Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2020 Jun 29;10(7):323. doi: 10.1007/s13205-020-02321-2

Improving the catalytic thermostability of Bacillus altitudinis W3 ω-transaminase by proline substitutions

Zihao Xie 1,#, Lixin Zhai 1, Di Meng 1, Qiaopeng Tian 1, Zhengbing Guan 1, Yujie Cai 1, Xiangru Liao 1,✉
PMCID: PMC7324462  PMID: 32656056

Abstract

As a green biocatalyst, transaminase with high thermostability can be better employed to synthesize many pharmaceutical intermediates in industry. To improve the thermostability of (R)-selective amine transaminase from Bacillus altitudinis W3, related mutation sites were determined by multiple amino acid sequence alignment between wild-type ω-transaminase and four potential thermophilic ω-transaminases, followed by replacement of the related amino acid residues with proline by site-directed mutagenesis. Three stabilized mutants (D192P, T237P, and D192P/T237P) showing the highest stability were obtained and used for further analysis. Comparison with the wild-type enzyme revealed that the double mutant D192P/T237P exhibited the largest shift in thermostability, with a 2.5-fold improvement of t1/2 at 40 °C, and a 6.3 °C increase in T1550, and a 5 °C higher optimal catalytic temperature. Additionally, this mutant exhibited an increase in catalytic efficiency (kcat/Km) relative to the wild-type enzyme. Modeling analysis indicated that the improved thermostability of the mutants could be associated with newly formed hydrophobic interactions and hydrogen bonds. This study shown that proline substitutions guided by sequence alignment to improve the thermostability of (R)-selective amine transaminase was effective and this method can also be used to engineering other enzymes.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02321-2) contains supplementary material, which is available to authorized users.

Keywords: Amino transferase, Protein engineering, Proline substitutions, Enzyme thermostability, Hydrophobic interactions, Hydrogen bonds

Introduction

Most medicines are chiral amines or their derivatives, including neurological, cardiovascular, antihypertensive, and anti-infective drugs and vaccines (Malik et al. 2012; Ghislieri and Turner 2014; Simon et al. 2014; Patil et al. 2018). Biological synthesis is a green and highly selective tool for chiral synthesis due to its advantages of high efficiency, and selectivity, mild reaction conditions, and green environmental protection of biocatalysts (Bornscheuer et al. 2012; Fuchs et al. 2014; Guo and Berglund 2016). Therefore, production of chiral amines by biocatalysts is more attractive and competitive than the use of traditional chemical catalysis and has a broad application prospect. Recent rapid developments in the chiral amine industry, have increased the need for research on the use of transaminases (TAs) for chiral compound synthesis (Ferrandi and Monti 2018).

Transaminases can produce chiral amines by transferring amino groups on amino donors to an amino acceptor with the help of pyridoxal 5′-phosphate (PLP, coenzyme) (Koszeleivski et al. 2008; Savile et al. 2010; Mathew and Yun 2012). The demand for (R)-selective amine compounds continues to increase relative to the more exploited (S)-ω-TAs (Jiang et al. 2015; Kim et al. 2018; Tang et al. 2019). However, most enzymes in nature are mesophilic, which only retain maximum activity under moderate temperatures and lose activity in a short period of time at high temperatures. For example, xylanases from a wide range of biological sources are widely used in a variety of industrial processes, but only thermostable xylanases are widely used; therefore, protein engineering methods have been employed to explore their potential (Baweja et al. 2016; Kumar et al. 2018). Given the harsh catalytic conditions in industrial production, improved thermostability of (R)-ω-TAs would be beneficial for the preparation of chiral amines (Martin et al. 2007; Xie et al. 2019). Compared with traditional directed evolution, the use of rational design to improve protein thermostability is both targeted and highly efficient, but requires clear understanding of the relationship between structure and function (Yang et al. 2015; Jones et al. 2017). There is currently multiple rational or semi-rational design methods used to improve enzyme thermostability. The double mutant S255K/S340P of low-temperature α-amylase from Pseudoalteromonas haloplanktis was obtained by molecular dynamics simulation and energy optimization, and resulted in improved thermal stability was sharply improved (Li et al. 2020). Additionally, B-factor analysis, allowed determination of mutation sites in lipases from Rhizopus chinensis, resulting in a mutant showing improved thermostability, a 48.2% increase in residual enzyme activity after 2 h at 50 °C and a 3.0-fold increase in half-life at 60 °C relative to the WT enzyme (Jiang et al. 2020). Moreover, after introducing a disulfide bond and proline substitution in chitinase from Paenibacillus pasadenensis CS0611, the resulting mutant showed a ~ 26.3-fold increase in half-life at 50 °C and a 7.9 °C increase in half-inactivation temperature relative to WT (Xu et al. 2020). These findings indicate the efficacy of improving enzyme improve thermostability by rational design methods. Multiple studies report that introduction of proline into a structure can enhance conformational rigidity and significantly improve thermostability (Allen et al. 1998; Suzuki 1999; Goihberg et al. 2007; Wang et al. 2014; Huang et al. 2015). Because the frequency of proline residues in thermophilic enzymes is higher than in mesophilic enzymes (Land et al. 2019), residues possibly related to thermostability can be identified by homologous multiple sequence alignment of thermophilic enzymes and mesophilic enzymes. However, few strategies involving proline substitution guided by sequence alignment to improve ω-TAs thermostability have been reported.

To determine whether a proline substitution guided by sequence alignment in a WT enzyme is effective at improving thermostability, we performed site-directed mutagenesis to screen (R)-ω-TAs mutants exhibiting with high thermostability.

Materials and methods

Materials

PrimeSTAR Max DNA, isopropyl-β-d-thiogalactopyranoside (IPTG) and all other experimental products were obtained from TaKaRa (Otsu, Japan). Ampicillin sodium salt was purchased from Molekula Ltd. (Gillingham, UK). Polymerase chain reaction (PCR) primer synthesis and DNA sequencing were conducted by Yixin Biotech. Corp. (Wuxi, China). The BeaverBeads His-tag protein purification kit was purchased from BEAVER (Suzhou, China).

Site selection for site-directed mutagenesis

To identify residues potentially related to thermostability in WT mesophilic enzymes, the sequence of ω-TA from Bacillus altitudinis W3 (ω-BPTA) (GeneBank No: CP011150.1) (Zhai et al. 2019), was used for alignment with amino acid sequences of potential thermophilic ω-TAs (TA-465, TA-W1, TA-2542, TA-20745) from Geobacillus thermodenitrificans subsp. Thermodenitrificans DSM465 (GeneBank No: KT719298.1), Sphaerobacter thermophilus DSM 20745 (GeneBank No: CP001823.1), Parageobacillus thermoglucosidasius DSM2542 (GeneBank No: CP012712.1) and Geobacillus thermoleovorans ARTRW1 (GeneBank No: CP042251.1) using DNAMAN software (https://www.lynnon.com/). Site-directed mutagenesis was used to introduce proline into ω-BPTA sites to screen ω-TA mutants with high thermostability.

Construction of site-directed mutagenesis

All primers used for mutagenesis are listed in Table S1. The reaction mixture (50 µL) contained 1 μL of 100 ng DNA template (pCold II-ota3, screened and cloned in our laboratory), 1 μL each of 10 μM forward and reverse primers, 10 μL of 5× PrimeSTAR buffer (with Mg2+), 4 μL of dNTP mixture, 0.5 μL of PrimeSTAR HS DNA polymerase (2.5 U μL−1) and 32.5 μL of autoclaved water. PCR conditions were as follows: 30 cycles of denaturation for 15 s at 98 °C, followed by annealing for 15 s at 55 °C and extension for 5.3 min at 72 °C. PCR products were digested using DpnI for 1 h at 37 °C to remove methylated templates and then used to transform competent Escherichia coli JM109 cells. To obtain the desired positive transformants, all selected mutants were verified by further DNA sequencing.

Purification of ω-BPTA and mutant enzymes

Escherichia coli BL21 (DE3) cells transformed with WT pCold II-ota3 and mutants were grown in Luria–Bertani medium containing 100 μg mL−1 ampicillin at 37 °C until reaching an optical density at 600 nm of between 0.4 and 0.6. The culture was then cooled to 15 °C for 30 min and protein expression was induced by adding IPTG at a final concentration of 0.4 mM, followed by incubation with shaking at 37 °C for 24 h. The cells were then harvested by centrifugation at 8000g for 10 min at 4 °C, the precipitate was resuspended in lysis buffer [20 mM sodium phosphate buffer and 0.1 mM PLP(pH 7.0)], cells were lysed by ultrasonication in an ice bath using a sonicator (sonication for 3 s and cooling for 2 s for a total time of 15 min; VCX130; Sonics, Newtown, CT, USA). After centrifugation at 12,000g at 4 °C for 10 min, the collected supernatant was used for further purification by Ni-affinity chromatography using a BeaverBeads His-tag protein purification kit according to manufacturer instructions. Purified enzymes were desalted using a 5 mL HiTrap desalting column and concentrated using an ultrafiltration tube. The relative molecular weights of the purified proteins were determined by employing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970).

Enzyme activity assay

The TA activities of the WT and mutants were measured according to a previously described method (Zhai et al. 2019), with enzyme activity defined according to acetophenone yield. All experiments were performed in triplicate.

The kinetic parameters of the WT and mutant TAs were measured under optimal conditions. (R)-α-phenethylamine [(R)-PEA] was used as the substrate at different concentrations in order to determine the Km value, which was calculated by plotting a nonlinear fitting curve using Origin Pro software (v.9.0; OriginaLab Corp., Northampton, MA, USA). Each group of experiments was performed in triplicate.

Thermostability measurement

The T1550 value, defined as the temperature at which 50% of enzyme activity is lost after a 15-min incubation time, of the WT and mutants were determined after incubation for 15 min at increasing temperatures from 20 to 60 °C, followed by measurement of residual activities under optimal conditions. Data were analyzed using the sigmoidal Boltzmann equation in Origin Pro 9.0 software. The half-life (t1/2) value, defined as the time at which 50% of enzyme activity is lost after incubation at 40 °C, of the WT and mutants was determined after incubation for different time periods (0–60 min) at 40 °C. Data were analyzed using the equation y = exp(−kd·t) in Origin Pro9.0 software. Residual activities were measured under optimal conditions. Enzyme activity–temperature profiles were determined by measuring relative activities from 25 to 65 °C, with maximal activity at 100%. All experiments were performed in triplicate.

Analysis of protein stability at the molecular level

The three-dimensional (3D) homology model of ω-BPTA was generated using SWISS-MODEL software (http://www.expasy.org/swissmod/). To assess the influence of intermolecular interactions, the Protein Interaction calculator (http://pic.mbu.iisc.ernet.in/job.html) (Tina et al. 2007) was used to predict changes in hydrogen bonds and hydrophobic interactions between the WT and mutant enzymes. Changes in intermolecular interactions before and after mutation were visualized using PyMOL (http://pymol.org/).

Results and discussion

Design and preliminary screening of TA mutants

Based on the multiple sequence alignment (Fig. 1), five residues (A191, D192, E225, T237, and G270) in the WT enzyme were selected for mutation to proline. Following purifications of the WT and mutants incubation for 30 min at 50 °C, we calculated the residual activity of WT at 20.5% as compared with that of D192P and T237P (27.7% and 32.0%, respectively). Therefore, we chose D192P, T237P, and the double mutant D192P/T237P for analysis of thermostability.

Fig. 1.

Fig. 1

Open in a new tab

Multiple sequence alignment of ω-TAs from Bacillus altitudinis W3 and Geobacillus thermodenitrificans subsp. Thermodenitrificans DSM465, Sphaerobacter Thermophilus DSM 20745, Parageobacillus thermoglucosidasius DSM2542 and Geobacillus thermoleovorans ARTRW1 using DNAMAN software. Five sites at the wild-type marked by black frames prepared for mutant. The red Arabic numbers represent the positions of amino acids in wild-type enzymes

Thermostability analysis of ω-BPTA and mutants

Purification and SDS-PAGE analysis of WT ω-BPTA, and the D192P, T237P, and D192P/T237P mutant revealed four protein bands at ~ 33.4 kDa, which was consistent with the theoretical molecular weight (Fig. S1).

We then determined their thermostability according to T1550 values and t1/2 values. We found T1550 and t1/2 values for WT of 39.3 °C and 12.7 min, respectively (Fig. 2a, b), whereas the T1550 values for the D192P and T237P mutants increased to 40.7 °C and 41.7 °C, respectively, and their t1/2 values increased to 16.9 min and 20.4 min, respectively. However, the D192P/T237P mutant showed the highest degree of improved thermostability, with T1550 and t1/2 values of 45.6 °C and 31.7 min at 40 °C, respectively. Additionally, evaluation of the optimal reaction temperature revealed 45 °C for the WT enzyme, with decreases in relative activity to < 30% above or below this temperature (Fig. 2c). By contrast, the optimal temperature for the two single point mutants was also 45 °C, but no rapid decreases in relative activity were observed outside of this temperature. For the double mutant, the optimal temperature was 50 °C, with relative activity maintained at > 50% up to 65 °C. These results suggested that proline introduction at the chosen sites improved ω-BPTA thermostability.

Fig. 2.

Fig. 2

Open in a new tab

Thermostability of WT ω-BPTA and mutant enzymes. a Thermal inactivation of WT ω-BPTA, mutant D192P, T237P and D192P/T237P at different temperatures over 15 min (T1550). b Thermal inactivation half-life (t1/2) of WT ω-BPTA, mutant D192P, T237P and D192P/T237P at 40 °C. c The effect of catalytic temperature on the WT ω-BPTA, mutant D192P, T237P and D192P/T237P

Kinetic properties of the WT and mutant enzymes

(R)-PEA was used as the substrate to determine the kinetic parameters of the enzymes (Table 1). Among the three mutants, the Km values of T237P increased, whereas those of D192P and D192P/T237P decreased relative to that of WT, indicating that the substrate affinity of D192P and D192P/T237P increased. Additionally, all three mutants showed higher catalytic efficiency (kcat/Km) relative to WT, with the highest value observed for D192P/T237P at a range of 0.38 min−1 mM−1 to 0.45 min−1 mM−1. These results showed that mutation outside of conserved domains improved both the thermostability and catalytic efficiency of the enzyme.

Table 1.

The steady-state kinetic constants (Km, kcat and kcat/Km) of wild-type and three mutants ((R)-PEA was used to the substrate)

Protein Km (mM) Kcat (min−1) Kcat/Km (min−1 mM−1)
Wild-type 12.88 ± 0.20 4.90 ± 0.15 0.38
D192P 12.66 ± 0.14 5.36 ± 0.12 0.42
T237P 13.91 ± 0.08 5.58 ± 0.11 0.40
D192P/T237P 11.74 ± 0.16 5.27 ± 0.12 0.45
Open in a new tab

Structural analysis

Recent studies show that formation of new hydrogen bonds and hydrophobic interactions are important for increasing enzyme thermostability (Hua et al. 2020; Wang et al. 2020). Additionally, Jiang et al. (2020) reported that a quadruple mutations in lipase result in loss of an α-helix proximal to the mutation site due to the loss of the hydrogen bond network, which reduced enzyme thermostability.

Here, to investigate the mechanism associated with the observed increase in thermostability of the mutants, we evaluated changes in intramolecular interactions involving hydrogen bonds and hydrophobic interactions using the 3D structures of the enzymes (Figs. 3 and 4). The results showed that in the WT enzyme, O atoms of Thr 237 formed hydrogen bonds with N atoms of His 239, Asp240 and Val241 at distances of 3.4 Å, 3.2 Å, and 3.3 Å, respectively (Fig. 3a). In the D192P and D192P/T237P mutants, we observed a newly formed hydrogen bond between the O atom of Pro192 and the N atom of Gly189 (2.8 Å) (Fig. 3b, d). Notably, the hydrogen bonds with Thr237 observed in the WT enzyme were maintained in all three mutants (Fig. 3b, d). It shown that the hydrogen bond at 237 residue made little contribution to the stability of this enzyme.

Fig. 3.

Fig. 3

Open in a new tab

Hydrogen bonds in wild-type BPTA, mutant D192P, T237P and D192P/T237P. a Wild-type ω-BPTA: the O atom of Thr237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å). b D192P: a new hydrogen bond has been formed between the O atom of Pro192 and the N atom of Gly189 (2.8 Å). c T237P: the O atom of Pro237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å). d D192P/T237P: a new hydrogen bond has been formed between the O atom of Pro192 and the N atom of Gly189 (2.8 Å) and the O atom of Pro237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å)

Fig. 4.

Fig. 4

Open in a new tab

Hydrophobic interaction in mutant D192P, T237P and D192P/T237P. a D192P: two new hydrophobic interactions have been formed between Asp192 and Leu249 (5.1 Å), Ala254 (6.1 Å). b T237P: a new hydrophobic interaction has been formed between Phe236 and Pro237 (4.8 Å). c D192P/T237P: three new hydrophobic interactions have been formed between Asp192 and Leu249 (5.1 Å), Ala254 (6.1 Å) and between Phe236 and Pro237 (4.8 Å)

For hydrophobic interactions, Asp192 and Thr237 in the WT enzyme were not close enough to form hydrophobic interactions with other side chains, whereas we observed two new hydrophobic interactions in the D192P mutant (between Asp192 and Leu249 and Asp192 and Ala254 at 5.1 Å and 6.1 Å, respectively) (Fig. 4a). Moreover, the T237P mutant showed one additional interaction, between Phe236 and Pro237 (4.8 Å) (Fig. 4b), and the D192P/T237P mutant showed two additional interactions (Pro192 in D192P and Pro237 in T237P, respectively) (Fig. 4c).

Site-directed mutagenesis can change the amino acid sequence and affected protein structure to improve the thermostability (Taylor and Vaisman 2010). In the present study, proline substitutions created additional hydrogen bonds, as well as hydrophobic interactions proximal to the mutation sites and increased overall structural stability residues. To sum up, in this research, hydrophobic interaction and hydrogen bond jointly promoted the protein stability. These results are consistent with a previous finding that the contribution of hydrophobic interactions (60 ± 4%) to protein stability is more significant than that of hydrogen bonds (40 ± 4%) (Pace et al. 2011).

Conclusion

In this study, we showed that introduction of proline residues effectively improved the thermostability. The T1550 and t1/2 values in all three mutants relative to WT, although the double mutant D192P/T237P exhibited the highest increases in thermostability, and relative activity, as well as a significantly increased catalytic efficiency. Our analysis suggested that the formation of new hydrogen bonds and hydrophobic interactions might have contributed to these results. These findings indicated that proline substitutions at sites identified by multiple sequence alignment is efficacious for improving protein thermostability.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 14 kb) (14.1KB, docx)
Supplementary material 2 (DOCX 219 kb) (219.3KB, docx)

Acknowledgements

This work was financially supported by the Collaborative Innovation Involving Production, Teaching & Research Funds of Jiangsu Province (BY2014023–28) and the Agricultural Support Project, Wuxi Science & Technology Development (CLE01N1310). We thank for the financial support by the project fund from Science and Technology Project of Taizhou (No. 1801gy24), and Nature Science Funding Sponsored by Zhejiang Province (HX2019069, HX2019078).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Zihao Xie—First author.

References

  1. Allen MJ, Coutinho PM, Ford CF. Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations. Protein Eng. 1998;11:783–788. doi: 10.1093/protein/11.9.783. [DOI] [PubMed] [Google Scholar]
  2. Baweja M, Nain L, Kawarabayasi Y, Shukla P. Current technological improvements in enzymes toward their biotechnological applications. Front Microbiol. 2016;7:13. doi: 10.3389/fmicb.2016.00965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bornscheuer UT, Huisman GW, Kazlauskas RJ, et al. Engineering the third wave of biocatalysis. Nature. 2012;485:185–194. doi: 10.1038/nature11117. [DOI] [PubMed] [Google Scholar]
  4. Ferrandi EE, Monti D. Amine transaminases in chiral amines synthesis: recent advances and challenges. World J Microbiol Biotechnol. 2018;34:13. doi: 10.1007/s11274-017-2395-2. [DOI] [PubMed] [Google Scholar]
  5. Fuchs CS, Simon RC, Riethorst W, et al. Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media. Bioorg Med Chem. 2014;22:5558–5562. doi: 10.1016/j.bmc.2014.05.055. [DOI] [PubMed] [Google Scholar]
  6. Ghislieri D, Turner NJ. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top Catal. 2014;57:284–300. doi: 10.1007/s11244-013-0184-1. [DOI] [Google Scholar]
  7. Goihberg E, Dym O, Tel-Or S, et al. A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase. Proteins-Struct Funct Bioinform. 2007;66:196–204. doi: 10.1002/prot.21170. [DOI] [PubMed] [Google Scholar]
  8. Guo F, Berglund P. Transaminase biocatalysis: optimization and application. Green Chem. 2016;19:333–360. doi: 10.1039/C6GC02328B. [DOI] [Google Scholar]
  9. Hua YJ, Lyu CJ, Liu CY, et al. Improving the thermostability of glutamate decarboxylase from Lactobacillus brevis by consensus mutagenesis. Appl Biochem Biotechnol. 2020 doi: 10.1007/s12010-020-03283-0. [DOI] [PubMed] [Google Scholar]
  10. Huang J, Jones BJ, Kazlauskas RJ. Stabilization of an α/β-hydrolase by introducing proline residues: salicylic acid binding protein 2 from tobacco. Biochemistry. 2015;54:4330–4341. doi: 10.1021/acs.biochem.5b00333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jiang J, Chen X, Zhang D, et al. Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast. Appl Microbiol Biotechnol. 2015;99:2613–2621. doi: 10.1007/s00253-014-6056-1. [DOI] [PubMed] [Google Scholar]
  12. Jiang ZB, Zhang CB, Tang MY, et al. Improving the thermostability of rhizopus chinensis lipase through site-directed mutagenesis based on B-factor analysis. Front Microbiol. 2020;11:8. doi: 10.3389/fmicb.2020.00346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones BJ, Lim HY, Huang J, Kazlauskas RJ. Comparison of five protein engineering strategies for stabilizing an α/β-hydrolase. Biochemistry. 2017;56:6521–6532. doi: 10.1021/acs.biochem.7b00571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim E-M, Park JH, Kim B-G, Seo J-H. Identification of (R)-selective ω-aminotransferases by exploring evolutionary sequence space. Enzyme Microb Technol. 2018;110:46–52. doi: 10.1016/j.enzmictec.2017.12.002. [DOI] [PubMed] [Google Scholar]
  15. Koszeleivski D, Lavandera I, Clay D, et al. Formal asymmetric biocatalytic reductive amination. Angew Chemie-Int Ed. 2008;47:9337–9340. doi: 10.1002/anie.200803763. [DOI] [PubMed] [Google Scholar]
  16. Kumar V, Dangi AK, Shukla P. Engineering thermostable microbial xylanases toward its industrial applications. Mol Biotechnol. 2018;60:226–235. doi: 10.1007/s12033-018-0059-6. [DOI] [PubMed] [Google Scholar]
  17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Land H, Campillo-Brocal JC, Humble MS, Berglund P. B-factor guided proline substitutions in chromobacterium violaceum amine transaminase: evaluation of the proline rule as a method for enzyme stabilization. ChemBioChem. 2019;20:1297–1304. doi: 10.1002/cbic.201800749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Q, Yan Y, Liu X, et al. Enhancing thermostability of a psychrophilic alpha-amylase by the structural energy optimization in the trajectories of molecular dynamics simulations. Int J Biol Macromol. 2020;142:624–633. doi: 10.1016/j.ijbiomac.2019.10.004. [DOI] [PubMed] [Google Scholar]
  20. Malik MS, Park ES, Shin JS. Features and technical applications of omega-transaminases. Appl Microbiol Biotechnol. 2012;94:1163–1171. doi: 10.1007/s00253-012-4103-3. [DOI] [PubMed] [Google Scholar]
  21. Martin AR, DiSanto R, Plotnikov I, et al. Improved activity and thermostability of (S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine. Biochem Eng J. 2007;37:246–255. doi: 10.1016/j.bej.2007.05.001. [DOI] [Google Scholar]
  22. Mathew S, Yun H. ω-Transaminases for the production of optically pure amines and unnatural amino acids. ACS Catal. 2012;2:993–1001. doi: 10.1021/cs300116n. [DOI] [Google Scholar]
  23. Pace CN, Fu H, Fryar KL, et al. Contribution of hydrophobic interactions to protein stability. J Mol Biol. 2011;408:514–528. doi: 10.1016/j.jmb.2011.02.053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patil MD, Grogan G, Bommarius A, Yun H. Recent advances in ω-transaminase-mediated biocatalysis for the enantioselective synthesis of chiral amines. Catalysts. 2018;8:254. doi: 10.3390/catal8070254. [DOI] [Google Scholar]
  25. Savile CK, Janey JM, Mundorff EC, et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science. 2010;329:305–309. doi: 10.1126/science.1188934. [DOI] [PubMed] [Google Scholar]
  26. Simon RC, Richter N, Busto E, Kroutil W. Recent developments of cascade reactions involving ω-transaminases. ACS Catal. 2014;4:129–143. doi: 10.1021/cs400930v. [DOI] [Google Scholar]
  27. Suzuki Y. The proline rule: a strategy for protein thermal stabilization. Proc Jpn Acad Ser B. 1999;75:133–137. doi: 10.2183/pjab.75.133. [DOI] [Google Scholar]
  28. Tang X, Zhang N, Ye G, Zheng Y. Efficient biosynthesis of (R)-3-amino-1-butanol by a novel (R)-selective transaminase from Actinobacteria sp. J Biotechnol. 2019;295:49–54. doi: 10.1016/j.jbiotec.2019.02.008. [DOI] [PubMed] [Google Scholar]
  29. Taylor TJ, Vaisman II. Discrimination of thermophilic and mesophilic proteins. BMC Struct Biol. 2010;10(Suppl 1):S5. doi: 10.1186/1472-6807-10-s1-s5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tina KG, Bhadra R, Srinivasan N. PIC: protein interactions calculator. Nucleic Acids Res. 2007;35:W473–W476. doi: 10.1093/nar/gkm423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang K, Luo H, Tian J, et al. Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues. Appl Environ Microbiol. 2014;80:2158–2165. doi: 10.1128/aem.03458-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang H, Zhang L, Wang Y, et al. Engineering the heparin-binding pocket to enhance the catalytic efficiency of a thermostable heparinase III from Bacteroides thetaiotaomicron. Enzyme Microb Technol. 2020;137:109549. doi: 10.1016/j.enzmictec.2020.109549. [DOI] [PubMed] [Google Scholar]
  33. Xie D, Yang J, Lv C, et al. Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis. J Biotechnol. 2019;293:8–16. doi: 10.1016/j.jbiotec.2019.01.007. [DOI] [PubMed] [Google Scholar]
  34. Xu P, Ni Z-F, Zong M-H, et al. Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design. Int J Biol Macromol. 2020;150:9–15. doi: 10.1016/j.ijbiomac.2020.02.033. [DOI] [PubMed] [Google Scholar]
  35. Yang H, Liu L, Li J, et al. Rational design to improve protein thermostability: recent advances and prospects. Chembioeng Rev. 2015;2:87–94. doi: 10.1002/cben.201400032. [DOI] [Google Scholar]
  36. Zhai L, Yang S, Lai Y, et al. Mining of aminotransferase gene ota3 from Bacillus pumilus W3 via genome analysis, gene cloning and expressing for compound bioamination. Gene. 2019;686:21–28. doi: 10.1016/j.gene.2018.10.082. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (DOCX 14 kb) (14.1KB, docx)
Supplementary material 2 (DOCX 219 kb) (219.3KB, docx)

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES