碱性淀粉酶的异源表达及分子改造
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摘要
碱性淀粉酶是一种在碱性环境（pH9.0~11.0）下稳定且可以通过裂解α-1,4-糖苷键高效水解淀粉的水解酶。碱性淀粉酶可以应用于纺织退浆、洗涤剂添加等领域。研究发现，碱性淀粉酶在纺织退浆领域中应用可以节省大量时间、降低环境污染，同时可以将对纺织织物本身造成的损坏降至最低程度。这极大提高了纺织物前处理过程中的效率，实现最大生产经济效益，是推动未来纺织物前处理工艺快速发展的关键酶制剂之一。
国内外对碱性淀粉酶的研究主要集中在生产菌株筛选、酶的分离纯化和酶学性质研究方面。碱性淀粉酶在纺织退浆工艺和洗涤剂添加过程中应用时，需要碱性淀粉酶具有强耐氧化特性和高催化效率。但野生型碱性淀粉酶的耐氧化性能差，催化效率低，需要通过分子改造提高其耐氧化性能及催化效率。本论文实现了碱性淀粉酶基因（Accession No. AY268953）分别在大肠杆菌（Escherichia coli）、枯草芽孢杆菌（Bacillus subtilis）、毕赤酵母（Pichia pastoris）表达系统中的异源表达，并在此基础上通过对碱性淀粉酶进行分子改造提高了其耐氧化特性和催化效率。主要结果如下：
1.根据不同宿主密码子偏好性分别优化了碱性淀粉酶密码子，并在宿主E. coli、B.subtilis、P. pastoris中成功表达。纯化后，测定碱性淀粉酶动力学参数，分析发现该酶的催化效率与已报道其他碱性淀粉酶相比较偏高，适合进一步应用研究。碱性淀粉酶的最适反应pH值为9.5，稳定pH范围为8.0~11.0。酶的最适反应温度为50℃，活化能（Ea）为36.1kJ/mol。酶在50和60℃温度下半衰期（t1/2）分别为15.5和3.2min，酶的熔解温度（Tm值）为64.3℃。1mM Na~+对碱性淀粉酶酶活力有激活作用，但其他金属离子对酶活力均没有促进作用。非离子表面活性剂对碱性淀粉酶稳定性影响较小，但阴离子表面活性剂SDS（十二烷基硫酸钠）可完全抑制酶的活性。液态洗涤剂和固态皂类洗涤剂对碱性淀粉酶酶活力抑制作用较强，但固态洗衣粉对酶活力影响较小。
2.通过在线模拟软件Swiss Model对碱性淀粉酶AmyK进行同源模拟，获得碱性淀粉酶3-D结构。分析酶3-D结构后，确定了活性位点周围5个关键甲硫氨酸残基（Met145、Met214、Met229、Met247、Met317）。采用单点突变方式，将关键氨基酸分别突变成亮氨酸后，突变体耐氧化性显著提高，但仅突变体M247L催化效率有提高。同时，突变体M247L的pH稳定性和热稳定性增强。单点突变对酶表面活性剂耐受性没有显著影响。突变体M247L与商业洗涤剂（洗衣粉）的兼容性增强。复合突变后，突变体的耐氧化性显著提高，特别是含有Met247位点突变方式产生的突变体。复合突变体M145-214L、M145-214-229L、M145-229-247L、M214-229-247L、M145-214-229-317L的底物结合能力加强。
3.基于同源模拟获得的3-D结构，通过单点突变方式分别将5个关键氨基酸突变成苏氨酸、丙氨酸、丝氨酸、异亮氨酸。突变体的耐氧化性显著增强。突变体M247T、M145I、M229T的kcat/Km值分别提高至改造前酶的1.3、1.5和2.1倍。通过对单点突变体耐氧化和催化效率变化综合分析，确定了8种正向单点突变方式（M145A、M145I、M214A、M229A、M229T、M247T、M247L、M317I）。通过上述8种突变方式系统进行复合突变，共获得8个五点突变体。其中，M145I-214A-229T-247T-317I的酶学性质提高最为显著：kcat/Km值提高为改造前酶的3.2倍；耐氧化性、耐碱性、热稳定性、表面活性剂稳定性和固态洗涤剂兼容性均增强。
4.将6条具有不同性质的寡肽与碱性淀粉酶N端分别融合后，在宿主E. coli BL21（DE3）中成功表达。融合寡肽1（AEAEAKAKAEAEAKAK）后，AmyK::OP1的比酶活提高为融合前酶的2.4倍。融合蛋白的Km值均减小，说明酶底物结合能力增强。AmyK::OP1的kcat值提高为融合前酶的3.4倍；同时，AmyK::OP1的kcat/Km值提高为融合前酶的5.4倍。融合后，融合蛋白的耐碱性增强。寡肽1、3、4或5与碱性淀粉酶融合表达后，酶的最适反应温度提高。融合蛋白AmyK::OP1的热稳定性和耐氧化性增强。洗衣粉对AmyK::OP1的酶活力具有激活作用；同时，其他融合蛋白与洗衣粉的兼容性也较高。融合蛋白在固态皂类和液态洗涤剂存在条件下稳定性均较低。
5.基于对碱性淀粉酶结构区域和氨基酸序列解析，确定了12种酶C端或N端不同截断方式，并在截断突变体N端融合表达寡肽1，共产生了24个突变体。碱性淀粉酶C端或N端截断后，突变体AmyKΔC500-587和AmyKΔC492-587的比酶活分别提高为截断前酶的3.2和2.7倍。AmyKΔC500-587和AmyKΔC492-587的kcat值均提高为截断前酶的2倍。AmyKΔC500-587和AmyKΔC492-587的kcat/Km值分别提高为截断前酶的3.4和2.6倍。截断后N端寡肽融合突变体AmyKΔC500-587::SOP和AmyKΔC492-587::SOP的kcat/Km值分别提高为截断融合前酶的29.7和22.5倍。C端淀粉结合区域对碱性淀粉酶降解玉米淀粉具有重要意义，但N端序列截断对碱性淀粉酶降解玉米淀粉能力没有影响。寡肽1融合表达对C端截断突变体降解玉米淀粉具有促进作用，但对N端截断突变体降解玉米淀粉的能力基本没有影响。随机截断和截断后融合表达对碱性淀粉酶pH稳定性和温度稳定性几乎没有影响。突变体AmyKΔC500-587、AmyKΔC492-587、AmyKΔC500-587::SOP、AmyKΔC492-587::SOP的耐氧化性增强。随机截断和截断后融合表达对碱性淀粉酶表面活性剂稳定性没有显著影响。突变体AmyKΔC500-587、AmyKΔC492-587、AmyKΔC500-587::SOP、AmyKΔC492-587::SOP与固态洗涤剂（洗衣粉）的兼容性增强。
Alkaline amylase is a kind of hydrolase that is stable at alkaline condition (pH9.0~11.0)and can hydrolyze starch by cleaving α-1,4-glucosidic linkages. It is being widely used intextile and detergents industries. It was reported that the application of alkaline amylase indesizing of cotton fiber can save time, decrease environmental pollution, and minimize thedamage to textile fabric, and this can greatly improve the efficiency of the textilepre-treatment to achieve maximum economical benefits. Overall, alkaline amylase is a keyenzyme that can promote the rapid development of textile fabric pre-treatment process.The current studies mainly focus on strain selection, purification and characterization ofalkaline amylase. When alkaline amylase is used in textile and detergents industries, itshould possess the characteristics of anti-oxidation and high catalytic efficiency and so on.However, the native alkaline amylases have some disadvantages, such as easy oxidation,low catalytic efficiency and low yield and so on. So far, there is no report about themolecular engineering of alkaline amylases to improve oxidative stability and catalyticefficiency.
In this thesis, we successfully expressed the alkaline amylase gene (Accession No.AY268953) in Escherichia coli, Bacillus subtilis, and Pichia pastoris, respectively. On thebasis of this, the anti-oxidation and catalytic efficiency of alkaline amylase were improved bymolecular engineering. The main results are listed as follows:
1. Based on the different preferred codons of different expression hosts, the codons ofalkaline amylase was firstly optimized and expressed in E. coli, B. subtilis and P. pastoris,respectively. After purification, the kinetic parameters of alkaline amylase weredetermined. It was found that its catalytic efficiency was higher than those reported andcan be used for further study. The relative activity was only18%after incubation with500mM H2O2for30min. The optimum pH was9.5, and the stable pH range was8.0~11.0.The optimum temperature was50oC, and Eawas36.1kJ/mol. The half-life (t1/2) at50and60oC was15.5and3.2min, respectively, and the melting temperature (Tm) was64.3oC.1mM Na+could enhance the activity of alkaline amylase, but the other ions could notimprove the activity. Non-ions surfactants had little influence on the stability of alkalineamylase, but anion surfactant SDS (sodium dodecyl sulfate) could completely inhibit theenzyme activity. The activity of alkaline amylase was inhibited by the liquid detergentsand solid soaps, while solid washing powders had little influence on the enzyme activity.
2. The3-D structure of alkaline amylase was modeled by online software Swiss Model. Byanalyzing the3-D structure, it was found that five key amino acids (Met145, Met214,Met229, Met247and Met317) around the active sites were important for theanti-oxidation of the enzyme. When these five key amino acids were reperatively replacedby leucine, the anti-oxidation ability of mutants was enhanced. But only the catalyticefficiency of M247L was improved. The pH and thermal stabilities of M247L were alsoenhanced. The single mutation did not affect the stability of enzyme on the surfactants.The compatibility of M247L with commercial detergents (washing powder) was improved.After compositive mutation, the anti-oxidation of mutants was significantly enhanced, especially mutants with Met247. The substrate binding ability of compositive mutants(M145-214L, M145-214-229L, M145-229-247L, M214-229-247L andM145-214-229-317L) was improved.
3. Based on the3-D structure, five key amino acids were replaced with threonine, alanine,serine or isoleucine by single mutation, respectively. After mutation, the anti-oxidation ofmutants was significantly enhanced. The kcat/Kmvalues of M247T, M145I and M229Twere increased by1.3-,1.5-and2.1-fold, respectively. Based on the comprehensiveanalysis about the anti-oxidation and catalytic efficiency of single mutants, eight positivesingle mutations (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I)were determined for the next step of compositive mutation. After compositive mutation,eight five-point mutants were obtained. Among all the mutants, the characteristics ofM145I-214A-229T-247T-317I were most significantly enhanced: kcat/Kmvalue increasedby3.2-fold; anti-oxidation, pH stability, thermal stability, anti-surfactants andcompatibility with solid detergents were also improved.
4. After fusion with six oligopeptides of different characteristics at N-terminal domain ofalkaline amylase, recombinant enzymes were expressed in E. coli BL21(DE3). Afterfusion with oligopeptide1(AEAEAKAKAEAEAKAK), the specific activity ofAmyK::OP1was increased by2.4-fold compared to that before fusion. The Kmvalues ofall fused enzymes decreased, and the ability of substrate binding was enhanced. The kcatvalue of AmyK::OP1was increased by3.4-fold compared to that before fusion. Thekcat/Kmvalue of AmyK::OP1was increased by5.4-fold compared to that before fusion.After fusion, the alkaline stability of fused enzymes was enhanced. After fusion witholigopeptide1,3,4or5, the optimum temperature of fusion proteins was improved. Thethermal stability and anti-oxidation of AmyK::OP1were enhanced. The activity ofAmyK::OP1could be activated by washing powder, and the compatibility of other fusionproteins was also high. But fusion proteins had low stability under the incubation withsolid soaps and liquid detergents.
5. Based on the analysis of the domains and amino acid sequence of alkaline amylase, twelverandom truncations at C-or N-terminus were done, and twelve truncated mutants wereobtained. And then, after fusion with oligopeptide1at N-terminus of mutants, anothertwelve truncation-fusion mutants were obtained. After truncation, the specific activity ofmutants AmyKΔC500-587and AmyKΔC492-587was increased by3.2-and2.7-fold,respectively. The kcatvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby2-fold. The kcat/Kmvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby3.4-and2.6-fold, respectively. The kcat/Kmvalues of AmyKΔC500-587::SOP andAmyKΔC492-587::SOP were increased by29.7-and22.5-fold, respectively. The starchbinding domain at C-terminus was important for hydrolyzing corn starch, but N-terminaltruncation did not affect hydrolysis of corn starch. Fusion with oligopeptide1couldenhance hydrolysis of trunctation mutants at C-terminus of corn starch. However, fusionwith oligopeptide1did not affect the hydrolysis of corn starch. The pH and thermalstabilities of mutants were not changed compared to those before trunctation and fusion.The anti-oxidation of AmyKΔC500-587, AmyKΔC492-587, AmyKΔC500-587::SOP and AmyKΔC492-587::SOP was enhanced. The stability against surfactants of mutants wasnot changed. The compatibility of AmyKΔC500-587, AmyKΔC492-587,AmyKΔC500-587::SOP and AmyKΔC492-587::SOP with solid detergents (washingpowder) was enhaned.
国内外对碱性淀粉酶的研究主要集中在生产菌株筛选、酶的分离纯化和酶学性质研究方面。碱性淀粉酶在纺织退浆工艺和洗涤剂添加过程中应用时，需要碱性淀粉酶具有强耐氧化特性和高催化效率。但野生型碱性淀粉酶的耐氧化性能差，催化效率低，需要通过分子改造提高其耐氧化性能及催化效率。本论文实现了碱性淀粉酶基因（Accession No. AY268953）分别在大肠杆菌（Escherichia coli）、枯草芽孢杆菌（Bacillus subtilis）、毕赤酵母（Pichia pastoris）表达系统中的异源表达，并在此基础上通过对碱性淀粉酶进行分子改造提高了其耐氧化特性和催化效率。主要结果如下：
1.根据不同宿主密码子偏好性分别优化了碱性淀粉酶密码子，并在宿主E. coli、B.subtilis、P. pastoris中成功表达。纯化后，测定碱性淀粉酶动力学参数，分析发现该酶的催化效率与已报道其他碱性淀粉酶相比较偏高，适合进一步应用研究。碱性淀粉酶的最适反应pH值为9.5，稳定pH范围为8.0~11.0。酶的最适反应温度为50℃，活化能（Ea）为36.1kJ/mol。酶在50和60℃温度下半衰期（t1/2）分别为15.5和3.2min，酶的熔解温度（Tm值）为64.3℃。1mM Na~+对碱性淀粉酶酶活力有激活作用，但其他金属离子对酶活力均没有促进作用。非离子表面活性剂对碱性淀粉酶稳定性影响较小，但阴离子表面活性剂SDS（十二烷基硫酸钠）可完全抑制酶的活性。液态洗涤剂和固态皂类洗涤剂对碱性淀粉酶酶活力抑制作用较强，但固态洗衣粉对酶活力影响较小。
2.通过在线模拟软件Swiss Model对碱性淀粉酶AmyK进行同源模拟，获得碱性淀粉酶3-D结构。分析酶3-D结构后，确定了活性位点周围5个关键甲硫氨酸残基（Met145、Met214、Met229、Met247、Met317）。采用单点突变方式，将关键氨基酸分别突变成亮氨酸后，突变体耐氧化性显著提高，但仅突变体M247L催化效率有提高。同时，突变体M247L的pH稳定性和热稳定性增强。单点突变对酶表面活性剂耐受性没有显著影响。突变体M247L与商业洗涤剂（洗衣粉）的兼容性增强。复合突变后，突变体的耐氧化性显著提高，特别是含有Met247位点突变方式产生的突变体。复合突变体M145-214L、M145-214-229L、M145-229-247L、M214-229-247L、M145-214-229-317L的底物结合能力加强。
3.基于同源模拟获得的3-D结构，通过单点突变方式分别将5个关键氨基酸突变成苏氨酸、丙氨酸、丝氨酸、异亮氨酸。突变体的耐氧化性显著增强。突变体M247T、M145I、M229T的kcat/Km值分别提高至改造前酶的1.3、1.5和2.1倍。通过对单点突变体耐氧化和催化效率变化综合分析，确定了8种正向单点突变方式（M145A、M145I、M214A、M229A、M229T、M247T、M247L、M317I）。通过上述8种突变方式系统进行复合突变，共获得8个五点突变体。其中，M145I-214A-229T-247T-317I的酶学性质提高最为显著：kcat/Km值提高为改造前酶的3.2倍；耐氧化性、耐碱性、热稳定性、表面活性剂稳定性和固态洗涤剂兼容性均增强。
4.将6条具有不同性质的寡肽与碱性淀粉酶N端分别融合后，在宿主E. coli BL21（DE3）中成功表达。融合寡肽1（AEAEAKAKAEAEAKAK）后，AmyK::OP1的比酶活提高为融合前酶的2.4倍。融合蛋白的Km值均减小，说明酶底物结合能力增强。AmyK::OP1的kcat值提高为融合前酶的3.4倍；同时，AmyK::OP1的kcat/Km值提高为融合前酶的5.4倍。融合后，融合蛋白的耐碱性增强。寡肽1、3、4或5与碱性淀粉酶融合表达后，酶的最适反应温度提高。融合蛋白AmyK::OP1的热稳定性和耐氧化性增强。洗衣粉对AmyK::OP1的酶活力具有激活作用；同时，其他融合蛋白与洗衣粉的兼容性也较高。融合蛋白在固态皂类和液态洗涤剂存在条件下稳定性均较低。
5.基于对碱性淀粉酶结构区域和氨基酸序列解析，确定了12种酶C端或N端不同截断方式，并在截断突变体N端融合表达寡肽1，共产生了24个突变体。碱性淀粉酶C端或N端截断后，突变体AmyKΔC500-587和AmyKΔC492-587的比酶活分别提高为截断前酶的3.2和2.7倍。AmyKΔC500-587和AmyKΔC492-587的kcat值均提高为截断前酶的2倍。AmyKΔC500-587和AmyKΔC492-587的kcat/Km值分别提高为截断前酶的3.4和2.6倍。截断后N端寡肽融合突变体AmyKΔC500-587::SOP和AmyKΔC492-587::SOP的kcat/Km值分别提高为截断融合前酶的29.7和22.5倍。C端淀粉结合区域对碱性淀粉酶降解玉米淀粉具有重要意义，但N端序列截断对碱性淀粉酶降解玉米淀粉能力没有影响。寡肽1融合表达对C端截断突变体降解玉米淀粉具有促进作用，但对N端截断突变体降解玉米淀粉的能力基本没有影响。随机截断和截断后融合表达对碱性淀粉酶pH稳定性和温度稳定性几乎没有影响。突变体AmyKΔC500-587、AmyKΔC492-587、AmyKΔC500-587::SOP、AmyKΔC492-587::SOP的耐氧化性增强。随机截断和截断后融合表达对碱性淀粉酶表面活性剂稳定性没有显著影响。突变体AmyKΔC500-587、AmyKΔC492-587、AmyKΔC500-587::SOP、AmyKΔC492-587::SOP与固态洗涤剂（洗衣粉）的兼容性增强。
Alkaline amylase is a kind of hydrolase that is stable at alkaline condition (pH9.0~11.0)and can hydrolyze starch by cleaving α-1,4-glucosidic linkages. It is being widely used intextile and detergents industries. It was reported that the application of alkaline amylase indesizing of cotton fiber can save time, decrease environmental pollution, and minimize thedamage to textile fabric, and this can greatly improve the efficiency of the textilepre-treatment to achieve maximum economical benefits. Overall, alkaline amylase is a keyenzyme that can promote the rapid development of textile fabric pre-treatment process.The current studies mainly focus on strain selection, purification and characterization ofalkaline amylase. When alkaline amylase is used in textile and detergents industries, itshould possess the characteristics of anti-oxidation and high catalytic efficiency and so on.However, the native alkaline amylases have some disadvantages, such as easy oxidation,low catalytic efficiency and low yield and so on. So far, there is no report about themolecular engineering of alkaline amylases to improve oxidative stability and catalyticefficiency.
In this thesis, we successfully expressed the alkaline amylase gene (Accession No.AY268953) in Escherichia coli, Bacillus subtilis, and Pichia pastoris, respectively. On thebasis of this, the anti-oxidation and catalytic efficiency of alkaline amylase were improved bymolecular engineering. The main results are listed as follows:
1. Based on the different preferred codons of different expression hosts, the codons ofalkaline amylase was firstly optimized and expressed in E. coli, B. subtilis and P. pastoris,respectively. After purification, the kinetic parameters of alkaline amylase weredetermined. It was found that its catalytic efficiency was higher than those reported andcan be used for further study. The relative activity was only18%after incubation with500mM H2O2for30min. The optimum pH was9.5, and the stable pH range was8.0~11.0.The optimum temperature was50oC, and Eawas36.1kJ/mol. The half-life (t1/2) at50and60oC was15.5and3.2min, respectively, and the melting temperature (Tm) was64.3oC.1mM Na+could enhance the activity of alkaline amylase, but the other ions could notimprove the activity. Non-ions surfactants had little influence on the stability of alkalineamylase, but anion surfactant SDS (sodium dodecyl sulfate) could completely inhibit theenzyme activity. The activity of alkaline amylase was inhibited by the liquid detergentsand solid soaps, while solid washing powders had little influence on the enzyme activity.
2. The3-D structure of alkaline amylase was modeled by online software Swiss Model. Byanalyzing the3-D structure, it was found that five key amino acids (Met145, Met214,Met229, Met247and Met317) around the active sites were important for theanti-oxidation of the enzyme. When these five key amino acids were reperatively replacedby leucine, the anti-oxidation ability of mutants was enhanced. But only the catalyticefficiency of M247L was improved. The pH and thermal stabilities of M247L were alsoenhanced. The single mutation did not affect the stability of enzyme on the surfactants.The compatibility of M247L with commercial detergents (washing powder) was improved.After compositive mutation, the anti-oxidation of mutants was significantly enhanced, especially mutants with Met247. The substrate binding ability of compositive mutants(M145-214L, M145-214-229L, M145-229-247L, M214-229-247L andM145-214-229-317L) was improved.
3. Based on the3-D structure, five key amino acids were replaced with threonine, alanine,serine or isoleucine by single mutation, respectively. After mutation, the anti-oxidation ofmutants was significantly enhanced. The kcat/Kmvalues of M247T, M145I and M229Twere increased by1.3-,1.5-and2.1-fold, respectively. Based on the comprehensiveanalysis about the anti-oxidation and catalytic efficiency of single mutants, eight positivesingle mutations (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I)were determined for the next step of compositive mutation. After compositive mutation,eight five-point mutants were obtained. Among all the mutants, the characteristics ofM145I-214A-229T-247T-317I were most significantly enhanced: kcat/Kmvalue increasedby3.2-fold; anti-oxidation, pH stability, thermal stability, anti-surfactants andcompatibility with solid detergents were also improved.
4. After fusion with six oligopeptides of different characteristics at N-terminal domain ofalkaline amylase, recombinant enzymes were expressed in E. coli BL21(DE3). Afterfusion with oligopeptide1(AEAEAKAKAEAEAKAK), the specific activity ofAmyK::OP1was increased by2.4-fold compared to that before fusion. The Kmvalues ofall fused enzymes decreased, and the ability of substrate binding was enhanced. The kcatvalue of AmyK::OP1was increased by3.4-fold compared to that before fusion. Thekcat/Kmvalue of AmyK::OP1was increased by5.4-fold compared to that before fusion.After fusion, the alkaline stability of fused enzymes was enhanced. After fusion witholigopeptide1,3,4or5, the optimum temperature of fusion proteins was improved. Thethermal stability and anti-oxidation of AmyK::OP1were enhanced. The activity ofAmyK::OP1could be activated by washing powder, and the compatibility of other fusionproteins was also high. But fusion proteins had low stability under the incubation withsolid soaps and liquid detergents.
5. Based on the analysis of the domains and amino acid sequence of alkaline amylase, twelverandom truncations at C-or N-terminus were done, and twelve truncated mutants wereobtained. And then, after fusion with oligopeptide1at N-terminus of mutants, anothertwelve truncation-fusion mutants were obtained. After truncation, the specific activity ofmutants AmyKΔC500-587and AmyKΔC492-587was increased by3.2-and2.7-fold,respectively. The kcatvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby2-fold. The kcat/Kmvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby3.4-and2.6-fold, respectively. The kcat/Kmvalues of AmyKΔC500-587::SOP andAmyKΔC492-587::SOP were increased by29.7-and22.5-fold, respectively. The starchbinding domain at C-terminus was important for hydrolyzing corn starch, but N-terminaltruncation did not affect hydrolysis of corn starch. Fusion with oligopeptide1couldenhance hydrolysis of trunctation mutants at C-terminus of corn starch. However, fusionwith oligopeptide1did not affect the hydrolysis of corn starch. The pH and thermalstabilities of mutants were not changed compared to those before trunctation and fusion.The anti-oxidation of AmyKΔC500-587, AmyKΔC492-587, AmyKΔC500-587::SOP and AmyKΔC492-587::SOP was enhanced. The stability against surfactants of mutants wasnot changed. The compatibility of AmyKΔC500-587, AmyKΔC492-587,AmyKΔC500-587::SOP and AmyKΔC492-587::SOP with solid detergents (washingpowder) was enhaned.
引文
[1] Nikitkova A E, Haase E M, Scannapieco FA. Taking the starch out of oral biofilm formation:Molecular basis and functional significance of salivary alpha-amylase binding to oral Streptococci[J]. Appl Environ Microbiol,2013,79(2):416-423.
[2] Reddy P S, Rani D J, Sulthana S. Amylases as a tool for industrial application: A review [J]. J PureAppl Microbiol,2011,5(1):167-171.
[3] Zajoncova L&Sebela M. Amylases-significance of determination of their activity [J]. ChemListy,2007,101(1):36-43.
[4] Wang N, Zhang Y H, Wang Q H, et al. Gene cloning and characterization of a novel α-amylasefrom alkaliphilic Alkalimonas amylolytica [J]. Biotechnol J,2006,1(11):1258-1265.
[5]吴襟,彭平,沈文等.嗜碱性芽孢杆菌碱性α-淀粉酶的纯化和性质[J].中国生物化学与分子生物学报,2005,21(6):852-856.
[6] Malhotra R, Noorwez S, Satyanarayana T. Production and partial characterization of thermostableand calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54[J]. Lett Appl Microbiol,2000,31(5):378-384.
[7] Murakami S, Nishimoto H, Toyama Y, et al. Purification and characterization of two alkaline,thermotolerant α-amylases from Bacillus halodurans38C-2-1and expression of the cloned gene inEscherichia coli [J]. Biosci Biotechnol Biochem,2007,71(10):2393-2401.
[8] Hashim S O, Delgado O, Hatti-Kaul R, et al. Starch hydrolysing Bacillus halodurans isolates froma Kenyan soda lake [J]. Biotechnol Lett,2004,26(10):823-828.
[9] Murakami S, Nagasaki K, Nishimoto H, et al. Purification and characterization of five alkaline,thermotolerant, and maltotetraose-producing alpha-amylases from Bacillus halodurans MS-2-5,and production of recombinant enzymes in Escherichia coli [J]. Enzyme Microb Technol,2008,43(4-5):321-328.
[10] Tawaba J-CB, Bera F, Thonart P. Modelling the beta-amylase activity during red sorghum maltingwhen Bacillus subtilis is used to control mould growth [J]. J Cereal Sci,2013,57(1):115-119.
[11] Derde L J, Gomand S V, Courtin C M, et al. Characterisation of three starch degrading enzymes:Thermostable beta-amylase, maltotetraogenic and maltogenic alpha-amylases [J]. Food Chem,2012,135(2):713-721.
[12] Li X&Yu H Y. Extracellular production of beta-amylase by a halophilic isolate, Halobacillus sp.LY9[J]. J Ind Microbiol Biotechnol,2011,38(11):1-7.
[13] Daba T, Kojima K, Inouye K. Characterization and solvent engineering of wheat beta-amylase forenhancing its activity and stability [J]. Enzyme Microb Technol,2012,51(5):245-251.
[14] Wang J, Zhao G, Li Y, et al. Reversible immobilization of glucoamylase onto magnetic chitosannanocarriers [J]. Appl Microbiol Biotechnol,2013,97(2):681-692.
[15] Tamayo-Ramos J A, Barends S, de Lange D, et al. Enhanced production of Aspergillus nigerlaccase-like multicopper oxidases through mRNA optimization of the glucoamylase expressionsystem [J]. Biotechnol Bioeng,2013,110(2):543-551.
[16] Pandey A, Nigam P, Soccol C, et al. Advances in microbial amylases [J]. Biotechnol ApplBiochem,2000,31:135-152.
[17] Bayramoglu G, Altintas B, Arica M Y. Immobilization of glucoamylase onto polyaniline-graftedmagnetic hydrogel via adsorption and adsorption/cross-linking [J]. Appl Microbiol Biotechnol,2013,97(3):1149-1159.
[18] Hagihara H, Igarashi K, Hayashi Y, et al. Novel alpha-amylase that is highly resistant to chelatingreagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38[J]. Appl EnvironMicrobiol,2001,67(4):1744-1750.
[19] Burhan A, Nisa U, G khan C, et al. Enzymatic properties of a novel thermostable, thermophilic,alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6[J]. ProcessBiochem,2003,38(10):1397-1403.
[20] Kuilderd H&Wu G. Applied Technology-simultaneous desizing and scouring withenzymes-simultaneous fabric desizing and scouring, using alkaline alpha-amylase and an alkalinescouring enzyme, reduces water [J]. AATCC Rev-Am Assoc Text Chem Color,2008,8(6):33-36.
[21] Prakash O&Jaiswal N. Alpha-amylase: An ideal representative of thermostable enzymes [J]. ApplBiochem Biotechnol,2010,160(8):2401-2414.
[22] Kuddus M, Roohi, Arif J M, et al. Structural adaptation and biocatalytic prospective of microbialcold-active alpha-amylase [J]. Afr J Microbiol Res,2012,6(2):206-213.
[23] Siddiqui K S, Parkin D M, Curmi P M G, et al. A novel approach for enhancing the catalyticefficiency of a protease at low temperature: Reduction in substrate inhibition by chemicalmodification [J]. Biotechnol Bioeng,2009,103(4):676-686.
[24] Valls C, Rojas C, Pujadas G, et al. Characterization of the activity and stability of amylase fromsaliva and detergent: Laboratory practicals for studying the activity and stability of amylase fromsaliva and various commercial detergents [J]. Biochem Mol Biol Educ,2012,40(4):254-265.
[25] Sivaramakrishnan S, Gangadharan D, Nampoothiri K M, et al. Alpha-amylases from microbialsources-An overview on recent developments [J]. Food Technol Biotechnol,2006,44(2):173-184.
[26] Rana N, Walia A, Gaur A. Alpha-amylases from microbial sources and its potential applications invarious industries [J]. Nat Acad Sci Lett-India,2013,36(1):9-17.
[27] Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective[J]. Process Biochem,2003,38(11):1599-1616.
[28] Ray R R. Beta-amylases from various fungal strains. A review [J]. Acta Microbiol etImmunologica Hungarica,2004,51(1-2):85-95.
[29] Bernhardsdotter E, Ng J D, Garriott O K, et al. Enzymic properties of an alkaline chelator-resistantalpha-amylase from an alkaliphilic Bacillus sp isolate L1711[J]. Process Biochem,2005,40(7):2401-2408.
[30] Burhan A, Nisa U, Gokhan C, et al. Enzymatic properties of a novel thermostable, thermophilic,alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp isolate ANT-6[J]. ProcessBiochem,2003,38(10):1397-1403.
[31] Shirai T I K, Ozawa T, Hagihara H, et al. Ancestral sequence evolutionary trace and crystalstructure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378to clarify the alkalineadaptation process of proteins [J]. Proteins Struct Funct Bioinf,2007,66(3):600-610.
[32]田新玉,马延和,周培瑾等.嗜碱菌碱性淀粉酶的研究[J].微生物学报,1991,31(5):364-370.
[33] Pancha I, Jain D, Shrivastav A, et al. A thermoactive alpha-amylase from a Bacillus sp. isolatedfrom CSMCRI salt farm [J]. Int J Biol Macromol,2010,47(2):288-291.
[34] Hmidet N, Bayoudh A, Berrin J G, et al. Purification and biochemical characterization of a novelalpha-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression ofamyN gene in Escherichia coli [J]. Process Biochem,2008,43(5):499-510.
[35] Hashim S O, Delgado O D, Martínez M A, et al. Alkaline active maltohexaose-formingalpha-amylase from Bacillus halodurans LBK34[J]. Enzyme Microb Technol,2005,36(1):139-146.
[36] Syed D G, Agasar D, Pandey A. Production and partial purification of α-amylase from a novelisolate Streptomyces gulbargensis [J]. J Ind Microbiol Biotechnol,2009,36(2):189-194.
[37] Yang H Q, Liu L, Li J H, et al. Heterologous expression, biochemical characterization, andoverproduction of alkaline alpha-amylase from Bacillus alcalophilus in Bacillus subtilis [J].Microb Cell Fact,2011,10:77.
[38] Zhao J, Lan X J, Su J, et al. Isolation and identification of an alkaliphilic Bacillus flexus XJU-3and analysis of its alkaline amylase [J]. Acta Microbiol Sin,2008,48(6):750.
[39]罗志刚,杨景峰,罗发兴等. α-淀粉酶的性质及应用[J].食品研究与开发,2007,28(8):163-167.
[40]刘春莉,张文学，杨瑞.特殊α-淀粉酶的应用研究现状和前景展望[J].四川食品与发酵,2002,38(4):5-9.
[41] Medda S&Chandra AK. New strains of Bacillus licheniformis and Bacillus coagulans producingthermostable α-amylase active at alkaline pH [J]. J Appl Microbiol,1980,48(1):47-58.
[42]田小群.嗜碱性芽孢杆菌（Bacillus sp）NT-39及其所产碱性淀粉酶的初步研究[J].武汉大学学报（自然科学版）,1990,(2):105-111.
[43]贾士儒,赵树欣, Tevec FG.耐碱性芽孢杆菌碱性淀粉酶研究I-菌种的分离与筛选[J].天津轻工业学院学报,1994,(2):1-6.
[44] Saxena R K, Dutt K, Agarwal L, et al. A highly thermostable and alkaline amylase from a Bacillussp. PN5[J]. Bioresour Technol,2007,98(2):260-265.
[45]刘建龙,刘建军,杨连生.碱性淀粉酶菌株的紫外线和He-Ne激光复合诱变及其产酶条件的研究[J].激光杂志,2005,26(3):83-84.
[46] G. Dastager S, Li W J, Agasar D, et al. Streptomyces gulbargensis sp. nov., isolated from soil inKarnataka, India [J]. Antonie Van Leeuwenhoek,2007,91(2):99-104.
[47] Ogbonnaya N&Odiase A. Influence of media composition on the production of alkalinealpha-amylase from Bacillus subtilis CB-18[J]. Acta Sci Pol Technol Aliment,2012,11(3):231-238.
[48] Manabe K, Kageyama Y, Tohata M, et al. High external pH enables more efficient secretion ofalkaline alpha-amylase AmyK38by Bacillus subtilis [J]. Microb Cell Fact,2012,11:74.
[49] Murakami S, Nagasaki K, Nishimoto H, et al. Purification and characterization of five alkaline,thermotolerant, and maltotetraose-producing alpha-amylases from Bacillus halodurans MS-2-5,and production of recombinant enzymes in Escherichia coli [J]. Enzyme Microb Technol,2008,43(4-5):321-328.
[50] Murakami S, Nishimoto H, Toyama Y, et al. Purification and characterization of two alkaline,thermotolerant alpha-amylases from Bacillus halodurans38C-2-1and expression of the clonedgene in Escherichia coli [J]. Biosci Biotechnol Biochem,2007,71(10):2393-2401.
[51]王平,王强.棉织物生物酶前处理润湿性评价[J].印染,2002,28(12):28-31.
[52]蒲宗耀,陈松,卢涛等.耐碱耐温淀粉酶性能及退浆工艺试验研究[J].纺织科技进展,2006,(1):24-26.
[53]赵海军,刘志军,王佩军等.液体宽温退浆酶在前处理中的应用[J].印染,2003,29(8):12-14.
[54]冯碧波,许海育,蔡再生.棉织物的生物酶退练一浴连续工艺探讨[J].印染,2004,30(1):7-14.
[55] Igarashi K, Hagihara H, Ito S. Protein engineering of detergent alpha-amylases [J]. TrendsGlycosci Glycotechnol,2003,15(82):101-114.
[56] Nielsen J E&Borchert T V. Protein engineering of bacterial alpha-amylases [J]. Biochim etBiophys Acta (BBA)-Protein Struct Molecul Enzymol,2000,1543(2):253-274.
[57] Declerck N, Machius M, Joyet P, et al. Engineering the thermostability of Bacillus licheniformisalpha-amylase [J]. Biol-Bratislava,2002,57(SUP/2):203-212.
[58] Ali M B, Khemakhem B, Robert X, et al. Thermostability enhancement and change in starchhydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100strain [J]. Biochem J,2006,394(Pt1):51-56.
[59] Khemakhem B, Ali M B, Aghajari N, et al. Engineering of the α-amylase from Geobacillusstearothermophilus US100for detergent incorporation [J]. Biotechnol Bioeng,2009,102(2):380-389.
[60] E G-F. Lactic acid bacteria: a promising alternative for recombinant protein production [J]. MicrobCell Fact,2012,11:157.
[61] Punt P J, Van Biezen N, Conesa A, et al. Filamentous fungi as cell factories for heterologousprotein production [J]. Trends Biotechnol,2002,20(5):200-206.
[62] Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond [J].Biotechnol Adv,2012,30(5):1102-1107.
[63] Vitikainen M, Hyyryl inen H L, Kivim ki A, et al. Secretion of heterologous proteins in Bacillussubtilis can be improved by engineering cell components affecting post-translocational proteinfolding and degradation [J]. J Appl Microbiol,2005,99(2):363-375.
[64] Liu Y P, Pan J F, Wei P L, et al. Efficient expression and purification of recombinant alcoholoxidase in Pichia pastoris [J]. Biotechnol Bioprocess Eng,2012,17(4):693-702.
[65]夏雨.枯草芽孢杆菌食品级表达系统的构建和分泌表达研究[D]:[博士学位论文].无锡:江南大学食品学院,2007.
[66]诸葛斌.碱性果胶质裂解酶基因工程菌的构建、高表达及酶学性质研究[D]:[博士毕业论文].无锡:江南大学生物工程学院,2007.
[67] Kim T U, Gu B G, Jeong J Y, et al. Purification and characterization of a maltotetraose-formingalkaline alpha-amylase from an alkalophilic Bacillus strain, GM8901[J]. Appl Environ Microbiol,1995,61(8):3105-3112.
[68] Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as thesubstrate [J]. J Biochem,1954,41(5):583-603.
[69] Fuhrmann G F&V lker B. Misuse of graphical analysis in nonlinear sugar transport kinetics byEadie-Hofstee plots [J]. Biochim et Biophys Acta (BBA)-Biomembr,1993,1145(1):180-182.
[70] Roy J K, Rai S K, Mukherjee A K. Characterization and application of a detergent-stable alkalinealpha-amylase from Bacillus subtilis strain AS-S01a [J]. Int J Biol Macromol,2012,50(1):219-229.
[71] Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective[J]. Process Biochem,2003,38(11):1599-1616.
[72] Bravo Rodríguez V, Jurado Alameda E, Martínez Gallegos J F, et al. Modification of the activity ofan alpha-amylase from Bacillus licheniformis by several surfactants [J]. Electron J Biotechnol,2006,9(5):0717-3458.
[73] Tan T C, Mijts B N, Swaminathan K, et al. Crystal structure of the polyextremophilic α-amylaseAmyB from Halothermothrix orenii: Details of a productive enzyme-substrate complex and an Ndomain with a role in binding raw starch [J]. J Mol Biol,2008,378(4):852-870.
[74] Vogt W. Oxidation of methionyl residues in proteins: Tools, targets, and reversal*1[J]. FreeRadical Biol Med,1995,18(1):93-105.
[75] Khemakhem B, Ali M B, Aghajari N, et al. Engineering of the α-amylase from Geobacillusstearothermophilus US100for detergent incorporation [J]. Biotechnol Bioeng,2009,102(2):380-389.
[76] Lin L L, Lo H F, Chiang W Y, et al. Replacement of methionine208in a truncated Bacillus sp.TS-23α-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide [J].Curr Microbiol,2003,46(3):211-216.
[77] Liu Y-H, Lu F-P, Li Y-L, et al. Acid stabilization of Bacillus licheniformis alpha amylase throughintroduction of mutations [J]. Appl Microbiol Biotechnol,2008,80(5):795-803.
[78] Brosnan M P, Kelly C T, Fogarty W M. Investigation of the mechanisms of irreversiblethermoinactivation of Bacillus stearothermophilus α-amylase [J]. Eur J Biochem,1992,203(1-2):225-231.
[79] Russell G L&Britton L N. Use of certain alcohol ethoxylates to maintain protease stability in thepresence of anionic surfactants [J]. J Surfactants Deterg,2002,5(1):5-10.
[80] Binter A, Staunig N, Jelesarov I, et al. A single intersubunit salt bridge affects oligomerization andcatalytic activity in a bacterial quinone reductase [J]. FEBS J,2009,276(18):5263-5274.
[81] Gumpena R, Kishor C, Ganji R J, et al. Glu121-Lys319salt bridge between catalytic andN-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N[J]. Protein Sci,2012,21(5):727-736.
[82] Vogt G, Woell S, Argos P. Protein thermal stability, hydrogen bonds, and ion pairs [J]. J Mol Biol,1997,269(4):631-643.
[83] Vogt G&Argos P. Protein thermal stability: hydrogen bonds or internal packing?[J]. Fold Des,1997,2: S40-S46.
[84] Brosnan M P, Kelly C T, Fogarty W M. Investigation of the mechanisms of irreversiblethermoinactivation of Bacillus stearothermophilus alpha-amylase [J]. Eur J Biochem/FEBS,1992,203(1-2):225-231.
[85] Guo H&Karplus M. Solvent influence on the stability of the peptide hydrogen bond: asupramolecular cooperative effect [J]. J Phys Chem,1994,98(29):7104-7105.
[86] Wu W, Xing L, Zhou B H, et al. Active protein aggregates induced by terminally attachedself-assembling peptide ELK16in Escherichia coli [J]. Microb Cell Fact,2011,10:9.
[87] Xing L, Wu W, Zhou B H, et al. Streamlined protein expression and purification using cleavableself-aggregating tags [J]. Microb Cell Fact,2011,10:42.
[88] Zhang S, Lockshin C, Herbert A, et al. Zuotin, a putative Z-DNA binding protein inSaccharomyces cerevisiae [J]. EMBO J,1992,11(10):3787.
[89] Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementaryoligopeptide to form a stable macroscopic membrane [J]. PNAS,1993,90(8):3334-3338.
[90] Soliman W, Bhattacharjee S, Kaur K. Adsorption of an antimicrobial peptide on self-assembledmonolayers by molecular dynamics simulation [J]. J Phys Chem B,2010,114(34):11292-11302.
[91] Lazar K L, Miller-Auer H, Getz G S, et al. Helix-turn-helix peptides that form α-helical fibrils:Turn sequences drive fibril structure [J]. Biochem,2005,44(38):12681-12689.
[92] Anantharamaiah G, Jones J, Brouillette C, et al. Studies of synthetic peptide analogs of theamphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine [J]. J Biol Chem,1985,260(18):10248-10255.
[93] Uitdehaag J C M, Mosi R, Kalk K H, et al. X-ray structures along the reaction pathway ofcyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family [J]. Nat Struct MolBiol,1999,6(5):432-436.
[94] Davies G&Henrissat B. Structures and mechanisms of glycosyl hydrolases [J]. Struct,1995,3(9):853-859.
[95] Jacobson M A&Colman R F. Distance relationships between the catalytic site labeled with4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase [J]. Biochem,1984,23(17):3789-3799.
[96] Wind R D, Uitdehaag J, Buitelaar R M, et al. Engineering of cyclodextrin product specificity andpH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacteriumthermosulfurigenes EM1[J]. J Biol Chem,1998,273(10):5771-5779.
[97] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism, substratespecificity, and stability [J]. Plant Mol Biol,1994,25(2):141-157.
[98] Liu Y H, Hu B, Xu Y J, et al. Improvement of the acid stability of Bacillus licheniformis alphaamylase by error-prone PCR [J]. J Appl Microbiol,2012,113(3):541-549.
[99] Wen S, Tan T W, Zhao H M. Improving the thermostability of lipase lip2from Yarrowia lipolytica[J]. J Biotechnol,2012,164(2):248-253.
[100] Lin L L, Huang C C, Lo H F. Impact of Arg210-Ser211deletion on thermostability of a truncatedBacillus sp. strain TS-23alpha-amylase [J]. Process Biochem,2008,43(5):559-565.
[101] Shaw A&Bott R. Engineering enzymes for stability [J]. Curr Opin Struct Biol,1996,6(4):546-550.
[102] Chi M C, Chen Y H, Wu T J, et al. Engineering of a truncated alpha-amylase of Bacillus sp. strainTS-23for the simultaneous improvement of thermal and oxidative stabilities [J]. J Biosci Bioeng,2009,109(6):531-538.
[103] Hong S Y, Lee J S, Cho K M, et al. Construction of the bifunctional enzymecellulase-β-glucosidase from the hyperthermophilic bacterium Thermotoga maritima [J].Biotechnol Lett,2007,29(6):931-936.
[104] Stoner M R, Dale D A, Gualfetti P J, et al. Ca2+-surfactant interactions affect enzyme stability indetergent solutions [J]. Biotechnol Prog,2005,21(6):1716-1723.
[105] Carbone M N&Arnold F H. Engineering by homologous recombination: exploring sequence andfunction within a conserved fold [J]. Curr Opin Struct Biol,2007,17(4):454-459.
[106] Joucla G, Pizzut S, Monsan P, et al. Construction of a fully active truncated alternansucrasepartially deleted of its carboxy-terminal domain [J]. FEBS Lett,2006,580(3):763-768.
[107] Huang H Q, Yang P L, Luo H Y, et al. High-level expression of a truncated1,3-1,4-β-D-glucanasefrom Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation [J].Appl Microbiol Biotechnol,2008,78(1):95-103.
[108] Wen T N, Chen J L, Lee S H, et al. A truncated Fibrobacter succinogenes1,3-1,4-β-D-glucanasewith improved enzymatic activity and thermotolerance [J]. Biochem,2005,44(25):9197-9205.
[109] Sumitani J, Tottori T, Kawaguchi T, et al. New type of starch-binding domain: the direct repeatmotif in the C-terminal region of Bacillus sp. no.195alpha-amylase contributes to starch bindingand raw starch degrading [J]. Biochem J,2000,350(Pt2):477-484.
[110] Yen C-C, Malmis C C, Lee G-C, et al. Site-specific saturation mutagenesis on residues132and450of Candida rugosa LIP2enhances catalytic efficiency and alters substrate specificity invarious chain lengths of triglycerides and esters [J]. J Agric Food Chem,2010,58(20):10899-10905.
[111] Hagihara H, Hayashi Y, Endo K, et al. Deduced amino-acid sequence of a calcium-free α-amylasefrom a strain of Bacillus [J]. Eur J Biochem,2001,268(14):3974-3982.
[2] Reddy P S, Rani D J, Sulthana S. Amylases as a tool for industrial application: A review [J]. J PureAppl Microbiol,2011,5(1):167-171.
[3] Zajoncova L&Sebela M. Amylases-significance of determination of their activity [J]. ChemListy,2007,101(1):36-43.
[4] Wang N, Zhang Y H, Wang Q H, et al. Gene cloning and characterization of a novel α-amylasefrom alkaliphilic Alkalimonas amylolytica [J]. Biotechnol J,2006,1(11):1258-1265.
[5]吴襟,彭平,沈文等.嗜碱性芽孢杆菌碱性α-淀粉酶的纯化和性质[J].中国生物化学与分子生物学报,2005,21(6):852-856.
[6] Malhotra R, Noorwez S, Satyanarayana T. Production and partial characterization of thermostableand calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54[J]. Lett Appl Microbiol,2000,31(5):378-384.
[7] Murakami S, Nishimoto H, Toyama Y, et al. Purification and characterization of two alkaline,thermotolerant α-amylases from Bacillus halodurans38C-2-1and expression of the cloned gene inEscherichia coli [J]. Biosci Biotechnol Biochem,2007,71(10):2393-2401.
[8] Hashim S O, Delgado O, Hatti-Kaul R, et al. Starch hydrolysing Bacillus halodurans isolates froma Kenyan soda lake [J]. Biotechnol Lett,2004,26(10):823-828.
[9] Murakami S, Nagasaki K, Nishimoto H, et al. Purification and characterization of five alkaline,thermotolerant, and maltotetraose-producing alpha-amylases from Bacillus halodurans MS-2-5,and production of recombinant enzymes in Escherichia coli [J]. Enzyme Microb Technol,2008,43(4-5):321-328.
[10] Tawaba J-CB, Bera F, Thonart P. Modelling the beta-amylase activity during red sorghum maltingwhen Bacillus subtilis is used to control mould growth [J]. J Cereal Sci,2013,57(1):115-119.
[11] Derde L J, Gomand S V, Courtin C M, et al. Characterisation of three starch degrading enzymes:Thermostable beta-amylase, maltotetraogenic and maltogenic alpha-amylases [J]. Food Chem,2012,135(2):713-721.
[12] Li X&Yu H Y. Extracellular production of beta-amylase by a halophilic isolate, Halobacillus sp.LY9[J]. J Ind Microbiol Biotechnol,2011,38(11):1-7.
[13] Daba T, Kojima K, Inouye K. Characterization and solvent engineering of wheat beta-amylase forenhancing its activity and stability [J]. Enzyme Microb Technol,2012,51(5):245-251.
[14] Wang J, Zhao G, Li Y, et al. Reversible immobilization of glucoamylase onto magnetic chitosannanocarriers [J]. Appl Microbiol Biotechnol,2013,97(2):681-692.
[15] Tamayo-Ramos J A, Barends S, de Lange D, et al. Enhanced production of Aspergillus nigerlaccase-like multicopper oxidases through mRNA optimization of the glucoamylase expressionsystem [J]. Biotechnol Bioeng,2013,110(2):543-551.
[16] Pandey A, Nigam P, Soccol C, et al. Advances in microbial amylases [J]. Biotechnol ApplBiochem,2000,31:135-152.
[17] Bayramoglu G, Altintas B, Arica M Y. Immobilization of glucoamylase onto polyaniline-graftedmagnetic hydrogel via adsorption and adsorption/cross-linking [J]. Appl Microbiol Biotechnol,2013,97(3):1149-1159.
[18] Hagihara H, Igarashi K, Hayashi Y, et al. Novel alpha-amylase that is highly resistant to chelatingreagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38[J]. Appl EnvironMicrobiol,2001,67(4):1744-1750.
[19] Burhan A, Nisa U, G khan C, et al. Enzymatic properties of a novel thermostable, thermophilic,alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6[J]. ProcessBiochem,2003,38(10):1397-1403.
[20] Kuilderd H&Wu G. Applied Technology-simultaneous desizing and scouring withenzymes-simultaneous fabric desizing and scouring, using alkaline alpha-amylase and an alkalinescouring enzyme, reduces water [J]. AATCC Rev-Am Assoc Text Chem Color,2008,8(6):33-36.
[21] Prakash O&Jaiswal N. Alpha-amylase: An ideal representative of thermostable enzymes [J]. ApplBiochem Biotechnol,2010,160(8):2401-2414.
[22] Kuddus M, Roohi, Arif J M, et al. Structural adaptation and biocatalytic prospective of microbialcold-active alpha-amylase [J]. Afr J Microbiol Res,2012,6(2):206-213.
[23] Siddiqui K S, Parkin D M, Curmi P M G, et al. A novel approach for enhancing the catalyticefficiency of a protease at low temperature: Reduction in substrate inhibition by chemicalmodification [J]. Biotechnol Bioeng,2009,103(4):676-686.
[24] Valls C, Rojas C, Pujadas G, et al. Characterization of the activity and stability of amylase fromsaliva and detergent: Laboratory practicals for studying the activity and stability of amylase fromsaliva and various commercial detergents [J]. Biochem Mol Biol Educ,2012,40(4):254-265.
[25] Sivaramakrishnan S, Gangadharan D, Nampoothiri K M, et al. Alpha-amylases from microbialsources-An overview on recent developments [J]. Food Technol Biotechnol,2006,44(2):173-184.
[26] Rana N, Walia A, Gaur A. Alpha-amylases from microbial sources and its potential applications invarious industries [J]. Nat Acad Sci Lett-India,2013,36(1):9-17.
[27] Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective[J]. Process Biochem,2003,38(11):1599-1616.
[28] Ray R R. Beta-amylases from various fungal strains. A review [J]. Acta Microbiol etImmunologica Hungarica,2004,51(1-2):85-95.
[29] Bernhardsdotter E, Ng J D, Garriott O K, et al. Enzymic properties of an alkaline chelator-resistantalpha-amylase from an alkaliphilic Bacillus sp isolate L1711[J]. Process Biochem,2005,40(7):2401-2408.
[30] Burhan A, Nisa U, Gokhan C, et al. Enzymatic properties of a novel thermostable, thermophilic,alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp isolate ANT-6[J]. ProcessBiochem,2003,38(10):1397-1403.
[31] Shirai T I K, Ozawa T, Hagihara H, et al. Ancestral sequence evolutionary trace and crystalstructure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378to clarify the alkalineadaptation process of proteins [J]. Proteins Struct Funct Bioinf,2007,66(3):600-610.
[32]田新玉,马延和,周培瑾等.嗜碱菌碱性淀粉酶的研究[J].微生物学报,1991,31(5):364-370.
[33] Pancha I, Jain D, Shrivastav A, et al. A thermoactive alpha-amylase from a Bacillus sp. isolatedfrom CSMCRI salt farm [J]. Int J Biol Macromol,2010,47(2):288-291.
[34] Hmidet N, Bayoudh A, Berrin J G, et al. Purification and biochemical characterization of a novelalpha-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression ofamyN gene in Escherichia coli [J]. Process Biochem,2008,43(5):499-510.
[35] Hashim S O, Delgado O D, Martínez M A, et al. Alkaline active maltohexaose-formingalpha-amylase from Bacillus halodurans LBK34[J]. Enzyme Microb Technol,2005,36(1):139-146.
[36] Syed D G, Agasar D, Pandey A. Production and partial purification of α-amylase from a novelisolate Streptomyces gulbargensis [J]. J Ind Microbiol Biotechnol,2009,36(2):189-194.
[37] Yang H Q, Liu L, Li J H, et al. Heterologous expression, biochemical characterization, andoverproduction of alkaline alpha-amylase from Bacillus alcalophilus in Bacillus subtilis [J].Microb Cell Fact,2011,10:77.
[38] Zhao J, Lan X J, Su J, et al. Isolation and identification of an alkaliphilic Bacillus flexus XJU-3and analysis of its alkaline amylase [J]. Acta Microbiol Sin,2008,48(6):750.
[39]罗志刚,杨景峰,罗发兴等. α-淀粉酶的性质及应用[J].食品研究与开发,2007,28(8):163-167.
[40]刘春莉,张文学，杨瑞.特殊α-淀粉酶的应用研究现状和前景展望[J].四川食品与发酵,2002,38(4):5-9.
[41] Medda S&Chandra AK. New strains of Bacillus licheniformis and Bacillus coagulans producingthermostable α-amylase active at alkaline pH [J]. J Appl Microbiol,1980,48(1):47-58.
[42]田小群.嗜碱性芽孢杆菌（Bacillus sp）NT-39及其所产碱性淀粉酶的初步研究[J].武汉大学学报（自然科学版）,1990,(2):105-111.
[43]贾士儒,赵树欣, Tevec FG.耐碱性芽孢杆菌碱性淀粉酶研究I-菌种的分离与筛选[J].天津轻工业学院学报,1994,(2):1-6.
[44] Saxena R K, Dutt K, Agarwal L, et al. A highly thermostable and alkaline amylase from a Bacillussp. PN5[J]. Bioresour Technol,2007,98(2):260-265.
[45]刘建龙,刘建军,杨连生.碱性淀粉酶菌株的紫外线和He-Ne激光复合诱变及其产酶条件的研究[J].激光杂志,2005,26(3):83-84.
[46] G. Dastager S, Li W J, Agasar D, et al. Streptomyces gulbargensis sp. nov., isolated from soil inKarnataka, India [J]. Antonie Van Leeuwenhoek,2007,91(2):99-104.
[47] Ogbonnaya N&Odiase A. Influence of media composition on the production of alkalinealpha-amylase from Bacillus subtilis CB-18[J]. Acta Sci Pol Technol Aliment,2012,11(3):231-238.
[48] Manabe K, Kageyama Y, Tohata M, et al. High external pH enables more efficient secretion ofalkaline alpha-amylase AmyK38by Bacillus subtilis [J]. Microb Cell Fact,2012,11:74.
[49] Murakami S, Nagasaki K, Nishimoto H, et al. Purification and characterization of five alkaline,thermotolerant, and maltotetraose-producing alpha-amylases from Bacillus halodurans MS-2-5,and production of recombinant enzymes in Escherichia coli [J]. Enzyme Microb Technol,2008,43(4-5):321-328.
[50] Murakami S, Nishimoto H, Toyama Y, et al. Purification and characterization of two alkaline,thermotolerant alpha-amylases from Bacillus halodurans38C-2-1and expression of the clonedgene in Escherichia coli [J]. Biosci Biotechnol Biochem,2007,71(10):2393-2401.
[51]王平,王强.棉织物生物酶前处理润湿性评价[J].印染,2002,28(12):28-31.
[52]蒲宗耀,陈松,卢涛等.耐碱耐温淀粉酶性能及退浆工艺试验研究[J].纺织科技进展,2006,(1):24-26.
[53]赵海军,刘志军,王佩军等.液体宽温退浆酶在前处理中的应用[J].印染,2003,29(8):12-14.
[54]冯碧波,许海育,蔡再生.棉织物的生物酶退练一浴连续工艺探讨[J].印染,2004,30(1):7-14.
[55] Igarashi K, Hagihara H, Ito S. Protein engineering of detergent alpha-amylases [J]. TrendsGlycosci Glycotechnol,2003,15(82):101-114.
[56] Nielsen J E&Borchert T V. Protein engineering of bacterial alpha-amylases [J]. Biochim etBiophys Acta (BBA)-Protein Struct Molecul Enzymol,2000,1543(2):253-274.
[57] Declerck N, Machius M, Joyet P, et al. Engineering the thermostability of Bacillus licheniformisalpha-amylase [J]. Biol-Bratislava,2002,57(SUP/2):203-212.
[58] Ali M B, Khemakhem B, Robert X, et al. Thermostability enhancement and change in starchhydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100strain [J]. Biochem J,2006,394(Pt1):51-56.
[59] Khemakhem B, Ali M B, Aghajari N, et al. Engineering of the α-amylase from Geobacillusstearothermophilus US100for detergent incorporation [J]. Biotechnol Bioeng,2009,102(2):380-389.
[60] E G-F. Lactic acid bacteria: a promising alternative for recombinant protein production [J]. MicrobCell Fact,2012,11:157.
[61] Punt P J, Van Biezen N, Conesa A, et al. Filamentous fungi as cell factories for heterologousprotein production [J]. Trends Biotechnol,2002,20(5):200-206.
[62] Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond [J].Biotechnol Adv,2012,30(5):1102-1107.
[63] Vitikainen M, Hyyryl inen H L, Kivim ki A, et al. Secretion of heterologous proteins in Bacillussubtilis can be improved by engineering cell components affecting post-translocational proteinfolding and degradation [J]. J Appl Microbiol,2005,99(2):363-375.
[64] Liu Y P, Pan J F, Wei P L, et al. Efficient expression and purification of recombinant alcoholoxidase in Pichia pastoris [J]. Biotechnol Bioprocess Eng,2012,17(4):693-702.
[65]夏雨.枯草芽孢杆菌食品级表达系统的构建和分泌表达研究[D]:[博士学位论文].无锡:江南大学食品学院,2007.
[66]诸葛斌.碱性果胶质裂解酶基因工程菌的构建、高表达及酶学性质研究[D]:[博士毕业论文].无锡:江南大学生物工程学院,2007.
[67] Kim T U, Gu B G, Jeong J Y, et al. Purification and characterization of a maltotetraose-formingalkaline alpha-amylase from an alkalophilic Bacillus strain, GM8901[J]. Appl Environ Microbiol,1995,61(8):3105-3112.
[68] Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as thesubstrate [J]. J Biochem,1954,41(5):583-603.
[69] Fuhrmann G F&V lker B. Misuse of graphical analysis in nonlinear sugar transport kinetics byEadie-Hofstee plots [J]. Biochim et Biophys Acta (BBA)-Biomembr,1993,1145(1):180-182.
[70] Roy J K, Rai S K, Mukherjee A K. Characterization and application of a detergent-stable alkalinealpha-amylase from Bacillus subtilis strain AS-S01a [J]. Int J Biol Macromol,2012,50(1):219-229.
[71] Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective[J]. Process Biochem,2003,38(11):1599-1616.
[72] Bravo Rodríguez V, Jurado Alameda E, Martínez Gallegos J F, et al. Modification of the activity ofan alpha-amylase from Bacillus licheniformis by several surfactants [J]. Electron J Biotechnol,2006,9(5):0717-3458.
[73] Tan T C, Mijts B N, Swaminathan K, et al. Crystal structure of the polyextremophilic α-amylaseAmyB from Halothermothrix orenii: Details of a productive enzyme-substrate complex and an Ndomain with a role in binding raw starch [J]. J Mol Biol,2008,378(4):852-870.
[74] Vogt W. Oxidation of methionyl residues in proteins: Tools, targets, and reversal*1[J]. FreeRadical Biol Med,1995,18(1):93-105.
[75] Khemakhem B, Ali M B, Aghajari N, et al. Engineering of the α-amylase from Geobacillusstearothermophilus US100for detergent incorporation [J]. Biotechnol Bioeng,2009,102(2):380-389.
[76] Lin L L, Lo H F, Chiang W Y, et al. Replacement of methionine208in a truncated Bacillus sp.TS-23α-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide [J].Curr Microbiol,2003,46(3):211-216.
[77] Liu Y-H, Lu F-P, Li Y-L, et al. Acid stabilization of Bacillus licheniformis alpha amylase throughintroduction of mutations [J]. Appl Microbiol Biotechnol,2008,80(5):795-803.
[78] Brosnan M P, Kelly C T, Fogarty W M. Investigation of the mechanisms of irreversiblethermoinactivation of Bacillus stearothermophilus α-amylase [J]. Eur J Biochem,1992,203(1-2):225-231.
[79] Russell G L&Britton L N. Use of certain alcohol ethoxylates to maintain protease stability in thepresence of anionic surfactants [J]. J Surfactants Deterg,2002,5(1):5-10.
[80] Binter A, Staunig N, Jelesarov I, et al. A single intersubunit salt bridge affects oligomerization andcatalytic activity in a bacterial quinone reductase [J]. FEBS J,2009,276(18):5263-5274.
[81] Gumpena R, Kishor C, Ganji R J, et al. Glu121-Lys319salt bridge between catalytic andN-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N[J]. Protein Sci,2012,21(5):727-736.
[82] Vogt G, Woell S, Argos P. Protein thermal stability, hydrogen bonds, and ion pairs [J]. J Mol Biol,1997,269(4):631-643.
[83] Vogt G&Argos P. Protein thermal stability: hydrogen bonds or internal packing?[J]. Fold Des,1997,2: S40-S46.
[84] Brosnan M P, Kelly C T, Fogarty W M. Investigation of the mechanisms of irreversiblethermoinactivation of Bacillus stearothermophilus alpha-amylase [J]. Eur J Biochem/FEBS,1992,203(1-2):225-231.
[85] Guo H&Karplus M. Solvent influence on the stability of the peptide hydrogen bond: asupramolecular cooperative effect [J]. J Phys Chem,1994,98(29):7104-7105.
[86] Wu W, Xing L, Zhou B H, et al. Active protein aggregates induced by terminally attachedself-assembling peptide ELK16in Escherichia coli [J]. Microb Cell Fact,2011,10:9.
[87] Xing L, Wu W, Zhou B H, et al. Streamlined protein expression and purification using cleavableself-aggregating tags [J]. Microb Cell Fact,2011,10:42.
[88] Zhang S, Lockshin C, Herbert A, et al. Zuotin, a putative Z-DNA binding protein inSaccharomyces cerevisiae [J]. EMBO J,1992,11(10):3787.
[89] Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementaryoligopeptide to form a stable macroscopic membrane [J]. PNAS,1993,90(8):3334-3338.
[90] Soliman W, Bhattacharjee S, Kaur K. Adsorption of an antimicrobial peptide on self-assembledmonolayers by molecular dynamics simulation [J]. J Phys Chem B,2010,114(34):11292-11302.
[91] Lazar K L, Miller-Auer H, Getz G S, et al. Helix-turn-helix peptides that form α-helical fibrils:Turn sequences drive fibril structure [J]. Biochem,2005,44(38):12681-12689.
[92] Anantharamaiah G, Jones J, Brouillette C, et al. Studies of synthetic peptide analogs of theamphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine [J]. J Biol Chem,1985,260(18):10248-10255.
[93] Uitdehaag J C M, Mosi R, Kalk K H, et al. X-ray structures along the reaction pathway ofcyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family [J]. Nat Struct MolBiol,1999,6(5):432-436.
[94] Davies G&Henrissat B. Structures and mechanisms of glycosyl hydrolases [J]. Struct,1995,3(9):853-859.
[95] Jacobson M A&Colman R F. Distance relationships between the catalytic site labeled with4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase [J]. Biochem,1984,23(17):3789-3799.
[96] Wind R D, Uitdehaag J, Buitelaar R M, et al. Engineering of cyclodextrin product specificity andpH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacteriumthermosulfurigenes EM1[J]. J Biol Chem,1998,273(10):5771-5779.
[97] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism, substratespecificity, and stability [J]. Plant Mol Biol,1994,25(2):141-157.
[98] Liu Y H, Hu B, Xu Y J, et al. Improvement of the acid stability of Bacillus licheniformis alphaamylase by error-prone PCR [J]. J Appl Microbiol,2012,113(3):541-549.
[99] Wen S, Tan T W, Zhao H M. Improving the thermostability of lipase lip2from Yarrowia lipolytica[J]. J Biotechnol,2012,164(2):248-253.
[100] Lin L L, Huang C C, Lo H F. Impact of Arg210-Ser211deletion on thermostability of a truncatedBacillus sp. strain TS-23alpha-amylase [J]. Process Biochem,2008,43(5):559-565.
[101] Shaw A&Bott R. Engineering enzymes for stability [J]. Curr Opin Struct Biol,1996,6(4):546-550.
[102] Chi M C, Chen Y H, Wu T J, et al. Engineering of a truncated alpha-amylase of Bacillus sp. strainTS-23for the simultaneous improvement of thermal and oxidative stabilities [J]. J Biosci Bioeng,2009,109(6):531-538.
[103] Hong S Y, Lee J S, Cho K M, et al. Construction of the bifunctional enzymecellulase-β-glucosidase from the hyperthermophilic bacterium Thermotoga maritima [J].Biotechnol Lett,2007,29(6):931-936.
[104] Stoner M R, Dale D A, Gualfetti P J, et al. Ca2+-surfactant interactions affect enzyme stability indetergent solutions [J]. Biotechnol Prog,2005,21(6):1716-1723.
[105] Carbone M N&Arnold F H. Engineering by homologous recombination: exploring sequence andfunction within a conserved fold [J]. Curr Opin Struct Biol,2007,17(4):454-459.
[106] Joucla G, Pizzut S, Monsan P, et al. Construction of a fully active truncated alternansucrasepartially deleted of its carboxy-terminal domain [J]. FEBS Lett,2006,580(3):763-768.
[107] Huang H Q, Yang P L, Luo H Y, et al. High-level expression of a truncated1,3-1,4-β-D-glucanasefrom Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation [J].Appl Microbiol Biotechnol,2008,78(1):95-103.
[108] Wen T N, Chen J L, Lee S H, et al. A truncated Fibrobacter succinogenes1,3-1,4-β-D-glucanasewith improved enzymatic activity and thermotolerance [J]. Biochem,2005,44(25):9197-9205.
[109] Sumitani J, Tottori T, Kawaguchi T, et al. New type of starch-binding domain: the direct repeatmotif in the C-terminal region of Bacillus sp. no.195alpha-amylase contributes to starch bindingand raw starch degrading [J]. Biochem J,2000,350(Pt2):477-484.
[110] Yen C-C, Malmis C C, Lee G-C, et al. Site-specific saturation mutagenesis on residues132and450of Candida rugosa LIP2enhances catalytic efficiency and alters substrate specificity invarious chain lengths of triglycerides and esters [J]. J Agric Food Chem,2010,58(20):10899-10905.
[111] Hagihara H, Hayashi Y, Endo K, et al. Deduced amino-acid sequence of a calcium-free α-amylasefrom a strain of Bacillus [J]. Eur J Biochem,2001,268(14):3974-3982.