Heterologous Expression And Characterization Of Cold-adapted Xylanase And ?-glucosidase From Bacillus Cellulosilyticus

The ?-glucosidase and xylanase play an important role in some fields,such as food,biomass and feed industry.The temperature characteristics of the enzyme have considered as the key parameter for industrial application.At present,many researches have focused on moderate and thermostable enzymes,however,the cold-adapted enzyme was preference for the heat-sensentiveprocess.Cold-adapted enzymes have very high catalytic activity at low and room temperatures,and can be easily removed by heat treatment,it shoes the excellent prospect in biotechnology filed.At present,for cold-adapted enzyme,it is difficulty to improve the yield,activity and stability.So,the cold-adapted enzyme has attracted more and more attention.The genome for Bacillus cellulosilyticus DSM 2522,a soil microorganism that is both alkaliphilic and halophilic,has recently been revealed.The result indicated that it contains a large amount of glycosyl hydrolase,which can be called as a carbohydrate enzyme library.This study used E.coli BL21(DE3)as a medium host to express ?-glucosidase and xylanase genes and have achieved a successful expression,the following investigation was then focused on their enzymatic properties.The major research results are as follows:1.The study has shown that cold-adapted and salt-tolerant is the most significant characteristic of ?-glucosidase(Bc BGl A)and xylanase(Xyn10A).Recombinant Bc BGl A belong to glycosyl hydrolase family 1.The optimal temperature and p H of the purified Xyn10 A were 40? and 7.0,respectively.Bc BGl A is retaining 27.5% of the optimal activity 4?,respectively.Recombinant Xyn10 A belong to glycosyl hydrolase family 10.The optimal temperature and p H of the purified Xyn10 A were 40? and 8.0,respectively.Its product showed the highest identity(67%)among the characterized xylanases.The Xyn10 A was sensitive to heat and showed obvious coldadapted activity,retaining 38.3% and 55.7% of the optimal activity 4? and 20?,respectively.Under the optimal conditions with cellobiose as substrate,the Kcat/Km was 45.56/s/m M,respectively.2.The high glucose tolerance exhibited by Bc BGl A in presence study provides an added advantage to relieve catabolic repression of endoglucanase and cellobiohydrolase caused by the accumulation of cellobiose during the bioconversion process.The hydrolysis properties could enable Xyn10 A to be used as an effective and powerful for large-scale production of XOs.3.Bc BGl A showed a high level of Na Cl.The highest activity was observed with 0.2M Na Cl.Xyn10 A also showed a high level of Na Cl.The highest activity was observed with 0.5M Na Cl.When the salt concentration reached 3M,the residual activity remaining was still 66.5%.The Bc BGl A and Xyn10 A have good stability and high catalytic activity at low temperature.The enzymes show the prospects in the field of food and biomass energy industry.