The L(+)-form of tartaric acid(I(+)-TA)exists extensively in nature,and is widely used in the food,chemical,textile,building,and pharmaceutical in-dustries(Su et al.,2001).The main method for L(+)-TA production is mic...The L(+)-form of tartaric acid(I(+)-TA)exists extensively in nature,and is widely used in the food,chemical,textile,building,and pharmaceutical in-dustries(Su et al.,2001).The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase(CESH),which can catalyze the asymmetric hydrolysis of cis epoxysuccinic acid or its salts to TA or tartrate(Bao et al.,2019).Seventeen species containing CESH have been isolated so far.However,most species for L(+)-TA production have been reported from bacteria(Xuan and Feng,2019).The only fungus isolated from soil by our lab recently,that could be used as catalyst for the process under acidic condition,is Aspergillus niger WH-2(Bao et al,2020).In order to find strains with new characteristics,this study attempted to isolate a new CESH source from fungi and investigate its application value.展开更多
Objective:This study aimed to clone and characterize the oxiranedicarboxylate hydrolase(ORCH) from Labrys sp.WH-1.Methods:Purification by column chromatography,characterization of enzymatic properties,gene cloning by ...Objective:This study aimed to clone and characterize the oxiranedicarboxylate hydrolase(ORCH) from Labrys sp.WH-1.Methods:Purification by column chromatography,characterization of enzymatic properties,gene cloning by protein terminal sequencing and polymerase chain reaction(PCR),and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.Results:The ORCH from Labrys sp.WH-1 was purified 26-fold with a yield of 12.7%.It is a monomer with an isoelectric point(pl) of 8.57 and molecular mass of 30.2 kDa.It was stable up to 55℃with temperature at which the activity of the enzyme decreased by 50% in 15 min(T5015) of 61℃and the half-life at 50℃(t1/2,50℃) of 51 min and was also stable from pH 4 to 10,with maximum activity at 55℃and pH 8.5.It is a metal-independent enzyme and strongly inhibited by Cu2+,Ag+,and anionic surfactants.Its kinetic parameters(Km,kcat,and kcat/Km) were 18.7 mmol/L,222.3 s-1,and 11.9 mmol/(L s),respectively.The ORCH gene,which contained an open reading frame(ORF) of 825 bp encoding 274 amino acid residues,was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.Conclusions The catalytic efficiency and thermal stability of the ORCH from Labrys sp.WH-1 were the best among the reported ORCHs,and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.展开更多
基金Zhejiang Provincial Natural Science Foundation of China(No.LQ19C200001)he Zhejiang Xinmiao Talent Project(No.2020R415014),China。
文摘The L(+)-form of tartaric acid(I(+)-TA)exists extensively in nature,and is widely used in the food,chemical,textile,building,and pharmaceutical in-dustries(Su et al.,2001).The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase(CESH),which can catalyze the asymmetric hydrolysis of cis epoxysuccinic acid or its salts to TA or tartrate(Bao et al.,2019).Seventeen species containing CESH have been isolated so far.However,most species for L(+)-TA production have been reported from bacteria(Xuan and Feng,2019).The only fungus isolated from soil by our lab recently,that could be used as catalyst for the process under acidic condition,is Aspergillus niger WH-2(Bao et al,2020).In order to find strains with new characteristics,this study attempted to isolate a new CESH source from fungi and investigate its application value.
基金Project supported by the Zhejiang Provincial Natural Science Foundation of China(No.LQ19C200001)the Education Department of Zhejiang Province Scientific Research Project(No.Y201737925)+1 种基金the Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products(No.2016KF0048)the Key Laboratory of Bioorganic Synthesis of Zhejiang Province(No.20170110),China
文摘Objective:This study aimed to clone and characterize the oxiranedicarboxylate hydrolase(ORCH) from Labrys sp.WH-1.Methods:Purification by column chromatography,characterization of enzymatic properties,gene cloning by protein terminal sequencing and polymerase chain reaction(PCR),and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.Results:The ORCH from Labrys sp.WH-1 was purified 26-fold with a yield of 12.7%.It is a monomer with an isoelectric point(pl) of 8.57 and molecular mass of 30.2 kDa.It was stable up to 55℃with temperature at which the activity of the enzyme decreased by 50% in 15 min(T5015) of 61℃and the half-life at 50℃(t1/2,50℃) of 51 min and was also stable from pH 4 to 10,with maximum activity at 55℃and pH 8.5.It is a metal-independent enzyme and strongly inhibited by Cu2+,Ag+,and anionic surfactants.Its kinetic parameters(Km,kcat,and kcat/Km) were 18.7 mmol/L,222.3 s-1,and 11.9 mmol/(L s),respectively.The ORCH gene,which contained an open reading frame(ORF) of 825 bp encoding 274 amino acid residues,was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.Conclusions The catalytic efficiency and thermal stability of the ORCH from Labrys sp.WH-1 were the best among the reported ORCHs,and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.
