Expression Of Optimized Designed Endoglucanase Ends Gene In Pichia Pastoris

Cellulase include many enzymes which are called endoglucanase,exoglucanase and P-glucosidase respectily.Cellulase- one of the glycoside hydrolases- is responsible for conversion of renewable cellulosic biomass to simple sugars for fermentation to ethanol, which can settle the issue of agriculture, renewable energy and environmental pollution etc. Cellulase also has been broadly used in a lot of industries like food,textile,forage and pharmacy. The low activity of cellulase limit its application,so it is high time to improve the output of cellulase. With the deep research of enzymatic hydrolysis of cellulase, the cellulase of higher specific activity for industrialization will be found as the rapid developments of many kinds of cross-subjects such as biochemistry, molecular biology and gene engineering etc.To obtain high production of celluase, we optimized the End gene and synthesized the gene Ends without altering the protein sequence according to Pichia Pastoris codon usage bias. DNAstars and Rensselaer-Wadsworth Bioinformatics Center (BiC) (www.bioinfo.rpi.edu) are used in designing DNA. As a result, the Ends gene was expressed successfully in Pichia Pastoris.The biology informations of End gene (Genbank No:DQ782954) were analized. The result shows that the End gene without sigal peptide was 1413bp in length and encoded 470-amino acide long polypeptide.The (G+C)% content was 44.70% in the End gene and the the (G+C)% content of the third site of codon was 44.40%. When theα-factor singal peptide sequence was added in the End gene,the (G+C)% content altered to 44.00% and 42.00% respectively.△G of the folding mRNA was -555.40 kcal/mol of the original mRNA.The nucleic acid sequence of Ends was designed from the amimo acid sequence of endoglucanase based on the pichia pastoris preferred codons.The synthesized 1413bp Ends gene showed 87.90% homology with the original End gene. The (G+C)% content was 45.00% in the Ends gene and the the (G+C)% content of the third site of codon was50.50%. When theα-factor singal peptide sequence was added in the Ends gene,the (G+C)% content altered to44.30% and47.10% respectively.△G of the folding mRNA was-548.90 kcal/mol of the original mRNA. The endoglucanase gene expression vector, pPIC9K-Ends, was constructed successfully after sequencing and the endoglucanase was expressed successfully.The E. coli DH5a including pPIC9K-Ends was obtained. The plasmid was Extracted,purified and recycled. The pPIC9K-Ends which was linearized by SalⅠwas used for transformation.The GS115 made competent was transformed with SalⅠ-linearized pPIC9K-Ends by electroporation. After selection by these culture plates of MM and MD,cellucase activity measurement and PCR, some colonies which exhibited the high endoglucanase activity were achieved. After methanol induction, the endoglucanase activity of pPIC9k-Ends-19 was 1034.7 U, which was the 1.2 times of P.pastoris GS115 pPIC-End. According to SDS-PAGE, the expressed endoglucanase of P.pastoris GS115 pPIC-Ends had a molecular size of approximately 79.82 KD which was the same as the expressed endoglucanase of P.pastoris GS115 pPIC9K-End. These enzymes from pPIC9K-Ends-19 and pPIC9K-End had the same enzyme property.