The two major challenges in industrial enzymatic catalysis are the limited number of chemical reaction types that are catalyzed by enzymes and the instability of enzymes under harsh conditions in industrial catalysis....The two major challenges in industrial enzymatic catalysis are the limited number of chemical reaction types that are catalyzed by enzymes and the instability of enzymes under harsh conditions in industrial catalysis.Expanding enzyme catalysis to a larger substrate scope and greater variety of chemical reactions and tuning the microenvironment surrounding enzyme molecules to achieve high enzyme performance are urgently needed.In this account,we focus on our efforts using the de novo approach to synthesis hybrid enzyme catalysts that can address these two challenges and the structure-function relationship is discussed to reveal the principles of designing hybrid enzyme catalysts.We hope that this account will promote further efforts toward fundamental research and wide applications of designed enzyme hybrid catalysts for expanding biocatalysis.展开更多
The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahiliza...The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahilization 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 cell- bound glutamate decarboxylase (GAD) activity of recombinant Escherichia coil (BL21 (DE3)-pET28a-gadB) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or suffactants to the culture media. Ampidllin was the most effective at improving cell-bound GAD activity of the BL21 (DE3)-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 pg. ml-1. Using this concentration, the cell hiomass did decrease by 40.58%, but the cell-bound GAD activity of BL21 (DE3)-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment ofBL21 (DE3)-pET28a-gadB cells with 5 tag.ml 1 ampicillin 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 transfor- mation rate of γ-aminobutyric acid (GABA) at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4 ℃.展开更多
Desymmetrization of prochiral 3-substituted glutaronitriles offers a new approach to access (S)-Pregabalin and (R)-Baclofen. A number of nitrilases from diverse sources were screened with 3-isobutylglutaronitriles...Desymmetrization of prochiral 3-substituted glutaronitriles offers a new approach to access (S)-Pregabalin and (R)-Baclofen. A number of nitrilases from diverse sources were screened with 3-isobutylglutaronitriles (1a) or 3-(4'-chlorophenyl)glutaronitriles (1b) as the substrate. Some nitrilases were found to catalyze the desymmetric hydrolysis of la and lb to form optically active 3-(cyanomethyl)-5-methylhexanoic acid (2a) and 3-(4'-chlorophenyl)-4-cyanobutanoic acid (2b) with high enantiomeric excesse (ee), respectively. This cannot be achieved using traditional chemical hydrolysis. Among them, AtNIT3 generated (R)-2b whereas BjNIT6402 and HsN1T produced the opposite (S)-enantiomer with high conversions and ee values. Not only the nitrilases showed different activities and stereoselectivities toward these 3-substituted glutaronitriles, the 3-substitueut of the substrates also exerted great effect on the enzyme activity and stereoselectivity. (S)-2a and (S)-2b were prepared with high yields and ee values using BjNIT6402 and HsNIT as the biocatalysts, respectively. A straightforward Curtius rearrangement of (S)-2a and (S)-2b, followed by the acidic hydrolysis, afforded (S)-Pregabalin and (R)-Baclofen. This offers a new platform methodology for the synthesis of optically active β-substituted T-amino acids of pharmaceutical importance.展开更多
The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates...The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.展开更多
Natural enzymes as biological catalysts possess remarkable advantages,especially their highly efficient and selective catalysis under mild conditions.However,most natural enzymes are proteins,thus exhibiting an inhere...Natural enzymes as biological catalysts possess remarkable advantages,especially their highly efficient and selective catalysis under mild conditions.However,most natural enzymes are proteins,thus exhibiting an inherent low durability to harsh reaction conditions.Artificial enzyme mimetics have been pursued extensively to avoid this drawback.Quite recently,some inorganic nanoparticles(NPs) have been found to exhibit unique enzyme mimetics.In addition,their much higher stability overcomes the inherent disadvantage of natural enzymes.Furthermore,easy mass-production and low cost endow them more benefits.As a new member of artificial enzyme mimetics,they have received intense attention.In this review article,major progress in this field is summarized and future perspectives are highlighted.展开更多
文摘The two major challenges in industrial enzymatic catalysis are the limited number of chemical reaction types that are catalyzed by enzymes and the instability of enzymes under harsh conditions in industrial catalysis.Expanding enzyme catalysis to a larger substrate scope and greater variety of chemical reactions and tuning the microenvironment surrounding enzyme molecules to achieve high enzyme performance are urgently needed.In this account,we focus on our efforts using the de novo approach to synthesis hybrid enzyme catalysts that can address these two challenges and the structure-function relationship is discussed to reveal the principles of designing hybrid enzyme catalysts.We hope that this account will promote further efforts toward fundamental research and wide applications of designed enzyme hybrid catalysts for expanding biocatalysis.
基金Supported by the grants from the National Natural Science Foundation of China(21176220,20876143,31470793)the Natural Science Foundation of Zhejiang Province(Z13B060008)the Key Technology Research and Development Project of Ningbo(2011C11023)
文摘The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahilization 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 cell- bound glutamate decarboxylase (GAD) activity of recombinant Escherichia coil (BL21 (DE3)-pET28a-gadB) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or suffactants to the culture media. Ampidllin was the most effective at improving cell-bound GAD activity of the BL21 (DE3)-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 pg. ml-1. Using this concentration, the cell hiomass did decrease by 40.58%, but the cell-bound GAD activity of BL21 (DE3)-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment ofBL21 (DE3)-pET28a-gadB cells with 5 tag.ml 1 ampicillin 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 transfor- mation rate of γ-aminobutyric acid (GABA) at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4 ℃.
基金financially supported by the Chinese Academy of Sciences (KSZD-EW-Z-015)the CAS Agenda to Provide S&T Support and Services for the National Strategic Emerging Industries
文摘Desymmetrization of prochiral 3-substituted glutaronitriles offers a new approach to access (S)-Pregabalin and (R)-Baclofen. A number of nitrilases from diverse sources were screened with 3-isobutylglutaronitriles (1a) or 3-(4'-chlorophenyl)glutaronitriles (1b) as the substrate. Some nitrilases were found to catalyze the desymmetric hydrolysis of la and lb to form optically active 3-(cyanomethyl)-5-methylhexanoic acid (2a) and 3-(4'-chlorophenyl)-4-cyanobutanoic acid (2b) with high enantiomeric excesse (ee), respectively. This cannot be achieved using traditional chemical hydrolysis. Among them, AtNIT3 generated (R)-2b whereas BjNIT6402 and HsN1T produced the opposite (S)-enantiomer with high conversions and ee values. Not only the nitrilases showed different activities and stereoselectivities toward these 3-substituted glutaronitriles, the 3-substitueut of the substrates also exerted great effect on the enzyme activity and stereoselectivity. (S)-2a and (S)-2b were prepared with high yields and ee values using BjNIT6402 and HsNIT as the biocatalysts, respectively. A straightforward Curtius rearrangement of (S)-2a and (S)-2b, followed by the acidic hydrolysis, afforded (S)-Pregabalin and (R)-Baclofen. This offers a new platform methodology for the synthesis of optically active β-substituted T-amino acids of pharmaceutical importance.
基金2011 Select Project in Scientific and Technological Activities for Returned Scholars of Chongqing Personnel Bureau, and the Doctoral Foundation ofSouthwest University (SWU112019)
文摘The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.
基金supported by the National Natural Science Foundation of China (Grant No. 20773032)the National Basic Research Program of China (Grant No. 2011CB932802)
文摘Natural enzymes as biological catalysts possess remarkable advantages,especially their highly efficient and selective catalysis under mild conditions.However,most natural enzymes are proteins,thus exhibiting an inherent low durability to harsh reaction conditions.Artificial enzyme mimetics have been pursued extensively to avoid this drawback.Quite recently,some inorganic nanoparticles(NPs) have been found to exhibit unique enzyme mimetics.In addition,their much higher stability overcomes the inherent disadvantage of natural enzymes.Furthermore,easy mass-production and low cost endow them more benefits.As a new member of artificial enzyme mimetics,they have received intense attention.In this review article,major progress in this field is summarized and future perspectives are highlighted.