Cloning And Protein Expression Of The XYL1 Gene Encoding Xylose Reductase From Candida Parapsilosis

Lignocellulose is the most abundant and cheapest renewable resource on the earth, and is supposed to be the most important successive resource for fuel alcohol production. Lignocellulose is composed of cellulose, hemicellulose and lignin. D-xylose is the most abundant monosaccharide in lignocellulose hydrolyzed product after glucose. About 85% to 90% hydrolyzed product of natural hemicellulose such as xylan is xylose. Cost-effective production of fuel ethanol from lignocellulose feedstocks requires the efficient ethanolic fermentation of the the hemicellulose fraction. For this reason, ethanolic fermentation of the pentose sugar xylose has received considerable attention.Based on xylose metabolic engineering ecology, a new xylose reductase (XR) gene (xyl1) which is a key enzyme gene in xylose metabolic engineering had been cloned from Candida parapsilosis by molecular biology technology. The xyl1 gene encoding xylose reductase was amplified by polymerase chain reaction,and then inserted into the downstream of the alpha-mating factor signal of the P.pastoris expression vector pGAPZαA. After transformation, the recombinant plasmid was integrated into the reginons of homology within the yeast genome . XYL1 P.pastoris expression vector was successfully constructed and recombinant xyl1 was expressed. The main contents run as follows:By GeneBank database, a degenerate primers and other primers were designed according to the result of homologous analysis of nucleotide sequences encoding XR of Candida sp.. A 1061bp cDNA fragment which encodes the partial sequences for xylose reductase (XR) was amplified by RACE technology from the total RNA of Candida parapsilosis. BLAST search showed that this sequence contained homology with Candida albicans aldose reductase XM₇15658 and the homologous rate was 80%. This gene should be a new XR gene(Genebank accession:EF033247).The open reading frame of xyl1 was inserted into the downstream of the alpha-mating factor signal of the P.pastoris expression vector pGAPZαA and was expressed. The recombinant XR has a (His)6 tags fusion expression and it can be detected by His-tag western. After SDS polyacrylamide gel electrophoresis, the proteins are then transferred to PVDF membrane , where they are probed (detected) using primary antibody and secondary antibody specific to the target protein.Then BCIP/NBT is bound to the secondary antibody and detection a blue color. The recombinant protein is XR which was expressed in our experiment. It has dual coenzyme specificity.In this study, the Candida parapsilosis xyl1 gene was cloned, characterized, and transformed into P.pastoris . It is a good candidate for studying cofactor specificity as well as for industrial applications such as using as a novel biocatalyst for the enzymatic synthesis of sugar alcohols to reduce downstream purification, which played an important role in application of lignocelluloses fuel alcohol production.