Expression And Application Of Thermostable Glycosidases From Thermotoga Naphthophila RUK-10

Glycoside compounds are widely distributed in nature, ranging from animal,plants and microorganism. They normally play important roles in diverse biologicalprocesses, such as the interaction between cells; because of pharmacological activitiesthey possessed, some of glycoside compounds have become the research hotspots inrecent years, such as ginsenoside, arbutin and salidroside. Glycoside hydrolases (EC3.2.1.-) are a widespread group of enzymes, which hydrolyse the glycosidic bondbetween two or more carbohydrates or between a carbohydrate and anon-carbohydrate moiety. Not only hydrolysis activity they possessed, most ofglycoside hydrolases also have synthetic activity to transfer glycosyl groups toalcohols, saccharides and other compounds, resulting in the formation ofalkyl-glycosides, oligosaccharides and other glycoside compounds. The currentbiotechnolog has now paid more attention to glycoside hydrolases from thermophilicorganisms. In addition to the excellent thermo stability they have, thermophilicenzymes are more favourable of catalyzing reactions at high temperatures due to thehigher initial reaction rate, higher substrate solubility and the inhibition of microbialcontamination.Herein, we cloned two new gene Tnap1577and Tnap0602corresponding toputative β-galactosidase and β-glucosidase from Thermotoga naphthophila RUK-10,a hyperthermophilic bacterium with optimum cell growth at80oC. Both of them wereexpressed in Escherichia coli BL21(DE3) in a soluble form with a His-tag at theN-terminus and purified to homogeneity, and then the properties of two enzymes werecharacterized in detail.For β-galactosidase Tnap1577, the optimum temperatures for hydrolysis ofo-nitrophenyl-β-D-galactoside (oNPGal) and lactose by the recombinantβ-galactosidase were found to be90and70oC, respectively. The correspondingoptimum pH values were6.8and5.8, respectively. Thermostability studies showed that the half-lives of the recombinant enzyme (0.2mg/ml) at75,80,85and90oCwere10.5,4,1, and0.3h, respectively. Kinetic studies on the recombinantβ-galactosidase revealed that Kmand Vmaxvalues for the hydrolysis of oNPGal at80oC were1.31mM and3385.67μmol min-1mg-1respectively. The Kmand Vmaxvaluefor the hydrolysis of lactose at70oC were1.43mM and2.67μmol min-1mg-1respectively.β-Glucosidase and β-galactosidase hydrolysis activity were both found inTnap0602. The optimum temperatures for hydrolysis of p-Nitrophenyl-β-D-glucopyranoside (pNPGlu) and oNPGal by the recombinant β-glucosidase were bothabove95oC, and the corresponding optimum pH values were both7.0. The optimumtemperature and pH for lactose hydrolysis were85oC and5.5respectively.Thermostability studies showed that the half-lives of the recombinant enzyme (0.2mg/ml) at75,80,85and90oC were84,32,14, and3h, respectively, and it was quitestable over the pH range5.0-10.0. The Kmvalues for pNPGlu and oNPGal hydrolysisat80oC were0.13and0.63mM respectively, and the Vmaxvalues were18389.10and6250μmol min-1mg-1of each. For the hydrolysis of lactose, the Kmand Vmaxvalueswere0.04mM and28.03μmol min-1mg-1at85oC respectively.These two glycosidases were both homodimer with subunit mass of70and51kDa respectively, and both of them showed transglycosylation activity. With lactoseas the sugar donor and alkyl alcohols as sugar acceptor, Tnap1577could catalyze thesynthesis of butyl galactopyranoside, hexyl galactopyranoside and octylgalactopyranoside in aqueous/organic solvent system. Galacto-oligosaccharide couldbe synthesed in aqueous system catalyzed by Tnap0602when lactose was used as thetranglycosyltaion substrate. Our research suggested that Tnap1577and Tnap0602might be candidate for industrial applications, not only for the hydrolysis of lactose,but also for the synthesis of glycoside compounds.