Improving thermostability of (<i>R</i>)‐selective amine transaminase from <i>Aspergillus terreus</i> through introduction of disulfide bonds

Xie, Dong-Fang; Fang, Hui; Mei, Jiaqi; Gong, Jinyan; Wang, Hongpeng; Shen, Xiaofeng; Huang, Jun; Mei, Lehe

doi:10.1002/bab.1572

Cited by 21 publications

(8 citation statements)

References 37 publications

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“…The root mean square deviation (RMSD) was calculated for the protein backbone using least-squares fitting, and the root mean square fluctuation (RMSF) was calculated using the coordinates derived from the MD trajectories of the last 5 ns timescale. The distributions of RMSD values were determined statistically with Origin 2018 (OriginLab, Northampton, MA, USA) [ 52 ]. The hydrogen bond analysis was performed using VMD [ 53 ].…”

Section: Methodsmentioning

confidence: 99%

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

Zhao

Chen

Wang

et al. 2021

IJMS

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D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

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Section: Methodsmentioning

confidence: 99%

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

Zhao

Chen

Wang

et al. 2021

IJMS

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“…Other characteristics have been improved through protein engineering, such as enhanced thermostability of an R-selective TAm from Aspergillus terreus through the introduction of disulphide bonds (Xie et al 2018). Indeed, rational design has even proved capable of changing the enantiopreference of a number of TAms.…”

Section: Protein Engineeringmentioning

confidence: 99%

Transaminases for industrial biocatalysis: novel enzyme discovery

Kelly

Mix

Moody

et al. 2020

Appl Microbiol Biotechnol

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Transaminases (TAms) are important enzymes for the production of chiral amines for the pharmaceutical and fine chemical industries. Novel TAms for use in these industries have been discovered using a range of approaches, including activity-guided methods and homologous sequence searches from cultured microorganisms to searches using key motifs and metagenomic mining of environmental DNA libraries. This mini-review focuses on the methods used for TAm discovery over the past two decades, analyzing the changing trends in the field and highlighting the advantages and drawbacks of the respective approaches used. This review will also discuss the role of protein engineering in the development of novel TAms and explore possible directions for future TAm discovery for application in industrial biocatalysis. Key Points• The past two decades of TAm enzyme discovery approaches are explored.• TAm sequences are phylogenetically analyzed and compared to other discovery methods.• Benefits and drawbacks of discovery approaches for novel biocatalysts are discussed.• The role of protein engineering and future discovery directions is highlighted.

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“…Protein engineering plays a vital role in enhancing the thermal stability of (R)selective At-ATA to expand its applicability in industrial processes (Liu et al, 2019). To date, the rational design of protein engineering involves many factors, such as surface electrostatic interactions, hydrophobic interactions, B-factor values, consensus mutagenesis, disulphide bridges, coevolution networks, and hydrogen bonding interactions (Pace et al, 2011;Wang et al, 2014;Zhang et al, 2014Zhang et al, , 2020Huang et al, 2017;Xie et al, 2018Xie et al, , 2019Moon et al, 2019;Zhu et al, 2019;Cao et al, 2020). All of these have been employed to develop stable proteins.…”

Section: Introductionmentioning

confidence: 99%

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

Cao

Fan

et al. 2021

Front. Chem.

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Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in T5010. Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.

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Improving thermostability of (R)‐selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds

Cited by 21 publications

References 37 publications

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

Transaminases for industrial biocatalysis: novel enzyme discovery

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

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