Characterization And Construction The Engineered Strain Secreted Xylanase

Xylan, a major component of hemicellulose, is the second most abundant nutural polysaccharide after cellulose. It is also the most abundant renewable resources in the nature. As a hydrolytic enzyme,it can degrade xylan into xylo-oligosaccharides or xylose. Over the past decade, it has been showed the great prospect for applications in paper and pulp industry food industry and feed industry. Xylanase has become one of the hot spots of modern enzymology. In this research paper, the gene encoding xylanase from Thermomyces lanuginosus SY-2 had been cloned and expressed in Pichia pastoris.In this study, the gene of xylanase (xynA) was amplified by RT- PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2 with primers designed according to published gene xynA sequences. Then the product of PCR was inserted into vector pMD18-T. The sequence analysis showed that gene coding region of mature peptide contained 585bp, which coded 194 amino acids, Theoretical molecular weight was 24.4KDa (GenBank Accession number: GU166389). The DNA sequences and putative amino acids sequences of xylanase from T.lanuginosus SY2 were 98.97% and 99.49% identical to other T.lanuginosus [GenBank: U35436] by BLAST alignment in GeneBank. So , this newly cloned enzyme was identified as xylanase.A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into P. pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. Transformants were screened and identified by MD/MM plates, PCR technique and enzyme digestion. By measuring enzyme activity of the fermentation supernatant, a recombinant yeast named XY-79 with higher protein content was elected. By the study on the growth and fermentation conditions of the XY-79, it was found that the genetic stability of the gene was so well that the rate of gene losing is still zero after 10 generation of breeding, even in the absence of selection pressure.The expression level of flask ferment supernatant induced for 216 hours achieved 113. 5 IU/mL and 889.7μg/mL. The optimal pH value and temperature of the enzyme activity was 5.5 and 65℃by studying the enzymatic properties. The enzyme activity retained above 85% while the enzyme was incubating at 55°C. The highest activity was measured at 65°C, but incubated 40 min,the enzyme was remained 45% activity this temperature. The enzyme of recombinant protein was rather stable.The activity retained above nearly 80% from 2.5 to 8.5 for 48 h. Magnesian ion and calcium ion promote xylanase when concentration of metallic ion was 1mM.Sodion and potassium ion promoted xylanase slightly.Most metallic ion controled xylanase in some degree when concentration of metallic ion was 10mM. Sodion hardly had any influence for xylanase. Manganese ion, silver ion and copper ion restrained xylanase fairly remarkably. More consistent with the enzymatic properties of the original strain, indicating that its enzymatic properties of eukaryotic expression not affected enzymatic properties.