopic 12
properties of the wild-type Amy7C and its mutants during the purification and characterization processes. All the mutants showed similar typical but narrower bell-shaped
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pH-activity profiles and shifted the pH-activity profiles toward acidity with decreased optimum pH values, in comparison to the wild type (Figs. 3&A1). The wild type showed
the maximum enzymatic activity at pH 6.5 and kept more than 75% of the maximum activity between pH 4.5 and pH 7.5. N271H reduced the optimum pH by 1.5 units and
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shifted the pH-activity range (>75% maximum activity) to pH 4.0-6.5. The combination of
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A270K and N271H further decreased the optimum pH to 4.5 and shifted the pH range (>75%
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maximum activity) at the acidic limb to pH 3.5. However, the pH optimum and pH range
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(>75%) of R172K/N271H fell in between that of their deconvolved single mutants, R172K
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and N271H.
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Mutations of R172K, A270K and N271H were sufficient to markedly shift pH-activity profiles and to increase the relative activity at acidic pH values (Table 2), while these
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rationally designed mutations here distributed within the range of 4.5 Å to the active site
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residues (Figs. 1&2b). These substitutions and their distances from the catalytic residues seemed to be different from the experimental results of Neilson et al. [6, 14] and Liu et al.
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[7], but were partially in line with the structural investigation of Sajedi et al. [17] Neilson et al. changed the pH-activity profiles of Bacillus α-amylase Ba2 by neutral → neutral and neutral → charged mutations at positions within 7-18 Å of the active site residues [14]. Liu et al. [7] reported two acid-stabilizing mutations L134R and S320A that located on the 13
surface and far away from the active site. Sajedi et al. performed a comparative structural investigation between a homology model of Ca2+-independent α-amylase from Bacillus sp.
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KR-8104 (KRA) with similar acidic pH-profile to the wild-type Amy7C and the crystal structure of Bacillus licheniformis BLA (PDB ID: 1BLI), then observed that substitutions with altered charge and decreased hydrophobic effects around the catalytic nucleophile of
KRA correspond thoroughly with the acidic pH-profile, and further speculated that these
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substitutions compared to the BLA might affect the pKa values of catalytic residues and
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consequently shift its pH profile [17]. However, all the observed 26 amino acid
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substitutions located 7.4-25.6 Å from the catalytic nucleophile (Asp231 numbering in
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KRA), which are much longer than the distance of less than 4.5 Å in this study, and neither
(Fig. 3)
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of the substitutions was the same as the three mutations of Amy7C reported here.
All the mutants except A270K performed the best activities at the same experimental
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temperature of 63.2oC as the wild-type with very similar temperature-activity profiles (Figs.
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4&A2). A270K enhanced the optimum temperature by 7.1oC, while the double mutant A270K/N271H did not inherit this thermostability improvement. To the best of our
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knowledge, this is the first report of the simultaneous improvement of optimum temperature (7.1oC increase) and optimum pH (1 unit reduction) achieved by only one single point mutation. The optimum temperature increase of 7.1oC is also one of the best ever accessed by introducing up to two point mutations into the α-amylases [23]. 14
(Fig. 4) 3.3. Comparison of kinetic parameters of purified α-amylases
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All the wild-type and mutant α-amylases displayed classic Michaelis-Menten kinetics using soluble starch as substrate under respective pH optima and temperature conditions. As shown in Table 2, compared to the wild-type, all three single mutants decreased the
turnover numbers (kcat) and catalytic efficiency (kcat/Km) values, while N271H reduced both
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the kcat and Km to nearly the same extent, bringing out a moderately changed kcat/Km value
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of 93% of the wild-type (see Fig. A3 for Lineweaver-Burk plots). A270K decreased the
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substrate affinity (1.35 fold Km), while both N271H and R172K increased the affinity for
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soluble starch (lower Km values). However, the double mutant A270K/N271H decreased
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the Km by 58% and increased the kcat by 84%, resulting in about 3.94-fold increase of
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catalytic efficiency (Table A8, see Supplementary materials). Up to date, the best acidic engineering result of α-amylase was achieved by a double
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mutant BLA (L134R/S320A), which decreased the optimum pH of BLA by 2 units and
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shifted the stable range pH from pH 5.5-7.0 to pH 4.0-6.5 [13]. The engineering of Ca-independent α-amylase first reported here has also successfully decreased the optimum
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pH by 2 unit and shifted stable range pH from 4.5-7.5 to pH 3.5-6.5. Compared to the previously reported Bacillus sp. KR-8104 acidic and Ca-independent α-amylase [31], which was optimally active at pH 4.0-6.0 and has kcat of 54 s-1 and kcat/ Km of 146 g-1.L.s-1, A270K/N271H showed better performance with optimum pH of 4.5, kcat of 2319.41±66.51 15
s-1 and kcat/ Km of 1878.83 g-1.L.s-1, which are among the best parameters ever reported [32]. (Table 2)
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3.4. Structural insight into acidic adaptation of mutant α-amylases Asn271 follows the α-helix harboring the transition state stabilizer residue, Asp267. As shown in Figs. 1c&5a the predicted active sites of wild-type and N271H mutant, the side
chain of His271 adopted a rotamer pointing away from the active site cleft and more
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solvent accessible than the Asn271, and eliminated the hydrogen bond Asn271↔ Glc2 of
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maltopentaose (Glc2 represents the second glucose unit in the substrate of maltopentose
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from the reducing end, the same below). Meanwhile, the N271H retained the hydrogen
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bonding network mediated by the substrate: Asp174 ↔ Glc3 of maltopentaose ↔ Glu206,
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and the hydrogen bond formed by main chain of His271 with catalytic Asp267 (Figs. 1b,
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1c&2a, Tables A2&A3). The local hydrogen bonding network interactions influenced dramatically the pKa values of catalytic residues [33]. With the substitution, the pKa values
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of Asp174, Glu206 and Asp267 were shifted to 3.68, 7.28 and 4.57 from 8.05, 6.14 and
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2.70, respectively (Tables 1, A2, A3&A4). The pKa values of proton donor (Asp 174) and nucleophile (Glu 206) in N271H were far lower than that of wild-type Amy7C. This would
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be the right reason for the shift of pH-activity profile toward acidity (Fig. 2&A2). In addition, the elimination of the acid-labile side chain amide group of Asn and formation of a stronger α-helix capping by the imidazole group of His may also benefit the acidic adaptation of N271H. 16
(Fig. 5) Ala270 is the last residue of the α-helix harboring the transition state stabilizer residue
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Asp267 in the predicted models of Amy7C and its mutants. The positively charged side chain amino group of Lys270 was deeply buried in a “π-cavity”, that was surrounded by five amino acid residues, Trp15, Tyr269, Phe302, Pro305 and Phe315 in A270K (Fig. 5b). The distance of less than 4.5 Å between the cationic moiety (side chain) of Lys270 and the
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faces of aromatic rings of “π-cavity” ensures considerable cation-π interactions [34], which
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would compensate the impairment by the small and hydrophobic (Ala) to the bulky and
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hydrophilic (Lys) substitution. Meanwhile, the location in random coils of Trp15, Pro305
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and Phe317 and the fulfillment of the cavity surrounding the A270 in the wild-type Amy7C
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by the bulky and hydrophilic side-chain of Lys270 may also contribute to the tolerance of
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the substitution. A structure-based multiple sequence alignment analysis showed that the naturally occurring residues at A270 position of α-amylases include arginine,
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proline, aspartic acid, threonine (not shown). For example, there is a hydrophobic and
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bulky amino acid, proline, in the counterpart of Lys270 of mutant A270K in naturally occurring thermostable α-amylases from the Bacillus licheniformis BLA (PDB ID: 1BLI)
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and Bacillus amyloliquefaciens BAA (PDB ID: 3BH4). Especially, another positively charged side chain amino group of Arg is also deeply buried in a “π-cavity” in the Alteromonas haloplanctis alpha-amylase AHA (PDB ID: 1AQH), Aspergillus oryzae alpha-amylase TAKA (PDB ID: 2TAA) and pig pancreatic alpha-amylase PPA (PDB ID: 17
3L2L). These observations may explain why the optimum temperature of A270K was increased by 7.1oC and the temperature-activity profile was shifted to higher temperatures
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(Figs. 4&A2). Because the electrostatic interactions between charged and polar amino acids can be felt at a significant distance, the positively charged Lys270 affected the pKa values
of active site residues in the positively charged environment [14]. The predicted pKa value of Asp174 was decreased by 0.03, while Glu206 and Asp267 were increased by respective
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0.04 and 2.01 in comparison to that of the wild-type (Table 1). Among the pKa changes, all
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the 0.03 decrease of Asp174, 0.04 increase of Glu206 and 2.61 increase of Asp267 came
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from coulombic interactions (Tables A2, A3&A4). These pKa shifts probably explain the
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one unit decrease of the optimum pH, the acidic shift of the acidic and basic limbs of
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pH-activity profile, and the alterations of kcat, Km values of A270K (Table 2).
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Arg172 is the third residue of fourth β stand of TIM barrel in the interior domain A, and is the antepenultimate residue before the nucleophile residue Asp174. Arg172 is the most
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conserved residue other than the three catalytic residues in α-amylase family, to which the
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only naturally occurring substitution is Lysine [35, 36]. We unexpectedly identified this mutation by only structure-based pKa analysis. The side chain of Arg 172 is involved in a
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hydrogen bonding network: Asp174↔Arg172↔Glc3 of maltopentaose (Figs. 1&5c). However, the side chain of Lys172 does not participate in any hydrogen bonding. The loss of hydrogen bonds by Lys172 made Asp174 more solvent accessible and higher pKa value (Table 1). The loss of hydrogen bond between Glc3 and Lys172 would strengthen the 18
hydrogen bond between Glu206 and Glc3, which decreased the pKa value of Glu206 (Tables 1, A3&A5). These were in good accordance with the environmental perturbations
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changing the pKa value of D174 site in R172K based on PROPKA analysis results, which divide the pKa increase of 3.72 (11.77 minus 8.05) into 0.16 desolvation effects, 0.09
unfavorable electrostatic reorganization energies, 1.52 side-chain hydrogen-bond interactions, and 2.03 Coulombic interactions (Table A2). The pKa decrease of 2.38 of
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Glu206 consists of 0.63 desolvation effects, 0.65 unfavorable electrostatic reorganization
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energies, -1.84 side-chain hydrogen-bond interactions, -0.51 backbone hydrogen-bond
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interactions, and -1.34 Coulombic interactions (Table A3). Such pKa value reduction of
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proton donor (Glu 206) and increase of nucleophile (Asp 174) would shift the acidic limb
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to more acidic values and the basic limb to more alkaline values for mutant R172K.
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Experimental results showed that R172K decreased the optimum pH by 0.5 unit and shifted the basic limb toward more acidity, but almost did not shift the acidic limb (Fig. 3&Table
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2). Meanwhile, mutant R172K decreased the kcat (4%), Km (38%) and kcat/Km (11%) values
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(Table 2). These results are consistent with previous mutational study on Bacillus stearothermophilus α-amylase [35], of which the counterpart R232K mutation resulted in
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lowered specific activity (around 12% of parental enzyme) but much broader optimum pH for activity. Numerous studies have shown strong non-additive effects when combining stabilizing mutations, especially likely for mutations in close proximity and mutations that would force 19
backbone movement, while some observed that stabilizing mutations could contribute independently to the overall stability of proteins [37, 38]. This study showed a cumulative
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effect between mutations shifting the pH-activity profiles in double mutant A270K/N271H (Table 2 and Fig. 3), suggesting relative independence between Lys 270 and His 271 (Figs.
5a, 5b&6a). However, A270K/N271H increased the catalytic activity greatly without
inheriting the thermostability of A270K substitution (Table 2). These results suggested an
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intricate reorientation of active site residues, substrate and a more optimized catalysis
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process of A270K/N271H, as evidenced by the alteration of kcat, Km values and substrate
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hydrolysis pattern (Table 2&Fig. A4).
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Double mutant R172K/N271H displayed moderate changes of pH-activity profile,
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optimum pH and enzymatic parameters in comparison to parental single mutations (Fig. 3
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&Table 2), despite the pKa values of D174, E206, D267, K172 and H271 of R172K/N271H were nearly the same as that in respective single mutants (Table 1), as indicated by their
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similar interactions around the mutations (Figs. 1c, 1e, 1g, 5a, 5c&6b, Tables A2-A7). In
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contrast to A270K/N271H, the additional incorporation of R172K mutation neither improved the acidic adaption property, nor increased the catalytic properties, compared to
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N271H. In addition, the simultaneous combination of three mutations offset the impact of each other, yielding a worse triple mutant (<5% activity, optimum pH=4.5) than R172K/N271H (data not shown). Obviously, the underlying structural basis for obviation of “cumulative effect” of R172K/N271H and triple mutant requires further investigation in the 20
future. (Fig. 6)
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4. Conclusions The acidic adaptation of B. subtilis Ca-independent α-amylase was for the first time
successfully engineered by rationally decreasing the pKa values of catalytic residues through mutations located within 4.5 Å of active site. All mutants enhanced acidic
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adaptation with decreased pH optima and shifted pH-activity profiles toward lower pH
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values. A270K/N271H showed 2 pH units decrease in optimum pH and about 3.94-fold
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increase of catalytic efficiency, yielding the best previously unreported Ca-independent and
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acidic α-amylase. Protein engineering of α-amylase for acidic adaptation here provides a
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successful example of the extent to which mutations near active site and computational
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models can be used for industrial enzyme improvements.
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Conflict of Interest
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The authors have no conflicts of interest.
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Acknowledgements This work was supported by grants from the Major Program of Natural Science Foundation of Guangxi [grant number 2016GXNSFEA380003], the Guangxi BaGui Scholars of Guangxi Zhuang Autonomous Region of China, the Chinese National Basic Research 21
Program (“973”)[grant 2009CB724703] and National Science and Technology Support
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Program [grant 2007BAD75B05].
Appendix A. Supplementary data
Additional experimental material and results, including olignonucleotide primers used for site-directed mutagenesis, normalized enzymatic parameters, normalized pH-activity
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profiles, normalized temperature-activity profiles and HPLC analysis of hydrolysis
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A
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products of soluble starch by purified recombinant wild-type and mutant α-amylases.
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References [1] M. Vihinen, P. Mantsala, Microbial amylolytic enzymes, Crit Rev Biochem Mol Biol, 24 (1989) 329-418. [2] M. Violet, J.C. Meunier, Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase, Biochem J, 263 (1989) 665-670.
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[3] S. Sivaramakrishnan, D. Gangadharan, K.M. Nampoothiri, C.R. Soccol, A. Pandey, α-amylases from microbial sources- An overview on recent development, Food Technol Biotechnol, 44 (2006) 173-184.
[4] W.D. Crabb, J.K. Shetty, Commodity scale production of sugars from starches, Curr Opin Microbiol, 2 (1999) 252-256.
[5] A. Pandey, P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh, R. Mohan, Advances in microbial amylases, Biotechnol Appl Biochem, 31 ( Pt 2) (2000) 135-152.
[6] J.E. Nielsen, L. Beier, D. Otzen, T.V. Borchert, H.B. Frantzen, K.V. Andersen, A. Svendsen, Electrostatics in the active site of an alpha-amylase, Eur J Biochem, 264 (1999) 816-824.
[7] Y.H. Liu, F.P. Lu, Y. Li, J.L. Wang, C. Gao, Acid stabilization of Bacillus licheniformis alpha amylase through
U
introduction of mutations, Appl Microbiol Biotechnol, 80 (2008) 795-803.
N
[8] Q.G. Zhang, Y. Han, H.Z. Xiao, Microbial alpha-amylase: A biomolecular overview, Process Biochemistry, 53 (2017) 88-101.
A
[9] R. Priyadharshini, P. Gunasekaran, Site-directed mutagenesis of the calcium-binding site of alpha-amylase of Bacillus licheniformis, Biotechnol Lett, 29 (2007) 1493-1499.
M
[10] M. Ghollasi, K. Khajeh, H. Naderi-Manesh, A. Ghasemi, Engineering of a Bacillus alpha-amylase with improved thermostability and calcium independency, Appl Biochem Biotechnol, 162 (2010) 444-459. [11] T.B. Dey, A. Kumar, R. Banerjee, P. Chandna, R.C. Kuhad, Improvement of microbial alpha-amylase
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stability: Strategic approaches, Process Biochemistry, 51 (2016) 1380-1390. [12] A. Shaw, R. Bott, A.G. Day, Protein engineering of alpha-amylase for low pH performance, Curr Opin
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Biotechnol, 10 (1999) 349-352.
[13] Y.H. Liu, F.P. Lu, Y. Li, X.B. Yin, Y. Wang, C. Gao, Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600, Appl Microbiol Biotechnol, 78 (2008) 85-94.
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[14] J.E. Nielsen, T.V. Borchert, G. Vriend, The determinants of alpha-amylase pH-activity profiles, Protein Eng, 14 (2001) 505-512.
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[15] C. Bessler, J. Schmitt, K.H. Maurer, R.D. Schmid, Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity, Protein Sci, 12 (2003) 2141-2149. [16] A. Sharma, T. Satyanarayana, Microbial acid-stable α-amylases: Characteristics, genetic engineering and applications, Process Biochemistry, 48 (2013) 201-211.
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[17] R.H. Sajedi, M. Taghdir, H. Naderi-Manesh, K. Khajeh, B. Ranjbar, Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+-independent alpha-amylase with acidic pH-profile, J Biochem Mol Biol, 40 (2007) 315-324. [18] G. Davies, B. Henrissat, Structures and mechanisms of glycosyl hydrolases, Structure, 3 (1995) 853-859. [19] Z. Fujimoto, K. Takase, N. Doui, M. Momma, T. Matsumoto, H. Mizuno, Crystal structure of a catalytic-site mutant alpha-amylase from Bacillus subtilis complexed with maltopentaose, J Mol Biol, 277
23
(1998) 393-407. [20] Y. Matsuura, M. Kusunoki, W. Harada, M. Kakudo, Structure and possible catalytic residues of Taka-amylase A, J Biochem, 95 (1984) 697-702. [21] J.E. Nielsen, T.V. Borchert, Protein engineering of bacterial alpha-amylases, Biochim Biophys Acta, 1543 (2000) 253-274.
SC RI PT
[22] J. Kyte, Mechanism in protein chemistry, Garland, New York, 1995. [23] C. Wang, R. Huang, B. He, Q. Du, Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis, BMC Bioinformatics, 13 (2012) 263.
[24] C. Wang, R. Huang, Q. Wang, N. Shen, D. Chen, Z. Li, Z. Huang, Alpha-amylase truncated body and application thereof. CN Patent 201210079326 [P]. 2012-03-23.
[25] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-MODEL: homology modelling of protein structures and complexes, Nucleic Acids Res, 46 (2018) W296-W303.
[26] T. Schwede, J. Kopp, N. Guex, M.C. Peitsch, SWISS-MODEL: An automated protein homology-modeling
U
server, Nucleic Acids Res, 31 (2003) 3381-3385.
N
[27] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal Chem, 31 (1959) 426-428.
A
[28] H. Imamura, S. Fushinobu, M. Yamamoto, T. Kumasaka, B.S. Jeon, T. Wakagi, H. Matsuzawa, Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor, J Biol
M
Chem, 278 (2003) 19378-19386.
[29] D.C. Bas, D.M. Rogers, J.H. Jensen, Very fast prediction and rationalization of pKa values for protein-ligand complexes, Proteins, 73 (2008) 765-783.
D
[30] B.W. Matthews, Structural and genetic analysis of protein stability, Annu Rev Biochem, 62 (1993) 139-160.
TE
[31] A. Salimi, F. Yousefi, M. Ghollasi, S. Daneshjou, H. Tavoli, S. Ghobadi, K. Khajeh, Investigations on possible roles of C-terminal propeptide of a Ca-independent alpha-amylase from bacillus, J Microbiol Biotechnol, 22 (2012) 1077-1083.
EP
[32] I. Schomburg, A. Chang, D. Schomburg, BRENDA, enzyme data and metabolic information, Nucleic Acids Res, 30 (2002) 47-49.
CC
[33] H. Li, A.D. Robertson, J.H. Jensen, Very fast empirical prediction and rationalization of protein pKa values, Proteins, 61 (2005) 704-721. [34] J.P. Gallivan, D.A. Dougherty, Cation-pi interactions in structural biology, Proc Natl Acad Sci U S A, 96 (1999) 9459-9464.
A
[35] M. Vihinen, P. Ollikka, J. Niskanen, P. Meyer, I. Suominen, M. Karp, L. Holm, J. Knowles, P. Mantsala, Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues, J Biochem, 107 (1990) 267-272. [36] S. Janecek, How many conserved sequence regions are there in the α-amylase family?, Biologia, 57 (2002) 29-41. [37] M. Matsumura, S. Yasumura, S. Aiba, Cumulative effect of intragenic amino-acid replacements on the
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thermostability of a protein, Nature, 323 (1986) 356-358. [38] L. Serrano, A.G. Day, A.R. Fersht, Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability, J Mol Biol,
A
CC
EP
TE
D
M
A
N
U
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233 (1993) 305-312.
25
Figure captions
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Fig.1 2D representation of the alpha-amylase-maltopentaose complexes: (a) Bacillus subtilis alpha-amylase BSUA (PDB ID: 1BAG); (b) Amy7C; (c) N271H; (d) A270K; (e) R172K; (f) A270KN271H; (g) R172KN271H. The dotted outline surrounding the ligand denotes the proximity contour, meaning distance to the active site interior. The blue
U
smudges that are drawn behind some of the ligand atoms denote ligand solvent exposure,
N
indicating the extent of solvent exposure. The turquoise discs that are drawn behind some
A
of the residues denote receptor solvent exposure, indicating the difference in solvent
M
exposure due to the presence of the ligand. The catalytic residues, Asp176, Glu208 and
D
Asp269 acting as respective nucleophile, general acid/base and transition state stabilizer,
TE
and the hydrogen bonds formed with the glucosidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the non-reducing end glucose residue of the substrate) in 1BAG (a)
EP
are well superimposed in Amy7C (b) and its mutants (c-g), indicating a similar catalytic
CC
mechanism. The 2D molecular graphics were generated by MOE software under the default
A
settings (Molecular Operation Environment, version2008.10).
26
SC RI PT U N A M D TE EP
Fig. 2 Mutation positions in Amy7C. Domains A, B and C of Amy7C are shown in light
CC
grey, cyan and yellow respectively. The maltopentaose substrate is shown in purple sticks.
A
The active site amino acid residues, Asp174, Glu206 and Asp267, which serve as respective nucleophile, catalytic acid/base and transition state stabilizer, are shown in red ball and stick form. The chosen mutation sites of Arg172, Ala270 and Asn271 are indicated in green ball and stick form. The dot lines represent hydrogen bonds. (a) overall distribution of
27
mutations in cartoon form; (b) close-up view of mutations in molecular surface form, the
U
SC RI PT
hydrophobic and hydrophilic residues are colored in green and blue respectively.
N
Fig.3 pH-activity profiles for the wild-type Amy7C and its mutants. The activities of
A
α-amylases were evaluated at protein concentrations of approximately 1 μg.mL−1 except
M
R172K (10 μg.mL−1) and R172K/N271H (5 μg.mL−1) with 5 g/L soluble starch at the
D
indicated pH under their optimum temperatures for 5 min. The buffer solutions were 50
TE
mM phosphate citrate (pH 2.5-6.0) and 50 mM sodium phosphate (pH 6.0-8.0). All the
EP
α-amylases were assayed at 63.2oC except A270K at 70.3oC. One activity unit (U) was defined as the amount of enzyme required to release 1 μmol dextrose equivalents per
CC
minute under test conditions. All the assays were implemented in triplicate and represented
A
by mean ± standard deviations.
28
SC RI PT
U
Fig.4 Temperature-activity profiles for the wild-type Amy7C and its mutants. The activities
N
of α-amylases were evaluated at protein concentrations of approximately 1 μg.mL−1 except
M
A
R172K (10 μg.mL−1) and R172K/N271H (5 μg.mL−1) with 5 g/L soluble starch at the indicated temperatures under their optimum pH values for 5 min. Amy7C, R172K, A270K
D
and R172K/N271H, N271H and A270K/N271H were assayed in pH 6.5, pH 6.0, pH 5.5,
TE
pH 5.0 and pH 4.5, respectively. One activity unit (U) was defined as the amount of
EP
enzyme required to release 1 μmol dextrose equivalents per minute under test conditions. All the assays were implemented in triplicate and represented by mean ± standard
A
CC
deviations.
29
SC RI PT
U
Fig. 5 Superimpositions of wild-type Amy7C and single mutants N271H (a), A270K (b)
N
and R172K (c). The wild-type residues are shown in grey (C atom), blue (N atom) and red
M
A
(C atom) spheres and sticks. The mutations are shown in green spheres. The maltopentaose substrate is shown in purple sticks. Hydrogen bonds existing only in wild-type and in both
D
wild-type and mutants are shown in blue and black dashed lines, respectively. The
A
CC
EP
orange sticks.
TE
“π-cavity” amino acid residues Trp15, Tyr269, Phe302, Pro305 and Phe315 are shown in
30
SC RI PT U N A M D
TE
Fig. 6 Superimpositions of wild-type Amy7C and double mutants A270K/N271H (a) and R172K/N271H (b). The wild-type residues are shown in grey (C atom), blue (N atom) and
EP
red (C atom) spheres and sticks. The mutations are shown in green spheres. The
CC
maltopentaose substrate is shown in purple sticks. Hydrogen bonds existing only in wild-type and in both wild-type and mutants are shown in blue and black dashed lines,
A
respectively. The “π-cavity” amino acid residues Trp15, Tyr269, Phe302, Pro305 and Phe315 are shown in orange sticks.
31
32
D
TE
EP
CC
A
SC RI PT
U
N
A
M
Table 1. Predicted pKa values of catalytic residues and mutations of wild-type Amy7C and its mutants pKa a
Enzyme
Wild-type
8.05
6.14
2.70
-
18.24 -
-
N271H
3.68
7.28
4.57
-
18.04 -
6.62
A270K
8.02
6.18
4.71
-
18.22 3.81
-
R172K
11.77
3.76
2.86
14.31 -
A270K/N271H 3.68
7.28
4.59
-
R172K/N271H 11.23
3.58
2.70
14.24 -
-
U
18.02 3.82 -
-
6.62 6.63
N
pKa values were calculated by the PROPKA sever (http://propka.ki.ku.dk/) using the
A
a
SC RI PT
D174 b E206 b D267 b K172 R172 K270 H271
M
theoretical PDB files of the wild-type Amy7C and its mutants in protein-ligand complex format. b D174, E206 and D267 were the catalytic residues playing the roles as nucleophile,
A
CC
EP
TE
D
catalytic acid/base, and transition state stabilizer, respectively. “-” indicates no value.
33
N U SC RI PT
Table 2. Enzymatic parameters of wild-type and mutant α-amylases pHopt a
Enzyme
Topt(oC) a
pHpred b
Tpred b
pHopt b
Topt (oC) b
Molecular
Vmax d
Km d
Activity e
kcat d
kcat/Km d
(95% CI)
weight c
(U/mg)
(g/L)
(U/mg)
(s-1)
(L.g-1.s-1)
63.36-65.29
47575
1589.76±88.56
3.31±0.0.5
956.54±27.65
1260.55±70.22
380.33
4.99-5.32
60.74-64.32
47598
1016.96±33.97
2.28±0.03
698.09±16.42
806.75±26.95
353.71
A
(95% CI) 6.5
63.2
6.32
64.33
N271H
5.0
63.2
5.16
62.53
A270K
5.5
70.3
5.39
70.53
5.28-5.50
69.42-71.64
47632
905.08±70.01
4.47±0.07
477.94±9.49
718.51±55.57
159.74
R172K
6.0
63.2
5.55
64.11
5.45-5.65
63.01-65.21
47547
63.63±2.78
1.26±0.04
50.84±2.88
50.42±2.20
41.84
A270K/N271H
4.5
63.2
5.06
61.81
4.88-5.24
61.17-62.46
47655
2920.25±83.74
1.36±0.03
2296.84±122.65
2319.41±66.51
1878.83
R172K/N271H
5.5
63.2
5.73
62.61
5.48-5.98
61.63-63.60
47570
254.39±10.55
2.61±0.04
167.03±4.88
201.69±8.36
76.07
ED
PT
CC E a pH
opt
b pH
6.14-6.50
M
Wild-type
and Topt were shown as the experimental pH and temperature optima, which were used to determine the kinetic parameters in this study.
pred,
Tpred, pHopt (95% CI) and Topt (95% CI) indicted the predicted mean pH optima, mean temperature optima, the 95% confidence intervals
A
when the pH-activity and temperature-activity relationship data were fitted as Gaussian functions. c
Molecular weight were deduced from the protein sequence.
d
Vmax, Km, kcat and kcat/Km values were assayed with different concentrations of soluble starch (0.4 g/L-9 g/L) in 50 mM phosphate citrate (pH
2.5-6.0) and 50 mM sodium phosphate (pH 6.0-8.0) for 5 min under the optimum pH and temperature. The Vmax, Km, kcat values were averaged from
34
N U SC RI PT
A
CC E
PT
ED
M
A
three independent experiments and shown as mean ± standard deviations. The kcat/Km values were calculated by dividing the means of kcat and
35
Km values. One activity unit (U) was defined as the amount of enzyme required to release 1 μmol dextrose equivalents per minute under test conditions, as described in “Material and methods”. e
Activity values were assayed at 5 g /L soluble starch in 50 mM phosphate citrate at optimum pH and
A
CC
EP
TE
D
M
A
N
U
SC RI PT
temperature values. All the experiments were carried out in triplicate.
36