【作者】 刘洋；

【导师】 王正祥； 石贵阳； 沈微；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 中温α-淀粉酶是重要的工业酶制剂之一,广泛用于淀粉糖生产、味精生产、啤酒和酒精生产中辅料的加工、织物退浆、以及其它酿造、有机酸和医药行业。目前,国内主要的工业生产菌株为BF-7658和Bacillus sp. M13。近20年来,我国学者一直未对中温α-淀粉酶生产菌株进行有效的遗传改良,使得其发酵生成水平落后于世界先进水平。因此,通过现代育种技术对中温α-淀粉酶工业生产菌株进行遗传改良,将有助于提升我国中温α-淀粉酶的发酵生产水平,实现其高效低成本制备。本课题以中温α-淀粉酶工业生产菌株B. amyloliquefaciens CICIM B2125为研究对象,纯化了该菌株产生的中温α-淀粉酶并进行了酶学性质研究;克隆了中温α-淀粉酶全长基因并验证了该基因的功能;对中温α-淀粉酶基因进行了定点突变,改变了中温α-淀粉酶的酶学性质;建立了中温α-淀粉酶工业生产菌株的遗传转化方法,并通过提高中温α-淀粉酶基因拷贝数显著增强了α-淀粉酶的分泌。1.纯化了B. amyloliquefaciens CICIM 2125产生的α-淀粉酶(BAA)并首次确定了纯酶的酶学性质。通过纯化,从发酵液中获得了电泳纯的BAA。酶回收率为20.1%,纯化倍数为26.9。对纯化的BAA的酶学性质进行了测定,该酶的分子量为58 kDa,最适反应温度为55℃,最适反应pH为6.0-9.0。pH稳定范围为7.0-10.0。添加10 mmoL/L Ca2+,可以使酶在55℃下保温1 h,还残留85%左右的活力。无Ca2+情况下,该酶在55℃下保温15 min,丧失活力。Ca2+,Mn2+,Co2+可以显著增强酶的活力。薄层层析分析表明:酶的淀粉水解产物主要为糊精和一些寡糖。2.克隆了BAA全长编码基因amyQ。该基因由启动子区(220 bp),结构基因(1544 bp)及终止序列(320 bp)构成。在启动子区有3个碱基序列发生了改变,分别位于-49位的碱基发生改变(C→T),-79和-80位的碱基由(TA→AT),结构基因与已报到的BAA编码基因(GenBank登录号:J01542)一致。3.克隆的amyQ基因在大肠杆菌和枯草芽孢杆菌中获得活性表达。将无信号肽的BAA结构基因(amyQ’)克隆入表达载体pET-28a,构建了重组载体pET-28a-amyQ’,通过IPTG诱导,在大肠杆菌中表达,重组菌的α-淀粉酶活力为2.8 U/mL培养液。重组酶的相对分子量为58 kDa。将携带有全长基因amyQ的重组载体pLY-amyQ,转化B. subtilis 1A510后,实现amyQ的分泌表达,所表达的重组BAA经纯化后,分析其酶学性质,发现其与天然BAA在酶学性质上没有明显差异。4.进一步选取了BAA中Ca2+结合位点上的3个氨基酸残基(231,233,438)进行定点突变,对突变体的酶学性质进行比较。与天然BAA相比,突变体D233N的比酶活下降了的84.8%;Km上升了55.6%,Kcat值下降了85%;热稳定性明显下降,最适反应温度范围也减小了10℃;最适反应pH下降了0.5个单位,pH稳定范围下降了3-4个单位。与对照相比,突变体D231N和D438G的比酶活分别下降了6.3%和3.5%,Km和Kcat值与对照相比没有显著变化;最适反应温度,热稳定性及最适反应pH及pH稳定性变化不显著。5.成功构建了重组BAA工业生产菌株,产酶水平显著提升。建立了BAA生产菌株优化的电转化方法,转化效率可达4.8×102。将pLY-amyQ转化B. amyloliquefaciens CICIM B2125,获得了携带多拷贝BAA编码基因的重组菌。重组菌在无选择压的培养基上传代100代,质粒稳定性保持在80%左右,可以基本满足工业生产对菌株的遗传稳定性的要求。重组菌的BAA分泌能力与出发菌株相比,在摇瓶水平上提高了50%以上,在30吨生产规模提高了30%左右,发酵周期缩短了8-12 h左右。更多还原

【Abstract】 Mesophilicα-Amylase is one of important industrial enzymes, which has extensive applications in starch processing, brewing, desizing, organic acid and pharmaceutical industries. BF-7658 and Bacillus. sp M13 are the two main mesophilicα-amylase industrial producing strains by far in china. The production ability of the strain was not effectively improved during these twenty years and was very low compared with the most advanced technology in the world. Therefore, it is very important to enhance the secretion ability of the industrial producing strain by genetic improving the strain through modern breeding technology and lower the cost of production.The mesophilicα-amylase was purified to homogeneity from theα-amylase industrial producing strain B. amyloliquefaciens CICIM B2125 and the enzyme characterizations were studied in this project. The full length gene encoding forα-amylase was cloned and the function of the gene was validated. More over, the enzyme properties were changed by site-directed mutagenesis. An optimized eletroporation transformation method was developed for B. amyloliquefaciens CICIM B2125. The secretion ability was improved significantly by increasing theα-amylase gene copies of the industrial strain.1. Theα-amylase from B. amyloliquefaciens CICIM B2125 (BAA) was purified to homogeneity and the enzyme properties were revealed for the first time. The recover rate of the enzyme was 20.1% and fold of purification was 26.9. The molecular weight of the enzyme was 58 kDa. The optimum temperature and pH were 55℃and 6.0-9.0, respectively. The enzyme was stable at the pH range of 7.0-10.0. It kept stable at 55℃for 1 h in the presence of 10 mmoL/L Ca2+ and remained 85% residue activity. The enzyme activities were significantly activated by Ca2+, Mn2+ and Co2+. The main endo-products to soluble starch were oligosaccharides and dextrin analyzed by thinner layer chromatography.2. The full-length gene encoding for mesophilicα-amylase (amyQ) was cloned from B. amyloliquefaciens CICIM B2125. The gene consists of 220 bp of promoter region, 1544 bp of structural gene fragment and 320 bp of terminal sequence. Three bases were altered in the promoter region at the site of -49 (C→T) and -79,-80 (TA→AT), however, the structural gene sequence of amyQ was the same as the amylase gene (GenBank accession No. J01542). 3. The clonedα-amylase gene was expressed in E. coli and B. subtilis. Theα-amylase gene fragment devoid of signal peptide sequence (amyQ′) was cloned into the plasmid pET-28a. The degenerated plasmid pET28a-amyQ′was introduced into E. coli to expressα-amylase and induced with IPTG. The enzyme activity was 2.8 U/mL in the supernatant of culture medium. The full-length gene (amyQ) was cloned into plasmid pLY121 and the recombinant plasmid pLY-amyQ was transformed into B. subtilis 1A510. Theα-amylase gene was expressed and the recombinant enzyme was purified by two-phase system. The characterizations of the recombinant enzyme were almost same compared with the native enzyme.4. Role of the calcium-binding residues Asp231, Asp233 and Asp438 of Bacillus amyloliquefaciensα-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233 and Asp438 were replaced with Asn, Asn and Gly to produce muants D231N, D233N and D438G, respectively. The specific activity for D233N was decreased by 84.8%, while D231N, D438G showed a decrease of 6.3%, 3.5% to that of the wild-type enzyme respectively. No significant changes in the Km value, thermo-stability, optimum temperature and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (Kcat/Km) and thermo-stability at 60oC. The ranges of optimum temperature and optimum pH for D233N were also reduced about 10oC and 3~4 units respectively.5. The enzyme secretion ability was significantly enhanced by the recombinant strain B. amyloliquefaciens CICIM B2125. An optimized electroporation transformation method was established for B. amyloliquefaciens CICIM B2125. The transformation efficiency was satisfied for genetic manipulating the strain. The recombinant plasmid pLY-amyQ was transformed into B. amyloliquefaciens CICIM B2125. About 80% of the recombinant plasmid pLY-amyQ was kept in B. amyloliquefaciens CICIM B2125 during 100 generation growth in the medium without antibiotic. It could meet the requirement of genetic stability of recombinant strain during industrial production. The secretion ability of the recombinant strain was increased by 50% and 30% in flask and 30 ton fermentor respectively, compared with the parental strain. The process of theα-amylase production was reduced about 8-12 h.更多还原