| Glycoside compounds are a kind of functional molecules widespread in natural environment,which play significant roles in various biochemical and physiological processes,including cell recognition,growth regulation,signal transmission,and structure support.Glycoconjugates,deriving from glycoside compounds,attracted more and more attention and were studied widely recently for their special properties containing high surface activity,solubility,biocompatibility and antibacterial property.Glycoside hydrolases(EC 3.2.1),which belongs to hydrolase super family,can hydrolyze the glycosidic bonds in both glycoside compounds and glycoconjugates specifically.Based on the researches of hydrolysis mechanism,it was found that the transglycosylation would be performed by the most of glycosidases when controlled the ratio between substrates and water.Compared with glycosyltransferases,synthesizing the novel glycoconjugates from glycoside compounds with glycoside hyrolases would be cheaper and easier to perform.Thermophilic glycosidases,which come from thermophilic microorganism,are more appropriate to be applied into industrial production for their high thermostability and tolerance of organic solvents.Especially in the synthesis process of glycoconjugates,thermophilic glycosidases have obviously advantages for their tolerances to high temperature,which can enhance the reaction rate,decrease the system viscosity,and avoid the pollution caused by microorganism.Thermophilic anaerobe Dictyoglomus thermophilum H-6-12,which was isolated from the hot spring in Japan,can grow regularly at 70 ?with lignocellulose.There were many genes encoding glycosidases in its genome,and among them,a gene with genbank number ACI18764.1 was predicted to encode a ?-glucosidase which belonged to GH3.According to the Blast analysis,we made sure it's a real gene which can be translated into a ?-glucosidase,and amplified it successfully with PCR techniques.Then it was cloned into p ET-28 a vectors and been transferred into E.coli Transetta strain successfully with molecular biology methods.Through the heterologous expression in engineering bacteria and the metal ion affinity chromatography with Ni-NTA beads,we got the relatively pure recombinant ?-gulosidase.The characterization of the recombinant ?-glucosidase Dt0262 indicated that the enzyme could hydrolyze both p NPGlc and cellobiose.For p NPGlc,its optimal catalytic conditions are gained at 80?,p H 6.0,and for cellobiose,the same parameters are gained at 75?,p H 5.5.Kinetic parameters showed that the Km and kcat of recombinant Dt0262 to both substrates were 0.23 m M,0.64s-1 and 3.57 m M,0.88s-1,respectively.According to the value of kcat/Km,recombinant Dt0262 preferred to hydrolyze p NPGlc.The study of stabilities demonstrated that this enzyme showed a better thermostability at 70 where the ? t1/2 could reach to 92 h than 80(20.3h)and ?90(21.8min).In addition,Dt0262 still showed an obvious thermal activation which ?could improve its activities to 132%(70?)and 146%(80?),respectively.Also,Dt0262 could keep active from p H4.5 to p H 7.5.The metal ion dependency research showed that recombinant Dt0262 did not depend on metal ions.According to the characterization results above,we can make sure that the Dt0262 is a thermophilic ?-glucosidase,which has a potential in application to the synthesis of glycoconjugates. |
PDF Full Text Request