Hot Cut Inside Clostridium Fiber Cellulose Glucanase In Saccharomyces Cerevisiae Cells On The Surface Of The Display And Its Properties

Cellulose is the most abundant and renewable resource. However, because its structure ishighly crystalline and contains a lot of lignin, it is very difficult for cellulose to be degraded intoglucoses which are some small available molecules, so that it also led to the utilization rate ofcellulose very lower. Cellulolytic enzymes are the generic name of enzyme which catalyze thehydrolysis of cellulose into glucoses and are consist of endoglucanases(EG [EC3.2.1.4]),cellobiohydrolases(CBH [EC3.2.1.91]) and β-glucosidases(BG [EC3.2.1.21]). But it is ratherimportant and difficult to improve the two troublesome problems that the cost is very higher andthe activity is very lower. With the development of genetic engineering, the expression ofrecombinant protein was a new way to resolve the two problems by different expression vectors.First of all, the primers were designed according to endoglucanase gene sequence depositedin GenBank and the multiple cloning site of the plasmid pYD1, which is a vector used forprotein surface display on Saccharomyces cerevisiae cells. Then we successfully cloned theendoglucanase from clostridium thermocellum stored in our laboratory, has the ability to producehigh level of cellulases, by PCR using total genomic DNA as template, clone the target gene intoPmd18-T vector and the recombined plasmid transform into E.coli DH5α, and the target gene aresequenced and compared. In the next part, the PCR product was inserted into the plasmid pYD1,and the construct was transformed into the yeast strain EBY100. then we got the positivetransformants, and it was confirmed that we successfully cloned the target gene into the yeastgenome by yeast colony PCR. Subsequently, after the target gene in the transformants wereinduced with2%galactose, the enzyme activity was detected by Congo-Red Staining methed.Finally, the characteristics of the enzyme was analysed by DNS method, and was compared withthe original enzyme activity of the clostridium thermocellum, respectively.According to the study results of the characteristics of the recombinase，we know thatoptimal temperature and pH of the displayed enzyme were50℃and4.6; after the target gene inthe transformants were induced with2%galactose,the activity of the recombinase was thehighest and was32IU/ml, its activity is even higher than natural endoglucanase’s. Differentmental ions had different effects on the activity of the recombinase: effects of activation orinhibition. The same mental ions in the different density had different effects on the activity ofthe recombinase.