Heterologous Expression And Enzymology Characteristics Research Of Glycoside Hydrolase

Glycoside hydrolase play an important role in the process that every living organisms need carbohydrate metabolism to live.As is known to all,there are more than 2500 kinds of glycoside hydrolases were found in nature.However,these glycoside hydrolases are usually found some deficiencies,such as low expression,poor stability,poor substrate specificity,low catalytic activity and so on.Thus,the modification of the character of enzymes is particularly urgent.This paper expresses and characterizes the properties of endo-?-1,4-glucanase(EC3.2.1.4)and endoinulinase(EC3.2.1.7)..Endo-?-1,4-glucanase Cell2A(EG3)belongs to glycoside hydrolase GH12 family,which can randomly hydrolyze ?-1,4-glycosidic bond and produce cellulose oligosaccharides.The cel12A genes from Penicillium oxalate 114-2,Trichoderma reesei QM6a and Neurospora crassa were choosed to research.These proteins were expressed by Pichia protein expression system.Three Cel12A protein were expressed successfully,and mixed with the crude enzyme from T1x1.Avicel,DCCR and CCR were choosed as the substrate for saccharification and the degradation of the substrates in high concentration by Cel12A were analyzed.It was found that the mixed enzyme played specific role in the degradation.This provided significance in guiding the application of Cel12A protein in the future.However,it is still need to optimize the amount of enzyme and the saccharification conditions.Endoinulinase belongs to glycoside hydrolase GH32 family,which can hydrolyze the inulin in jerusalem artichoke to fructose or fructo-oligosaccharides.Due to the fructo-oligosaccharides has many health functions,so the endoinulinase gene which came from Aspergillus Niger was choosed and restructured in T.reesei H1?ligD.The crude enzyme from the transformant was used in Saccharification.Inulin was used as substrate in this study,it was found that the activity of inulinase enzyme was low.It may be due to the enzyme is not purified and the enzyme composition is too complex.Also,it may be due to CBH ? is one kind of induced promoter.Although the inulinase was expressed,the quantity is limited,which resulted the saccharification effect is low.So,the transformant need be reconstructed in the future.