Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10  

作　　者：YANG Jingwen DI Xiangjun WANG Man GAO Renjun 

机构地区：[1]Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, School of Life Science, Jilin University, Changchun 130012, P. R. China

出　　处：《Chemical Research in Chinese Universities》2015年第4期564-568,共5页高等学校化学研究（英文版）

摘　　要：We cloned and expressed a new recombinant β-galactosidase（TN0949） from Thermotoga naphthophila RKU-10 with the pET28a（＋） vector system in Escherichia coli BL21（DE3）, and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2＋-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. TN0949 can hydrolyze o-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH（3.0 to 7.0） after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.We cloned and expressed a new recombinant β-galactosidase（TN0949） from Thermotoga naphthophila RKU-10 with the pET28a（＋） vector system in Escherichia coli BL21（DE3）, and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2＋-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. TN0949 can hydrolyze o-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH（3.0 to 7.0） after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.

关 键 词：Β-GALACTOSIDASE THERMOSTABLE TRANSGLYCOSYLATION 

分 类 号：Q785[生物学—分子生物学] TS252.1[轻工技术与工程—农产品加工及贮藏工程]