| Thermophiles are kinds of microorganisms living in high temperature environment.Research on the thermophiles has important significance,for example,the origin of life,biological evolution,adaptation high temperature mechanisms and thermophilic enzymes excavation etc.With the development of genome sequencing technology,a lot of thermophilic microbial genomes were sequenced.This provided good resources for the excavation and development of novel enzymes.The optimum growth temperature of Anoxybacillus flavithernus subsp.yunnanensis E13T is 55? and its genome sequencing was completed in 2014.In this paper,based on the analysis of glycosidases in the genome,we chose three glycoside hydrolases that were not reported and were similar to those of known homologous enzymes no more than 60%,and also carried out clone expression and enzymatic properties research.Three enzymes socalled ?-glucosidase(EC 3.2.1.21,Bg1P),?-galactosidase(EC 3.2.1.23,BgalP)and a-galactosidase(EC 3.2.1.22,AgalP).The gene of ?-glucosidase BglP was cloned into the expression vector pET22b,and obtained soluble expression in E.coli.The purified protein molecular weight of 52 kDa,consistent with the theoretical molecular weight.The optimum pH and temperature were 7.0 and 60?,respectively.BglP possess excellent thermal stability with the half-life is up t0 10 h at 60?.It is also very stable in neutral and weak alkaline environments(pH 7.0-8.5).Specific activity with p-nitrophenyl-?-D-glucopyranoside(pNPG)as substrate was 842±15.5 U/mg,Km and Vmax were 0.29±0.01 mM and 771±39 ?mol/min.mg.With cellobiose as substrate,Km and Vmax were 12.3±0,mMand 107±5.6?mol/min.mg.This indicated that BglP is a highly efficient enzyme for catalysis.BglP can be activated by glucose and xylose.When the concentrations of glucose and xylose were higher than 2.2 M and 1.4 M,Bglp's activity was inhibited.But within this concentration,activity was significantly improved.When the concentrations of glucose and xylose were 0.4 M and 0.6 M,Bglp's activity was raised to the highest,respectively,compared to 2.6 times and 1.8 times.Glucose can not only improved the Bglp's activity,but also significantly improved the temperature stability of BglP.These properties indicated BglP was a novel enzyme with e:xcellent resource potential.Under the coptimal concditions,the hydrolysis rate of BglP to daidzein and genistein were 98.5%and 96.8%.So BglP demonstrated its good application potential in the soy isoflavones industry.BglP also had a significant application advantage in cellulose degradation.To Celluclast 1.5L degradation bagasse,for example,BglP can improve the conversion rate of 1.9 times.BglP can increase the conversion of 3.4 and 3.0 times when there was a high concentration of glucose(500 mM)or xylose(200 mM)in the reaction system.The gene of ?-galactosidase BgalP was cloned into the expression vector pET32a,the purified protein molecular weight of 76 kDa,consistent with the theoretical molecular weight.The optimum temperature of ?-galactosidase BgalP was 55?,the optimum pH 6.5.BgalP had good thermal stability,the half-life at 65 ? was 8 h.Specific activity with oNPG as substrate was 188 U/mg,exceed other bacteria in the same family of p-galactosidase.In addition,BgalP had a good tolerance to common organic solvents,and the current study of organic solvent tolerance of ?-galactosidase was rarely reported,so BgalP can be used in industrial environments containing organic solvents.The gene of ?-galactosidase AgalP was cloned into the expression vector pET32a,protein molecular weight of 86 kDa,formation of inclusion bodies under conventional expression conditions.The refolding method by Ni-NTA column was used to renature AgalP,the final recovery rate of soluble protein was 25%.The optimum temperature of AgalP was 65 ?.It had excellent thermal stability,the half-life at 65 ? was 5.5 h.The optimum pH was 7.0.In the pH range of 5-10 can maintain good acid and alkali stability.Specific activity with pNPGal as substrate was 22 U/mg,Km was 0.143 mM,Vmax was 5.2?umol/min.mg.Compared with other a-galactosidase,the activity level of AgalP was found to be low. |
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