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Design of salt-bridge cyclization peptide tags for stability and activity enhancement of enzymes
Process Biochemistry 81 (2019) 39–47
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Design of salt-bridge cyclization peptide tags for stability and activity enhancement of enzymes
T
⁎
Lingjun Tanga,1, Ji Yanga,1, Jie Chena, Jing Zhanga, Huimin Yua,b, , Zhongyao Shena a b
Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Salt-bridge cyclization peptide tag (SbCPT) Insertion mutagenesis Molecular dynamics simulations Nitrile hydratase Nitrilase
The terminus is usually the most flexible and fluctuating part that affects the enzyme stability. In this study, we proposed an enzyme terminus stabilization strategy based on the attachment of a salt-bridge cyclization peptide tag (SbCPT) without activity loss. SbCPTs of different lengths, as well as different subunit termini of nitrile hydratases from Rhodococcus ruber TH (NHaseM-TH) for SbCPT insertion, were investigated by molecular dynamics (MD) simulations and mutagenesis experiments. The investigation revealed that a smaller SbCPT would be favorable and that the SbCPT should only be attached on the enzyme terminus that is extensible and also far from any special functional area due to the minimal influences on the enzyme structure. The stability of different SbCPTs types (GKPEG, GKPDG, GRPEG, GRPDG) was identified as RD > RE > KD > KE. By insertion of an optimal SbCPT (GRPDG) to the C-terminus of the α-subunit of NHase, the SbCPT-attached NHase variants demonstrated improved stability by a maximum of 32.7%, as well as the overall catalytic competence. Additionally, the effect of SbCPT with enhanced stability and non-reduced activity was also verified in nitrilase from Rhodococcus rhodochrous tg1-A6, indicating a general applicability of the convenient SbCPT strategy for better industrial applications.
1. Introduction For an industrial enzyme with a defined substrate, activity and stability are the two most important properties [1–3]. However, the trade-off between activity and stability generally limits the biotechnological applications of enzymes [4]. Industrial bio-catalytic processes, which are increasingly important in the production of diverse bulk or fine chemicals and medicine intermediates, have seen considerable growth and are in need of enzymes with both high efficiency and stability. Although several methodologies have been developed, such as directed evolution [5–7], rational design [8,9], and immobilization [3,10], there remains a need for a novel strategy of both simplicity and effectiveness to enhance the activity and stability of target enzymes. The salt-bridge is a strong interaction connecting positively and negatively charged amino acids. Its intensity generally falls between that of covalent bonds and hydrogen bonds. It participates in the folding and formation of the tertiary structure of enzymes and it is insensitive to temperature change or a polar environment, thus it plays a
key role in stabilizing the enzyme structures [11,12]. Under stress conditions such as high temperature, inappropriate pH and organic solvents, the structure of well-folded enzymes will be deformed, which will consequently cause activity loss of the enzymes [13]. Comparative studies on the structures of mesophilic and thermophilic enzymes showed that some special strong interactions, including the disulfide bond, salt bridge and hydrogen bond, are of great significance for the outstanding stability of the thermophilic enzymes [14]. Particularly, extremely thermophilic enzymes generally have more salt bridges compared to mesophilic enzymes [15–17]. Several studies have investigated the function of salt-bridges in different parts of enzymes, including the surface, the vicinity of active site, and the terminus of the enzymes (Table S1). Strop et al. observed the contribution of the salt bridge to the stability of the super thermophilic enzyme PFRD-XC4 [18]. Kumara et al. showed that more salt bridges are found near the active site in the thermophilic glutamate dehydrogenase [19]. Cobucci-Ponzano et al. reported that the salt bridge network at the C-terminus of the β-glycosidase from the
Abbreviations: SbCPT, salt-bridge cyclization peptide tag; NHase, nitrile hydratases; MD simulations, molecular dynamics simulations; NHaseM-TH, NHase from Rhodococcus ruber TH; Nit-tg1A6, nitrilase from R. rhodochrous tg1-A6; PMF, potential-of-mean-force; ABF, Adaptive Biasing Force ⁎ Corresponding author. E-mail address: [email protected] (H. Yu). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.procbio.2019.03.002 Received 15 November 2018; Received in revised form 6 February 2019; Accepted 6 March 2019 Available online 08 March 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. Molecular dynamics simulations
hyperthermophilic archaeon Sulfolobus solfataricus plays an important role in the thermal stabilization of the quaternary structure [20]. Foglia et al. also reported on the importance of N-terminal salt bridges to the stability of thermophilic esterase 2 [21]. A short peptide loop was applied to cyclize the polypeptide backbone of enzymes to enhance the stability [22,23]. Nitrilase hydratase (NHase) and nitrilase are two remarkable nitrileconverting enzymes that are widely used to hydrolyze the available nitrile to corresponding amides and acids, respectively. Previous studies regarding the modification of NHase and nitrilase for stability improvement usually caused activity loss. For example, Shi et al. increased the thermal stability of NHase by stabilizing the rigid α-helix structure, but this resulted in a 30–50% activity loss [24]. Cui et al. presented a site targeted amino recombination method that enhanced both the thermal stability and acrylamide tolerance of NHase, but its activity was decreased by 20% [25]. Liu et al. fused NHase with two self-assembling peptides (EAK16-AEAEAKAKAEAEAKAK and ELK16-LELELKLKLELELKLK) and obtained stability-enhanced enzymes, but the activity was not simultaneously enhanced either [26]. Yang et al. introduced an amphipathic short peptide 18 A (EWLKAFYEKVLEKLKELF) to the C-terminus of a nitrilase, which resulted in higher stability but lower activity [27]. Our previous study investigated the thermal sensitive regions of NHase through molecular dynamics (MD) simulations and found that the N- and C-terminus are the most unstable regions that are responsible for the initial heat denaturation of NHase [28]. For the NHase from the Rhodococcus ruber TH (NHaseM-TH) [29], the global stability was significantly enhanced when it was introduced by a salt-bridge module into the C-terminus of its β-subunit [30]. Enlightened by these studies, this work describes a novel idea to design a salt-bridge cyclization peptide tag (SbCPT) for enzyme stability enhancement. The SbCPT, similar to the widely used His-tag for fast protein purification, can be conveniently attached to the subunit terminus of an enzyme. We proposed that the SbCPT would prevent large fluctuations of the sensitive subunit terminus and stabilize the target enzyme under elevated thermal or other environmental stress. Meanwhile, the SbCPT would also enhance, or at least would not be detrimental to, the activity of the enzyme. Using Co-type NHaseM–TH from R. ruber TH [31,32] and aliphatic nitrilase from R. rhodochrous tg1-A6 (Nit-tg1 A6) [33,34] as model enzymes, a series of SbCPTs were designed and investigated both computationally and experimentally.
All simulations reported in this study were performed with the NAMD program [37]. Each system was first minimized for 200 conjugate gradient steps, followed by equilibration for 0.5 ns. Then, different types of production simulations were performed for different purposes. These simulations were conducted in an isobaric-isothermal ensemble with a temperature of 310 K and a pressure of 1 atm using softly damped Langevin dynamics and the Langevin piston [38]. All bonds involving hydrogen were constrained by the SHAKE algorithm. The nonbonded van der Waals cutoff was set to 12 Å and switched smoothly from 10 Å. For calculations of long-range electrostatic interactions, the PME algorithm was utilized [39]. Short- and long-range interactions were calculated with time steps of 2 and 4 fs, respectively, using a multiple-time step algorithm [40]. To calculate the potential-of-mean-force (PMF) along the collective variable (CV), which was the distance between the side chain of the basic residue and that of the acidic residue in the SbCPT, and to identify the SbCPT conformation that required the minimum energy, Adaptive Biasing Force (ABF) simulations were performed. The advantage of ABF is that the sampling can rapidly become uniform, which in turn greatly improves statistical precision of the calculated free energy [41]. The calculation along the CV was stratified into four serial windows, ranging from 0 to 20 Å, with a grid spacing of 0.1 Å. For each window, an 80-ns ABF simulation was performed to obtain the converged gradients, which were continuous across the simulation bins. The structures and trajectories from the MD simulations were visualized and analyzed using visual molecular dynamics (VMD) software [42]. The density maps of the amino hydrogen in the backbone of position i+2 (for SB1) or i+3 (for SB2 and SB3) were plotted with the VMD software by first aligning the corresponding backbone. The density maps calculated from the ABF simulations were reweighted based on their biased energies. 2.3. Homology modeling Homology modeling of nitrile hydratase (NHase) from Rhodococcus ruber TH (NHaseM-TH) was carried out by I-TASSER, which was ranked as the No 1 server for protein structure prediction in CASP7, CASP8, CASP9, CASP10, CASP11, and CASP12 experiments [43,44]. Obvious mistakes in the relative positions of the α-subunit and β-subunit would occur if the whole sequence of NHaseM-TH was submitted to the ITASSER server altogether. Thus, the structures of the α- and β-subunits of NHaseM-TH were first constructed by separately submitting their respective sequences to the I-TASSER server. The structures of the two subunits were then docked together based on the template of the NHase from Pseudonocardia thermophila JCM 3095 (PDB ID: 1UGP), which was the communal template in the top five predictions produced by ITASSER. Further structural optimization was performed by FG-MD to obtain the final whole structure of NHaseM-TH [45].
2. Materials and methods 2.1. Models and systems The structures of SbCPTs of different lengths (GRPEG, GRGPGEG, and GRGGPGGEG) and different types (GRPEG, GRPDG, GKPEG, and GKPDG) were constructed. Their N-termini and C-termini were capped with neutral acetyl group (CH3CO-) and a methylamino group (-NHCH3), respectively, to avoid the electrostatic interactions between the charged residues and the termini of the SbCPTs. For example, GRPEG SbCPT was constructed as CH3CO-Gly-Arg-Pro-Glu-Gly-NHCH3. All the peptide bonds were in the trans conformation (ω = 180°). The initial main structure of SbCPT was in an extended conformation (φ = -180°, ψ = 180°) except for the Pro residue (φ = -70°, ψ = -180° or 70°). Counter ions (Na + or Cl−) at a concentration of ˜100 mM were added into the SbCPT system. Each system was solvated with a cubic TIP3P water box [35]. For the SbCPT of a length of five amino acids, the dimension of the periodic cell was 30 × 30 × 30 Å3, while the lengths of the cubic periodic cells for SbCPTs of seven (GRGPGEG) and nine residues (GRGGPGGEG) were 36 Å and 45 Å, respectively. The all-atom CHARMM force field [36] was used for each system.
2.4. Bacterial strains, plasmids and chemicals E. coli TOP-10F’ (Invitrogen) was used for cloning and amplification of the mutant plasmids. E. coli BL21(DE3) (Tiangen, China) was used for the expression of the NHase-THM, Nit-tg1-A6, and all of their engineered mutants with different SbCPTs. Seed cultures of the strains were all grown at 37 °C and 200 rpm in LB medium containing kanamycin (100 μg/mL). Cultivation of all of the recombinant E. coli strains for NHase or nitrilase expression was carried out in a conical flask. The NHase expression in E. coli BL21(DE3) was induced by 2% of 0.5 mol/L lactose and 0.2% of 0.5 mol/L CoCl2 in LB medium for 8 h when the OD600 reached 2.0. The nitrilase expression in E. coli BL21(DE3) was induced by 2% of 0.5 mol/L lactose in LB medium for 8 h when OD600 reached 2.0. The cell density was measured spectrophotometrically at 600 nm (OD600). After expression, the cells were collected by 40
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quantitative analysis using Quantity-One Software v. 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA). The Kcat/Km value were calculated using the apparent Vmax and the target proteins concentrations. Nittg1 A6 and its variants were also using the similar method to obtain their apparent enzymology parameters.
centrifugation at 6000 × g for 10 min at 4 °C and washed twice, with 50 mM phosphate-buffered saline (3 × PBS buffer: NaCl 8.0 g/L, KCl 0.2 g/L, Na2HPO4 1.15 g/L, KH2PO4 0.2 g/L, pH 7.0). All plasmids and strains used in this study are listed in Table S2 and Table S3. Plasmids pETNHM and pET-Nit were constructed as described previously [46]. T4 DNA ligase and Q5 DNA polymerase were purchased from NEB China. Restriction endonuclease, Pfu DNA polymerase, T4 Polynucleotide Kinase, and lactose were obtained from Takara Biotechnology Dalian Co. Ltd. The PCR product clean-up kit and plasmid DNA purification kit were purchased from Biomiga (Shanghai, China).
3. Results 3.1. Design and evaluation of SbCPTs with different lengths SbCPTs of different lengths were investigated to find the optimal size and structure for enzyme terminal stabilization. An arginine (Arg, R) and a glutamic acid (Glu, E) were employed in the SbCPT as the basic and acidic amino acids, respectively, to form a salt bridge. The turn structure was introduced by a proline (Pro, P) residue. SbCPTs of 5, 7, and 9 amino acid lengths were chosen and named as follows: SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG). The MD simulations of the three SbCPTs showed that all were able to form pseudocyclic structures constrained by the Arg-Glu salt bridge (Figure S2). The SB2 and SB3 formed β-turn structures, while the SB1 formed a nonclassical turn structure mainly involving three residues (Arg, Pro, and Glu) to retain the curvature. The detailed information about the potential-of-mean-force (PMF) energy referred to the SbCPT stabilization was provided by Adaptive Biasing Force (ABF) MD simulations. In the ABF simulations, the collective variable (CV) was chosen as the distance between the side chain of the basic residue and that of the acidic residue in the SbCPT (Fig. 1A). The distance at the PMF minimum of SB1 was 3.5 Å (Fig. 1B), indicating that a stable salt bridge was formed in the natural state of SB1 (N). At the distance of 11.3 Å, there was another local PMF minimum, which could be treated as the unfolded state of SB1 (D). The SB1 structure with the PMF maximum between N and D was the transition state (E≠), only through which the N and D can convert to each other (Fig. 1B). The higher the energy barrier is between the transition state E≠ and natural state N, the more difficult it is for N to kinetically convert to the denatured state D, indicating a more stable salt bridge formed in the SbCPT. Thus, the energy barrier can be regarded as a measurement of SbCPT stability. According to the PMF energies obtained by the ABF MD simulations SB2 and SB3 were slightly more stable than SB1, whether kinetically or thermodynamically (Fig. 1C and Table S5). Experimental insertion mutagenesis of SB1-SB3 to the C-terminus of the NHaseM–TH α-subunit was further performed. The results showed that the attachment of SB2 or SB3 to NHaseM–TH (NH-SB2 or NH-SB3) caused a drastic decrease in enzyme activity. However, the insertion of SB1 (NH-SB1), the shortest SbCPT among the three SbCPTs, was not detrimental to the enzyme activity, and on the contrary resulted in small enhancement of the NH-SB1 activity (Fig. 2). Accordingly, the SbCPT with the five amino acid length (GRPEG) was subsequently selected to minimize the impact of SbCPT insertion on the local structure of the enzyme.
2.5. Construction of SbCPT-attached NHase and nitrilase mutants The parental enzyme NHaseM-TH, expressed by the plasmid pETNHM, contains the initiation codon mutation from GTG to ATG [45]. The procedure for the construction of SbCPT-attached NHase mutants is shown in Figure S1, taking the construction of plasmid pETNHM-βXX (XX is RE, RD, KE, or KD) as an example. The engineered plasmid containing SbCPT-attached NHase was constructed by sense and antisense primers based on the template plasmid pETNHM. The mutant plasmid was then transformed into the E. coli BL21(DE3) pLysS to express the NHase mutant. The Nit-tg1 A6 mutants were constructed in a similar way. Gene cloning operations, such as polymerase chain reaction (PCR) and DNA electrophoresis were carried out by standard procedures. The genes were sequenced by SinoGenoMax. All primers were synthesized by Invitrogen Biotech (Beijing) Co. Ltd. and are listed in Table S4. 2.6. NHase or nitrilase activity assay and SDS-PAGE electrophoresis The enzyme activity assays and SDS-PAGE analysis were performed as described by Chen et al. [30]. In brief, NHase activity was assayed at 28 °C in a 5-mL reaction mixture containing 100 μL of acrylonitrile and 0.25 g/L free cells in 50 mM PBS buffer for 5 min. Nitrilase activity was assayed at 28 °C in a 1.12 mL reaction mixture containing 20 μL of acrylonitrile and 0.5 g/L free cells for 10 min. 100 μL of 3 M HCl was added to stop the reaction. The concentration of acrylamide (for the NHase activity assay) or ammonium acrylate (for the nitrilase activity assay) in the supernatant of the reaction mixture were measured by gas chromatography (Trace1300, Thermo Fisher Scientific, America) to determine the NHase or nitrilase activity. Protein expression results were observed by SDS-PAGE analysis with a 5% stacking gel and 12% running gel stained with the Coomassie brilliant blue R-250. One unit (U) of NHase or nitrilase activity corresponds to the amount of the enzyme producing 1 μ mol product per min at optimal reaction conditions. 2.7. Thermal stability assessment The cells containing NHase variant in 50 mM PBS were soaked in a water bath at 42 °C, and the activity was measured by GC at room temperature. The cells containing the nitrilase variant were soaked in a water bath at 50 °C for a certain time, and the activity was measured by GC at 28 °C. The activity value at the start time was defined as 100%.
3.2. Insertion of the SbCPT into different subunit terminus of NHaseM–TH Attachment of SB1 (renamed as RE hereafter according to the types of its charged residues) on the N- or C-terminus of the NHase α- or βsubunit caused evident changes in enzyme activity. As shown in Fig. 3A and B, the insertion of RE in the C-terminus of the NHase α- or βsubunit (NH-αC or NH-βC) increased the enzyme activity to some extent, while the insertion of RE in the N-terminus (NH-αN or NH-βN) led to a considerable decrease in activity. Protein electrophoresis showed that the expressed bands of NH-αN, NH-αC and NH-βC were similar to the wild-type (WT) NHaseM–TH, while NH-βN was almost undetectable (Fig. 3C). This may be one of the reasons why NH-βN had the lowest activity among the NHase variants. According to the structure of NHaseM–TH constructed by homology
2.8. Apparent enzymology parameter measurement NHaseM-TH and the SbCPT-attached variants activity was measured at gradient substrate concentration for different reaction time (5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 40 s). The initial reaction rate was obtained as the slope of the reaction curve through the linear fit. Vmax and Km were obtained using Matlab V. R2015b by directly fitting the activity data to the Michaelis-Menten equation. The total soluble protein concentrations of NHaseM-TH and the SbCPT-attached variants were measured by BCA protein assay (Promega, Madison, USA). The proportions of target proteins were determined by SDS-PAGE electrophoresis band 41
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Fig. 1. Interpretation of potential-of-mean-force (PMF) as a measurement of the stability of the salt-bridge cyclization peptide tag (SbCPT). A. The collective variable (CV) chosen in the Adaptive Biasing Force (ABF) MD simulations was the distance between the side chain of the basic residue (Arg in this case) and that of the acidic residue (Glu in this case). B. Variation in the PMF energy along with the CV distance. N is the natural state, which adopts the lowest free energy when a salt bridge is formed. D is the denatured state, which adopts a minimum energy when the salt bridge is unfolded. E≠ is the transition state between N and D. The energy barrier and energy difference between N and D are also labeled on the chart. C. PMF variation along with the CV distance change in the SB1, SB2, and SB3 SbCPTs. SbCPTs of 5, 7, and 9 amino acid lengths were chosen and named as follows: SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG).
attachment of a SbCPT, the N-terminus of α-subunit is also likely to clash with the scaffold of NHaseM–TH and cause a great loss of activity. Among the four termini, the C-terminus of the β-subunit is the most extensible and located totally outside of the enzyme scaffold. An additional SbCPT in NH-βC will not disturb the infrastructure of the enzyme and also stabilize the flexible C-terminus of its β-subunit. In addition to the local steric hindrance, enzyme activity may also be affected by obstruction of substrate and product transport tunnel.
modeling (Figure S3), the local steric hindrances around the subunit termini are in the order: βN > αN > > αC > βC (Figs. 3D and S4), which correlates highly with the enzyme activities after SbCPT attachment (Fig. 3B). Especially, the N-terminus of the β-subunit is located at a cavity that is closely surrounded by other parts of the NHaseM–TH. Thus, even a small peptide attached to this terminus will not fit in the original cavity, leading to the disturbance of the enzyme folding, which may consequently affect the expression of NH-βC. With an
Fig. 2. Insertion experiment of salt-bridge cyclization peptide tags (SbCPTs) with different lengths. A. SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG) SbCPTs were attached to the C-terminus of the nitrile hydratases (NHase) α-subunit. B. Enzyme activities of the NHase variants. The experiments were performed in triplicate. NH indicates the control NHaseM–TH. NH-SB1, NH-SB2, and NH-SB3 are the recombinant NHases in BL21(DE3)SB1, BL21(DE3)-SB2, and BL21(DE3)-SB3, respectively.
42
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Fig. 3. Insertion experiment of salt-bridge cyclization peptide tags (SbCPTs) into different terminus of nitrile hydratases from Rhodococcus ruber TH (NHaseM–TH). A. RE SbCPT (a pentapeptide: GRPEG) was attached at the N- or C-terminus of the nitrile hydratases (NHase) α- or β-subunit. B. Enzyme activities of the NHase variants. Experiments were performed in triplicate. C. Protein electrophoresis of the four SbCPT-attached NHase variants. NH indicates the control NHaseM–TH. NH-αN, NHαC, NH-βN, and NH-βC are the recombinant NHase in BL21(DE3)-αN, BL21(DE3)-αC, BL21(DE3)-βN, and BL21(DE3)-βC, respectively. D. The termini of the α- and β-subunit in NHaseM–TH. The structure of NHaseM–TH except for the four termini is shown as the grey surface. The termini of α- and β-subunits are shown as blue and purple ribbons, respectively.
The N-terminus of the β-subunit partially constitutes the entrance of the tunnel B and the N-terminus of the α-subunit is near that tunnel (Figure S3). Adequate flexibility is necessary for a functional tunnel and, therefore, the stabilization effect by SbCPT attachment on these two termini would be another reason for the decrease in enzyme activity. The C-terminus of either the α- or β-subunit is far from the two possible tunnels, thus its change will have little effect on the tunnel(s). Considering both the local steric hindrance and flexibility of the functional tunnel, the C-terminus of the α- or β-subunit in NHaseM–TH was chosen as the subsequent target for SbCPT insertion to improve enzyme activity and stability.
charged residues were close to each other, indicating that a salt bridge was formed in their most stable conformation (Fig. 4 and Table S6). RE and RD exhibited especially larger energy differences between their salt-bridge formed state and unfolded state (1.09 and 2.13 kcal/mol, respectively), ensuring that their conformation would be firmly constrained by the salt bridge even in a fluctuating environment, such as the local high temperature, which would make for easier conversion of the salt-bridge formed state to the unfolded state across the transition state. In contrast to these SbCPTs, the lowest free energy of KE occupied an undesirable unfolded conformation in which the distance between the side chains of the two charged residues was approximately 12 Å. However, if the KE could initially fold into a salt-bridge formed state, this state could be stably maintained and separated from the unfolded state by an energy barrier of 1.58 kcal/mol (≈ 2.6 kBT). Taking account both the kinetic parameter (energy barrier) and the thermodynamic parameter (energy difference), the stability of the four SbCPTs was in the order RD > RE > KD > KE (Table S6).
3.3. Computational investigation of different types of SbCPTs There are four types of SbCPT with the sequence GXPXG (X denotes a charged residue), these include RE (GRPEG), RD (GRPDG), KE (GKPEG), and KD (GKPDG). The PMF energies obtained by the ABF MD simulations of the four SbCPTs showed that the lowest free energies of RE, RD, and KD took the conformation when the side chains of the two 43
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Fig. 4. Potential-of-mean-force (PMF) energy variation along with the change in CV distance in the RE, RD, KE, and KD SbCPTs. RE, RD, KE and KD were named according to the types of its charged residues. RE: GRPEG, RD: GRPDG, KE: GKPEG, KD: GKPDG.
subsequently assessed in term of the retention activity after incubation at 42 °C for 3 h. For most of the variants, no obvious change of stability was found with respect to WT NHaseM-TH (Fig. 5B). Two exceptions were NH-αRD for which the retention activity increased by 32.7%, and NH-αKE that observed the retention activity decrease by 19%. Compared with the results of the ABF MD simulations, RD and KE, which stabilized and destabilized the NHase when attached at the C-terminus of the α-subunit, respectively, were also found to be the most and least stable SbCPTs respectively, as estimated by PMF energy. Purified NHase was tried to harvest through adding the His-tag to the N terminal of the β subunit of the NHase. But a significant activity decrease was observed, due to the formation of inclusion body (Figure S6). Considering that the cellular catalysts are used in industrial applications of NHase, in this study we further analyzed the performance of intracellular enzyme as indicated by its apparent enzymology parameters. From the results of the apparent Km, Vmax and Kcat/Km of the enzymes analyzed in Table 1, we found that for the SbCPT-attached variants, the Vmax was all increased, which is in consistent with the result in Fig. 5A. Especially, the Kcat/Km of NH-αRD was enhanced by 17.6% relative to the control, indicating that the NH-αRD catalyzed the hydration of acrylonitrile to acrylamide was faster than NHase alone.
3.4. Assessment of the NHase activity and stability with different SbCPTs The NHase variants (NH-αRE, NH-αRD, NH-αKE, NH-αKD, NHβRE, NH-βRD, NH-βKE, and NH-βKD) with one of the four SbCPTs (RE, RD, KE, and KD) attached to the C-terminus of the α- or β-subunit were constructed to investigate their enzyme activities and stabilities. The overall catalytic competence of all the SbCPT-attached NHases were greater than or at least comparable to that of the WT NHaseM-TH, especially the activity of NH-αKD, which was increased by 54% (Fig. 5A). Thermal stabilities of these SbCPT-attached NHase variants were
3.5. Assessment of the nitrilase activity and stability with different SbCPTs The Nit-tg1 A6 variants (Nit-CKE, Nit-CKD, Nit-CRD, Nit-NKE, NitNKD, Nit-NRD) with one of the three SbCPTs (KE, KD and RD) attached to the C- or N-terminus were constructed to further test their properties. It can be seen that the enzyme activities of the Nit-tg1 A6 variants with SbCPT attached to the C-terminus were distinctly enhanced compared with the WT Nit-tg1 A6, while activity of the N-terminal attached variants was severely reduced (Fig. 6A). Although both the N- and Ctermini are far from the active site in Nit-tg1 A6, the C-terminus sticks out from the main structure, while the N-terminus remains close to the Table 1 Apparent enzymology parameters for NHase and its variants modified by SbCPTs on the C terminal of the α subunit of the NHase. Fig. 5. Insertion of different salt-bridge cyclization peptide tag (SbCPT) into the C-terminus of nitrile hydratases from Rhodococcus ruber TH (NHaseM–TH). A. Enzyme activities of the nitrile hydratases (NHase) variants. B. The thermal stability of the NHase variants was assessed in PBS buffer (50 mM, pH 7.0) at 42 °C for 3 h. NH indicates the control NHaseM–TH. Experiments were performed in triplicate. 44
Strain
Km/ (g/L)
NHase NH-α KE NH-α KD NH-α RE NH-α RD
5.20 ± 4.64 ± 4.70 ± 4.42 ± 4.07 ±
0.10 0.05 0.10 0.04 0.15
Vmax/ (U/mL)
Kcat/ Km/(U·L·g−1/ μmol Enzyme)
114.9 ± 126.6 ± 125.0 ± 112.4 ± 135.1 ±
3784 3662 3509 3027 4450
2.0 1.5 4.0 3.0 2.5
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glycine residues were introduced into SB1 (GRPEG), SB2 (GRGPGEG) and SB3 (GRGGPGGEG) to change the length of SbCPTs. According to the PMF energies obtained by ABF MD simulations, the energy difference of SB1-SB3 finally did not change systematically (the order is SB2 > SB3 > SB1) (Fig. 1C and Table S5). The reason we deduced is that not only the peptide length, but also the amino acid composition such as the quantity of glycine residues affect the stability of SbCPTs. On the one hand, with the increase of peptide length, more glycine residues introduced would reduce the space-resistance against the turnstructure formation, help easily form a more stable salt-bridge; on the other hand, however, more and more glycine residues would also cause an unfavorable entropy-driven effect that prone to break ionic interactions in hydrophilic environment. When the number of glycine residues increased to the extent that the negative instable effect played the major role, the overall stability of the SbCPT would reduce conversely. In addition, the applicability of SB1, SB2 and SB3 should also consider the effect of the SbCPTs with different length on the 3D structure of a target enzyme. From the experimental results of Fig. 2, we can find that inserting large SbCPTs such as SB2 and SB3 into the terminus of NHase significantly reduced the enzyme activity, indicating that there existed negative impact of long SbCPTs on the local structure of NHase. It might be the steric hindrance in the subunit terminus, leading to a clash or distortion of the terminal structure and thereby a decrease of the enzyme activity. To maintain enzyme activity while improving stability, a relatively smaller SbCPT should be selected. Thus, SbCPT with a five amino acid length was postulated to be suitable for a universal application since a turn structure with a salt bridge could be formed while minimizing the negative effect on the structure. Additionally, proline-rich structures usually do significant contribution to stabilizing enzymes as well [48]. For the function of proline residue in SbCPTs of this work, however, it only acted as a key amino acid assisting formation of the turn-structure. Investigation of the SbCPT insertion position by experiments and homology modeling showed that the local structural environment of the SbCPT-attached terminus was crucial for the mutational activity. Insertion of a SbCPT to the terminus in a confining space would cause a clash with the infrastructure of the enzyme and inevitably decrease the enzyme’s activity (Fig. 3). Moreover, although the termini of enzymes are generally far from the active site, they may still constitute or approach other functional regions such as the substrate and product transport tunnel (e.g., the N-terminus of the NHase β-subunit in this study), a hot spot for protein-protein recognition, a subunit surface for electron transport, etc. To preserve the full function of the enzyme and maintain its activity, it is better to avoid a large change (e.g., SbCPT insertion) in the functional terminus. Thus, SbCPT should only be attached on the enzyme terminus that is extensible and also far from any special functional area. Without loss of activity, the SbCPT insertion into such a terminus would even generally enhance the total enzyme activity probably due to stabilization of the flexible enzyme terminus from degradation [49] or enhancement of specific enzyme activity from the change of the NHase variant structure. For the four different types of SbCPTs, the PMF energies calculated by the ABF MD simulations indicated that their stabilities are in the order: RD > RE > KD > KE. This means that residue R with guanidine group can form a more stable non-classical turn structure with D or E than residue K. Experimental results of SbCPT attached on the Cterminus of the α-subunit were in the order: RD > RE ≈ KD > KE (Fig. 5B), which were approximately consistent with the computational results, in which the stability of NH-αRD was highly enhanced while that of NH-αKE, in contrast, was even reduced. The experimental results of SbCPT attached on the C-terminus of β-subunit, however, made the explanation complicated. On the one hand, the molecular sizes of SbCPTs containing RD or KD were smaller than that of RE or KE, respectively, thereby prone to form a relatively more stable salt-bridge due to less space resistance of the SbCPT alone. On the other hand, the spatial position and amino acid sequence of the specific terminus
Fig. 6. Applicability assessment of salt-bridge cyclization peptide tag (SbCPT)insertion in nitrilase from R. rhodochrous tg1-A6 (Nit-tg1 A6). A. Enzyme activities of the nitrilase variants with SbCPTs inserted into the C- or N-terminus. B. Thermal stability of nitrilase variants with C- terminus insertion of SbCPTs. Nitrilase activities were measured after being at 50 °C for 15 min. “Nitrilase” indicates the control Nit-tg1 A6. Experiments were performed in triplicate.
surface (Figure S5). Insertion of a SbCPT to the N-terminus of nitrilase may impair its interactions with the surface, thus counteracting the stabilization benefit from SbCPT attachment, or even destabilizing the N-terminus structure. Inserting the His-tag to the N-terminus of nitrilase Nit-tg1-A6 showed similar results, and no purified nitrilase could be successfully obtained (Figure S7). For better understanding the influences of the SbCPTs on Nit-tg1-A6 properties, we also used free cells to measure the apparent enzymology parameters (Table S7). The Vmax of the SbCPT-attached variants was also enhanced corresponding to the results shown in Fig. 6. Analyzing the SDS-PAGE results using software named Quantity One (Figure S8), we found that the expression of the target protein was increased. Comparing with the Nit-tg1-A6 (52% in total soluble protein expression), the expression of the Nit-KE, Nit-RD and Nit-KD in total soluble protein ratio was 63.4%, 60.6% and 60.8%, respectively. Thermal stabilities of the C-terminal SbCPT-attached Nit-tg1 A6 variants were further measured in terms of the retention activity after incubation at 50 °C for 15 min. It was shown that the stability of all the C-terminal SbCPT-attached variants was enhanced, especially that of Nit-CRD, which was increased by 30.3% (from 50.1% in WT Nit-tg1 A6 to 65.3% in Nit-CRD) (Fig. 6B).
4. Discussion Several studies have shown that a salt bridge in the terminus of an enzyme would enhance the enzyme stability [20,21,30,47]. Here, we proposed an enzyme stabilization strategy by terminal insertion of a SbCPT. The core idea is to enable an oligo-peptide to form a cyclization structure via salt-bridge interaction and apply it to reduce the extensional freedom of the enzyme terminus, and thereby universally enhance stability and activity of diverse enzymes. Design of SbCPTs with different length was first concerned. Several 45
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carrying the SbCPT will also affect the activity and stability of target enzyme after SbCPT attachment. The local spatial hindrance around the C-terminus of β-subunit, as well as the interaction of SbCPT with the enzyme scaffold, could both exert an intricate influence on enzyme activity and stability, besides the structure of SbCPT alone. In light of these considerations, we suggested that RD could be used as a universal SbCPT attaching to the enzyme terminus to enhance both the total activity and stability. In contrast to rational design [8], only very little information about the enzyme structure (i.e., the local steric environment of the enzyme termini) is required with the SbCPT strategy. This would be of great value when the enzyme structure is difficult to obtain or only has low accuracy, especially for the industrial biocatalysts. Additionally, unlike random mutagenesis [7], the SbCPT strategy only requires a small number of experiments, but a relatively higher rate of success can be achieved. The general applicability of the SbCPT strategy was further shown with Nit-tg1 A6, which indicated that terminal attachment of the SbCPT could be used as a universal strategy for the stability enhancement of industrial enzymes without activity loss.
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Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21476126 and 21776157), National Key Basic Research Project 973 (2013CB733600) and the China Postdoctoral Science Foundation (No. 2015M581110). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2019.03.002. References [1] J.M. Choi, S.S. Han, H.S. Kim, Industrial applications of enzyme biocatalysis: Current status and future aspects, Biotechnol. Adv. 33 (2015) 1443–1454, https:// doi.org/10.1016/j.biotechadv.2015.02.014. [2] A. Madhavan, R. Sindhu, P. Binod, R.K. Sukumaran, A. Pandey, Strategies for design of improved biocatalysts for industrial applications, Bioresour. Technol. 245 (2017) 1304–1313, https://doi.org/10.1016/j.biortech.2017.05.031. [3] C. Berna, K. Rodriguez, R. Martinez, Integrating enzyme immobilization and protein engineering: an alternative path for the development of novel and improved industrial biocatalysts, Biotechnol. Adv. 36 (2018) 1470–1480, https://doi.org/10. 1016/j.biotechadv.2018.06.002. [4] N. Tokuriki, F. Stricher, L. Serrano, D.S. Tawfik, How protein stability and new functions trade off, PLoS Comput. Biol. (2008), https://doi.org/10.1371/journal. pcbi.1000002. [5] C.A. Denard, H. Ren, H. Zhao, Improving and repurposing biocatalysts via directed evolution, Curr. Opin. Chem. Biol. 25 (2015) 55–64, https://doi.org/10.1016/j. cbpa.2014.12.036. [6] M.S. Packer, D.R. Liu, Methods for the directed evolution of proteins, Nat. Rev. Genet. 16 (2015) 379–394, https://doi.org/10.1038/nrg3927. [7] R. Frey, T. Hayashi, R.M. Buller, Directed evolution of carbon-hydrogen bond activating enzymes, Curr. Opin. Biothenol. 60 (2019) 29–38, https://doi.org/10. 1016/j.copbio.2018.12.004. [8] P.S. Huang, S.E. Boyken, D. Baker, The coming of age of de novo protein design, Nature. 537 (2016) 320–327, https://doi.org/10.1038/nature19946. [9] Q. Liu, G. Xun, Y. Feng, The state-of-the-art strategies of protein engineering for enzyme stabilization, Biotechnol. Adv. (2018), https://doi.org/10.1016/j. biotechadv.2018.10.011 In Press. [10] M.C.P. Goncalves, T.G. Kieckbusch, R.F. Perna, J.T. Fujimoto, S.A.V. Morales, J.P. Romanelli, Trends on enzyme immobilization researched based on bibliometric analysis, Process Biochem. 76 (2019) 95–110, https://doi.org/10.1016/j.procbio. 2018.09.016. [11] C. Lee, H. Wang, J.H. wang, C. Tseng, Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study, PLoS One 9 (2014) e112751, , https://doi.org/10.1371/journal.pone.0112751. [12] N. Zhang, Y. Wang, L. An, X. Song, Q. Huang, Z. Liu, L. Yao, Entropy drives the formation of salt bridges in the protein GB3, Angew. Chem. Int. Ed. 129 (2017) 7601–7604, https://doi.org/10.1002/ange.201702968.
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Design of salt-bridge cyclization peptide tags for stability and activity enhancement of enzymes
T
⁎
Lingjun Tanga,1, Ji Yanga,1, Jie Chena, Jing Zhanga, Huimin Yua,b, , Zhongyao Shena a b
Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Salt-bridge cyclization peptide tag (SbCPT) Insertion mutagenesis Molecular dynamics simulations Nitrile hydratase Nitrilase
The terminus is usually the most flexible and fluctuating part that affects the enzyme stability. In this study, we proposed an enzyme terminus stabilization strategy based on the attachment of a salt-bridge cyclization peptide tag (SbCPT) without activity loss. SbCPTs of different lengths, as well as different subunit termini of nitrile hydratases from Rhodococcus ruber TH (NHaseM-TH) for SbCPT insertion, were investigated by molecular dynamics (MD) simulations and mutagenesis experiments. The investigation revealed that a smaller SbCPT would be favorable and that the SbCPT should only be attached on the enzyme terminus that is extensible and also far from any special functional area due to the minimal influences on the enzyme structure. The stability of different SbCPTs types (GKPEG, GKPDG, GRPEG, GRPDG) was identified as RD > RE > KD > KE. By insertion of an optimal SbCPT (GRPDG) to the C-terminus of the α-subunit of NHase, the SbCPT-attached NHase variants demonstrated improved stability by a maximum of 32.7%, as well as the overall catalytic competence. Additionally, the effect of SbCPT with enhanced stability and non-reduced activity was also verified in nitrilase from Rhodococcus rhodochrous tg1-A6, indicating a general applicability of the convenient SbCPT strategy for better industrial applications.
1. Introduction For an industrial enzyme with a defined substrate, activity and stability are the two most important properties [1–3]. However, the trade-off between activity and stability generally limits the biotechnological applications of enzymes [4]. Industrial bio-catalytic processes, which are increasingly important in the production of diverse bulk or fine chemicals and medicine intermediates, have seen considerable growth and are in need of enzymes with both high efficiency and stability. Although several methodologies have been developed, such as directed evolution [5–7], rational design [8,9], and immobilization [3,10], there remains a need for a novel strategy of both simplicity and effectiveness to enhance the activity and stability of target enzymes. The salt-bridge is a strong interaction connecting positively and negatively charged amino acids. Its intensity generally falls between that of covalent bonds and hydrogen bonds. It participates in the folding and formation of the tertiary structure of enzymes and it is insensitive to temperature change or a polar environment, thus it plays a
key role in stabilizing the enzyme structures [11,12]. Under stress conditions such as high temperature, inappropriate pH and organic solvents, the structure of well-folded enzymes will be deformed, which will consequently cause activity loss of the enzymes [13]. Comparative studies on the structures of mesophilic and thermophilic enzymes showed that some special strong interactions, including the disulfide bond, salt bridge and hydrogen bond, are of great significance for the outstanding stability of the thermophilic enzymes [14]. Particularly, extremely thermophilic enzymes generally have more salt bridges compared to mesophilic enzymes [15–17]. Several studies have investigated the function of salt-bridges in different parts of enzymes, including the surface, the vicinity of active site, and the terminus of the enzymes (Table S1). Strop et al. observed the contribution of the salt bridge to the stability of the super thermophilic enzyme PFRD-XC4 [18]. Kumara et al. showed that more salt bridges are found near the active site in the thermophilic glutamate dehydrogenase [19]. Cobucci-Ponzano et al. reported that the salt bridge network at the C-terminus of the β-glycosidase from the
Abbreviations: SbCPT, salt-bridge cyclization peptide tag; NHase, nitrile hydratases; MD simulations, molecular dynamics simulations; NHaseM-TH, NHase from Rhodococcus ruber TH; Nit-tg1A6, nitrilase from R. rhodochrous tg1-A6; PMF, potential-of-mean-force; ABF, Adaptive Biasing Force ⁎ Corresponding author. E-mail address: [email protected] (H. Yu). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.procbio.2019.03.002 Received 15 November 2018; Received in revised form 6 February 2019; Accepted 6 March 2019 Available online 08 March 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. Molecular dynamics simulations
hyperthermophilic archaeon Sulfolobus solfataricus plays an important role in the thermal stabilization of the quaternary structure [20]. Foglia et al. also reported on the importance of N-terminal salt bridges to the stability of thermophilic esterase 2 [21]. A short peptide loop was applied to cyclize the polypeptide backbone of enzymes to enhance the stability [22,23]. Nitrilase hydratase (NHase) and nitrilase are two remarkable nitrileconverting enzymes that are widely used to hydrolyze the available nitrile to corresponding amides and acids, respectively. Previous studies regarding the modification of NHase and nitrilase for stability improvement usually caused activity loss. For example, Shi et al. increased the thermal stability of NHase by stabilizing the rigid α-helix structure, but this resulted in a 30–50% activity loss [24]. Cui et al. presented a site targeted amino recombination method that enhanced both the thermal stability and acrylamide tolerance of NHase, but its activity was decreased by 20% [25]. Liu et al. fused NHase with two self-assembling peptides (EAK16-AEAEAKAKAEAEAKAK and ELK16-LELELKLKLELELKLK) and obtained stability-enhanced enzymes, but the activity was not simultaneously enhanced either [26]. Yang et al. introduced an amphipathic short peptide 18 A (EWLKAFYEKVLEKLKELF) to the C-terminus of a nitrilase, which resulted in higher stability but lower activity [27]. Our previous study investigated the thermal sensitive regions of NHase through molecular dynamics (MD) simulations and found that the N- and C-terminus are the most unstable regions that are responsible for the initial heat denaturation of NHase [28]. For the NHase from the Rhodococcus ruber TH (NHaseM-TH) [29], the global stability was significantly enhanced when it was introduced by a salt-bridge module into the C-terminus of its β-subunit [30]. Enlightened by these studies, this work describes a novel idea to design a salt-bridge cyclization peptide tag (SbCPT) for enzyme stability enhancement. The SbCPT, similar to the widely used His-tag for fast protein purification, can be conveniently attached to the subunit terminus of an enzyme. We proposed that the SbCPT would prevent large fluctuations of the sensitive subunit terminus and stabilize the target enzyme under elevated thermal or other environmental stress. Meanwhile, the SbCPT would also enhance, or at least would not be detrimental to, the activity of the enzyme. Using Co-type NHaseM–TH from R. ruber TH [31,32] and aliphatic nitrilase from R. rhodochrous tg1-A6 (Nit-tg1 A6) [33,34] as model enzymes, a series of SbCPTs were designed and investigated both computationally and experimentally.
All simulations reported in this study were performed with the NAMD program [37]. Each system was first minimized for 200 conjugate gradient steps, followed by equilibration for 0.5 ns. Then, different types of production simulations were performed for different purposes. These simulations were conducted in an isobaric-isothermal ensemble with a temperature of 310 K and a pressure of 1 atm using softly damped Langevin dynamics and the Langevin piston [38]. All bonds involving hydrogen were constrained by the SHAKE algorithm. The nonbonded van der Waals cutoff was set to 12 Å and switched smoothly from 10 Å. For calculations of long-range electrostatic interactions, the PME algorithm was utilized [39]. Short- and long-range interactions were calculated with time steps of 2 and 4 fs, respectively, using a multiple-time step algorithm [40]. To calculate the potential-of-mean-force (PMF) along the collective variable (CV), which was the distance between the side chain of the basic residue and that of the acidic residue in the SbCPT, and to identify the SbCPT conformation that required the minimum energy, Adaptive Biasing Force (ABF) simulations were performed. The advantage of ABF is that the sampling can rapidly become uniform, which in turn greatly improves statistical precision of the calculated free energy [41]. The calculation along the CV was stratified into four serial windows, ranging from 0 to 20 Å, with a grid spacing of 0.1 Å. For each window, an 80-ns ABF simulation was performed to obtain the converged gradients, which were continuous across the simulation bins. The structures and trajectories from the MD simulations were visualized and analyzed using visual molecular dynamics (VMD) software [42]. The density maps of the amino hydrogen in the backbone of position i+2 (for SB1) or i+3 (for SB2 and SB3) were plotted with the VMD software by first aligning the corresponding backbone. The density maps calculated from the ABF simulations were reweighted based on their biased energies. 2.3. Homology modeling Homology modeling of nitrile hydratase (NHase) from Rhodococcus ruber TH (NHaseM-TH) was carried out by I-TASSER, which was ranked as the No 1 server for protein structure prediction in CASP7, CASP8, CASP9, CASP10, CASP11, and CASP12 experiments [43,44]. Obvious mistakes in the relative positions of the α-subunit and β-subunit would occur if the whole sequence of NHaseM-TH was submitted to the ITASSER server altogether. Thus, the structures of the α- and β-subunits of NHaseM-TH were first constructed by separately submitting their respective sequences to the I-TASSER server. The structures of the two subunits were then docked together based on the template of the NHase from Pseudonocardia thermophila JCM 3095 (PDB ID: 1UGP), which was the communal template in the top five predictions produced by ITASSER. Further structural optimization was performed by FG-MD to obtain the final whole structure of NHaseM-TH [45].
2. Materials and methods 2.1. Models and systems The structures of SbCPTs of different lengths (GRPEG, GRGPGEG, and GRGGPGGEG) and different types (GRPEG, GRPDG, GKPEG, and GKPDG) were constructed. Their N-termini and C-termini were capped with neutral acetyl group (CH3CO-) and a methylamino group (-NHCH3), respectively, to avoid the electrostatic interactions between the charged residues and the termini of the SbCPTs. For example, GRPEG SbCPT was constructed as CH3CO-Gly-Arg-Pro-Glu-Gly-NHCH3. All the peptide bonds were in the trans conformation (ω = 180°). The initial main structure of SbCPT was in an extended conformation (φ = -180°, ψ = 180°) except for the Pro residue (φ = -70°, ψ = -180° or 70°). Counter ions (Na + or Cl−) at a concentration of ˜100 mM were added into the SbCPT system. Each system was solvated with a cubic TIP3P water box [35]. For the SbCPT of a length of five amino acids, the dimension of the periodic cell was 30 × 30 × 30 Å3, while the lengths of the cubic periodic cells for SbCPTs of seven (GRGPGEG) and nine residues (GRGGPGGEG) were 36 Å and 45 Å, respectively. The all-atom CHARMM force field [36] was used for each system.
2.4. Bacterial strains, plasmids and chemicals E. coli TOP-10F’ (Invitrogen) was used for cloning and amplification of the mutant plasmids. E. coli BL21(DE3) (Tiangen, China) was used for the expression of the NHase-THM, Nit-tg1-A6, and all of their engineered mutants with different SbCPTs. Seed cultures of the strains were all grown at 37 °C and 200 rpm in LB medium containing kanamycin (100 μg/mL). Cultivation of all of the recombinant E. coli strains for NHase or nitrilase expression was carried out in a conical flask. The NHase expression in E. coli BL21(DE3) was induced by 2% of 0.5 mol/L lactose and 0.2% of 0.5 mol/L CoCl2 in LB medium for 8 h when the OD600 reached 2.0. The nitrilase expression in E. coli BL21(DE3) was induced by 2% of 0.5 mol/L lactose in LB medium for 8 h when OD600 reached 2.0. The cell density was measured spectrophotometrically at 600 nm (OD600). After expression, the cells were collected by 40
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quantitative analysis using Quantity-One Software v. 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA). The Kcat/Km value were calculated using the apparent Vmax and the target proteins concentrations. Nittg1 A6 and its variants were also using the similar method to obtain their apparent enzymology parameters.
centrifugation at 6000 × g for 10 min at 4 °C and washed twice, with 50 mM phosphate-buffered saline (3 × PBS buffer: NaCl 8.0 g/L, KCl 0.2 g/L, Na2HPO4 1.15 g/L, KH2PO4 0.2 g/L, pH 7.0). All plasmids and strains used in this study are listed in Table S2 and Table S3. Plasmids pETNHM and pET-Nit were constructed as described previously [46]. T4 DNA ligase and Q5 DNA polymerase were purchased from NEB China. Restriction endonuclease, Pfu DNA polymerase, T4 Polynucleotide Kinase, and lactose were obtained from Takara Biotechnology Dalian Co. Ltd. The PCR product clean-up kit and plasmid DNA purification kit were purchased from Biomiga (Shanghai, China).
3. Results 3.1. Design and evaluation of SbCPTs with different lengths SbCPTs of different lengths were investigated to find the optimal size and structure for enzyme terminal stabilization. An arginine (Arg, R) and a glutamic acid (Glu, E) were employed in the SbCPT as the basic and acidic amino acids, respectively, to form a salt bridge. The turn structure was introduced by a proline (Pro, P) residue. SbCPTs of 5, 7, and 9 amino acid lengths were chosen and named as follows: SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG). The MD simulations of the three SbCPTs showed that all were able to form pseudocyclic structures constrained by the Arg-Glu salt bridge (Figure S2). The SB2 and SB3 formed β-turn structures, while the SB1 formed a nonclassical turn structure mainly involving three residues (Arg, Pro, and Glu) to retain the curvature. The detailed information about the potential-of-mean-force (PMF) energy referred to the SbCPT stabilization was provided by Adaptive Biasing Force (ABF) MD simulations. In the ABF simulations, the collective variable (CV) was chosen as the distance between the side chain of the basic residue and that of the acidic residue in the SbCPT (Fig. 1A). The distance at the PMF minimum of SB1 was 3.5 Å (Fig. 1B), indicating that a stable salt bridge was formed in the natural state of SB1 (N). At the distance of 11.3 Å, there was another local PMF minimum, which could be treated as the unfolded state of SB1 (D). The SB1 structure with the PMF maximum between N and D was the transition state (E≠), only through which the N and D can convert to each other (Fig. 1B). The higher the energy barrier is between the transition state E≠ and natural state N, the more difficult it is for N to kinetically convert to the denatured state D, indicating a more stable salt bridge formed in the SbCPT. Thus, the energy barrier can be regarded as a measurement of SbCPT stability. According to the PMF energies obtained by the ABF MD simulations SB2 and SB3 were slightly more stable than SB1, whether kinetically or thermodynamically (Fig. 1C and Table S5). Experimental insertion mutagenesis of SB1-SB3 to the C-terminus of the NHaseM–TH α-subunit was further performed. The results showed that the attachment of SB2 or SB3 to NHaseM–TH (NH-SB2 or NH-SB3) caused a drastic decrease in enzyme activity. However, the insertion of SB1 (NH-SB1), the shortest SbCPT among the three SbCPTs, was not detrimental to the enzyme activity, and on the contrary resulted in small enhancement of the NH-SB1 activity (Fig. 2). Accordingly, the SbCPT with the five amino acid length (GRPEG) was subsequently selected to minimize the impact of SbCPT insertion on the local structure of the enzyme.
2.5. Construction of SbCPT-attached NHase and nitrilase mutants The parental enzyme NHaseM-TH, expressed by the plasmid pETNHM, contains the initiation codon mutation from GTG to ATG [45]. The procedure for the construction of SbCPT-attached NHase mutants is shown in Figure S1, taking the construction of plasmid pETNHM-βXX (XX is RE, RD, KE, or KD) as an example. The engineered plasmid containing SbCPT-attached NHase was constructed by sense and antisense primers based on the template plasmid pETNHM. The mutant plasmid was then transformed into the E. coli BL21(DE3) pLysS to express the NHase mutant. The Nit-tg1 A6 mutants were constructed in a similar way. Gene cloning operations, such as polymerase chain reaction (PCR) and DNA electrophoresis were carried out by standard procedures. The genes were sequenced by SinoGenoMax. All primers were synthesized by Invitrogen Biotech (Beijing) Co. Ltd. and are listed in Table S4. 2.6. NHase or nitrilase activity assay and SDS-PAGE electrophoresis The enzyme activity assays and SDS-PAGE analysis were performed as described by Chen et al. [30]. In brief, NHase activity was assayed at 28 °C in a 5-mL reaction mixture containing 100 μL of acrylonitrile and 0.25 g/L free cells in 50 mM PBS buffer for 5 min. Nitrilase activity was assayed at 28 °C in a 1.12 mL reaction mixture containing 20 μL of acrylonitrile and 0.5 g/L free cells for 10 min. 100 μL of 3 M HCl was added to stop the reaction. The concentration of acrylamide (for the NHase activity assay) or ammonium acrylate (for the nitrilase activity assay) in the supernatant of the reaction mixture were measured by gas chromatography (Trace1300, Thermo Fisher Scientific, America) to determine the NHase or nitrilase activity. Protein expression results were observed by SDS-PAGE analysis with a 5% stacking gel and 12% running gel stained with the Coomassie brilliant blue R-250. One unit (U) of NHase or nitrilase activity corresponds to the amount of the enzyme producing 1 μ mol product per min at optimal reaction conditions. 2.7. Thermal stability assessment The cells containing NHase variant in 50 mM PBS were soaked in a water bath at 42 °C, and the activity was measured by GC at room temperature. The cells containing the nitrilase variant were soaked in a water bath at 50 °C for a certain time, and the activity was measured by GC at 28 °C. The activity value at the start time was defined as 100%.
3.2. Insertion of the SbCPT into different subunit terminus of NHaseM–TH Attachment of SB1 (renamed as RE hereafter according to the types of its charged residues) on the N- or C-terminus of the NHase α- or βsubunit caused evident changes in enzyme activity. As shown in Fig. 3A and B, the insertion of RE in the C-terminus of the NHase α- or βsubunit (NH-αC or NH-βC) increased the enzyme activity to some extent, while the insertion of RE in the N-terminus (NH-αN or NH-βN) led to a considerable decrease in activity. Protein electrophoresis showed that the expressed bands of NH-αN, NH-αC and NH-βC were similar to the wild-type (WT) NHaseM–TH, while NH-βN was almost undetectable (Fig. 3C). This may be one of the reasons why NH-βN had the lowest activity among the NHase variants. According to the structure of NHaseM–TH constructed by homology
2.8. Apparent enzymology parameter measurement NHaseM-TH and the SbCPT-attached variants activity was measured at gradient substrate concentration for different reaction time (5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 40 s). The initial reaction rate was obtained as the slope of the reaction curve through the linear fit. Vmax and Km were obtained using Matlab V. R2015b by directly fitting the activity data to the Michaelis-Menten equation. The total soluble protein concentrations of NHaseM-TH and the SbCPT-attached variants were measured by BCA protein assay (Promega, Madison, USA). The proportions of target proteins were determined by SDS-PAGE electrophoresis band 41
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Fig. 1. Interpretation of potential-of-mean-force (PMF) as a measurement of the stability of the salt-bridge cyclization peptide tag (SbCPT). A. The collective variable (CV) chosen in the Adaptive Biasing Force (ABF) MD simulations was the distance between the side chain of the basic residue (Arg in this case) and that of the acidic residue (Glu in this case). B. Variation in the PMF energy along with the CV distance. N is the natural state, which adopts the lowest free energy when a salt bridge is formed. D is the denatured state, which adopts a minimum energy when the salt bridge is unfolded. E≠ is the transition state between N and D. The energy barrier and energy difference between N and D are also labeled on the chart. C. PMF variation along with the CV distance change in the SB1, SB2, and SB3 SbCPTs. SbCPTs of 5, 7, and 9 amino acid lengths were chosen and named as follows: SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG).
attachment of a SbCPT, the N-terminus of α-subunit is also likely to clash with the scaffold of NHaseM–TH and cause a great loss of activity. Among the four termini, the C-terminus of the β-subunit is the most extensible and located totally outside of the enzyme scaffold. An additional SbCPT in NH-βC will not disturb the infrastructure of the enzyme and also stabilize the flexible C-terminus of its β-subunit. In addition to the local steric hindrance, enzyme activity may also be affected by obstruction of substrate and product transport tunnel.
modeling (Figure S3), the local steric hindrances around the subunit termini are in the order: βN > αN > > αC > βC (Figs. 3D and S4), which correlates highly with the enzyme activities after SbCPT attachment (Fig. 3B). Especially, the N-terminus of the β-subunit is located at a cavity that is closely surrounded by other parts of the NHaseM–TH. Thus, even a small peptide attached to this terminus will not fit in the original cavity, leading to the disturbance of the enzyme folding, which may consequently affect the expression of NH-βC. With an
Fig. 2. Insertion experiment of salt-bridge cyclization peptide tags (SbCPTs) with different lengths. A. SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG) SbCPTs were attached to the C-terminus of the nitrile hydratases (NHase) α-subunit. B. Enzyme activities of the NHase variants. The experiments were performed in triplicate. NH indicates the control NHaseM–TH. NH-SB1, NH-SB2, and NH-SB3 are the recombinant NHases in BL21(DE3)SB1, BL21(DE3)-SB2, and BL21(DE3)-SB3, respectively.
42
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Fig. 3. Insertion experiment of salt-bridge cyclization peptide tags (SbCPTs) into different terminus of nitrile hydratases from Rhodococcus ruber TH (NHaseM–TH). A. RE SbCPT (a pentapeptide: GRPEG) was attached at the N- or C-terminus of the nitrile hydratases (NHase) α- or β-subunit. B. Enzyme activities of the NHase variants. Experiments were performed in triplicate. C. Protein electrophoresis of the four SbCPT-attached NHase variants. NH indicates the control NHaseM–TH. NH-αN, NHαC, NH-βN, and NH-βC are the recombinant NHase in BL21(DE3)-αN, BL21(DE3)-αC, BL21(DE3)-βN, and BL21(DE3)-βC, respectively. D. The termini of the α- and β-subunit in NHaseM–TH. The structure of NHaseM–TH except for the four termini is shown as the grey surface. The termini of α- and β-subunits are shown as blue and purple ribbons, respectively.
The N-terminus of the β-subunit partially constitutes the entrance of the tunnel B and the N-terminus of the α-subunit is near that tunnel (Figure S3). Adequate flexibility is necessary for a functional tunnel and, therefore, the stabilization effect by SbCPT attachment on these two termini would be another reason for the decrease in enzyme activity. The C-terminus of either the α- or β-subunit is far from the two possible tunnels, thus its change will have little effect on the tunnel(s). Considering both the local steric hindrance and flexibility of the functional tunnel, the C-terminus of the α- or β-subunit in NHaseM–TH was chosen as the subsequent target for SbCPT insertion to improve enzyme activity and stability.
charged residues were close to each other, indicating that a salt bridge was formed in their most stable conformation (Fig. 4 and Table S6). RE and RD exhibited especially larger energy differences between their salt-bridge formed state and unfolded state (1.09 and 2.13 kcal/mol, respectively), ensuring that their conformation would be firmly constrained by the salt bridge even in a fluctuating environment, such as the local high temperature, which would make for easier conversion of the salt-bridge formed state to the unfolded state across the transition state. In contrast to these SbCPTs, the lowest free energy of KE occupied an undesirable unfolded conformation in which the distance between the side chains of the two charged residues was approximately 12 Å. However, if the KE could initially fold into a salt-bridge formed state, this state could be stably maintained and separated from the unfolded state by an energy barrier of 1.58 kcal/mol (≈ 2.6 kBT). Taking account both the kinetic parameter (energy barrier) and the thermodynamic parameter (energy difference), the stability of the four SbCPTs was in the order RD > RE > KD > KE (Table S6).
3.3. Computational investigation of different types of SbCPTs There are four types of SbCPT with the sequence GXPXG (X denotes a charged residue), these include RE (GRPEG), RD (GRPDG), KE (GKPEG), and KD (GKPDG). The PMF energies obtained by the ABF MD simulations of the four SbCPTs showed that the lowest free energies of RE, RD, and KD took the conformation when the side chains of the two 43
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Fig. 4. Potential-of-mean-force (PMF) energy variation along with the change in CV distance in the RE, RD, KE, and KD SbCPTs. RE, RD, KE and KD were named according to the types of its charged residues. RE: GRPEG, RD: GRPDG, KE: GKPEG, KD: GKPDG.
subsequently assessed in term of the retention activity after incubation at 42 °C for 3 h. For most of the variants, no obvious change of stability was found with respect to WT NHaseM-TH (Fig. 5B). Two exceptions were NH-αRD for which the retention activity increased by 32.7%, and NH-αKE that observed the retention activity decrease by 19%. Compared with the results of the ABF MD simulations, RD and KE, which stabilized and destabilized the NHase when attached at the C-terminus of the α-subunit, respectively, were also found to be the most and least stable SbCPTs respectively, as estimated by PMF energy. Purified NHase was tried to harvest through adding the His-tag to the N terminal of the β subunit of the NHase. But a significant activity decrease was observed, due to the formation of inclusion body (Figure S6). Considering that the cellular catalysts are used in industrial applications of NHase, in this study we further analyzed the performance of intracellular enzyme as indicated by its apparent enzymology parameters. From the results of the apparent Km, Vmax and Kcat/Km of the enzymes analyzed in Table 1, we found that for the SbCPT-attached variants, the Vmax was all increased, which is in consistent with the result in Fig. 5A. Especially, the Kcat/Km of NH-αRD was enhanced by 17.6% relative to the control, indicating that the NH-αRD catalyzed the hydration of acrylonitrile to acrylamide was faster than NHase alone.
3.4. Assessment of the NHase activity and stability with different SbCPTs The NHase variants (NH-αRE, NH-αRD, NH-αKE, NH-αKD, NHβRE, NH-βRD, NH-βKE, and NH-βKD) with one of the four SbCPTs (RE, RD, KE, and KD) attached to the C-terminus of the α- or β-subunit were constructed to investigate their enzyme activities and stabilities. The overall catalytic competence of all the SbCPT-attached NHases were greater than or at least comparable to that of the WT NHaseM-TH, especially the activity of NH-αKD, which was increased by 54% (Fig. 5A). Thermal stabilities of these SbCPT-attached NHase variants were
3.5. Assessment of the nitrilase activity and stability with different SbCPTs The Nit-tg1 A6 variants (Nit-CKE, Nit-CKD, Nit-CRD, Nit-NKE, NitNKD, Nit-NRD) with one of the three SbCPTs (KE, KD and RD) attached to the C- or N-terminus were constructed to further test their properties. It can be seen that the enzyme activities of the Nit-tg1 A6 variants with SbCPT attached to the C-terminus were distinctly enhanced compared with the WT Nit-tg1 A6, while activity of the N-terminal attached variants was severely reduced (Fig. 6A). Although both the N- and Ctermini are far from the active site in Nit-tg1 A6, the C-terminus sticks out from the main structure, while the N-terminus remains close to the Table 1 Apparent enzymology parameters for NHase and its variants modified by SbCPTs on the C terminal of the α subunit of the NHase. Fig. 5. Insertion of different salt-bridge cyclization peptide tag (SbCPT) into the C-terminus of nitrile hydratases from Rhodococcus ruber TH (NHaseM–TH). A. Enzyme activities of the nitrile hydratases (NHase) variants. B. The thermal stability of the NHase variants was assessed in PBS buffer (50 mM, pH 7.0) at 42 °C for 3 h. NH indicates the control NHaseM–TH. Experiments were performed in triplicate. 44
Strain
Km/ (g/L)
NHase NH-α KE NH-α KD NH-α RE NH-α RD
5.20 ± 4.64 ± 4.70 ± 4.42 ± 4.07 ±
0.10 0.05 0.10 0.04 0.15
Vmax/ (U/mL)
Kcat/ Km/(U·L·g−1/ μmol Enzyme)
114.9 ± 126.6 ± 125.0 ± 112.4 ± 135.1 ±
3784 3662 3509 3027 4450
2.0 1.5 4.0 3.0 2.5
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glycine residues were introduced into SB1 (GRPEG), SB2 (GRGPGEG) and SB3 (GRGGPGGEG) to change the length of SbCPTs. According to the PMF energies obtained by ABF MD simulations, the energy difference of SB1-SB3 finally did not change systematically (the order is SB2 > SB3 > SB1) (Fig. 1C and Table S5). The reason we deduced is that not only the peptide length, but also the amino acid composition such as the quantity of glycine residues affect the stability of SbCPTs. On the one hand, with the increase of peptide length, more glycine residues introduced would reduce the space-resistance against the turnstructure formation, help easily form a more stable salt-bridge; on the other hand, however, more and more glycine residues would also cause an unfavorable entropy-driven effect that prone to break ionic interactions in hydrophilic environment. When the number of glycine residues increased to the extent that the negative instable effect played the major role, the overall stability of the SbCPT would reduce conversely. In addition, the applicability of SB1, SB2 and SB3 should also consider the effect of the SbCPTs with different length on the 3D structure of a target enzyme. From the experimental results of Fig. 2, we can find that inserting large SbCPTs such as SB2 and SB3 into the terminus of NHase significantly reduced the enzyme activity, indicating that there existed negative impact of long SbCPTs on the local structure of NHase. It might be the steric hindrance in the subunit terminus, leading to a clash or distortion of the terminal structure and thereby a decrease of the enzyme activity. To maintain enzyme activity while improving stability, a relatively smaller SbCPT should be selected. Thus, SbCPT with a five amino acid length was postulated to be suitable for a universal application since a turn structure with a salt bridge could be formed while minimizing the negative effect on the structure. Additionally, proline-rich structures usually do significant contribution to stabilizing enzymes as well [48]. For the function of proline residue in SbCPTs of this work, however, it only acted as a key amino acid assisting formation of the turn-structure. Investigation of the SbCPT insertion position by experiments and homology modeling showed that the local structural environment of the SbCPT-attached terminus was crucial for the mutational activity. Insertion of a SbCPT to the terminus in a confining space would cause a clash with the infrastructure of the enzyme and inevitably decrease the enzyme’s activity (Fig. 3). Moreover, although the termini of enzymes are generally far from the active site, they may still constitute or approach other functional regions such as the substrate and product transport tunnel (e.g., the N-terminus of the NHase β-subunit in this study), a hot spot for protein-protein recognition, a subunit surface for electron transport, etc. To preserve the full function of the enzyme and maintain its activity, it is better to avoid a large change (e.g., SbCPT insertion) in the functional terminus. Thus, SbCPT should only be attached on the enzyme terminus that is extensible and also far from any special functional area. Without loss of activity, the SbCPT insertion into such a terminus would even generally enhance the total enzyme activity probably due to stabilization of the flexible enzyme terminus from degradation [49] or enhancement of specific enzyme activity from the change of the NHase variant structure. For the four different types of SbCPTs, the PMF energies calculated by the ABF MD simulations indicated that their stabilities are in the order: RD > RE > KD > KE. This means that residue R with guanidine group can form a more stable non-classical turn structure with D or E than residue K. Experimental results of SbCPT attached on the Cterminus of the α-subunit were in the order: RD > RE ≈ KD > KE (Fig. 5B), which were approximately consistent with the computational results, in which the stability of NH-αRD was highly enhanced while that of NH-αKE, in contrast, was even reduced. The experimental results of SbCPT attached on the C-terminus of β-subunit, however, made the explanation complicated. On the one hand, the molecular sizes of SbCPTs containing RD or KD were smaller than that of RE or KE, respectively, thereby prone to form a relatively more stable salt-bridge due to less space resistance of the SbCPT alone. On the other hand, the spatial position and amino acid sequence of the specific terminus
Fig. 6. Applicability assessment of salt-bridge cyclization peptide tag (SbCPT)insertion in nitrilase from R. rhodochrous tg1-A6 (Nit-tg1 A6). A. Enzyme activities of the nitrilase variants with SbCPTs inserted into the C- or N-terminus. B. Thermal stability of nitrilase variants with C- terminus insertion of SbCPTs. Nitrilase activities were measured after being at 50 °C for 15 min. “Nitrilase” indicates the control Nit-tg1 A6. Experiments were performed in triplicate.
surface (Figure S5). Insertion of a SbCPT to the N-terminus of nitrilase may impair its interactions with the surface, thus counteracting the stabilization benefit from SbCPT attachment, or even destabilizing the N-terminus structure. Inserting the His-tag to the N-terminus of nitrilase Nit-tg1-A6 showed similar results, and no purified nitrilase could be successfully obtained (Figure S7). For better understanding the influences of the SbCPTs on Nit-tg1-A6 properties, we also used free cells to measure the apparent enzymology parameters (Table S7). The Vmax of the SbCPT-attached variants was also enhanced corresponding to the results shown in Fig. 6. Analyzing the SDS-PAGE results using software named Quantity One (Figure S8), we found that the expression of the target protein was increased. Comparing with the Nit-tg1-A6 (52% in total soluble protein expression), the expression of the Nit-KE, Nit-RD and Nit-KD in total soluble protein ratio was 63.4%, 60.6% and 60.8%, respectively. Thermal stabilities of the C-terminal SbCPT-attached Nit-tg1 A6 variants were further measured in terms of the retention activity after incubation at 50 °C for 15 min. It was shown that the stability of all the C-terminal SbCPT-attached variants was enhanced, especially that of Nit-CRD, which was increased by 30.3% (from 50.1% in WT Nit-tg1 A6 to 65.3% in Nit-CRD) (Fig. 6B).
4. Discussion Several studies have shown that a salt bridge in the terminus of an enzyme would enhance the enzyme stability [20,21,30,47]. Here, we proposed an enzyme stabilization strategy by terminal insertion of a SbCPT. The core idea is to enable an oligo-peptide to form a cyclization structure via salt-bridge interaction and apply it to reduce the extensional freedom of the enzyme terminus, and thereby universally enhance stability and activity of diverse enzymes. Design of SbCPTs with different length was first concerned. Several 45
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carrying the SbCPT will also affect the activity and stability of target enzyme after SbCPT attachment. The local spatial hindrance around the C-terminus of β-subunit, as well as the interaction of SbCPT with the enzyme scaffold, could both exert an intricate influence on enzyme activity and stability, besides the structure of SbCPT alone. In light of these considerations, we suggested that RD could be used as a universal SbCPT attaching to the enzyme terminus to enhance both the total activity and stability. In contrast to rational design [8], only very little information about the enzyme structure (i.e., the local steric environment of the enzyme termini) is required with the SbCPT strategy. This would be of great value when the enzyme structure is difficult to obtain or only has low accuracy, especially for the industrial biocatalysts. Additionally, unlike random mutagenesis [7], the SbCPT strategy only requires a small number of experiments, but a relatively higher rate of success can be achieved. The general applicability of the SbCPT strategy was further shown with Nit-tg1 A6, which indicated that terminal attachment of the SbCPT could be used as a universal strategy for the stability enhancement of industrial enzymes without activity loss.
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