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Planting under plastic-film mulches is widely used in spring maize production in arid-cold regions for water conservation and warming the soil. To ameliorate the associated issues such as plastic-film residues and additional labor during the “seedling release” in spring maize production, we have developed a plastic-film-side seeding (PSS) technology with the supporting machinery. In the semi-arid regions of Northwest China, a 7-year trial demonstrated that PSS increased plant number per hectare by 6 547 and maize yield by 1 686 kg ha–1 compared with the traditional method of seeding under plastic-film mulch (PM). Two-year experiments were conducted in two semi-arid regions to further understand the effects of PSS on three important aspects of production: (i) the moisture and temperature of soil, (ii) maize development, yield output, and water use efficiency (WUE), and (iii) the revenue and plastic-film residuals in comparison with that of flat planting (CK) and PM. Continuous monitoring of the soil status demonstrated that, compared with CK, the PSS treatment significantly increased the temperature and moisture of the 0–20 cm soil in the seeding row at the early stage of maize development, and it also promoted grain yield (at 884–1 089 kg ha–1) and WUE, achieving a similar effect as the PM treatment. Economically, the labor inputs of PSS were equal to CK, whereas the PM cost an additional 960 CNY ha–1 in labor for releasing the seedlings from below the film. Overall, the PSS system increased profits by 5.83% (547 CNY ha–1 yr–1) and 8.16% (748 CNY ha–1 yr–1) compared with CK and PM, respectively. Environmentally, PSS achieved a residual film recovery rate of nearly 100% and eliminated 96 to 130 kg ha–1 of residual plastic-film in PM in 3–5 years of maize production. Collectively, these results show that PSS is an eco-friendly technique for improving yield stability and incomes for the sustainable production of maize in semi-arid regions.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (k cat /K m ) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates., Competing Interests: Declaration of competing interest The authors declare on conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 MwTA M8 , which exhibited 79.2-fold higher activity than the chimeric scaffold MwTA 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vibegron is a novel, potent, highly selective β 3 -adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6 A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The K m of EaSDR6 A138L/S193A was decreased from 1.57 mM to 0.67 mM, k cat was increased by 2.17 folds, and the overall catalytic efficiency k cat /K m was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW ) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW ). The substrate concentration and spatiotemporal yield were higher than ever reported., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Enantiopure β-nitroalcohols, as an important class of nitro-containing compounds, are essential building blocks in pharmaceutical and organic chemistry, particularly for the synthesis of β-adrenergic blockers. In this study, we present the successful protein engineering of halohydrin dehalogenase HHDHamb for the enantioselective bio-nitration of various phenyl glycidyl ethers to the corresponding chiral β-nitroalcohols, using the inexpensive, commercially available, and safer nitrite as a nitrating agent. The chiral (R)- and (S)-1-nitro-3-phenoxypropan-2-ols were synthesized by the several enantiocomplementary HHDHamb variants through the whole-cell biotransformation, which showed good catalytic efficiency (up to 43% isolated yields) and high optical purity (up to >99% ee). In addition, we also demonstrated that the bio-nitration method was able to tolerate the substrate at a high concentration of 1000 mM (150 g/L). Furthermore, representative synthesis of two optically active enantiomers of the β-adrenergic blocker metoprolol was successfully achieved by utilizing the corresponding chiral β-nitroalcohols as precursors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g -1 . The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L -1 L-met and 0.60 g L -1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L -1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L -1 to 10.1 g L -1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production., Competing Interests: Declarations of interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In order to improve the degradation activity of β-glucosidase (CpBgl) from Coniophora puteana, the structural modification was conducted. The enzyme activity of mutants CpBgl-Q20C and CpBgl-A240S was increased by 65.75% and 58.58%, respectively. These mutants exhibited maximum activity under the same conditions as wild-type CpBgl (65 ℃ and pH 5.0), slightly improved stabilities compared that of the wild-type, and remarkably enhanced activities in the presence of Mn 2+ or Fe 2+ . The V max of CpBgl-Q20C and CpBgl-A240S was increased to 138.18 and 125.14 μmol/mg/min, respectively, from 81.34 μmol/mg/min of the wild-type, and the catalysis efficiency (k cat /K m ) of CpBgl-Q20C (335.79 min -1 /mM) and CpBgl-A240S (281.51 min -1 /mM) was significantly improved compared with that of the wild-type (149.12 min -1 /mM). When the mutant CpBgl-Q20C were used in the practical degradation of different biomasses, the glucose yields of filter paper, corncob residue, and fungi mycelia residue were increased by 17.68%, 25.10%, and 20.37%, respectively. The spatial locations of the mutation residues in the architecture of CpBgl and their unique roles in the enzyme-substrate binding and catalytic efficiency were probed in this work. These results laid a foundation for evolution of other glycoside hydrolases and the industrial bio-degradation of cellulosic biomass in nature., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase LsCR for the synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a "small and smart" mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant LsCR M3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) LsCR, the catalytic activity of LsCR M3 was increased by 4.7 times, the catalytic efficiency k cat /K M value was increased by 2.9 times, and the half-life t 1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% (v/v) isopropanol, LsCR M3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e.) in 11 h with a space-time yield (STY) as high as 809 g/L∙d., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In order to improve the catalytic efficiency of cellulase for more effective utilization of lignocellulose, a novel endoglucanase (CgEndo) from Colletotrichum graminicola was expressed by Pichia pastoris X33 and modified by site-directed mutagenesis. Two mutants, Y63S and N20D/S113T, with 62.31% and 57.14% increased enzyme activities were obtained, respectively. On this basis, their biochemical properties, kinetic parameters, structural information as well as the application in biomass degradation were investigated and compared with the wild-type CgEngo. The results indicated that the mutation Y63S and N20D/S113T resulted in an improvement of proximity between enzyme and substrate through conformational changes of the catalytic region, which might contribute to the higher enzyme activities and catalysis efficiency (K cat /K m ) of Y63S and N20D/S113T. These findings laid important foundation for the further engineering of this endoglucanase and practical application in efficient degradation of cellulosic biomass in nature., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the chemical-biological synthesis route of gabapentin, immobilized Escherichia coli cells harboring nitrilase are used to catalyze the biotransformation of intermediate 1-cyanocyclohexaneacetonitile to 1-cyanocyclohexaneacetic acid. Herein, we present a novel cell immobilization method, which is based on cell adsorption using 75 g/L Escherichia coli cells and 6 g/L zeolite, cell crosslinking using 3 g/L polyethylenemine and biomimetic silicification using 18 g/L hydrolyzed tetramethylorthosilicate. The constructed "hybrid biomimetic silica particles (HBSPs)" with core-shell structure showed a specific activity of 147.2 ± 2.3 U/g, 82.6 ± 2.8% recovery of nitrilase activity and a half-life of 19.1 ± 1.9 h at 55 °C. 1-Cyanocyclohexaneacetonitrile (1.0 M) could be completely hydrolyzed by 50 g/L of HBSPs at pH 7.5, 35 °C in 4 h, providing 92.1 ± 3.2% yield of 1-cyanocyclohexaneacetic acid. In batch reactions, the HBSPs could be reused for 13 cycles and maintained 79.9 ± 4.1% residual activity after the 10th batch, providing an average product yield of 92.6% in the first 10 batches with a productivity of 619.3 g/L/day. In addition, multi-layer structures consisting of silica coating and polyethylenemine/glutaraldehyde crosslinking were constructed to enhance the mechanical strength of immobilized cells, and the effects of coating layers on the catalytic properties of immobilized cells was discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co 2+ and Mg 2+ . The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased K m from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Echinocandin B deacylase (ECBD) from Actinoplanes utahensis can be applied to produce echinocandin B nucleus (ECBN), an essential intermediate of the echinocandins antifungal drugs such as anidulafungin. To date, the expression of ECBD has been limited to Streptomyces. To achieve the active expression of ECBD in Escherichia coli (E. coli), we constructed a plasmid carrying two subunits of ECBD for T7 RNA polymerase driven transcription of dicistron messenger after codon optimization. Subsequently, the introduction of peptide tags in the recombinant ECBD was adopted to reduce the formation of inclusion bodies and enhance the ECBD solubility. The peptide tags with the opposite electrostatic charge, hexa-lysine (6K) and GEGEG (GE), exhibited the best positive effect, which was verified by activity assay and structural simulation. After that, optimization of culture conditions and characterization of ECBD were conducted, the optimal pH and temperature were 7.0 and 60 °C. It is the first report concerning the functional expression of ECBD in the host E. coli. Our results reported here can provide a reference for the high-level expression of other deacylases with respect to a possible industrial application., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Comparative proteomes of Actinoplanes utahensis ZJB-03852 grown on various saccharides (glucose, maltotriose, maltose, glucose + maltose) were analyzed using 2D-DIGE and MALDI-TOF/TOF-MS. Acarbose was detected in all groups except in the glucose only culture. The abundance of acarbose synthesis proteins AcbV, AcbK, AcbL and AcbN was highest in the medium containing mixed glucose and maltose. The accumulation of Zwf and Xpk1 in acarbose-producing media indicated that the cyclitol moiety of acarbose was derived from pentose phosphate pathway. The elevation of GlnA supported that glutamine was a good nitrogen source of the nitrogen-atom in acarbose synthesis. SIGNIFICANCE: Non-insulin-dependent diabetes mellitus, also known as Type II diabetes, constitutes >90% of the diabetes mellitus worldwide. Acarbose is clinically utilized to treat Type II diabetes, but the fermentation process of acarbose-producing Actinoplanes is usually accompanied with structural analogues of acarbose. In this study, we compared the proteomics of Actinoplanes utahensis ZJB-03852 grown on various saccharides by 2D-DIGE and MALDI-TOF/TOF-MS. Our findings highlighted the importance of key proteins in the formation of acarbose and its analogues when A. utahensis was cultivated in various saccharides. These results revealed fundamental data to elucidate the complexity of formation of acarbose analogues., (Copyright © 2021 Elsevier B.V. All rights reserved.)

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an advanced chiral diol intermediate of the cholesterol-lowering drug atorvastatin. KmAKR M5 (W297H/Y296W/K29H/Y28A/T63M) constructed in our previous work, displayed good biocatalytic performance on (3R,5R)-2. In the present work, stepwise evolution was applied to further enhance the thermostability and activity of KmAKR M5 . For thermostability enhancement, N109 and S196 located far from the active site were picked out by structure-guided consensus engineering, and mutated by site-directed mutagenesis (SDM). For catalytic efficiency improvement, the residues A30 and T302 adjacent to the substrate-binding pocket were subjected to site-saturation mutagenesis (SSM). As a result, the "best" mutant KmAKR M9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C) was developed, of which T 50 15 and T m were 5.0 °C and 8.2 °C higher than those of KmAKR M5 . Moreover, compared to KmAKR M5 , KmAKR M9 displayed a 1.9-fold (846 vs 2436 min) and 6.7-fold (126 vs 972 min) longer half-lives at 40 and 50 °C, respectively. Structural analysis suggested that beneficial mutations introduced additional hydrophobic interactions and hydrogen bonds, contributing rigidification of the flexible loops and the increase of internal forces, hence increasing the thermostability and activity. 5 g DCW (dry cell weight) L -1 KmAKR M9 completely reduced 350 g L -1 t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate ((5R)-1), within 3.7 h at 40 °C, yielding optically pure (3R,5R)-2 (d.e. p  > 99.5%) with a space-time yield (STY) of 1.82 kg L -1 d -1 . Hence, KmAKR M9 is a robust biocatalyst for the synthesis of (3R,5R)-2., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective ω-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1+T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 g DCW /L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached ≈32 g/(L·d)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

6-(N-hydroxyethyl)-amino-6-deoxy-l-sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ± 3.0 g/L of 6NSL production with 94.5 ± 3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the "best" variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose K m , k cat /K m towards (5R)-1 was 0.66 mM and 210.77 s -1 mM -1 , respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L -1 Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L -1 Variant VI completely reduced (5R)-1 of up to 450 g L -1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e. p ) with a space-time yield (STY) of 1.24 kg L -1 day -1 , and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L -1 Variant VI completely reduced 400 g L -1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e. p ) with a STY of 1.34 kg L -1 day -1 . In summary, Variant VI displayed industrial application potential in statins biomanufacturing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

L-phosphinothricin (L-PPT) is a competitive and environmentally friendly herbicide. To develop an efficient approach for synthesis of l-PPT, a kinetic resolution route with a novel aminoacylase (SmAcy) mined from Stenotrophomonas maltophilia using N-acetyl-PPT as a substrate was first constructed. This SmAcy exhibited high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-acetyl-PPT. Optically pure l-PPT (> 99.9 % ee p ) was acquired with high conversion (> 49 %) within 4 h by the whole cells. On the basis of the docking analysis, a main reason for high enantioselectivity (E > 200) of SmAcy towards l-enantiomer would be that the D-N-acetyl-PPT cannot interact with the key general acid-base residue and the metal ions. A low-cost and simple preparation process of the substrate from commercially available racemic PPT for production of L-PPT was provided. A chemical racemization method of the unreacted D-enantiomer of substrate was also provided to recycle the unwanted substrate enantiomer. This study provides a potential route for the industrial production of L-PPT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A highly active secretory expression system in Bacillus subtilis was constructed for enhancing the heterogeneous secretory expression efficiency of a recombinant amidase from Bacillus megaterium (Bm-Ami). Initially, six promoters P lytR , P spoVG , P aprE , P yvyD , P ftsH , P amyE were screened out from 33 endogenous phase-dependent promoters via the strategy of promoter engineering which showed higher transcriptional levels than the original promoter P cdd . Afterwards, a secretory expression system pBSHdd2-20 containing a dual-promoter P amyE-cdd and a signal peptide Pac was constructed showing an extracellular Bm-Ami activity of 135.58 U mL -1 during shake-flask cultivation, which was 3.58-fold greater than that with plasmid pBSH1 (control). Finally, extracellular amidase activity of a strain containing the plasmid pBSHdd2-20 reached 430 U mL -1 (equal to 10.8 mg mL -1 ) and 10.72 U mg -1 DCW after 52 h in a scale-up fermentation, which was the highest secretory efficiency for Bm-Ami in all studies to date. In conclusion, a highly secretory system constructed in this study can be applied for the industrial production of amidase and provides a basis for the development of an excellent secretory expression system for similar proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important building-block of atorvastatin. In our previous work, a variant KlAKR-Y295W-W296 L (designated as M1) of Kluyveromyces lactis aldo-keto reductase (wild type (WT) KlAKR, M0) was developed, which possessed strict diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To further improve its catalytic performance, semi-rational engineering of M1 was performed in present work, and the "best" varaint KlAKR-Y295W-W296L-I125V-S30P-Q212R-I63W (M8) was developed. M8's K m B towards (5R)-1 was 2.02 mM, and the catalytic efficiency (k cat /K m B ) value was 36.31 s -1  mM -1 , which was 1.9-fold higher than that of the parent M1. Compared with M1, the half-life t 1/2 ,  T S50 50 and T P50 50 of M8 were improved. Under the optimized conditions, (5R)-1 at load of up to 80 g L -1 was completely reduced in 1.5 h by M8 along with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, producing (3R,5R)-2 in de p > 99.5% and space-time yields (STY) of 660.0 g L -1 d - 1 ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The K m values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate v max was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency k cat /K m of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e. P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nitrilase-catalyzed regioselective hydrolysis of 1-cyanocyclohexaneacetonitrile (1-CHAN) is a green and efficient approach for the preparation of 1-cyanocyclohexaneacetic acid (1-CHAA), a key precursor for the synthesis of gabapentin. Here, a mesoporous biosilica particles prepared by the ethyleneamine-mediated silicification have been used as carrier for the encapsulation of nitrilase from Acidovorax facilis (NitA). The silica-encapsulated NitA (NitA@silica) with triethylenetetramine as an initiator showed the highest immobilization efficiency (98.3%) and specific activity (672.6 U/g). Both free and encapsulated NitA were optimally active at 40 °C and pH 7.0, however, the encapsulated enzyme exhibited wider optimum temperature range, and enhanced thermal stability compared with the free enzyme. The kinetic parameters K m and V max for free and encapsulated NitA were calculated to be 141 mM and 9.97 mM min -1 , and 280 mM and 9.02 mM min -1 , respectively. The encapsulated NitA showed good reusability and retained about 94.2% of its initial activity even after 16 cycles of reaction. Also, the storage experiments revealed high activity maintenance of encapsulated NitA after 17-day storage at 4 °C. A preparative scale regioselective hydrolysis of 1-CHAN to 1-CHAA with encapsulated NitA as biocatalyst was carried out in a 2 L stirred bioreactor. The concentration of 1-CHAA reached 152 g/L after 8 h reaction and the conversion was 90.9%. These results showed that the encapsulation of NitA in ethyleneamine-mediated biosilica is an efficient and simple way for preparation of stable nitrilase and have a great potential for application in enzymatic production of carboxylic acids., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methionine is an essential sulfur-containing amino acid for organisms. The separation and purification are important for the production of l-methionine from fermentation broth. In this work, the adsorption properties for l-methionine separation of ten macroporous resins were firstly evaluated. Macroporous cation resin D72 showed the best adsorption capacity (52.37 mg/g) and desorption rate (99.12%). The adsorption kinetics of l-methionine on D72 resin followed the pseudo second-order model and adsorption isotherm fitted well to the Sips model, the adsorption process mainly followed a physisorption mechanism. D72 resin packed columns were then used in the batch separation of fermentation broth, the operation parameters were optimized during the dynamic adsorption and desorption experiments. Under the optimized conditions: fermentation broth adjusted to pH = 2, loading flow rate at 2 BV/h, loading volume 55 mL, 1 mol/L NH 3 ·H 2 O as eluent, elution flow rate at 2 BV/h, column height-diameter ratio at 14:1, excellent recovery and purity of l-methionine (82.37% and 85.69%, respectively) could be achieved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epoxide hydrolase-mediated biocatalysis has a great prospective in the biosynthesis of optically pure epoxides. The present work targets toward the thermo-stabilization of epoxide hydrolase by covalent conjugation with polysaccharide. An epoxide hydrolase from Agrobacterium radiobacter (ArEH) was modified by covalent coupling to seven oxidized polysaccharides with different chemical structures and characteristics. Among all conjugates, ArEH with ficoll exhibited both the highest specific activity (494 U/mg) and half-life at 60 °C (t 1/2  = 183 min). The conjugated enzyme also displayed wider optimum pH and temperature ranges, higher catalytic number (k cat ), and catalytic efficiency (k cat /K m ) as compared to its native counterpart. The conjugation significantly decreased the enthalpy of activation (ΔH*), free energy of transition state binding (ΔG* E-T ), and free energy of activation (ΔG*) for epichlorohydrin hydrolysis. Moreover, the conjugated ArEH showed enhanced thermal stability as evidenced by its longer half-life (t 1/2 ), lower thermal deactivation constant (k d ), and higher D values at 50-70 °C. Furthermore, the enzymatic activity of conjugated ArEH elevated significantly by Ca 2+ and it showed increased tolerance against Co 2+ , Fe 3+ and EDTA inhibitors. Finally, fluorescence and circular dichroism spectroscopic analysis were performed to confirm the noticeable conformational changes in ArEH structure after conjugation with ficoll, which could be responsible for the observed catalytic and stability enhancement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tyrosine phenol-lyase (TPL) naturally catalyzes the reversible β-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed. One of the substrate, sodium pyruvate was found to react with salicylaldehyde under alkali condition, forming a yellow color compound. The concentration of sodium pyruvate can be quantified according to the absorbance of the colorimetric compound at wavelength of 465 nm and the activity of TPL could be screened according to the decrease of the absorbance. After optimization of the colorimetric reaction conditions, the established high-throughput screening method was successfully used for screening of TPL with enhanced activity for l-DOPA synthesis. The confirmed sensitivity and accuracy demonstrated the feasibility and application potential of this screening method., (Copyright © 2018. Published by Elsevier Inc.)

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L -1 N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L -1 resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid., (Copyright © 2018. Published by Elsevier Inc.)

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L -1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L -1 h -1  g -1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co 2+ stimulated nitrilase activity, whereas Cu 2+ , Zn 2+ and Tween 80 showed a strong inhibitory effect. The V max and k cat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the K m was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg -1 . Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, β-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the k cat /K m value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL -1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b) is a valuable chiral synthon of atorvastatin calcium. A novel NADPH-specific aldo-keto reductase (AKR) was identified from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 by genome database mining, displaying t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1a) reducing activity and moderate diastereoselectivity (de p ∼80.5%). Molecular homology modeling and docking studies demonstrated that the side chain of Trp297 blocks binding of (5R)-1a to KmAKR. The mutation of Trp297 to His led to dramatic conformational changes and significant improvement in both diastereoselectivity and activity. In comparison with KmAKR, KmAKR-W297H displayed strict diastereoselectivity, and 2.8-fold, 3.9-fold improvement in k cat and k cat /K m toward (5R)-1a, which were 10.36s -1 and 6.56s -1 ·mM -1 respectively. Coupling KmAKR-W297H with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for coenzyme regeneration, 100mM (5R)-1a was completely reduced to (3R,5R)-1b within 12h, in a de p >99.5%., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A R-stereospecific amidase was purified from Brevibacterium epidermidis ZJB-07021 and characterized in detail. The amidase was purified to homogeneity by three chromatographic steps for up to 328.9-fold with specific activity of 31.9 U mg(-1). The enzyme was a homodimer with a molecular mass of 94 kDa. It exhibited maximum activity at 40 °C and pH 7.5. The enzyme was strongly inactivated by serine protease inhibitor PMSF. The values of Km and Vmax for racemic 2,2-dimethylcyclopropane carboxamide (DMCPCA) were 4.58 mM and 35.03 μmol min(-1) mg(-1) protein, respectively. The amidase showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides, but could hardly hydrolyze the bulky side-chain-containing amides. Furthermore, kinetic resolution of racemic DMCPCA by the amidase afforded S-DMCPCA in 46.3% yield and 99% ee with an average E-value of 67. These unique properties of the amidase imply that it is a promising biocatalyst for the production of chiral amides and carboxylic acids., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates. Dt-Ami 2 showed maximum activity near a quite alkaline pH (11.0) and exhibited opposite enantioselectivity to Dt-Ami 6. Furthermore, a novel bioprocess for hydrolysis of 1-cyanocyclohexaneacetamide was developed using Dt-Ami 6 as biocatalyst, resulting in >99% conversion within 1.5h at a substrate loading of 100g/L by 0.5g/L of Escherichia coli cells. On the other hand, nitrilase superfamily amidase only hydrolyzed aliphatic amides. The Km values of Dt-Ami 7 were considerably increased with the extension of chain length of aliphatic amides. The characterized enzymes from different families showed distinct biochemical characteristics and catalytic properties, leading to a better understanding of the two super amidase family members., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000mM) in a space-time yield per gram of biomass (DCW) of 22.9mmolL(-1)h(-1)gDCW(-1) at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)-calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)-calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (vmax), apparent Michaelis-Menten constant (Km) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (kcat) were calculated as 7.63 U mg(-1), 0.204 mM, 4.42 mM and 697.4 min(-1), respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An abamectin (ABM)-degrading bacterium, Stenotrophomonas maltophilia ZJB-14120, was isolated and identified. This strain is capable of degrading 84.82% of ABM at an initial concentration of 200 mg/L over a 48 h incubation period. This strain showed efficient biodegradation ability (7.81 mg/L/h) to ABM and high tolerance (1000 mg/L) to all macrolides tested. In addition to ABM, emamectin, erythromycin and spiramycin can also be degraded by this strain. Modifications involving either reduction of the double bond between C22-C23 or replacement of the C25-group of ABM with a cyclohexyl group can completely inhibit biodegradation of ABM. The ABM-degrading capability of strain ZJB-14120 is likely to be intrinsic to its metabolism and could be inhibited by incubating with erythromycin, azithromycin, spiramycin or rifampicin. A new and successive degradation pathway was proposed based on metabolite analysis. Although there is evidence for metabolite inhibition, this strain has high ABM degradation activity and reusability. Further investigation showed that activated macrolide efflux pump(s) and an undetermined mechanism for regulating the intracellular ABM concentration are responsible for normal uptake of essential metabolites while pumping out excess harmful compounds. Strain ZJB-14120 may provide efficient treatment of water and soil contaminated by toxic levels of abamectin and emamectin., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Echinocandin B (ECB), an echinocandin type of lipopeptide antibiotic produced by Aspergillus nidulans, is a precursor for the synthesis of novel anti-fungal drug - anidulafungin. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography was established for ECB purification from Aspergillus nidulans CCTCC M 2010275 fermentation broth. Among nine macroporous resin adsorbents tested, the non-polar resin HP-20 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Langmuir equation, and the adsorption kinetic followed the pseudo-second order model. The separation parameters of ECB from broth were optimised by dynamic adsorption/desorption experiments with the column packed with HP-20 resin. Under optimal conditions, the purity increased by 3.8-fold from 23.2% in broth to 88.5% in eluent with 87.1% recovery yield by a one-step treatment. Our study provided a one-step and effective method for large-scale production of ECB, and offered references for separating other echinocandins from broth., (Copyright © 2014 Elsevier B.V. All rights reserved.)

(2S, 3R)-methyl-2-benzamidomethyl-3-hydroxybutyrate (MBHB) is a key intermediate in the synthesis of 4-aceoxyazetidinone, a building block for the production of penems and carbapenems. More attentions have been paid to screen biocatalysts achieving asymmetric preparation of (2S, 3R)-MBHB. In this study, an improved chiral high-performance liquid chromatographic (HPLC) method was developed for the stereoselective determination of 2-benzamidomethyl-3-oxobutanoate (BMOB) and MBHB, and further employed into the biotransformation of BMOB. Chiral separation was achieved within 12 min on Chiralpak AY-H column, which was faster and more suitable for screening biocatalysts exhibited reduction activity and (2S, 3R)-stereospecificity toward BMOB than on other columns. Ultimately, a new strain, Burkholderia gladioli ZJB-12126 capable of reducing BMOB to (2S, 3R)-MBHB was successfully isolated based on this newly constructed HPLC method. Samples were prepared by liquid-liquid extraction system using ethyl acetate as the extractor solvent. The extraction recoveries of BMOB and MBHB isomers ranged from 91.6 to 94.1% with relative standard deviation (RSD) below 10%. Linear calibration curves were obtained in the concentration range of 50-5000 μg/mL for both BMOB and MBHB isomers, respectively. Intra-day and inter-day precisions and accuracy were below 15% for all isomers evaluated by RSDs and relative errors (REs), respectively. This novel method was demonstrated to be suitable for assessing the biotransformation process of BMOB., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The identification of potential cellular targets of small molecules is important in biomedical research and drug discovery, but has been challenging due to a lack of proteome-based methods that enable direct investigation of small molecule-protein interaction in live cells. This review summarizes some of the recent advances in target identification of bioactive molecules (including drugs and natural products) using in situ methods for cell-based proteome profiling of potential on and off targets., (Copyright © 2013 Elsevier Ltd. All rights reserved.)