Improved catalytic efficiency of catalase from Bacillus subtilis by rational mutation of Lys114

Accepted Manuscript Title: Improved catalytic efficiency of catalase from Bacillus subtilis by rational mutation of Lys114 Author: Wenlong Cao Zhen Kang Song Liu Long Liu Guocheng Du Jian Chen PII: DOI: Reference:

S1359-5113(14)00318-3 http://dx.doi.org/doi:10.1016/j.procbio.2014.05.016 PRBI 10149

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

1-2-2014 12-5-2014 27-5-2014

Please cite this article as: Cao W, Kang Z, Liu S, Liu L, Du G, Chen J, Improved catalytic efficiency of catalase from Bacillus subtilis by rational mutation of Lys114, Process Biochemistry (2014), http://dx.doi.org/10.1016/j.procbio.2014.05.016 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.

Improved catalytic efficiency of catalase from Bacillus subtilis by

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rational mutation of Lys114

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Wenlong Cao1,2,3,4, Zhen Kang1,2,3,4†, Song Liu2,3,4, Long Liu1,2,3,4, Guocheng Du1,3,4,6, Jian

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Chen1,3,4,5

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1. Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi

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214122, China

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2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan

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University, Wuxi 214122, China

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3. School of Biotechnology, Jiangnan University, Wuxi 214122, China

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4. Synergy Innovation Center of Modern Industrial Fermentation, Jiangnan University, Wuxi

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214122, China

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5. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University,

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Wuxi 214122, China

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6. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education,

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Jiangnan University, Wuxi 214122, China

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†Corresponding author,

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Zhen Kang, Phone: +86-510-85918307. Fax: +86-510-85918309. E-mail: [email protected]

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ABSTRACT

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Alkaline catalases with good properties are desirable in the textile industry. In the present

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work, by applying the PoPMuSiC algorithm for the calculation of the folding free energy,

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Lys114 of a Bacillus catalase was rationally selected and engineered to improve the

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thermostability. Interestingly, the Lys114Tyr, Lys114Val, Lys114Met and Lys114Ile variants

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showed higher catalytic efficiencies when compared with the wild-type protein. In particular,

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the Lys114Tyr variant showed the highest catalytic efficiency, which was 5.3-fold of the

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wild-type catalase. The production of the Lys114Tyr variant may represent an improved

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catalase suitable for industrial purposes.

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Keywords

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Catalase; Enzyme engineering; Site-directed mutagenesis; Catalytic efficiency

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1. Introduction Catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC1.11.1.6), is widely

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distributed in archaea, eubacteria, fungi, plants and animals. Catalases, as a central component

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of detoxification pathways, play an important role in nature because they prevent the

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formation of highly reactive oxygen species by catalyzing the dismutation of H2O2 into water

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and O2 [1]. Currently, more than 300 catalase sequences have been identified and reported [2],

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and variety of catalases have been purified, characterized [3-6] and produced [3, 7].

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According to enzymological properties, catalases could be classified into three general classes

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[2]: heme-containing monofunctional catalases, heme-containing bifunctional

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catalase-peroxidases and Mn-containing catalases. Monofunctional catalases from different

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sources have been extensively studied, including characterization of their structures [8] and

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catalytic mechanisms [9]. Although the monofunctional catalases can be clearly divided into

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three clades [8], they have similar structural properties and are composed of ~460 residues

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even in distantly related organisms. In addition, the active sites of monofunctional catalases

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are highly conserved and consist of three residues: His, Ser and Asn [2].

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Currently, heme-containing monofunctional catalases have been widely used in several commercial areas, including the food, textile, dairy, environmental protection, pulp and paper

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industries, as well as in clinical analysis [10]. To further expand its applications, intensive research has been focused on identification, overexpression and evaluation of new natural catalase genes [3, 7, 11, 12]. However, few studies towards catalase engineering have been reported [13]. In fact, many protein engineering approaches for improving the target performance have been developed and attracted significant attention. Over the past years, site-directed mutagenesis with the help of structural information and algorithm (including

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PoPMuSiC) analysis has most commonly been developed and applied for rational engineering

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[14-18]. Accordingly, many enzymes have been engineered and optimized in terms of

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catalytic efficiency and thermostability with site-directed mutagenesis [14, 19-22].

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In this work, residue Lys114 of a cloned Bacillus catalase was rationally selected and mutated to improve its enzymatic properties. Interestingly, mutation of Lys114 to Val, Met, 3 Page 3 of 22

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Trp, Ile and especially to Tyr substantially increased the catalytic efficiency that was 5.3-fold

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of the wild-type catalase.

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2. Materials and methods

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2.1. Bacterial strains and plasmids Escherichia coli JM109 purchased from TaKaRa (Dalian, China) was used as the host for

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molecular cloning and manipulation of plasmids. B. subtilis WSHDZ-01 (CCTCC M206062)

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and B. subtilis WB600 (Microbial Resources Data) were used as the source of genomic DNA

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and the host for the expression of the catalase, respectively. The shuttle vector pStop1622

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[23], supplied by Professor Dieter Jahn (Technical University Braunschweig, Germany),

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between E. coli and B. subtilis was used for gene cloning, site-directed mutagenesis and

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expression.

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2.2. Cloning of the katA gene and site-directed mutagenesis

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The katA gene was amplified from genomic DNA of B. subtilis WSHDZ-01 using primers katAF and katAR (Table 1), which were designed according to the published sequence [24].

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In particular, sequence alignment results showed that the cloned katA gene was completely

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identical to the published sequence (GenBank accession no. AB046412). After digestion with

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SpeI and SmaI, the fragment was ligated into the shuttle plasmid pStop1622 (induced by

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xylose) to generate pStop1622-katA, which was used as the template of site-directed mutagenesis. Using the designed oligonucleotides (Table 1), site-directed mutagenesis of the catalase gene katA was conducted using the QuickChange Site-Directed Mutagenesis Kit. The plasmids with the mutated sequences were digested with DpnI. After treatment with DpnI, mutated plasmids were transformed to E. coli JM109 for selection of the positive transformants. The plasmids extracted from positive transformants were further confirmed by

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DNA sequencing. The confirmed katA mutants Lys114Phe, Lys114Ile, Lys114Trp,

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Lys114Tyr, Lys114Val, Lys114Leu, Lys114Cys, Lys114Met and Lys114His were then

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transformed into B. subtilis WB600 to generate the recombinant strains B. subtilis 600F, 600I,

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600W, 600Y, 600V, 600L, 600C, 600M and 600H, respectively. The recombinant B. subtilis

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WB600WT (pStop1622-katA) containing the wild-type katA gene was used as the control. 4 Page 4 of 22

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2.3. Culture media and conditions Luria Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl, pH

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7.2) containing 100 μg/mL ampicillin was used for cultivating the recombinant E. coli strains.

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Recombinant B. subtilis strains were cultivated in 50 mL malt extract medium (6% malt

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extract, pH 7.5) supplemented with tetracycline (10 μg/mL) at 37 °C for 12 h. The seed

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culture was then inoculated into the flask at a ratio of 5% (v/v) for fermentation. The

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fermentation medium contained (g/L) glucose 20, NaNO3 10, KH2PO4 0.6, MgSO4·7H2O 0.5,

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Na2HPO4 9.52, FeSO4·7H2O 0.0025, pH 7.5. When the optical density at 600 nm (OD600)

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reached 1.0, 0.5% (w/v) xylose was added to induce the expression of the catalase. Cells were

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cultivated for 48 h with an agitation of 200 rpm at 37 °C and samples were centrifuged

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(10000 × g for 10 min at 4 °C) and the supernatant was filtered through a 0.22 μm syringe

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filter for analysis.

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2.4. Purification and SDS-PAGE analysis of the catalase For purification of the recombinant catalase, absolute ethanol was added to the

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supernatant to 60% (v/v) saturation at 0 °C. The precipitated protein was collected and

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dissolved in buffer A (20 mM potassium phosphate buffer, pH 7.0) and then dialyzed for 24 h

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against the same buffer. The sample was filtered (0.22 μm) and loaded onto the AKTA

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purifier (GE Healthcare, USA) equipped with an anion exchange column (Q-DEAE). The sample material was eluted at a flow rate of 1 mL/min with a five-column-volume linear gradient of 0 to 1 M NaCl in buffer A. After the fractions containing catalase activity were pooled, the purified catalases were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a vertical mini gel apparatus (Bio-Rad) at 150 V for 1 h. Protein bands were stained with a Coomassie brilliant blue R-250 solution. The protein

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concentration was estimated by the Bradford assay with bovine serum as the standard.

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2.5. Enzyme activity assays

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Catalase activity was assayed spectrophotometrically by monitoring the decrease in H2O2

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levels at 37 °C, as higher temperatures can accelerate the decomposition of H2O2 which will

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affect the measured results of enzyme activity. The standard reaction mixture contained 50 5 Page 5 of 22

mmol/L KH2PO4-K2HPO4 buffer (pH 7.0), 10 mmol/L H2O2, and 0.1 mL of the enzyme

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solution in a total volume of 3.0 mL. The reaction was initiated by the addition of enzyme and

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the conversion rate of H2O2 was measured using an UV-visible spectrophotometer (UV-2450)

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at 240 nm for 30 s. The published ε value of H2O2 at 240 nm (ε240, 43.6 mM−1 cm−1) was used

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in the calculation. One unit of catalase activity was defined as the amount of enzyme that

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decomposed 1 μmol H2O2 per min at 37 °C.

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2.6. Determination of the optimal temperature/pH and thermostability conditions

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To accurately determine the characteristic of variants, all mutant enzymes were diluted to

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the same concentration. The optimal temperature was measured in the range of 30–60 °C with

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an interval of 10 °C. According to previous research, the reaction was also conducted at 55 °C

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[25] (Fig. S1 in the Supplementary Material). The thermal inactivation half-life (t1/2) at 60 °C

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was determined by the residual activity versus incubation time that was deduced by linear

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regression [26]. The optimal pH for recombinant variants was determined in the following 50

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mM buffers: potassium phosphate (pH: 7, 8), Tris-HCl (pH: 9), Glycine-NaOH (pH: 10, 11)

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and Na2HPO4-NaOH (pH: 12) (Fig. S2 in the Supplementary Material).

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2.7. Estimation of kinetic parameters and protein

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Due to oxidative inhibition caused by a high concentration of substrate [27], the reaction

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would not proceed when the concentration of H2O2 was >100 mM. Consequently, assays were performed with active enzymes in different concentrations of H2O2 (2.5, 3.3, 5, 7.5, 10, 12.5, 15, 30, 40, 50, 60 and 80 mM). Eadie–Hofstee plots were used to calculate the kinetic parameters Km and Vmax, according to the enzyme reactions. The turnover number value, kcat, was calculated using the following equation: kcat = Vmax/[E0], where [E0] is the pure enzyme concentration.

2.8. Computer-aided structure modeling The theoretical structure of catalase was obtained using the crystal structure of the

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catalase from Enterococcus faecalis (PDB ID: 1si8) [28] by homology modeling using the

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Swiss-Model protein modeling server (http://swissmodel.expasy.org/) [29]. The sequence

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identity between the template and the target was 68.15% and the rmsd value for all atoms 6 Page 6 of 22

between the two proteins was 0.14 Å. Differences and mutation positions of the models were

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evaluated in Discovery Studio Client 2.5 by examining the secondary structure of the protein.

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All the wild-type and mutant proteins were subjected into PISA server

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(http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) for protein analysis [30]. All graphical

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molecular representations were generated using Accelrys Discovery Studio Client 2.5.

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3. Results

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3.1. Construction and overexpression of the catalase variants

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According to the PoPMuSiC computer-aided design program, all possible stabilizing

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point mutations of the catalase were evaluated by calculating the values of the folding free

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energy (Fig. S3 in the Supplementary Material), and most of the potential mutations were

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concentrated on residue Lys114, which is located within the β-barrel domain (Fig. 3B). As a

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result, nine variant candidates Lys114Phe, Lys114Ile, Lys114Trp, Lys114Tyr, Lys114Val,

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Lys114Leu, Lys114Cys, Lys114Met and Lys114His with lower minus values of –2.76, –2.68,

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–2.54, –2.51, –2.41, –2.29, –1.96, –1.50 and –1.49 kcal/mol were selected for experimental

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investigation. Applying site-directed mutagenesis, the variants were constructed and

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overexpressed by B. subtilis WB600 with the xylose-induced system [23]. To confirm the

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functional expression of all the variants, the corresponding recombinant strains 600F, 600I,

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600W, 600Y, 600V, 600L, 600C, 600M and 600H were cultivated with WB600WT as the control.

As shown in Fig. 1, all the catalase variants were secreted and functionally

overexpressed. In particular, mutation of Lys114 to Trp gave rise to an enzyme with a decreased activity when compared with the activity of the wild-type protein, whereas Phe, Leu, Cys or His mutations showed negligible change in catalytic activity. In contrast,

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replacement of Lys114 with Met, Tyr, Val and Ile significantly increased the extracellular

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catalase activities. The strains 600Y (Lys114Tyr) and 600V (Lys114Val) accumulated

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catalase up to 12,653 U/mL and 13,726 U/mL (Fig. 1), which were 2.3 and 2.5 times of the

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control strain WB600WT, respectively. However, no evident differences were observed

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regarding the concentration of extracellular catalases (data not shown). As a result, 7 Page 7 of 22

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differences among the variants may be attributed to intrinsic property changes to the catalase.

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3.2. Purification and characterization of the catalase variants To investigate the enzymatic properties, we purified all the constructed variants and the

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wild-type catalase by applying a two-step purification procedure with ethanol precipitation

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and anion exchange chromatography. As shown in Fig. 2, a purified single band was detected

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by SDS-PAGE, which was consistent with the calculated molecular mass (about 60 kDa).

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Moreover, it was found that the Lys114Tyr, Lys114Val, Lys114Met, Lys114Trp and

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Lys114Ile variants showed higher t1/2 values at 60 °C compared with the wild-type catalase

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(Table 2). However, no obvious changes were observed towards secondary structures (data

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not shown) and all the variants were subjected to the constant optimal temperature (55 °C,

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Supplementary Material Fig. S1) which was identical to that of the wild-type catalase.

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3.3. Improved catalytic efficiency with mutagenesis of Lys114

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Kinetic parameters of the wild-type catalase and nine variants were calculated and analyzed to uncover factors that affected enzymatic activity. As shown in Table 2, remarkable

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differences in kcat, Km and kcat/Km (indicated as the catalytic efficiency) were generated.

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Substitution of Lys114 with Val and Tyr increased the Km value to 118.8 and 41.3 mM, which

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were about 5.7-fold and 1.9-fold of the wild-type catalase Km value, respectively. In contrast,

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replacement with Trp decreased the Km value to 12.8 mM while the other mutations resulted in no significant change in the Km values. More importantly, mutation of Lys114 to Tyr, Val, Met and Ile remarkably improved the kcat/Km values, which were about 5.3, 3.5, 3.6 and 4.1 times of the wild-type catalase value, respectively (Table 2). Although the variant Lys114Trp had a higher affinity to the substrate, its catalytic efficiency decreased to 10.1 mM–1 s–1 due to a low value of kcat. As expected, the results herein were in accordance with the results of the

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extracellular catalase activities (Fig. 1). Besides, replacement with the hydrophobic Phe failed

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to prominently increase the catalytic efficiency (Table 2), indicating that further research

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would be required for revealing the mechanism.

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3.4. Modeling and analysis of the wild-type catalase and variants

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To explore the factors involved in the improved catalytic efficiency, 3D structures of the 8 Page 8 of 22

wild-type and variants were constructed based on the template (PDB: 1si8). Consistent with

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previous report [8], this heme-containing catalase is a homotetramer (Fig. 3A) and the

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polypeptide chain of single subunit could be divided into four regions: the N-terminal arm,

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the β-barrel, the wrapping region and the C-terminal helical region (Fig. 3B). Lys114, the

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residue that mutated is located in a β-sheet of the β-barrel domain which is the central feature

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of catalase fold. Furthermore, it could be found that Lys114 located on the interface connects

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the heme group and involves in the interaction of the subunits (Fig. S4 in the

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Supplementary Material), indicating its important role in products release [31]. Compared

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with the wild-type catalase (Fig. 3C), the variant Lys114Tyr showed fewer hydrogen bonds at

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the interface (Fig. 3D), which might cause a more flexible channel and eventually resulted in

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improved catalytic efficiency.

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4. Discussion

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In the present work, we used the PoPMuSiC-2.1 algorithm to calculate and identify potential amino acid substitutions for enhancing the thermostability of a catalase [32]. As a

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consequence, nine mutant candidates were constructed and five variants were experimentally

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validated to have remarkably increased t1/2 values. Interestingly, catalytic efficiency of the

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variant Lys114Tyr was also improved. Recently, by site-directed mutagenesis, thermostability

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and activity of lipase and protease from Bacillus pumilus were both enhanced, respectively [20, 21], indicating the wide applicability of the site-directed mutagenesis strategy. Although five variants Lys114Tyr, Lys114Val, Lys114Met, Lys114Trp and Lys114Ile

showed increased t1/2 values, no significant changes of secondary structures were observed (data not shown). In catalase, Lys114 is the sole hydrophilic charged residue that locates in a

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β-sheet of the β-barrel domain. Generally, it has been accepted that hydrophobicity of the

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region around the active site plays a crucial role in the thermostability of enzymes [33].

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Therefore, replacement of the hydrophilic Lys residue with hydrophobic amino acids Tyr, Val,

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Met, Trp and Ile increased the internal hydrophobicity, which might contributed to the

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increased t1/2 values. The variants especially Lys114Tyr regarding thermostability would be

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further studied in future to uncover the main factors. Additionally, we found that substitution of Lys114 with residue Tyr gave rise to significantly increased catalytic efficiency. Although the variant Lys114Tyr showed a

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decrease in substrate affinities, their catalytic efficiencies were significantly improved

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because of the higher kcat values (Table 2). In heme-containing monofunctional catalases, two

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minor channels exist and might be alternatives for products release (Fig. 3B). It has been

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reported that one channel connects the heme group with the interface between monomers

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while the other connects the heme group with NADP(H) binding site [31]. Previously, it has

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been reported that mutations that enlarge the minor channel connecting NADP(H) with heme

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group lead to increased catalytic efficiency [34]. Thus, substitution of Lys114 to Tyr

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improved the catalytic efficiency might be attributed to some changes involved in minor

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channels. Moreover, structural analysis showed that Lys114 forms two hydrogen bonds with

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Asp123 while Tyr114 forms only one hydrogen bond with Asn121 (Fig. 3C, 3D). As a result,

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the decreased hydrogen bonds might result in a more flexible structure for releasing the

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product, which also contributed to the increased catalytic efficiency.

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5. Conclusions

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In conclusion, calculated by the PoPMuSiC 2.1 algorithm, we rationally selected and

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mutated the residue Lys114 to Phe, Ile, Trp, Tyr, Val, Leu, Cys, Met and His. Unexpectedly, Five variants Lys114Tyr, Lys114Val, Lys114Met and Lys114Ile gave rise to higher catalytic efficiencies compared with the wild-type catalase. In particular, the variant Lys114Tyr exhibited the best properties whose value of catalytic efficiency was about 5.3-fold of the wild-type catalase. In addition, the variant Lys114Tyr also showed higher t1/2 value although no obvious changes were observed regarding to secondary structures. The results indicated

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that residues that form the β-barrel domain, which interacts with the heme-pocket (Fig. 1A)

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and main channel, could represent potential candidates for engineering. After all, the variant

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Lys114Tyr constructed in this work with significantly increased catalytic efficiency may have

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significant potential in the production of a catalase for industrial applications.

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Acknowledgements We would like to thank Professor Dieter Jahn (Technical University Braunschweig,

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Germany) for supplying the plasmid pStop1622. This work was financially supported by the

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National High Technology Research and Development Program of China (863 Program,

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2011AA100905, 2012AA022202), Program for Changjiang Scholars and Innovative

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Research Team in University (no. IRT1135), the 111 Project, the National Science

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Foundation for Post-doctoral Scientists of China (2013M540414) and the Jiangsu Planned

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Projects for Postdoctoral Research Funds (2013M540414).

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Appendix A. Supplementary data

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The following is the supplementary data to this article.

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catalase HPII of Escherichia coli. Protein Sci 1999;8:490-498.

371

M

372 373

377 378 379 380 381 382 383 384 385

te

376

Ac ce p

375

d

374

386 387 388 389 390

15 Page 15 of 22

391 392

Table 1 Primers used in this study Oligonucleotides

Sequence (5'-3')

Primers GGACTAGTAAAAGCTGTTACAACAAGTT

KatA-R

TCCCCCGGGGATGAGAAGCATACGCAATG

Lys114Tyr-F

ACCCGCGCGGATTTGCTGTTTATTTTTATACTGAAGAAGGAAA

Lys114Tyr-R

TTTCCTTCTTCAGTATAAAAATAAACAGCAAATCCGCGCGGGT

Lys114Val-F

CCCGCGCGGATTTGCTGTTGTATTTTATACTGAAGAAGG

Lys114Val-R

CCTTCTTCAGTATAAAATACAACAGCAAATCCGCGCGGG

Lys114Met-F

CCCGCGCGGATTTGCTGTTATGTTTTATACTGAAGAAGGAAA

Lys114Met-R

TTTCCTTCTTCAGTATAAAACATAACAGCAAATCCGCGCGGG

Lys114Ile-F

CCGCGCGGATTTGCTGTTATATTTTATACTGAAGAAGG

Lys114Ile-R

CCTTCTTCAGTATAAAATATAACAGCAAATCCGCGCGG

Lys114Phe-F

GACCCGCGCGGATTTGCTGTTTTTTTTTATACTGAAGAAGGAAAC

Lys114Phe-R

GTTTCCTTCTTCAGTATAAAAAAAAACAGCAAATCCGCGCGGGTC

Lys114Trp-F

GACCCGCGCGGATTTGCTGTTTGGTTTTATACTGAAGAAGGAAAC

Lys114Trp-R

GTTTCCTTCTTCAGTATAAAACCAAACAGCAAATCCGCGCGGGTC

Ac ce p

te

d

M

an

us

cr

ip t

KatA-F

Lys114Leu-F

CCCGCGCGGATTTGCTGTTCTATTTTATACTGAAGAAGG

Lys114Leu-R

CCTTCTTCAGTATAAAATAGAACAGCAAATCCGCGCGGG

Lys114His-F

GACCCGCGCGGATTTGCTGTTCATTTTTATACTGAAGAAGGAAAC

Lys114His-R

GTTTCCTTCTTCAGTATAAAAATGAACAGCAAATCCGCGCGGGTC

Lys114Cys-F

GACCCGCGCGGATTTGCTGTTTGTTTTTATACTGAAGAAGGAAAC

Lys114Cys-R

GTTTCCTTCTTCAGTATAAAAACAAACAGCAAATCCGCGCGGGTC

393 394 395 396 397 398

16 Page 16 of 22

399

Table 2 Thermostability and kinetic parameters of the wild-type catalase and its variants kcat (s-1)

Km (mM)

kcat/Km (mM-1 s-1)

Wild enzyme

20

306±14

20.9±0.2

14.6

Lys114Phe

26

367±16

23.9±0.9

Lys114Ile

42

1728±67

28.6±0.6

Lys114Trp

58

130±9

12.8±0.4

Lys114Tyr

65

3170±119

41.3±0.5

Lys114Val

36

6138±248

118.8±0.8

51.7

Lys114Leu

24

435±19

24.7±0.9

17.6

Lys114Cys

20

642±26

Lys114Met

60

1359±49

Lys114His

23

427±18

403 404 405 406 407 408 409 410

an

us

cr

60.4 10.2 76.8

30.9±1.2

20.8

25.6±0.3

53.1

21.8±0.9

19.6

d

Half-life (t1/2) of thermal inactivation at 60 °C was measured. Each value represents the

mean of three independent measurements.

te

402

a

15.4

Ac ce p

401

ip t

t1/2a (min)

Enzyme

M

400

411 412 413 414 415

17 Page 17 of 22

416

Figure legends

417

Fig. 1 Analysis of extracellular catalase activities. The strains listed are B. subtilis WB600WT

419

(wild-type KatA), 600F (Lys114Phe), 600I (Lys114Ile), 600W (Lys114Trp), 600Y

420

(Lys114Tyr), 600V (Lys114Val), 600L (Lys114Leu), 600C (Lys114Cys), 600M

421

(Lys114Met) and 600H (Lys114His). Each value represents the mean of three independent

422

measurements.

cr

ip t

418

us

423

Fig. 2 SDS-PAGE analysis of the catalase variants. Lane M, molecular weight marker; lane 1

425

the purified catalase; lane 2, crude extract of the recombinants.

an

424

426

Fig. 3 The model structure of the catalase and local model of interface between monomers.

428

(A) Homotetramer structure of catalase. Four monomers are shown in yellow, green, blue and

429

orange, respectively. The interface between monomers indicated was shown in dashed box;

430

(B) Overall fold of one catalase subunit. The N-terminal arm, β-barrel, wrapping region,

431

C-terminal helical region and the heme group were shown in green, cyan, purple, yellow and

432

red, respectively. The catalytic residues His54, Ser93 and Asn127 were shown in red ‘ball and

434 435 436 437 438

d

te

Ac ce p

433

M

427

stick’ representations. The position of the point mutation Lys114 was shown in purple ‘ball and stick’ representation; (C) Local model of the interface in the dashed box between monomers of the wild-type catalase. The amino acids were shown as ‘ball and stick’ representations. Hydrogen bonds were shown in green dotted lines; (D) Local model of the interface in the dashed box between monomers of the variant Lys114Tyr.

439 440 441 442 443

18 Page 18 of 22

444 445 446

ip t

16000 14000

cr us

10000 8000 6000

an

Enzyme activity (U/mL)

12000

4000

M

2000 0

448 449 450 451 452 453 454

W 600

Y 600

d

I 600

V 600

L 600

C 600

M 600

H 600

--

Strains

Ac ce p

447

F 600

te

WT 600 B W

455 456 457 458 459 460

19 Page 19 of 22

461 462 463

467 468 469 470 471 472 473

te

466

Ac ce p

465

d

M

an

us

cr

ip t

464

474 475 476 477 478 479

20 Page 20 of 22

480 481 482

484 485 486 487 488

Ac ce p

te

d

M

an

us

cr

ip t

483

489 490 491 492

21 Page 21 of 22

492

Highlights

493

•The PoPMuSiC algorithm was applied for rational engineering of the Bacillus catalase.

495

•Lys114 was mutated to increase the catalytic efficiency.

496

•The variant Lys114Tyr showed great potential in the production of industrial catalase.

ip t

494

cr

497

Ac ce p

te

d

M

an

us

498

22 Page 22 of 22