Cloning,expression and characterization of a recombinant thermophilic β-galactosidase from Dictyoglomus thermophilum H-6-12

【Author】

Xiaochen Chen;Xiaofu Sun;Xinye Han;Zuoming Zhang;Renjun Gao;

【Abstract】

As a kind of important glycosidase,(3-galactosidases,especially thermostable ones have been the focus of dairy industry and glycobiology.A novel thermophilic P-galactosidase gene(Dth0381)from hyperthermophilic obligate anaerobes Dictyoglomus thermophilum H-6-12,belong to GH42,was cloned into expression vector pET28a(+),and expressed successfully in Escherichia coli BL21(DE3)cells.After induced with 0.2mmol/L IPTG under 120rpm at 20℃for 12h,crude extract could be obtained by ultrasonication of the cell pellet.The recombinant Dth0381 was purified via Ni-NTA affinity chromatography,and its molecular mass was approximately 75kDa via SDS-PAGE analysis.The optimal temperature and pH of hydrolysis reaction to oNPG and lactose with recombinant Dt0381 was determined to be over 95℃at pH 6.8 and 85℃at pH 4.8,respectively.Kinetic study for recombinant Dt0381revealed the K_m and k_(cat)of this enzyme for hydrolyzing oNPG and lactose were 1.33mmol/L,18.86s"1,and55.50mmol/L,12.08s"1,respectively.The thermostability research of recombinant Dt0381 showed it could remain about 50%residual activity after incubated at 80℃for 11.7h and incubated at 70℃for 29h.In addition,the recombinant enzyme could tolerate many bivalent cations and keep stable under the pH range from 6.0 to 9.8.Transglycosylation assay showed that Dth0381 could be used to synthesis short chain alkyl galactosides,especially for the synthesizing of n-butyl galactoside at 80℃.The conversion ratio of n-butyl galactoside was over 50%.

【Keywords】

β-galactosidase;;Dictyoglomus thermophilum;;Transglycosylation

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