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With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, agricultural, and feedstuff industries. The development of synthetic methodologies for optically pure amino acids has been driven by their significant applications. Among the various synthesis methods for the production of chiral amino acids, enzymatic asymmetric synthesis is a unique preparation strategy that shows great potential. This review provides an overview of the reported methods for enzymatic asymmetric synthesis of chiral amino acids, including asymmetric reductive amination of keto acids, asymmetric transfer of an amino group to keto acids, enantioselective addition of ammonia to α,β-unsaturated acids, and aldol condensation of an amino acid to aldehydes.

Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of L-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.

Glycoside hydrolases (GHs) exhibit high activity and stability under harsh conditions, such as high temperatures and extreme pHs, given their wide use in industrial biotechnology. However, strategies for improving the acidophilic and alkalophilic adaptations of GHs are poorly summarized due to the complexity of the mechanisms of these adaptations. This review not only highlights the adaptation mechanisms of acidophilic and alkalophilic GHs under extreme pH conditions, but also summarizes the recent advances in engineering the pH performances of GHs with a focus on four strategies of protein engineering, enzyme immobilization, chemical modification, and medium engineering (additives). The examples described here summarize the methods used in modulating the pH performances of GHs and indicate that methods integrated in different protein engineering techniques or methods are efficient to generate industrial biocatalysts with the desired pH performance and other adapted enzyme properties. [ABSTRACT FROM AUTHOR]

2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from Rhodococcus zopfii (RzNIT/W167G) was constitutively overexpressed with Escherichia coli as host, which exhibited a onefold increase in enzymatic activity compared with inducible expression. Biosynthesis of 2-CA using whole cells harboring nitrilase as biocatalysts were investigated and 318.5 mM 2-CA was produced, which was the highest level for 2-CA production catalyzed by nitrilase to date. 2-CA was recovered from the reaction mixture through a simple acidification step with a recovery yield of 90%. This study developed an efficient bioprocess for 2-CA with great potential for industrial application. [ABSTRACT FROM AUTHOR]

Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application. [ABSTRACT FROM AUTHOR]

BACKGROUND: Non‐aqueous biocatalytic system has exhibited advantages including outstanding substrate solubility, facilitation for product purification, suppression of unwanted water‐dependent side reactions and rare wastewater discharge. However, limited by enzyme stability, selectivity and cofactor recycling, there has been no successful attempt to use carbonyl reductases in non‐aqueous phase. RESULTS: In this study, a non‐aqueous catalytic system of carbonyl reductase was developed for the first time with a carbonyl reductase–cofactor co‐immobilized biocatalyst. Using n‐hexane as the reaction medium, the key chiral diol intermediate of the top‐selling hypolipidemic drug rosuvastatin, tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate ((3R,5S)‐CDHH) was prepared in a continuous packed‐bed bioreactor, with a total yield of 95.51%, enantiomeric excess (e.e.) > 99.5% and diastereomeric excess (d.e.) > 99.5% in a 44‐batch reaction. The yield of per unit biocatalyst was 34.42 mg U−1, and the space–time yield was 91.01 g h−1 L−1. CONCLUSION: Our work corroborated the valuable potential of carbonyl reductases in non‐aqueous biosynthesis of chiral pharmaceuticals and fine chemicals. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Nitrilases are widely distributed in nature and are able to hydrolyze nitriles into their corresponding carboxylic acids and ammonia. In industry, nitrilases have been used as green biocatalysts for the production of high value-added products. To date, biocatalysts are considered to be important alternatives to chemical catalysts due to increasing environmental problems and resource scarcity. This review provides an overview of recent advances of nitrilases in aspects of distribution, enzyme screening, molecular structure and catalytic mechanism, protein engineering, and their potential applications in industry. [ABSTRACT FROM AUTHOR]

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit–subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted. [ABSTRACT FROM AUTHOR]

Enzyme membrane reactors (EMRs) are becoming increasingly interesting for application in bioconversion processes in food processing, pharmaceutics, biorefinery and wastewater treatment. Integrating the highly efficient enzymatic reaction with selectable membrane separation technology, the EMRs are able to reduce product inhibition, improve the stability of the enzyme, increase the number of reaction cycles and sustainably separate products from biotransformation solutions. Generally, in EMRs, enzymes can be either free in the solution, immobilized on an additional carrier or immobilized directly in/on a porous structure of the membrane, of which the system with the enzyme immobilized directly in/on the membrane (EIM) is a relatively simple and most widely applied method. The EIM system not only facilitates recycling of the enzymes but also in many cases additionally enhances enzyme properties such as the stability and viability. A membrane with a porous structure is usually considered as a potential carrier for enzyme immobilization. A series of immobilization methods has been developed to fix the enzymes on the membranes. The purpose of this review is to systemically summarize the immobilization methods of enzymes in/on membranes and the applications of the EIM system for biocatalysis. [ABSTRACT FROM AUTHOR]

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L−1. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and eep of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor. [ABSTRACT FROM AUTHOR]

Objective: Rational engineering of the crevice-like binding site of lipases for improvement of lipases' catalytic properties.Resuts: The residues located at the crevice-like binding site of four representative lipases including Thermomyces lanuginosus lipases (TLL and Lip), Rhizopus oryzae lipase (ROL), and Rhizomucor miehei lipase (RML) were identified through structural analysis. The residues at the bottom of the crevice-like binding site recognizing the substrates with short/medium carbon chain length and those located at the right-hand wall of the surface crevice region affecting the product release were changed by site-directed mutagenesis. The corresponding double mutants exhibited ~ 5 to 14-fold higher activity towards p-nitrophenyl esters than their wild types, and their substrate preference shifted to acyl moiety with shorter carbon chain length. In addition, the mutations led to an increase of B-factor, resulting in decrease of their optimum temperature by 10-20 °C.Conclusions: The key residues located at the crevice-like binding site play important roles in determining lipase activity, substrate preference and optimum temperature, which offers a useful new paradigm for facilitating rational design of lipases. [ABSTRACT FROM AUTHOR]

( S)-4-Chloro-3-hydroxybutyronitrile [( S)-CHBN] was used as a chiral building block for the preparation of atorvastatin. In this study, ( R, S)-epichlorohydrin [( R, S)-ECH] and 1,3-dichloro-2-propanol (1,3-DCP) were investigated to prepare ( S)-CHBN by using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1. Preparing ( S)-CHBN from ( R, S)-ECH gave a modest enantiomeric excess ( ee), whereas by using 1,3-DCP as the substrate, ( S)-CHBN was obtained with 97.3 % ee after pH optimization. However, a low ee value and low yield of ( S)-CHBN were obtained if the substrate concentration was increased to 10 g L−1. To obtain a higher ee value and yield, 16 mutants were constructed and screened. The variant W249F with improvements in activity and enantioselectivity was identified and applied at a 1,3-DCP loading of 10 g L−1, which gave ( S)-CHBN in 86 % yield with 97.5 % ee in 1 h. This is the first report of a one-step biocatalytic process for the preparation of ( S)-CHBN from prochiral 1,3-DCP. [ABSTRACT FROM AUTHOR]

( S)-4-chloro-3-hydroxybutanoate (( S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the ( S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65 U/L and 13.90 g DCW/L which was 3.24 and 2.60-folds compared with those in the 50 L fermentor. The biocatalytic process for the synthesis of ( S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-chloro-3-oxobutanoate concentration reached to 1.7 M, and the ( S)-CHBE with yield of 97.2 % and enantiomeric excess ( e.e.) of 99 % was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17 mM/h and 27.55 mM/h/g DCW for ( S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, ( S)-CHBE was extracted from the reaction mixture with 82 % of yield and 95 % of purity. This study paved the foundation for the upscale production of ( S)-CHBE by biocatalysis method. [ABSTRACT FROM AUTHOR]

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3 S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin. [ABSTRACT FROM AUTHOR]

In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN. [ABSTRACT FROM AUTHOR]

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0-7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni, Ba and Fe. The apparent K and V values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min mg, for 2-deoxyribose were 0.033 mM and 2.59 μmol min mg, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3 R, 5 S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3 R, 5 S)-6-chloro-2,4,6-trideoxyhexose. [ABSTRACT FROM AUTHOR]

Biocatalytic asymmetric preparation of ( R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent ( R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. ( R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave ( R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of ( R)-1,3-butanediol. [ABSTRACT FROM AUTHOR]

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l, 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application. [ABSTRACT FROM AUTHOR]

Abstract: Bioconversion of p-methoxyphenylacetonitrile (MOPAN) to p-methoxyphenylacetic acid (MOPAA) by immobilized cells of Bacillus subtilis ZJB-063, a nitrilase producing strain, was investigated in batch and repeated batch. Four entrapment matrixes were screened to search for a suitable support in terms of enzyme activity and reusability of the bead, and alginate exhibited significant process advantages over its other counterparts. The effects of sodium alginate concentration, bead diameter, CaCl2 concentration were examined. Maximum activity was achieved when the above parameters were set as 2.5% sodium alginate, 2mm of bead diameter, 4.0% CaCl2. An initial MOPAN concentration of 10mM was completely converted to MOPAA by entrapped cells after 8h reaction under the optimized conditions. Efficient reusability of the biocatalyst up to nine batches was achieved by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 18 batches. [Copyright &y& Elsevier]

Abstract: Valienamine, an aminocyclitol with similar configuration to α-glucose, has a strong inhibitory effect on α-glucosidase. α-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase was investigated. Our results show that valienamine inhibition of honeybee α-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee α-glucosidase in vitro with an IC50 value of 5.22×10−5 M and K i value of 3.54×10−4 M at pH 6.5, 45°C. Valienamine has the potential to be developed into novel insecticides. [Copyright &y& Elsevier]

Surface-crosslinked hydrogel particles encapsulating E. coli cells harboring nitrilase were constructed and used as a durable biocatalyst in the synthesis of gabapentin intermediate. E. coli cells (50 g/L) expressing nitrilase were encapsulated in 2% (w/v) alginate hydrogel, 0.5%(w/v) glutaraldehyde and 1%(w/v) polyethyleneimine were sequentially used as crosslinker. The constructed alginate particles with GA&PEI composite crosslinking had a similar catalytic capacity compared to equivalent free cells, which could completely hydrolyze 1.0 M substrate 1-cyanocyclohexaneacetonitrile within 4 h at pH 7.5 and 35 °C, and have a specific activity of 134.6 U/g, an optimum temperature at 55 °C and a corresponding half-life up to 19.1 ± 1.9 h. The particles showed excellent storage stability, reusability and mechanical strength, which retained 90.3% activity after 2-month storage at 4 °C, and 83.2% activity after 14-day storage at 35 °C. In batch reactions, 50 g/L particles could be reused for maximum 32 cycles, maintained 63.5% residual activity and 50.7% particle integrity, providing an average product yield of 94.6% in the first 30 batches and a productivity of 1467.03 g/L/day. Besides, the stability and catalytic activity of the particles were almost unaffected in several organic co-solvents and in purified water, showing their good industrial potential. [Display omitted] • Surface-crosslinked hydrogel particles encapsulating E. coli cells were prepared as biocatalysts. • The particles have similar catalytic capacity compared to equivalent free cells. • High activity and strength enable it to completely hydrolyze 1 M substrate within 4 h. • The particles retained 90.3% activity after 2 months of storage and could be reused up to 32 times. [ABSTRACT FROM AUTHOR]

The biosynthetic pathway of l -methionine in microorganisms was complex and regulated at multiple levels. In this study, a two-step method for l -methionine production combined fermentation and biocatalysis was realized in one pot. The O -succinyl- l -homoserine (OSH) producing strain Escherichia coli W3110(DE3) ΔIJB∗Trc metL /pTrc- metA fbr-Trc- thrA fbr- yjeH (ΔIJB) was constructed initially. OSH in the fermentation supernatant was then converted to l -methionine in the presence of O -succinyl- l -homoserine sulfhydrylase (OSHS) and sodium methanethiol. The titer of l -methionine could reach 21.1 g/L after 88 h (84 h fermentation and 4 h catalysis) in a two-step method (process 1). In a one-pot two-strain system (process 2), two strains ΔIJB and E. coli BL21(DE3)/pET28b–OSHS–cutinase were co-cultured, and 8.24 g/L l -methionine was obtained. In another one-pot one-strain system (process 3), strain E. coli ΔIJB/pET28b–OSHS–cutinase could co-express OSHS and cutinase during ΔIJB fermentation at the same time, obtaining 13.6 g/L l -methionine in a 5 L fermentor after 84 h. By comparing the three processes for l -methionine production based on the process 1, the simplified process in process 3 provided in this study showed potent in the large-scale production of l -methionine with convenient handling and production efficiency, but further works still need to be carried out to improve the l -methionine production. • High-yield O-succinyl- l -homoserine (OSH) producing strain was constructed in Escherichia coli W3110(DE3) (ΔIJB). • Enzyme O-succinyl- l -homoserine sulfhydrylase and cutinase were co-expressed in E. coli BL21(DE3) (OSHS–Cut). • A one-pot two-strain system was established for l -methionine production with co-cultivation of ΔIJB and OSHS–Cut. • A system combining of OSH production and enzymatic catalysis in one pot was established. [ABSTRACT FROM AUTHOR]

• A novel enzyme cascade-based deracemization of phosphinothricin was developed. • A "two stages in one-pot" strategy was developed to avoid by-product inhibition. • High yield and excellent enantiomeric excess of product were achieved. • No extra cofactor was required for the developed enzyme cascade. Deracemization of D,L-phosphinothricin (D,L-PPT) is one of the most promising routes for preparation of optically pure L-PPT. In this work, an efficient multi-enzyme redox cascade was developed for deracemization ofPPT, which includes oxidative reaction and reductive reaction. The oxidative reaction catalyzing oxidative deamination of D-PPT to 2-oxo-4-[(hydroxy)(-methyl)phosphinyl]butyric acid (PPO) was performed by a D-amino acid oxidase and a catalase for removing H 2 O 2. The reductive reaction catalyzing amination of PPO to L-PPT is achieved by a glufosinate dehydrogenase and a glucose dehydrogenase for cofactor regeneration. To avoid the inhibitory effect of glucose on the oxidative reaction, a "two stages in one-pot" strategy was developed to combine these two reactions in deracemization process. By using this strategy, the L-PPT was obtained with a high yield (89 %) and > 99 % enantiomeric excess at substrate loading of 300 mM in absence of addition of extra NADP+. These encouraging results demonstrated that the developed enzyme cascade deracemization process exhibits great potential and economical competitiveness for manufacture of L-PPT from D,L-PPT. [ABSTRACT FROM AUTHOR]

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant Mw TA M8 , which exhibited 79.2-fold higher activity than the chimeric scaffold Mw TA Mc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates. • Effective screening (R)-selective transaminase based on substrate walking strategy. • Simultaneously conferred (R)-selectivity and activity via motif swapping. • Semi-rational design for reshaping pocket achieve activity from scratch to strong. [ABSTRACT FROM AUTHOR]

Highlights • A novel (R)-selective As -TA was screened for biosynthesis of (R)-3-amino-1-butanol. • The catalytic process was optimized for enhanced production of (R)-3-amino-1-butanol. • High concentration of substrate could be effectively converted by a fed-batch method. • The product with a maximum yield of 29.6 g/L and e.e. value of 99.9% was obtained. Abstract (R)-3-amino-1-butanol is a key intermediate of Dolutegravir for the treatment of HIV/AIDS and its green and efficient biosynthesis has attracted a great deal of attention. Transaminases are currently used as promising biocatalyst for the synthesis of chiral amines. However, many transaminases have (S)-specificity and (R)-selective transaminases were less exploited and studied, making the production of (R)-amines remain challenging. In this study, a novel transaminase from Actinobacteria sp. (As -TA) was obtained and applied for the biosynthesis of (R)-3-amino-1-butanol by transferring the amino group from isopropylamine to 4-hydroxy-2-butanone. After optimization of the reaction condition and using a substrate fed-batch strategy, the conversion of 100, 200, 300, 400 and 500 mM 4-hydroxy-2-butanone reached 100%, 94.9%, 86.1%, 76.1% and 70.9%, respectively. (R)-3-amino-1-butanol with a maximum yield of 29.6 g/L and 99.9% e.e. value was obtained. This was the first time demonstrating the successful biosynthesis of (R)-3-amino-1-butanol with transaminase as biocatalyst and the obtained As -TA enriched the enzyme pool of transaminase with (R)-specificity. [ABSTRACT FROM AUTHOR]

Glucose isomerase (GI) responsible for catalyzing the isomerization from d -glucose to d -fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d -fructose concentration. [ABSTRACT FROM AUTHOR]

The scissile fatty acid binding site of lipases is divided into different sub-groups and plays an important role in the catalytic properties of the enzymes. In this study, the Talaromyces thermophilus lipase was engineered by altering its crevice-like binding site for efficient synthesis of chiral intermediate of Pregablin through kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE). The substitution of residues located at the crevice-like binding site with phenylalanine (Phe) resulted in significantly increased hydrolysis activity. The variant L206F/P207F/L259F exhibited a 37.23-fold and 47.02-fold improvement in the specific activity and turnover number ( k cat ) toward CNDE, respectively. Simultaneously, the optimum temperature and substrate preference were both altered in the variants. The study herein successfully engineered the TTL with improved catalytic properties for efficient biosynthesis of Pregablin intermediate. The investigation of structure-functional relationship provided important guidance for further modification of lipases with crevice-like binding site domain. [ABSTRACT FROM AUTHOR]

[Display omitted] • A strategy of reaction-kinetic model-guided biocatalyst engineering was developed. • The kinetic model of a dual-enzyme-catalyzed bioreaction was constructed. • The limiting factor was analyzed under the guide of reaction kinetic model. • A high space–time yield (1152 g/L d-1) was achieved for atorvastatin precursor. Current research on dual-enzyme-catalyzed bioreaction systems has focused on single-enzyme engineering and enzyme cascade development. However, few studies have investigated the bioreaction kinetic model, which creates a gap between the biocatalyst and reaction systems. To fill this gap, we developed a reaction-kinetic model-guided biocatalyst engineering strategy, including the iterative cycle of "kinetic model construction, limiting factor analysis, and biocatalyst engineering". In the proof-of-concept experiment, a dual-enzyme-catalyzed bioreaction of the atorvastatin precursor (3 R ,5 R)- 2 was selected and the iterative cycle was performed in two rounds. First, a double-substrate-ordered Bi–Bi kinetic model was constructed and validated. Thereafter, the limiting factors were analyzed using the constructed model and further confirmed experimentally. The limiting factor was glucose dehydrogenase total activity in the first round, whereas it changed to aldo–keto reductase total activity in the second round. Under this guidance, the bioreaction system was improved through enzyme selection, protein engineering, promoter optimization, and optimization of the expression conditions. Finally, the coupling efficiency of the two enzymes increased, resulting in high space–time yields (>1 kg/L d-1) of the atorvastatin precursor in both 250 mL flasks and 50 L bioreactors. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant Escherichia coli cells harbouring haloalcohol dehalogenase and epoxide hydrolase were successfully immobilized by adsorption onto perlite and used to prepare (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis in a specially designed reactor. Two-phase solution was used as the reaction system in order to improve the yield of epichlorohydrin. In the two-phase system containing 40% (v/v) cyclohexane, the yield of racemic epichlorohydrin formed in the first step was 73%, and the yield of (R)-epichlorohydrin with enantiomeric excess (ee) ≥99% increased from 19.2% to 25.1% in the second step. Ultimately, the yield of (R)-epichlorohydrin reached 26.4% by optimization of the flow rate of air and amount of immobilized cells. To our knowledge, this was the first report on production of (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase. [Copyright &y& Elsevier]

Abstract: In the current study, an epoxide hydrolase (EH) gene from Rhodosporidium toruloides was synthesized and expressed in Escherichia coli. After purification, we found that the optimal pH and temperature of this enzyme were 7.5 and 35°C, respectively. The recombinant EH obtained in this study was temperature-sensitive and the activity decreased significantly above 45°C. The values of apparent K m and V max were 0.5953mol/l and 0.0105mol/(L·min). In addition, enantiomeric excesses value of (R)-epichlorohydrin could reach 100% after 40-min reaction. Moreover, this EH showed a broad substrates specificity toward epoxides. To the best of our knowledge, this is the first time to report the application of R. toruloides EH in racemic resolution of (R,S)-epichlorohydrin to produce (R)-epichlorohydrin. [Copyright &y& Elsevier]

Enantioselective biotransformation of prochiral 1,3-DCP by halohydrin dehalogenases (HHDHs) is particularly attractive since 100% yield of chiral epichlorohydrin (ECH) may be obtained. HheC mutant (P175S/W249P) displayed greatly improved enantiomeric excess ( ee ) of ( S )-ECH from 5% to 95.3% in the catalyzed dehalogenation of 1,3-DCP at pH 8.0. ( S )-ECH was enantioselectively biotransformed from 40 mM 1,3-DCP with 92.3% ee and 93.2% yield at pH 10.0. To increase the ee of ( S )-ECH, the catalysis was carried out using HheC mutant coupled with epoxide hydrolase mutant and the maximum yield and ee of ( S )-ECH reached 91.2% and > 99%. [ABSTRACT FROM AUTHOR]

• Aqueous-organic two-phase system was applied for (S)-epichlorohydrin production. • Structure-based engineering was performed on halohydrin dehalogenases (HheC ps). • A zigzag tunnel and depressed cave region were key to the properties of HheC ps. • Positive mutants were obtained with increased activity and enantioselectivity. Halohydrin dehalogenases (HHDHs) represent as important biocatalysts for the production of valuable chiral epoxides and β-substituted alcohols. The HHDH from A. radiobacter (HheC) showed great potential for biosynthesis of (S)-epichlorohydrin ((S)-ECH). However, two bottlenecks including self-degradation and enzyme-mediated racemization of product in aqueous solution restricted its wide-spread applications. To solve the problems, the biocatalytic process was performed in aqueous-organic two-phase system and structure-based engineered of HheC was performed. A newly found zigzag tunnel connecting the active site region and outside space, as well as a depressed cave structure in formation of an extra three-pot herringbone channel were demonstrated to play a pivotal role affecting the activity and enantioselectivity of the enzyme. By mutation of the involved amino acid residues, two variants E255 T and E255 G showed significantly improved activity, with specific activity increased from 59.9 U/mg to 101.6 U/mg and 77.5 U/mg, respectively, while one variant Y93 L exhibited high enantioselectivity, with ee value of (S)-ECH maintained at >99 %. In addition, the synergic variant Y93 L/E255 T had both improved catalytic efficiency and enantioselectivity. The results not only facilitate the applicability of HheC for efficient production of (S)-ECH, but also give new insights into the structural-functional relationship of the enzyme. [ABSTRACT FROM AUTHOR]

• A novel covalent co-immobilization method of transaminase and PLP was constructed. • Addition of exogenous PLP was eliminated when harboring the biocatalyst. • The biocatalyst showed good operational stability in a 700-batch reaction. Transaminases (TAs, EC 2.6.1.X) are a class of promising catalysts for the synthesis of chiral amines. The addition of exogenous expensive cofactor, pyridoxal 5′-phosphate (PLP) leads to the high cost and complicated reaction composition and thus limits the industrial implementation of TAs. In this study, a novel self-sufficient biocatalyst was developed based on transaminase and PLP covalent co-immobilization with the activity recovery of 83.6% and the specific activity of 343.0 U/g catalyst. The self-sufficient BgTA biocatalyst was employed in the continuous biosynthesis of (R)-sitagliptin in a recirculating packed bed reactor (RPBR) for 700 batches reaction without extra addition of PLP. The yield and e.e. of (R)-sitagliptin for each batch were above 90% and 99% respectively. The space-time yield was 40.0 g/(L·h) which was higher than ever reported. This is the first report about the covalent co-immobilization of TA and PLP with high activity and stability, and our work also contributed to the economic flexibility of chiral amine pharmaceutical intermediates biosynthesis. [ABSTRACT FROM AUTHOR]