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Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. So, improving the tolerance of enzymes in organic solvents is essential. Herein, the method of regional random mutation combined with combinatorial mutation was used to improve the resistance of transaminase from Aspergillus terreus (AtATA) in organic solvents, and the best mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency toward 1-acetylnaphthalene and the stability were higher than the wild-type (WT) of AtATA. M3 also showed enhanced stability against six organic solvents with different oil-water partition coefficients (log P values). The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics simulations under different concentration DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Intra- and intermolecular interaction analysis indicated that the increased hydrogen bonds and hydrophobic interactions within monomers or at the interface of two monomers also strengthened the stability of the overall structure against organic solvents. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was and an intermediate of Cinacalcet Hydrochloride. Moreover, 3~10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 94.2~38.9% yield and a strict R-stereoselectivity within 10 h under 25% DMSO, which was higher than WT and expected to be a potential biocatalyst for industrial application.

4-Hydroxyphenylpyruvic acid (4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound l -amino acid deaminase (m l -AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the m l -AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from l -tyrosine. The secretory efficiency of m l -AAD conducted by its own twin-arginine signal peptide (twin-arginine translocation pathway, Tat) and integrated pelB (the general secretory pathway, Sec)-Tat signal peptide was determined and compared firstly, using two pET systems (pET28a and pET20b). It was found that the Tat pathway (pET28a-mlaad) resulted in higher cell-associated m l -AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts Bl21(DE3) and 0.05 mmol·L− 1 IPTG were found to be suitable for m l -AAD expression. The reaction conditions for m l -AAD were optimized and 72.72 mmol·L− 1 4-HPPA was obtained from 100 mmol·L− 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design (DbD) and Modelling of Disulfide Bonds in Proteins (MODIP) to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilised mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild-type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1-fold, and 3.6-fold increase in half-life (t1/2) at 40°C, and a 4.6°C, and 5.1°C increase in T5010. In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40°C and a 5.5°C increase in T5010. Molecular dynamics (MD) simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation (RMSD) for the overall residues at elevated temperature, and consequently increased the protein rigidity. The stabilised mutation of R131C-D134C was in the regions of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121–136. This article is protected by copyright. All rights reserved

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce &gamma, aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 &deg, C and 1.4 &deg, C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l -glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 A resolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM−1 S−1 versus 0.777 mM−1 S−1), which was the highest activity among all variants tested. The flexible loop (Tyr308–Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2) at 40 °C, and a 4.6 °C increase in T5010 among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T5010. Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T5010.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t