Clone Of Cbh1Gene From Trichoderma Harzianum And Expression In Saccharomyces Cerevisiae

Kitchen waste is the main component of municipal solid waste which includesstarch foods, plant fiber, animal protein, fat and other carbohydrate. Due to thecellulose is wrapped by the lignin and the characteristics of insoluble in water, it ishard to degrade cellulose into the small molecular substances such as themonosaccharide, so that the utilization of cellulose and the degration efficiency isvery low. Cellulase, which can catalyze the cellulosic degration into glucoses isconsist of Exocellobiohydrolase, endoglucanases and β-glucosidases. However, theability of natural cellulosic enzymes in the degration of cellulose is low and the costof the extraction of the cellulase is high. So, it comes into being of using the methodof genetic engineering to produce the cellulase.Yeast surface display system, as a kind of microbial surface display system,can display the target protein on the surface of the yeast cells. It also can completethe protein modification after translation. As a eukaryotic expression system, yeastsurface display system contains the protein secration and expression mechanism andhas been widely used due to the obvious advantages.In this research, we successfully cloned the gene of exocellobiohydrolase fromthe Trichoderma harzianum T88which was stored in our laborary. We designed therelavant primers according to the certain sequences in Genbank and the multitycloning sites of the plasmid pYD1. The gene ecoding cbh1was amplified by PCR,which was immediately inserted into the plasmid pYD1which is a vector used forprotein surface display on Saccharomyces cerevisiae cells. Then the recombinedplasmid pYD1-cbh1was transformed into the S. cerevisiae EBY100with themethod of lithium acetate transformation. The positive transformates were inducedwith galactose and then the enzyme activity and the protein concentration wereanalysed. The activity of the recombined enzyme was the highest after the strainwas induced for48h.Accoding to the study of the characteristics of the recombinase, the resultsshowed that the optimal temperature and the pH of the displayedexocellobiohydrolase were60℃and5.0, the surface displayed exocellobio-hydrolase was stable at pH4.0-6.0. The best reaction time is10min. By measuring the influence of metal irons on the rebinase, we find that Ca2+, Mn2+are theactivarors in the final concentration of1.0mM. Cu2+, Zn2+and Fe2+are theactivitors in the final concentration of5.0mM. However, Ca2+and Fe3+and Ag+arethe inhibitors in the same concentration. We also find that the cell surface displayenzyme shows good storage stability and genetic stability, this will has certaintheoretical and guiding significance on the degration of cellulose in the kitchengarbage.