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Pocket Modification of x-Amine Transaminase AtATA for Overcoming the Trade-Off Between Activity and Stability Toward 1-Acetonaphthone

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Engineering 2023年第11期 203-214页

作者： Jiaren Cao Fangfang Fan Changjiang Lyu Sheng Hu Weirui Zhao Jiaqi mei Shuai Qiu lehe mei Jun Huang Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical ManufacturingSchool of Biological and Chemical EngineeringZhejiang University of Science and TechnologyHangzhou 310023China School of Biological and Chemical Engineering Ningbo Tech UniversityNingbo 315100China Hangzhou Huadong Medicine Group Co.Ltd. Hangzhou 310011China College of Chemical and Biological Engineering Zhejiang UniversityHangzhou 310027China Jinhua Advanced Research Institute Jinhua 321019China

Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs.Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs.Herein,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA)to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone.We discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as the"best"mutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(k_(cat)/K_(m)^(1-acetonaphthone),where kcatis the constant of catalytic activities and is 10.1 min^(-1),K_(m)^(1-acetonaphthoneis) Michaelis-Menten constant and is 1.7 mmol·L^(-1))and half-life(t1/2)by 59-fold to 5.9 L·min^(-1)·mmol-1and by 1.6-fold to 46.9 min,respectively.Moreover,using M4 as the biocatalyst,we converted a 20 mmol·L^(-1)aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product,(R)-(+)-1(1-naphthyl)ethylamine((R)-NEA),with 78%yield and high enantiomeric purity(R>99.5%)within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs.The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability.Strengthening of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity.Moreover,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence similarity with AtATA.This research has important impl

关键词： Trade-off Co-evolution Amine transaminase (R)-(+)-1(1-naphthyl)ethylamine

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Biosynthesis of 4-hydroxyphenylpyruvic acid from L-tyrosine using recombinant Escherichia coli cells expressing membrane bound L-amino acid deaminase

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Chinese Journal of Chemical Engineering 2018年第2期26卷 380-385页

作者： Huanru Ding Weirui Zhao Changjiang Lu Jun Huang Sheng Hu Shanjing Yao lehe mei Jinbo Wang Jiaqi mei School of Biotechnology and Chemical Engineering Ningbo Institute of TechnologyZhejiang UniversityNingbo 315100China Department of Chemical and Biolagical Engineering Zhejiang UniversityHangzhou 310027China School of Biological and Chemical Engineering Zhejiang University of Science and TechnologyHarlgzhou 310023China Department of Chemical Engineering The University of utahSalt Lake City 84102America

4-Hydroxyphenylpyruvic acid(4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase(mL-AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H_2 O_2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide(twin-arginine translocation pathway, Tat) and integrated pelB(the general secretory pathway, Sec)-Tat signal peptide was determined and compared firstly, using two pET systems(pET28 a and pET20 b). It was found that the Tat pathway(pET28 a-mlaad) resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts B121(DE3) and 0.05 mmol ·L^(-1) IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol·L^(-1) 4-HPPA was obtained from 100 mmol·L^(-1) tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.

关键词：化学合成方法酷氨酸脱氨基房间酶膜反应条件优化条件

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Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

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Chinese Journal of Chemical Engineering 2016年第7期24卷 909-913页

作者： Weirui Zhao Sheng Hu Jun Huang Piyu Ke Shanjing Yao Yinlin Lei lehe mei Jinbo Wang School of Biotechnology and Chemical Engineering Ningbo Institute of Technology Zhejiang University Ningho 315100 China Department of Chemical and Biological Engineering Zhejiang University Hangzhou 310027 China School of Biological and Chemical Engineering Zhejiang University of Science and Technology Hangzhou 310023 China

The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase(GAD) activity of recombinant Escherichia coli(BL21(DE3)-p ET28a-gad B) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-p ET28a-gad B, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration,the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-p ET28a-gad B and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21(DE3)-p ET28a-gad B cells with 5 μg·ml-1ampicillin resulted in structural changes to the cell envelope,but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol(PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid(GABA) at the 10 th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after30 days of storage at 4 °C.

关键词：谷氨酸脱羧酶重组大肠杆菌生物催化剂氨苄西林全细胞酶活性通透性细胞膜结构

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High-level production of𝛾γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

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Engineering Microbiology 2023年第2期3卷 65-71页

作者： Lili Yao Changjiang Lyu Yuting Wang Sheng Hu Weirui Zhao Hongwei Cao Jun Huang lehe mei College of Life Science and Technology Heilongjiang Bayi Agricultural UniversityDaqing 163319China School of Biological and Chemical Engineering Zhejiang University of Science and TechnologyHangzhou 310023China School of Biological and Chemical Engineering Ningbo Tech UniversityNingbo 315100China College of Chemical and Biological Engineering Zhejiang UniversityHangzhou 310027China

Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.

关键词： Glutamate decarboxylase Antiporter γ-Aminobutyric acid E.coli Whole-cell biocatalysts

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口袋改造策略克服ω氨基转移酶AtATA对1-乙酰基萘活性和稳定性之间的“trade-off”效应（英文）

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Engineering 2023年第11期 203-214页

作者： Jiaren Cao Fangfang Fan Changjiang Lyu Sheng Hu Weirui Zhao Jiaqi mei Shuai Qiu lehe mei Jun Huang Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing School of Biological and Chemical Engineering Zhejiang University of Science and Technology School of Biological and Chemical Engineering Ningbo Tech University Hangzhou Huadong Medicine Group Co.Ltd. College of Chemical and Biological Engineering Zhejiang University Jinhua Advanced Research Institute

Amine transaminases(ATAs) catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs.Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs.Herein,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA) to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone.We discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as the "best" mutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(kcat/Km1-acetonaphthone,where kcatis the constant of catalytic activities and is 10.1 min-1,Km1-acetonaphthoneis Michaelis-Menten constant and is 1.7 mmol·L-1) and half-life(t1/2) by 59-fold to 5.9 L·min-1·mmol-1and by 1.6-fold to 46.9 min,respectively.Moreover,using M4 as the biocatalyst,we converted a 20 mmol·L-1aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product,(R)-(+)-1(1-naphthyl)ethylamine((R)-NEA),with 78% yield and high enantiomeric purity(R> 99.5%) within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs.The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability.Strengthening of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity.Moreover,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence similarity with AtATA.This research has important implications for overc

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Production of Indigo by Immobilization of E. coli BL21 （DE3） Cells in Calcium-Alginate Gel Capsules

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Chinese Journal of Chemical Engineering 2007年第3期15卷 387-390页

作者：陆燕梅乐和 Department of Chemical and Biochemical Engineering Zhejiang University Hangzhou 310027 China

The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobi- lized by entrapment in calcium-alginate gel capsules was examined,and various characteristics of immobilized cells were assessed.Optimum conditions for cells activity were not affected after immobilization,and pH and tempera- ture for both free and immobilized cells were found to be pH 7.5 and 35℃,respectively.The immobilized cells ex- hibited a markedly improved thermal stability than free cells.After five repeated experiments,the yield of indigo with the immobilized cells retained over 94%of their original activity,which indicated that the operational stability for recycling in batch processes was improved.

关键词：海澡酸钙胶囊重组生产方法靛蓝

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Purification and Stability of an Antithrombus Enzyme from Bacillus subtilis

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Chinese Journal of Chemical Engineering 2004年第5期12卷 707-710页

作者：关怡新姚善泾俞丽华梅乐和 Department of Chemical and Biochemical Engineering Zhejiang University Hangzhou 310027 China

An antithrombus enzyme (ATE) was precipitated by (NH4)2SO4 or ethanol from supernatant of Bacillus subtilis culture broth then purified using ion exchange chromatography on CM-sepharose fast flow. The effects of ionic strength and pH value on protein adsorption, the gradient elution at different flow rates and step elution were examined respectively. The recovery yield of the optimised process was 74.5% with a purification factor 8.1. The ATE molecular weight was estimated as 30ku by SDS-PAGE. The experimental results showed that the enzyme was stable in the range of pH 7 to pH11, and temperature 25℃ to 37℃.

关键词：溶解酶 ATE 分离纯化工艺稳定性血栓症心血管病生物技术血浆酶原

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Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

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Chinese Journal of Chemical Engineering 2007年第2期15卷 157-161页

作者：黄俊梅乐和盛清姚善泾林东强 Department of Chemical and Biochemical Engineering Zhejiang University College of Life Science Zhejiang Sci-Tech University

A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH4)2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel fil-tration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50min at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.

关键词：鲜乳分离短乳杆菌谷氨酸脱羧酶纯化特性

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A Two-stage pH and Temperature Control with Substrate Feeding Strategy for Production of Gamma-aminobutyric Acid by Lactobacillus brevis CGMCC 1306

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Chinese Journal of Chemical Engineering 2013年第10期21卷 1190-1194页

作者：彭春龙黄俊胡升赵伟睿姚善泾梅乐和 Department of Chemical and Biological Engineering Zhejiang University Hangzhou 310027 China School of Biological and Chemical Engineering Zhejiang University of Science and Technology Hangzhou 310023China School of Biological and Chemical Engineering Ningbo Institute of Technology Zhejiang University Ningbo 315100China

由乳杆菌 brevis CGMCC 1306 优化 gamma-aminobutyric 酸(伽马氨基丁酸) 的生产的方法被调查。结果显示房间生长在 pH 是最大的 5.0，当时 4.5 是的 pH 对伽马氨基丁酸更好形成。为细胞生长的最佳的温度(35 ????

关键词： γ-氨基丁酸温度控制优化生产短乳杆菌 pH值 GABA 底料细胞生长

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普那霉素发酵与吸附分离耦合过程动力学

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化工学报 2010年第3期61卷 668-675页

作者：郑喜金志华梅乐和张达浙江大学化学工程与生物工程学系浙江杭州310027 浙江大学宁波理工学院生物与化学工程分院浙江宁波315100

根据S.pristinaespiralis E-71普那霉素发酵过程的代谢特性提出了一个针对产物合成期的普那霉素合成动力学方程,结合液膜-孔扩散理论,建立了描述2.5L通气搅拌发酵罐中JD-1大孔吸附树脂吸附分离原位耦合发酵过程的动力学模型,并获得产物... 详细信息

根据S.pristinaespiralis E-71普那霉素发酵过程的代谢特性提出了一个针对产物合成期的普那霉素合成动力学方程,结合液膜-孔扩散理论,建立了描述2.5L通气搅拌发酵罐中JD-1大孔吸附树脂吸附分离原位耦合发酵过程的动力学模型,并获得产物生成速率常数、产物抑制常数及液膜传质和孔内扩散系数。结果表明该发酵-吸附耦合过程动力学模型能较好地描述这一原位分离耦合发酵生产普纳霉素过程。在此基础上,考察了树脂添加量及树脂半径对原位分离耦合发酵过程的影响,结果表明添加JD-1树脂能有效移走发酵液主体相中高达90%的普那霉素,且原位分离效果随树脂粒径减小而增强,在一定范围内随树脂添加量的增加而增强,当添加70g.L-1半径为0.195mm的树脂时,普纳霉素产量达到1.01g.L-1,其中被树脂吸附的高于95%。

关键词：始旋链霉菌普那霉素大孔吸附树脂发酵与分离耦合动力学模型

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