【作者】 李由然；

【导师】 石贵阳；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 异淀粉酶（EC3.2.1.68）能够水解支链淀粉和糖原内部的α-1,6-糖苷键而形成直链淀粉，为淀粉彻底水解所必须，在生产和科研领域俱有应用价值。微生物和植物都能够产生异淀粉酶，其中微生物来源的异淀粉酶由于生产效率高，易于提取以及成本低廉而更具工业前景。相比于另一类型的淀粉脱枝酶普鲁兰酶，异淀粉酶能同时从内部和外部水解支链淀粉的分支点，其催化反应具有不可逆性且催化活性不会被麦芽糖所抑制。然而现有的少数几种异淀粉酶因为产量过低或在工业淀粉水解条件下不稳定，仍无法取代普鲁兰酶而广泛应用。为此，本论文首先改良了地衣芽孢杆菌表达系统，通过温敏型传递质粒的两轮同源重组构建了淀粉酶和部分蛋白酶失活的表达宿主；接下来采用蛋白质谱鉴定-反向PCR的策略实现了新型芽孢杆菌异淀粉酶编码基因的克隆；同时通过分子生物学手段完成了该异淀粉酶在大肠杆菌中的表达和重组酶的纯化及功能鉴定；然后根据半理性设计的原则，选取了高保守位点周围的区域进行氨基酸点突变，提高了突变株的催化性能。本研究获得主要结果如下：1.改良了地衣芽孢杆菌表达系统，通过温敏型传递质粒的两轮同源重组构建了淀粉酶和部分蛋白酶失活的表达宿主。分别从地衣芽孢杆菌基因组DNA中扩增出α-淀粉酶编码基因amyL和一种主要蛋白酶的编码基因aprE的部分结构基因，以此为基础构建了传递质粒pNZTATN和pNZTPTS。将它们转化入地衣芽孢杆菌后，经过传代培养，促使传递质粒与基因组DNA发生两轮同源重组，将基因组DNA上的amyL和aprE基因替换为无功能的人工片段，获得重组菌从而实现α-淀粉酶和蛋白酶编码基因的失活。重组菌分泌的淀粉酶活为原始菌的0.9%，蛋白酶为原始菌的35%。该方法可以成为芽孢杆菌表达宿主功能改造的有效工具。2.对细菌Bacillus lentus CICIM304异淀粉酶基因进行了克隆、鉴定和大肠杆菌中的功能性表达。Bacillus lentus CICIM304异淀粉酶的基因序列国内外尚无报道。将该异淀粉酶使用胰蛋白酶酶解后进行液质色谱串联质谱分析，从中鉴定得到192个肽段，其中3个肽段与细菌普鲁兰酶同源性较高。基于所获得肽段的氨基酸序列，设计简并引物扩增出异淀粉酶编码基因内部1400bp的核苷酸片段。然后使用反向PCR技术进行染色体步移，克隆出长度为2658bp的异淀粉酶编码基因iam1，其编码产物含886个氨基酸残基，与已报道的异淀粉酶的同源性低于40%。以质粒载体pET-28a （+）为基础构建相关表达载体，实现了异淀粉酶在大肠杆菌中的功能性表达，重组菌细胞破碎液上清中酶活为17.6±0.5U/mL。3.纯化重组异淀粉酶并对其酶学性质进行了研究。使用亲和层析获得了重组异淀粉酶纯品，SDS-PAGE中显示其分子量约为100kDa。酶学性质研究结果显示，其最适反应温度为70oC，在此温度下保温1h后仍能保留90%以上的酶活。最适反应pH为6.5，在pH6.0-8.5的范围内能够保持较高的催化活性。Ca²⁺、Mg²⁺、Mn²⁺、Na⁺、K⁺、Co²⁺对该酶具有激活作用；Zn²⁺、Sn²⁺、Fe3+对酶活抑制超过50%。该酶对EDTA，柠檬酸盐和PMSF的敏感性较低。2mol/L的脲和30mmol/L的SDS都会造成酶的显著失活。20%的麦芽糖和20mmol/L的α-环糊精对酶活无影响，50mmol/L的α-环糊精也仅抑制4%的酶活。4.根据半理性设计的原则，选取了高保守位点周围的区域进行氨基酸点突变，提高了异淀粉酶的催化性能。通过基因定点突变，确定了异淀粉酶的结构域I, III和IV中的Asp509, His514和Glu609为关键氨基酸，它们的改变会导致酶的完全失活；而His698的突变对酶活有轻微影响。接着选取了高保守位点周围的非保守区域进行突变，通过间接影响保守位点，而避免因为直接修改保守位点导致的酶的结构与功能的大幅改变导致的活力完全丧失。在对三个突变位点的总共九个替代中多个突变体的Km值都有所降低，其中突变体R505E和G608V的最大反应速率（Vmax）比野生型异淀粉酶分别提高了11%和25%，比酶活分别提升了13%和33%。5.使用双基因失活的地衣芽孢杆菌表达宿主，实现了异淀粉酶的高效表达。使用双功能启动子PQ和来源于地衣芽孢杆菌amyL基因的信号肽介导异淀粉酶在芽孢杆菌中分泌表达。摇瓶发酵条件下在枯草芽孢杆菌1A717中表达了74U/mL的异淀粉酶活。分别使用电转化和原生质体方法将表达质粒转化入解淀粉芽孢杆菌和地衣芽孢杆菌。通过酶解支链淀粉以及制备特殊底物，验证了异淀粉酶在两种芽孢杆菌中实现功能表达。将重组质粒pHYQSIA转化入双基因失活的地衣芽孢杆菌APD1中，摇瓶条件下96h后产酶221U/mL。在15L发酵罐中进行放大实验，分批发酵条件下72h后酶活最高达到2140U/mL。更多还原

【Abstract】 Isoamylase （EC3.2.1.68） is one of the starch debranching enzymes that hydrolyzesα-1,6-glucosidic linkages in glycogen, amylopectin and their phosphorylase limit dextrins toyield amylose and oligosaccharides. Although isoamylase are found to be produced out ofboth botanical and microbial sources, microbial strains are often used for commercial purposedue to their high efficiency, easy extract and low cost. Compared with pullulanase, isoamylasehas three advantages: both endo-and exo-cleavage activity, higher efficient cleavage on α-D-（1,6）-glucoside linkages and less inhibition by its main product maltose. However, it is notavailable for industrial use either because of its low yield or because of its instability underthe conditions of current starch processing.Therefore, in this study, a Bacillus expression system was engineered to inactivateamylase and one of proteases, in order to meet the requirement for isoamylase expression.Then a novel isoamylase gene from Bacillus lentus CICIM304was cloned and expressed as afunctional enzyme. Moreover, a semi-rational designed direct evolution guided by structuralinformation was carried out to improve the catalytic properties of isoamylase.The main resultsare as follows:1. Bacillus licheniformis expression system was improved by genetic engineering.temperature-sensitive plasmid vectors were constructed to inactivate amyL and aprEgene of the host. An engineered Bacillus licheniformis with amyL and aprE gene inactivatedwas constructed. The gene inactivation was based on a highly temperature-sensitive plasmidvector. amyL and aprE, coding α-amylase and one of proteases, respectively, were clonedfrom genomic DNA of B. licheniformis. Two artificial nonfunction-copies of the genes wereconstructed by insertion of antibiotic genes. Then, the constructed delivery plasmidspNZTATN and pNZTPTS, carrying the artifical copies surrounded by DNA fragments thatflank the desired insertion site were transformed into B. licheniformis. After two-roundrecombinations stimulated by temperature shifting, the obtained mutant strain, B.licheniformis APD1had99.1%and65%reduction in extracellular amylase activity andprotease activity, respectively.2. The gene encoding isoamylase in Bacillus lentus CICIM304was cloned,characterized and functionally expressed in E. coli. As isoamylase gene in Bacillus lentusCICIM304has never been reported, a new strategy including MS/MS and inverse-PCRtechniques was adopted. Firstly, the purified enzyme was separated by SDS-PAGE followedby in-gel digestion of trypsin. The peptides released were subjected to nanoLC-ESI-Q-TOF/MS/MS analysis. Then degenerate primer DNAs were designed based on thecharacterized peptides sequences to amplify part of the gene. Genome-walking was performedby using inverse-PCRs and at the end of this experiment, a2658ORF of isoamylase encodinggene was identified, whose product had885amino acids with less than40%identity tomicrobial isoamylase ever reported. Finally, the gene was expressed in E. coli. by using thevector pET-28a （+） and the highest intracellular isoamylase activity was17.6±0.5U/mL. 3. The recombinant isoamylase was purified and characterized. The recombinantisoamylase produced by E. coli was purified by affinity chromatography, which showed asingle band on SDS-PAGE with molecular mass of around100kDa. The biochemicalproperties of the recombinant enzyme were characterized. Its maximal activity occurred at70oC, and1h of incubation at70oC would make it lose merely10%of its maximal activity.The enzyme exhibited good stability between pH6.0and8.5with an optimal activity at pH6.5. Ca²⁺、Mg²⁺、Mn²⁺、Na⁺、K⁺and Co²⁺slightly enhanced the enzyme activity as chloridesalts. Zn²⁺, Fe3+and Sn²⁺reduced the activity evidently to less than50%of control levels.EDTA, citrate and PMSF were not inhibitory, on the contrary,2mol/L urea and30mmol/LSDS led to significant loss of its activity. Maltose and α-cyclodextrin up to a concentration of20%and20mmol/L, respectively, did not influence the activity of the enzyme. A4%inhibition of the activity was observed in the presence of50mM α-Cyclodextrin.4. Semi-rational designed site-derected mutations guided by structural informationwere carried out to improve the catalytic properties of isoamylase. Asp509, His514andGlu609in Region I, III and IV were proved to be essential amino acids by site-directedmutagenesis. Replacement of His698had merely slight effects on activity. The sites near theessential amino acids were chosen as the candidates for mutation for the reason that theseamino acids were located in non-conserved regions so as not to affect the active/catalytic sitesdirectly. On the other hand, mutation of highly conserved region would lead to significantchange in structure which might blow the function of the enzyme strongly to a completeinactivation. Kmvalue of5mutants was reduced, from which R505P and G608V had animprovement in Vmaxof11%and25%, and their specific activity was also raised by13%and33%, respectively.5. Isoamylase was efficiently expressed in engineered B. licheniformis with amyLand aprE gene inactivated. Expression and secretion of the isoamylase in bacillus strainswere under the control of an expression cassette including an artificial bifunctional promoterPQ and S signal peptide region of B. licheniformis amyl gene. In Bacillus subtilis, the highestisoamylase expression level obtained was74U/mL in shaking flask. The expression plasmidpHYQSIA was transformed into B. amyloliquefaciens and B. licheniformis. The functionalexpression of the isoamylase in the two hosts was testified by preparation of maltose fromstarch. Recombinant enzymes obtained88%and92%of conversion yields, respectively,when they was used cooperatively with fungal malto-genic amylase. The results demonstratedthat isoamylase had been functionally expressed in the two hosts as the conversion yieldsobtained by enzymes of parent strain was only around60%. Isoamylase was functionallyexpressed in the two-genes inactivated mutant strain and the higest α-amylase productionlevels obtained was221U/mL under shake flask fermentation. The scale up fermentation wasperformed in a15L fermenter with batch form, and the highest expression level obtained was2140U/mL.更多还原