| China, as a big country breeding, feed production highest in the word. However, due to the special circumstances of our country, its relative lack of feed resources, the use of biotechnology to improve the utilization of conventional feed, and develop some unconventional feed for widening feed resources, it will be extremely important. In the past decades, xylan have been widely used in feed, paper pulp and food industries. In this study, based on important value of the xylan and broad prospects of developing new xylan in rumen, construct recombinant Bacillus subtilis WB800N containing high-copy xyalanse genes. After that, xylanase a were heterologously expressed by Solid state fermentation , And the characterization of enzyme was studied.The aim of this study is constructing the integrating vector pHT43/Spo0A. The foundation of vector pHT43/Spo0A is vector pHT43 which has been constructed in Mo Bi Tec. In Spo0A gene as a single exchange of homologous fragments using a restriction enzyme Kpn I site of insertion, be integrated vector pHT43/Spo0A.It was suggested by both previous study and our result, CBD of xynT was non-essential for catalysis. With the context, CBD was removed from xynT and the catalytic domain was used for construction of high-copy B.S. Then xynT_CBD gene were subeloned to Ecoli-B.subtilis shuttle vector pHT43.A pair of isocaudarners (Xho I and Sal I ) was employed to establish a suttle vector pHT43(+)/3 (xynT_CBD) , which contains three tandemly arranged expression cassettes (Pgrac+SamyQ+xynT_CBD)The resultant pHT43(+)/3 (xynT_CBD) plasmid was transformed into Bacillus subtilis WB800N and the gene copy number of the strain was confirmed by qPCR.Protein of xynT_CBD was overexpressed by IPTG induction. By SDS-PAGE and Western-Blotting verify its correctness, 21KD qualitatively verify the size of the specific protein bands exogenous xylanase protein. After Ni-NTA affinity chromatography purification, its enzymatic properties were analyzed. |
PDF Full Text Request