Cloning And Expression Of Aspergillus Niger Xylanase Gene In The Yeast Saccharomyces Cerevisiae

Xylan is one kind ingredient of plant that can be recycled in the nature. The xylanase can degrade the Xylan to become the xylose or the oligoxylose. It has the widespread use in the feed, the papermaking, food as well as in the energy industry. The research of xylanase and the xylanase gene is one hot spots in the field of current enzymology. At present mainly concentrates in the high production strain selective breeding and in the research of xylanase gene clone and expression. This research has been based upon Aspergillus niger MC062, cloning and the expression of xylanase gene . Main findings as follows:The XYN7 gene encoding endo-P-l,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from Aspergillus niger MC062. Using RT-PCR method, we cloned the gene for an G/11 xylanase from Aspergillus niger. The cDNA sequence was very similar to the sequence which cloned by scientists come from South Africa and Poland.The nucleotide sequence of the cDNA fragment was verified to contain a 631bp open reading frame that encodes a 210 amino acid residues.The XYN7 gene encoding a xylanase from Aspergillus niger has been cloned and expressed in Saccharomyces cerevisiae INVSc1. The XYN7 gene, located on multicopy shuttle vectors pYES6/CT, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the GALI promoter and CYC1 transcription termination signal , respectively. 24 hours after galactose induction , The top expression of recombinant fusion protein has fullfiled, xylanase activity was 62.5IU/mL.The enzyme following over-expression in Saccharomyces cerevisiae INVSc1 produced an recombinant enzyme with a temperature optimum of 55 °C which can efficiently bind and hydrolyse insoluble xylan, and this xylanase showed high activity on xylans from hardwoods and cereals .The pH optimum of the enzyme is 3.5, but it is active over a broad pH range.Metal ions at different concentration of 10 mM and 2 mM on xylanase activity was also determined. The enzyme was completely inhibited by Co2+, while strong inhibition was caused by Fe3+, Zn2+, and Mg2+ at high concentration of 10 mM (31.9%, 41.2%, and 51.4% residual activity, respectively). Mn2+ produced a small stimulating effect.SDS were strong inhibitors of the enzyme activity, either at 2 and 10 mM.A extreme Activation will been saw when Ascorbic acid was be added to the reaction mixture.