摘要
木聚糖酶是一类特异降解不同木聚糖的酶的总称,在制浆造纸工业、食品工业、饲料工业和纤维提取工业具有广泛的应用价值。自20世纪80年代对木聚糖酶进行研究以来,已筛选到很多能产木聚糖酶的资源,但到目前为止,木聚糖酶还没能大规模工业化生产,主要原因有产酶菌株产量不高、分离纯化困难、酶学性质存在应用缺陷等,因此,木聚糖酶高产菌株的筛选、分离纯化方法的优化和利用基因工程手段对产木聚糖酶菌株进行分子改造等仍是目前的研究热点。
本研究从筛选木聚糖酶高产菌株入手,系统地研究了枯草芽孢杆菌(Bacillus subtilis)木聚糖酶的分离纯化、酶学性质和木聚糖酶基因的多样性、克隆与表达、分子改造等内容。主要结果是：
1、筛选到1株木聚糖酶高产菌株,编号为Bacillus subtilis BE-91。BE-91菌株在优化的发酵条件下,发酵液中木聚糖酶活力达423.24 U／mL；两步纯化法从BE-91菌株发酵液中分离出SDS-PAGE纯木聚糖酶,回收率69.2%,纯化倍数18倍,比酶活28453.6 U／mg；酶学性质研究表明,其分子量为22.54 kD,等电点为9.63,稳定pH 3.4～6.4,稳定温度0-65℃, Fe2+、Co2+对其有激活作用,Cu2+有强烈的抑制作用,以燕麦木聚糖为底物时,Km和Vmax分别为0.5 mg／mL和533U；该木聚糖酶含有YNAPSIDGDR, TTFTQYWSVR、SDGGTYDIYTTR、RPTGS NATITFSNHVNAWK和SPLIEYYVVDSWGTYRPTGTYK等氨基酸序列。
2、从BE-91菌株中克隆到木聚糖酶基因xynA (GeneBank登录号为DQ845010)。xynA的ORF为642 bp,编码213个氨基酸,理论分子量为20 kD,可能N端第1-26位氨基酸为信号肽,与已知的Bacillus subtilis木聚糖酶基因xynA的同源性为98%；将xynA克隆进E. coli表达载体pET-28a(+)和pEASY-El,在E.coli BL21(DE3)中都得到特异性表达；重组菌pEASY-El-xynA-BL21在优化的诱导条件下,发酵液中木聚糖酶活力达1226 U／mL；用组氨酸标记纯化试剂盒对重组木聚糖酶进行分离纯化,获得SDS-PAGE纯重组酶,酶学性质研究表明其最适作用温度65℃,最适作用pH值6.4；同时,将xynA克隆进P. pastoris表达载体pPIC9k,在P.pastoris GS115中得到特异性表达,重组菌在甲醇诱导3 d后,发酵液中的木聚糖酶活力为27.45 U／mL。
3、采用改良鸟枪法构建了Bacillus subtilis基因组文库。根据特异性底物水解圈和酶谱分析从文库中筛选出含功能基因的阳性重组子,提取质粒进行序列测定、比对、分析,获得木聚糖酶基因xynA、xynB、xynC、xynD和xynP的核苷酸序列；将xynB、xynC、xynD和xynP分别与表达载体pEASY-E1连接,转化入E. coli BL21,获得特异性表达阳性重组菌；用重组菌降解木聚糖,对产物进行HPLC分析,结果表明,xynA、xynC和xynD编码木聚糖酶基因,xynB和xynP编码木糖苷酶基因,且xynA、xynC、xynD和xynP的核苷酸序列和氨基酸序列之间同源性极低。
4、采用缺失表达手段验证了木聚糖酶基因xynA表达木聚糖酶活性的关键位点。对木聚糖酶基因xynA的核苷酸序列和氨基酸序列进行比对、分析及分子结构预测,设计引物,PCR扩增出缺失部分序列的基因片段,与表达载体pET-28a(+)连接,转入E. coli BL21,获得多个特异性表达的重组菌；对重组菌的木聚糖酶表达情况进行分析,结果表明,BE-91菌株木聚糖酶基因xynA的5’-端第29-38位AA和3’-端第174～197位AA对木聚糖酶的分泌表达很关键。
上述研究结果,可为构建可商业化应用的木聚糖酶高效表达基因工程菌株提供重要科学依据。
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.
引文
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