| Xylanases(EC 3.2.1.8) are hydrolytic enzymes(O-glycoside hydrolases) that catalyze the hydrolysis of xylan into xylotriose, xylobiose, and xylose. Xylanases have found widespread industrial applications in the food, paper-pulp, feed industries. One particular GH family 10 xylanase(xynA, GenBank accession no. JX560161) from Streptomyces sp. FA1 was recently purified, characterized, cloned and expressed in Escherichia coli BL21(DE3) as a secreted protein. However, a substantial portion of the protein product was typically obtained as insoluble, misfolded inclusion bodies in E. coli, which limited the Xyn A expression level. P. pastoris expression systems are able to secrete protein in soluble form and produce large quantities of recombinant protein. By optimizing signal peptide, fermentation optimization, coexpression of protein disulfide isomerase(PDI) to get higher production. Finally, an application study was performed to investigate the effect of this enzyme on the quality of CSB quality. This study provides the basis for the application of Xyn A to the industrial production of Chinese steamed bread.(1) Recombinant yeast were constructed with the xylanase signal peptide and Saccharomyces cerevisiae α signal peptide to guide xylanase extracellular expression of recombinant yeast. After 120 h of induction, it showed the activity of recombinant XynA with Saccharomyces cerevisiae α signal peptide and xylanase signal peptide mediated were 130 U·m L-1 and 40.1 U·m L-1 respectively. Streptomyces sp. FA1 xylanase was purified to electrophoretic homogeneity using ammonium sulfate fractionation followed by SP Sepharose Fast Flow cation- exchange chromatography. The optimal pH and the optimal temperature of Xyn A were 5.5 and 60 °C, showed good pH stability, with more than 80% of the activity retaining after incubation at pH from 3.0 to 11.0. The specific activity of the purified Xyn A was 250.7 U·mg-1, Km and Vmax values were found to be 2.4 mg mL-1 and 299.3 μmol·min-1·mg-1 respectively. XynA was completely inhibited by a 10 mmo L·L-1 concentration of Fe3+ and partially inhibited by 10 mmoL·L-1 concentrations of Cr2+ and Mn2+.(2) To optimize the production of XynA, on the production of XynA using P. pastoris in a 3.6-L bioreactor were studied. When the induction temperature was 28 °C, the optimal initial induction cell density OD600 was 100, methanol concentration was 1.0%, the production of Xyn A activity in the culture supernatant reached 1374 U·mL-1 at 132 h.(3) Streptomyces sp. FA1 xylanase contains five pair of disulphide bonds. In present study, in order to improve the production of recombinant xylanase, PDI was selected to coexpress with the xylanase.The pdi gene was amplified by PCR from the genome of P. pastoris and cloned into the expression vector pPICZ A; then the recombinant plasmid was lineared and transformed into P. pastoris KM71/pPIC9K-xynA. The recombinant P. pastoris were cultivated in shake flasks, the activity of xylanase was 180 U·m L-1. Further optimization study were carried out in 3.6 L bioreactor. When the induction temperature was 28 °C, the optimal initial induction cell density OD600 was 200, methanol concentration was 1.0%, the production of Xyn A activity in the culture supernatant reached 1781 U·m L-1 at 132 h, which was the highest expression level of a Streptomyces sp. xylanase belonging to GH family 10 obtained in P. pastoris and 1.3-fold higher than that have not coexpressed.(4) To investigate its performance in the baking industry, the Xyn A and a commercial xylanase were used in attempts to improve the quality of CSB. Using XynA, the highest specific volume was 7.3% higher than the control. When the optimal commercial xylanse was used, the specific volume was 5.4% higher than the control.Under which condition the hardness of CSB decreased from 2031 to 1862 g, which was better than that with the commercial xylanase at its optimum dosage. This study provides the basis for the application of the enzyme in the baking industry. |
PDF Full Text Request