Li Zhoukun,Zheng Wenwen,Zhang Lei,Wang Yanxin,Zhang Yajuan,Qiao Yan,Luo Xue,Huang Yan,Cui Zhongli
The enzyme 4-α-glucanotransferase catalyzes the intramolecular and intermolecular transglycosylation of α-1,4-glucan, which plays an important role in starch metabolism and starch modification. In this study, the gene encoding a 4-α-glucanotransferase from Corallococcus sp. EGB ( ccGtase ) is cloned and expressed in Escherichia coli BL21 (DE3). CcGtase contains all the conserved amino acid residues of glycoside hydrolase family 77 (GH77), which form the active site environment of the enzymes in the α-amylase family, while sequence alignments show that 4-α-glucanotransferases from myxobacteria constitute an independent group along with the GH77 glucanotransferases. Product analysis shows that CcGtase is able to convert malto-oligosaccharides and starch into linear maltooligosaccharides with a low degree of polymerization (DP < 12) instead of cycloamylose. The transfer action toward starch is only observed in the presence of glucose, and delayed production of maltose from the transfer reaction is also observed. During the reaction, no apparent hydrolytic activity is observed. Based on the low sequence identity (9–35%) and unique characteristics, CcGtase can be regarded as a new member of the GH77 family.
Zhang Yajuan,Duan Yajuan,Zhong Lingli,Li Zhoukun,Cui Zhongli,Huang Yan
The asymmetric reduction of prochiral ketones is a promising process for synthesis of optically active alcohols. The aldo-keto reductase (AKR) is an attractive candidate of biocatalyst, due to its high enantioselectivity and environmentally friendly reaction conditions. In this work, nine putative AKR encoding genes from Corallococcus sp. EGB were cloned and expressed in Escherichia coli . Of these produced enzymes (CoAKRs), CoAKR7 exhibited reductive activity to various ketones and ketoesters, especially very high activity toward ethyl 4-chloro-3-oxobutanoate (COBE) with NADPH as the coenzyme. The CoAKR7 was optimally active at pH 7.0 and 50 °C. The apparent K m and V max for COBE was 14.18 U/mg and 0.269 mM, respectively. Moreover, CoAKR7 catalyzed an anti-Prelog reduction of COBE to ( S )-ethyl-4-chloro-3-hydroxybutanoate (CHBE) with e.e. >99%. Enzyme-substrate-cofactor docking analysis elucidated the molecular mechanism of the substrate stereospecificity, providing basis for protein engineering of these enzymes for applications in the synthesis of valuable chemicals.
Lei Zhang,Lingli Zhong,Juying Wang,Yuqiang Zhao,Yiwei Zhang,Yitong Zheng,Weiliang Dong,Xianfeng Ye,Yan Huang,Zhoukun Li,Zhongli Cui
The process of raw starch conversion has attracted extensive attention in recent years due to its high energy conversion efficiency and saving of operational costs. In this study, extensive hydrolysis of raw starch by α-amylase AmyM from Corallococcus sp. EGB was investigated with robust substrate and products tolerance. AmyM showed broad substrate specificity toward raw starches with different types, and kept active and stable over a wide range of temperatures (30–70 °C) and pH values (5.0–9.0) and recycling. The V max values of AmyM towards wheat and corn raw starch were 1111 and 741 μmol/min/mg without depending on Ca 2+ , respectively. SEM analysis showed that AmyM hydrolysis results in the formation of porous and creviced surfaces around the starch granules peripheries. The cost-effective conversion of raw starch for the production of malto-oligosaccharides (MOSs) was identified. AmyM achieved high hydrolysis degree of 90% for 30% wheat and corn raw starch after 20 h at 45 °C with 5 U per mg of raw starch, resulting in formation of 70% MOSs and 30% dextrin. During this process, maltose (G2), maltotriose (G3) and maltotetraose (G4) were produced synchronously with G9 and G11, indicating the potential application in enzymatic conversion of raw starch under low temperature.
危险品化学品经营许可证（带存储）