| Xylanase is the general term of a group of enzymes that can specifically degrade different xylans. It has a wide range of applications in papermaking, food processing, plant fiber extraction and feed additives. Since the 1980s, have started researching in xylanase, researchers have obtained many resources that can produce xylanase. The main reasons for xylanase can't large-scale industrialized were the low xylanase production, difficult to separation and purification, and limited application enzyme properties, so screening of high-yield xylanase-producing strains, optimization the methods of separation and purification, using genetic engineering means to molecular modify strains were currently the domestic and international research hotspots.Firstly, the study screened a high-yield xylanase strain, then systematically studied purification, enzyme properties of xylanase and the diversity, cloning and expression, molecular modification of the xylanase gene from the strain. Main results were as follows:1, The high-yield xylanase strain was Bacillus subtilis BE-91, with the xylanase activity of 423.24 U/mL at the optimization fermentation process. Electrophoretically pure xylanase was separated and purified from the fermentation broth with the recovery rate of 69.2%, purification factor of 18, and specific activity of 28453.6 U/mg. Its molecular weight was measured as 22.54 kD, isoelectric point 9.63, stabilizing pH 3.4-6.4, stabilizing temperature 0-65℃. It can be activated by Fe2+ and Co2+, and strongly inhibited by Cu2+; Km and Vmax were 0.5 mg/mL and 533 U, respectively, with oat spelt xylan as the substrate; Part amino acid sequences were YNAPSIDGDR, TTFTQYWSVR, SDGGTYDIYTTTR, RPTGSNATITFSNHVNAW and SPLIEYYVVDSWGTYRPTGTYK.2, The xylanase gene xynA was cloned from Bacillus subtilis BE-91 (Gene Bank accession number GQ845010). The complete ORF of xynA was 642 bp, coding 213 amino acids, theory molecular was 20 kD,1-26 amino acids was signal peptide; Compared with xylanase gene from Bacillus subtilis logged by others, homology was 98%; xynA was efficiently expressed in E. coli BL21 with pET-28a (+), pEASY-E1 vector, and the xylanase activity of recombinant pEASY-xynA-BL21 was 1226 U/mL at the optimization induce condition; SDS-PAGE pure recombinant xylanase was obtained by His-tagged purification kits, the optimal temperature of recombinant xylanase was 65℃, the optimal pH was 6.4; At the same time, xynA was also expressed in P. pastoris GS115 with the expression vector pPIC9k, and the recombinant xylanase activity was 27.45 U/mL with methanol induced 3 days.3, The genome library of Bacillus subtilis was constructed by improved shotgun method. The positive recons containing the function gene were extracted from the library using specific substrate enzyme hydrolysis and spectrum analysis, and determined the sequences of positive recons and compared with each other by bioinformatics analysis software, then obtained the nucleotide sequences of xynA, xynB, xynC, xynD and xynP. XynB, xynC, xynD, xynP were expressed in E. coli BL21 with pEASY-E1 vector, respectively. The products of recons degradated xylan were analysis by HPLC, and the results showed that xynA, xynC and xynD coding xylanase, xynB and xynP coding xylosidase, the nucleotide and amino acid sequences of xynA, xynC, xynD and xynB, xynP were extremely low homology.4, Determined the key sites for xylanase activity by delete expression. The nucleotide and amino acid sequences of xylanase gene xynA were analysed by bioinformatics analysis software, then designed primers and PCR amplificatied the genes with deleted part sequences, expressed in E. coli BL21 with pET 28a (+) vector, and obtained multiple recons. The xylanase activity of recons showed that the 5' 29～38 AA and 3'174～197 AA of xylanase gene xynA from Bacillus subtilis BE-91 were the key sites for xylanase activity.The results can provide important scientific basis for establishing efficiently genetic engineering strain to produce commercial xylanase.
|
PDF Full Text Request