Bacillus lentus CICIM304异淀粉酶的基因克隆、鉴定与表达
|
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。Ca~(2+)、Mg~(2+)、Mn~(2+)、Na~+、K~+、Co~(2+)对该酶具有激活作用；Zn~(2+)、Sn~(2+)、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。
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. Ca~(2+)、Mg~(2+)、Mn~(2+)、Na~+、K~+and Co~(2+)slightly enhanced the enzyme activity as chloridesalts. Zn~(2+), Fe3+and Sn~(2+)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.
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。Ca~(2+)、Mg~(2+)、Mn~(2+)、Na~+、K~+、Co~(2+)对该酶具有激活作用；Zn~(2+)、Sn~(2+)、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。
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. Ca~(2+)、Mg~(2+)、Mn~(2+)、Na~+、K~+and Co~(2+)slightly enhanced the enzyme activity as chloridesalts. Zn~(2+), Fe3+and Sn~(2+)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.
引文
[1] Rindlav-Westling As, Stading M, Hermansson A-M, et al. Structure, mechanical and barrier propertiesof amylose and amylopectin films[J]. Carbohydr Polym,1998,36(2):217-224.
[2] Manners DJ. Recent developments in our understanding of amylopectin structure[J]. Carbohydr Polym,1989,11(2):87-112.
[3] Moore R, Storey R, Uno G. Principles of Botany. In: New York: McGraw Hill;2001.
[4] Gupta R, Gigras P, Mohapatra H, et al. Microbial α-amylases: a biotechnological perspective[J].Process Biochem,2003,38(11):1599-1616.
[5] Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, et al. a-Amylases from microbial sources–anoverview on recent developments[J]. Food Technol Biotechnol,2006,44(2):173-184.
[6] Miguel A N S M, Martins-Meyer T S, Da Costa Figueiredo E R V S, et al. Enzymes in Bakery: Currentand Future Trends [J].2013,
[7] kerberg A, Liljeberg H, Bj rck I. Effects of amylose/amylopectin ratio and baking conditions onresistant starch formation and glycaemic indices[J]. J Cereal Sci,1998,28(1):71-80.
[8] Margareta Leeman A, Karlsson ME, Eliasson A-C, et al. Resistant starch formation in temperaturetreated potato starches varying in amylose/amylopectin ratio[J]. Carbohydr Polym,2006,65(3):306-313.
[9] Lynd LR, Cushman JH, Nichols RJ, et al. Fuel ethanol from cellulosic biomass[J].Science(Washington),1991,251(4999):1318-1323.
[10] Gunja ZH, Manners D, Maung K. Studies on carbohydrate-metabolizing enzymes.7. Yeastisoamylase[J]. Biochem J,1961,81(2):392.
[11] Ishizaki Y, Taniguchi H, Maruyama Y, et al. Debranching enzymes of potato tubers (Solanumtuberosum L.). I. Purification and some properties of potato isoamylase[J]. Agr Biol Chem,1983,47(4):771.
[12] Nakamura Y, Umemoto T, Ogata N, et al. Starch debranching enzyme (R-enzyme or pullulanase) fromdeveloping rice endosperm: purification, cDNA and chromosomal localization of the gene[J]. Planta,1996,199(2):209-218.
[13] Doman-Pytka M, Bardowski J. Pullulan degrading enzymes of bacterial origin[J]. Crit Rev Microbiol,2004,30(2):107-121.
[14] Hatada Y, Igarashi K, Ozaki K, et al. Amino acid sequence and molecular structure of an alkalineamylopullulanase from Bacillus that hydrolyzes α-1,4and α-1,6linkages in polysaccharides atdifferent active sites[J]. J Biol Chem,1996,271(39):24075-24083.
[15] Katsuya Y, Mezaki Y, Kubota M, et al. Three-dimensional structure of Pseudomonas isoamylase at2.2A resolution[J]. J Mol Biol,1998,281(5):885-897.
[16] Akai H, Yokobayashi K, Misaki A, et al. Complete hydrolysis of branching linkages in glycogen byPseudomonas isoamylase: distribution of linear chains[J]. Biochim Biophys Acta (BBA)-GeneralSubjects,1971,237(3):422-429.
[17] Yokobayashi K, Misaki A, Harada T. Purification and properties of pseudomonas isoamylase[J].Biochim Biophys Acta (BBA)-Enzymology,1970,212(3):458-469.
[18] Gunja-Smith Z, Marshall J, Mercier C, et al. A revision of the Meyer-Bernfeld model of glycogen andamylopectin[J]. FEBS letters,1970,12(2):101.
[19] Kobayashi S, Kainuma K, Suzuki S. Purification and some properties of Bacillus maceranscycloamylose (cyclodextrin) glucanotransferase[J]. Carbohydr Research,1978,61(1):229-238.
[20] Horwath RO, Lally JA, Rotheim P. Process for producing. alpha.-1,6glucosidases using thermophilicmicroorganisms. In: Google Patents;1977.
[21] Maruo B, Kobayashi T. Enzymic scission of the branch links in amylopectin[J]. Nature,1951,167,606-607.
[22] Lee EY, Carter J, Nielsen L, et al. Purification and properties of yeast amylo-1,6-glucosidase-oligo-1,4. far.1,4-glucantransferase[J]. Biochemistry,1970,9(11):2347-2355.
[23] Spencer-Martins I. Extracellular isoamylase produced by the yeast Lipomyces kononenkoae[J]. ApplEnviron Microbiol,1982,44(6):1253-1257.
[24] Odibo F, Okafor N, Tom M, et al. Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea[J]. World J Microbiol Biotechnol,1992,8(2):102-105.
[25] Harada T, Yokobayashi K, Misaki A. Formation of isoamylase by Pseudomonas[J]. Appl Microbiol,1968,16(10):1439-1444.
[26] Harada T, Misaki A, Akai H, et al. Characterization of Pseudomonas isoamylase by its actions onamylopectin and glycogen: Comparison with Aerobacter pullulanase[J]. Biochim Biophys Acta (BBA)-Enzymology,1972,268(2):497-505.
[27] Fujita M, Futai M, Amemura A. In vivo expression of the Pseudomonas stutzeri maltotetraose-formingamylase gene (amyP)[J]. J Bacteriol,1990,172(3):1595-1599.
[28] Nielsen GC, Diers IV, Outtrup H, et al. Debranching enzyme product, preparation and use thereof. In:Google Patents;1985.
[29] Van Der Maarel M J, Van Der Veen B, Uitdehaag J, et al. Properties and applications ofstarch-converting enzymes of the α-amylase family [J]. J Biotechnol,2002,94(2):137-55.
[30] Urlaub H, Wober G. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillusamyloliquefaciens[J]. Febs Letters,1975,57(1):1-4.
[31] Yusaku F, Shiosaka S, Ueda S. Isoamylase production by aerobacter aerogenes:(I) Effect of nitrogensource[J]. Soc Biosci Bioeng, Japan.1970,48:8-13.
[32] Spencer-Martins I. Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae[J]. ApplEnviron Microbiol,1982,44(6):1253-1257.
[33] Ara K, Saeki K, Ito S. Purification and characterization of an alkaline isoamylase from an alkalophilicstrain of Bacillus[J]. J Gen Microbiol,1993,139(4):781-786.
[34] Yamada Y, Sato T, Ohya T. Isoamylase and process for producing the same. In: Google Patents;1994.
[35] Castro G, Garcia G, Sineriz F. Extracellular isoamylase produced by Bacillus circulans MIR‐137[J]. JAppl Microbiol,1992,73(6):520-523.
[36] Ma YJ, Lin LL, Chien HR, et al. Efficient utilization of starch by a recombinant strain ofSaccharomyces cerevisiae producing glucoamylase and isoamylase[J]. Biotechnol Appl Biochem.2000,31(1):55-59.
[37] Prayitno N, Archibald A. Distribution of muropeptides in walls of Bacillus subtilis and atemperature-sensitive mutant[J]. World J Microbiol Biotechnol,1996,12(6):579-584.
[38] Ghosh B, Ray R. Saccharification of raw native starches by extracellular isoamylase of Rhizopusoryzae[J]. Biotechnology,2010,9(2):224-228.
[39] Krohn B, Barry G, Kishore G. An isoamylase with neutral pH optimum from a Flavobacterium species:cloning, characterization and expression of the iam gene[J]. Mol Gen Gen,1997,254(5):469-478.
[40] Gomes I, Gomes J, Steiner W. Highly thermostable amylase and pullulanase of the extremethermophilic eubacterium Rhodothermus marinus: production and partial characterization[J]. BioresourTechnol,2003,90(2):207-214.
[41] Woo E-J, Lee S, Cha H, et al. Structural insight into the bifunctional mechanism of theglycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus[J]. J Biol Chem,2008,283(42):28641-28648.
[42] Ueda S, Nanri N. Production of isoamylase by Escherichia intermedia[J]. Appl Microbiol,1967,15(3):492-496.
[43] Jeanningros R, Creuzet-Sigal N, Frixon C, et al. Purification and properties of a debranching enzymefrom Escherichia coli[J]. Biochim Biophys Acta,1976,438(1):186-199.
[44] Kato M. Trehalose production with a new enzymatic system from Sulfolobus solfataricus KM1[J]. JMol Catal B: Enzymatic,1999,6(3):223-233.
[45] Chen JH, Chen ZY, Chow TY, et al. Nucleotide sequence and expression of the isoamylase gene froman isoamylase-hyperproducing mutant, Pseudomonas amyloderamosa JD210[J]. Biochim Biophys Acta,1990,1087(3):309-315.
[46] Wu XC, Lee W, Tran L, et al. Engineering a Bacillus subtilis expression-secretion system with a straindeficient in six extracellular proteases[J]. J Bacteriol,1991,173(16):4952-4958.
[47] Amemura A, Chakraborty R, Fujita M, et al. Cloning and nucleotide sequence of the isoamylase genefrom Pseudomonas amyloderamosa SB-15[J]. J Biol Chem,1988,263(19):9271-9275.
[48] Chen P-H, Lin L-L, Hsu W-H. Expression of Pseudomonas amyloderamosa isoamylase gene inSaccharomyces cerevisiae[J]. Biotechnol Lett,1998,20(8):735-739.
[49] Tognoni A, Carrera P, Galli G, et al. Cloning and nucleotide sequence of the isoamylase gene from astrain of Pseudomonas sp[J]. J Gen Microbiol,1989,135(1):37-45.
[50] Abe J, Ushijima C, Hizukuri S. Expression of the isoamylase gene of Flavobacterium odoratum KU inEscherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis[J].Appl Environ Microbiol,1999,65(9):4163-4170.
[51] Barry G, Kishore G, Krohn B. Isoamylase gene from Flavobacterium sp. compositions containing itand methods using it. Patent CA2195786WO1996/003513. In;1996.
[52] Krohn BM, Barry GF, Kishore GM. An isoamylase with neutral pH optimum from a Flavobacteriumspecies: cloning, characterization and expression of the iam gene[J]. Mol Gen Genet,1997,254(5):469-478.
[53] Cho KM, Kim EJ, Math RK, et al. Cloning of isoamylase gene of Pectobacterium carotovorum subsp.carotovorum LY34and identification of essential residues of enzyme[J]. J Life Sci,2007,17:1182-1190.
[54] Haki G, Rakshit S. Developments in industrially important thermostable enzymes: a review [J].Bioresour Technol,2003,89(1):17-34.
[55] Wu X-C, Lee W, Tran L, et al. Engineering a Bacillus subtilis expression-secretion system with a straindeficient in six extracellular proteases [J]. J Bacteriol,1991,173(16):4952-8.
[56] Gu Rout-Fleury A-M, Frandsen N, Stragier P. Plasmids for ectopic integration in Bacillus subtilis [J].Gene,1996,180(1):57-61.
[57] Wells J A, Ferrari E, Henner D J, et al. Cloning, sequencing, and secretion of Bacillusamyloliquefaciens subtillisin in Bacillus subtilis [J]. Nucleic Acids Research,1983,11(22):7911-25.
[58] Jacobs M, Eliasson M, Uhl N M, et al. Cloning, sequencing and expression of subtilisin Carlsbergfrom Bacillus licheniformis [J]. Nucleic Acids Res,1985,13(24):8913-26.
[59] Kobayashi K, Ehrlich SD, Albertini A, et al. Essential Bacillus subtilis genes[J]. P Natl A Sci,2003,100(8):4678-4683.
[60] Wong S-L. Advances in the use of Bacillus subtilis for the expression and secretion of heterologousproteins[J]. Curr Opin Biotechnol,1995,6(5):517-522.
[61] Zhang X-Z, Cui Z-L, Hong Q, et al. High-Level expression and secretion of methyl parathionhydrolase in Bacillus subtilis WB800[J]. Appl Environ Microbiol,2005,71(7):4101-4103.
[62] Cho H-Y, Yukawa H, Inui M, et al. Production of minicellulosomes from Clostridium cellulovorans inBacillus subtilis WB800[J]. Appl Environ Microbiol,2004,70(9):5704-5707.
[63] Vagner V, Dervyn E, Ehrlich SD. A vector for systematic gene inactivation in Bacillus subtilis[J].Microbiology,1998,144(11):3097-3104.
[64] Koehler TM. Bacillus anthracis genetics and virulence gene regulation[J]. Curr Top MicrobiolImmunol,2002,271:143-164.
[65] Dai Z, Sirard JC, Mock M, et al. The atxA gene product activates transcription of the anthrax toxingenes and is essential for virulence[J]. Mol Microbiol,2006,16(6):1171-1181.
[66] Brown DP, Ganova‐Raeva L, Green BD, et al. Characterization of spo0A homologues in diverseBacillus and Clostridium species identifies a probable DNA‐binding domain[J]. Mol Microbiol,2006,14(3):411-426.
[67] Cendrowski S, Macarthur W, Hanna P. Bacillus anthracis requires siderophore biosynthesis for growthin macrophages and mouse virulence [J]. Mol Microbiol,2004,51(2):407-17.
[68] Biswas I, Gruss A, Ehrlich SD, et al. High-efficiency gene inactivation and replacement system forgram-positive bacteria[J]. J Bacteriol,1993,175(11):3628-3635.
[69] Weisblum B, Graham MY, Gryczan T, et al. Plasmid copy number control: isolation andcharacterization of high-copy-number mutants of plasmid pE194[J]. J Bacteriol,1979,137(1):635-643.
[70] Thorne CB, Stull HB. Factors affecting transformation of Bacillus licheniformis[J]. J Bacteriol,1966,91(3):1012-1020.
[71] Hanahan D. Studies on transformation of Escherichia coli with plasmids [J]. J Mol Biol,1983,166(4):557-80.
[72] Bott KF, Wilson GA. Development of competence in the Bacillus subtilis transformation system[J]. Jof Bacteriol,1967,94(3):562-570.
[73] Vehmaanper J. Transformation of Bacillus amyloliquefaciens by electroporation[J]. FEMS MicrobiolLett,1989,61(1–2):165-169.
[74] Waschkau B, Waldeck J, Wieland S, et al. Generation of readily transformable Bacillus licheniformismutants[J]. Appl Microbiol Biotechnol,2008,78(1):181-188.
[75] Chang S, Cohen S. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA[J].Mol Gen Gen,1979,168(1):111-115.
[76] Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Anal Chem,1959,31(3):426-8.
[77] Kuzminov A. DNA replication meets genetic exchange: Chromosomal damage and its repair byhomologous recombination[J]. P Natl A Sci,2001,98(15):8461-8468.
[78] Kogoma T. Stable DNA replication: interplay between DNA replication, homologous recombination,and transcription[J]. Microbiol Mol Biol Rev,1997,61(2):212-238.
[79] Maguin E, Prévost H, Ehrlich SD, et al. Efficient insertional mutagenesis in lactococci and othergram-positive bacteria[J]. J Bacteriol,1996,178(3):931-935.
[80] Zakataeva N, Nikitina O, Gronskiy S, et al. A simple method to introduce marker-free geneticmodifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains[J].Appl Microbiol Biotechnol,2010,85(4):1201-1209.
[81] Maguin E, Duwat P, Hege T, et al. New thermosensitive plasmid for gram-positive bacteria[J]. JBacteriol,1992,174(17):5633-5638.
[82] Framson PE, Nittayajarn A, Merry J, et al. New genetic techniques for group B streptococci:high-efficiency transformation, maintenance of temperature-sensitive pWV01plasmids, andmutagenesis with Tn917[J]. Appl Environ Microbiol,1997,63(9):3539-3547.
[83] Gutierrez JA, Crowley PJ, Brown DP, et al. Insertional mutagenesis and recovery of interrupted genesof Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displayingacid sensitivity and nutritional requirements[J]. J Bacteriol,1996,178(14):4166-4175.
[84] McCleary BV, Codd R. Measurement of β-amylase in cereal flours and commercial enzymepreparations[J]. J Cereal Sci,1989,9(1):17-33.
[85] Lizotte PA, Henson CA, Duke SH. Purification and characterization of pea epicotyl β-amylase[J].Plant Physiol,1990,92(3):615-621.
[86] Matsuura Y, Kusunoki M, Harada W, et al. Structure and possible catalytic residues of taka-amylaseA[J]. J Biochem,1984,95(3):697-702.
[87] Suganuma T, Matsuno R, Ohnishi M, et al. A study of the mechanism of action of taka-amylase A onlinear oligosaccharides by product analysis and computer simulation[J]. J Biochem,1978,84(2):293-316.
[88] Hyun HH, Zeikus JG. General biochemical characterization of thermostable extracellular β-amylasefrom Clostridium thermosulfurogenes[J]. Appl Environ Microbiol,1985,49(5):1162-1167.
[89] Mikami B, Adachi M, Kage T, et al. Structure of raw starch-digesting Bacillus cereus β-amylasecomplexed with maltose[J]. Biochemistry,1999,38(22):7050-7061.
[90] Kim IC, Cha JH, Kim JR, et al. Catalytic properties of the cloned amylase from Bacilluslicheniformis[J]. J Biol Chem,1992,267(31):22108-22114.
[91] Sauer S, Kliem M. Mass spectrometry tools for the classification and identification of bacteria[J]. NatRev Microbiol,2010,8(1):74-82.
[92] Facklam R, Elliott J. Identification, classification, and clinical relevance of catalase-negative,gram-positive cocci, excluding the streptococci and enterococci[J]. Clin Microbiol Rev,1995,8(4):479-495.
[93] Petti C, Polage C, Schreckenberger P. The role of16S rRNA gene sequencing in identification ofmicroorganisms misidentified by conventional methods[J]. J Clin Microbiol,2005,43(12):6123-6125.
[94] Klijn N, Weerkamp AH, de Vos WM. Identification of mesophilic lactic acid bacteria by usingpolymerase chain reaction-amplified variable regions of16S rRNA and specific DNA probes[J]. ApplEnviron Microbiol,1991,57(11):3390-3393.
[95] Laguerre G, Allard M-R, Revoy F, et al. Rapid identification of rhizobia by restriction fragment lengthpolymorphism analysis of PCR-amplified16S rRNA genes[J]. Appl Environ Microbiol,1994,60(1):56-63.
[96] Xu D, C té J-C. Phylogenetic relationships between Bacillus species and related genera inferred fromcomparison of3′end16S rDNA and5′end16S–23S ITS nucleotide sequences[J]. Int J Sys EvolMicrobiol,2003,53(3):695-704.
[97] Relman D, Schmidt T, MacDermott R, et al. Identification of the uncultured bacillus of Whipple'sdisease[J]. New Eng J Med,1992,327(5):293.
[98] Sachidanandam R, Weissman D, Schmidt SC, et al. A map of human genome sequence variationcontaining1.42million single nucleotide polymorphisms[J]. Nature,2001,409(6822):928-933.
[99] Abbott A. And now for the proteome[J]. Nature,2001,409(6822):747-747.
[100] Miranker A, Robinson CV, Radford SE, et al. Detection of transient protein folding populations bymass spectrometry[J]. Science (New York, NY),1993,262(5135):896.
[101] Kussmann M, Nordhoff E, Rahbek‐Nielsen H, et al. Matrix‐assisted laser desorption/ionizationmass spectrometry sample preparation techniques designed for various peptide and protein analytes[J].J Mass Spectr,1998,32(6):593-601.
[102] Dan S, Marton I, Dekel M, et al. Cloning, expression, characterization, and nucleophile identificationof family3, Aspergillus niger β-Glucosidase[J]. J Biol Chem,2000,275(7):4973-4980.
[103] Wilson GA, Young FE. Isolation of a sequence-specific endonuclease (BamI) from Bacillusamyloliquefaciens H[J]. J. Mol Biol,1975,97:123-125.
[104] Weisburg WG, Barns SM, Pelletier DA, et al.16S ribosomal DNA amplification for phylogeneticstudy[J]. J Bacteriol,1991,173(2):697-703.
[105] Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patternsproduced by pulsed-field gel electrophoresis: criteria for bacterial strain typing[J]. J Clin Microbiol,1995,33(9):2233.
[106] Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutationdetection by pyrosequencing[J]. J Mol Diag,2005,7(3):413-421.
[107] Sch gger H, Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for theseparation of proteins in the range from1to100kDa[J]. Anal Biochem,1987,166(2):368.
[108] Rosenfeld J, Capdevielle Jl, Guillemot JC, et al. In-gel digestion of proteins for internal sequenceanalysis after one-or two-dimensional gel electrophoresis[J]. Anal Biochem,1992,203(1):173.
[109] Wen B, Doneanu CE, Gartner CA, et al. Fluorescent photoaffinity labeling of cytochrome P4503A4by lapachenole: identification of modification sites by mass spectrometry[J]. Biochemistry.2005,44(6):1833-1845.
[110] Ma M, Wang J, Chen R, et al. Expanding the crustacean neuropeptidome using a multi-faceted massspectrometric approach[J]. J Proteome Res,2009,8(5):2426.
[111] Nielsen P, Rainey F A, Outtrup H, et al. Comparative16S rDNA sequence analysis of somealkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus [J]. FemsMicrobiol Lett,1994,117(1):61-5.
[112] Kuhn P, Knapp M, Soltis SM, et al. The0.78structure of a serine protease: Bacillus lentussubtilisin[J]. Biochemistry,1998,37(39):13446-13452.
[113] Betzel C, Klupsch S, Papendorf G, et al. Crystal structure of the alkaline proteinase Savinase fromBacillus lentus at1.4resolution[J]. J Mol Biol,.1992,223(2):427-445.
[114] Sabioni JG, Park YK. Production and characterization of cyclodextrin glycosyltransferase fromBacillus lentus[J]. Starch‐St rke,2006,44(6):225-229.
[115] Zhang X, Huang C, Tang X, et al. Identification of structural proteins from shrimp white spotsyndrome virus (WSSV) by2DE-MS[J]. Proteins: Struc Func Bioinform,2004,55(2):229-235.
[116] Ou K, Kesuma D, Ganesan K, et al. Quantitative profiling of drug-associated proteomic alterationsby combined2-nitrobenzenesulfenyl chloride (nbs) isotope labeling and2DE/MS identification[J]. JProteome Res,2006,5(9):2194-2206.
[117] Peng J, Elias JE, Thoreen CC, et al. evaluation of multidimensional chromatography coupled withtandem mass spectrometry (LC/LC MS/MS) for large-scale protein analysis: the yeast proteome[J]. JProteome Res,2002,2(1):43-50.
[118] Jemal M. High-throughput quantitative bioanalysis by LC/MS/MS[J]. Biomed Chromatog,2000,14(6):422-429.
[119] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Matrix effect in quantitative LC/MS/MSanalyses of biological fluids: a method for determination of finasteride in human plasma at picogramper milliliter concentrations[J]. Anal Chem,1998,70(5):882-889.
[120] Washburn MP, Ulaszek R, Deciu C, et al. Analysis of quantitative proteomic data generated viamultidimensional protein identification technology[J]. Anal Chem,2002,74(7):1650-1657.
[121] Wolters DA, Washburn MP, Yates JR. An automated multidimensional protein identificationtechnology for shotgun proteomics[J]. Anal Chem,2001,73(23):5683-5690.
[122] Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome by multidimensionalprotein identification technology[J]. Nature Biotechnol,2001,19(3):242-247.
[123] Keller A, Nesvizhskii AI, Kolker E, et al. Empirical statistical model to estimate the accuracy ofpeptide identifications made by MS/MS and database search[J]. Anal Chem,2002,74(20):5383-5392.
[124] Korfmacher WA, Cox KA, Bryant MS, et al. HPLC-API/MS/MS: a powerful tool for integratingdrug metabolism into the drug discovery process[J]. Drug Discovery Today,1997,2(12):532-537.
[125] Bairoch A, Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in2000[J]. Nucleic Acids Res,2000,28(1):45-48.
[126] Bairoch A, Boeckmann B. The SWISS-PROT protein sequence data bank[J]. Nucleic Acids Res,1992,20(suppl):2019-2022.
[127] Wong JH, Balmer Y, Cai N, et al. Unraveling thioredoxin-linked metabolic processes of cerealstarchy endosperm using proteomics[J]. FEBS Lett,2003,547(1–3):151-156.
[128] Severin A, Nickbarg E, Wooters J, et al. Proteomic analysis and identification of streptococcuspyogenes surface-associated proteins[J]. J Bacteriol,2007,189(5):1514-1522.
[129] Xu SB, Li T, Deng ZY, et al. Dynamic proteomic analysis reveals a switch between central carbonmetabolism and alcoholic fermentation in rice filling grains[J]. Plant Physiol,2008,148(2):908-925.
[130] Dahiyat B I, Mayo S L. De novo protein design: fully automated sequence selection [J]. Science,1997,278(5335):82-7.
[131] Dancik V, Addona T A, Clauser K R, et al. De novo peptide sequencing via tandem massspectrometry [J]. J Comput Biol,1999,6(3-4):327-42.
[132] Ochman H, Gerber A S, Hartl D L. Genetic applications of an inverse polymerase chain reaction [J].Genetics,1988,120(3):621-3.
[133] Shobha MS, Tharanathan RN. Nonspecific activity of Bacillus acidopullulyticus pullulanase ondebranching of guar galactomannan[J]. J Agr Food Chem,2008,56(22):10858-10864.
[134] Matuschek M, Burchhardt G, Sahm K, et al. Pullulanase of Thermoanaerobacteriumthermosulfurigenes EM1(Clostridium thermosulfurogenes): molecular analysis of the gene, compositestructure of the enzyme, and a common model for its attachment to the cell surface[J]. J Bacteriol,1994,176(11):3295-3302.
[135] Albertson GD, McHale RH, Gibbs MD, et al. Cloning and sequence of a type I pullulanase from anextremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus[J]. Biochimica etBiophysica Acta (BBA)-Gene Struc Expre,1997,1354(1):35-39.
[136] Bertoldo C, Duffner F, Jorgensen PL, et al. Pullulanase Type I from Fervidobacterium pennavoransVen5: Cloning, sequencing, and expression of the gene and biochemical characterization of therecombinant enzyme[J]. Appl Environ Microbiol,1999,65(5):2084-2091.
[137] Ben Messaoud E, Ben Ammar Y, Mellouli L, et al. Thermostable pullulanase type I from newisolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli[J].Enzym Microb Technol,2002,31(6):827-832.
[138] Zouari Ayadi D, Ben Ali M, Jemli S, et al. Heterologous expression, secretion and characterization ofthe Geobacillus thermoleovorans US105type I pullulanase[J]. Appl Microbiol Biotechnol,2008,78(3):473-481.
[139] Kuriki T, Park J-H, Okada S, et al. Purification and characterization of thermostable pullulanase fromBacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis[J].Appl Environ Microbiol,1988,54(11):2881-2883.
[140] Urlaub H, Wober G. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillusamyloliquefaciens[J]. FEBS Lett,1975,57(1):1-4.
[141] Gunja ZH, Manners DJ, Khin M. Studies on carbohydrate-metabolizing enzymes.3. Yeast branchingenzyme[J]. Biochem J,1960,75:441-450.
[142] Kitagawa H, Amemura A, Harada T. Studies on the inhibition and molecular properties of crystallinePseudomonas isoamylase[J]. Agr Biol Chem,1975,39.
[143] Marshall JJ. Inhibition of pullulanase by Schardinger dextrins[J]. FEBS Lett,1973,37(2):269-273.
[144] Jeon JH, Kim S-J, Lee HS, et al. Novel metagenome-derived carboxylesterase that hydrolyzesβ-lactam antibiotics[J].Appl Environ Microbiol,2011,77(21):7830-7836.
[145] Gruber AR, Lorenz R, Bernhart SH, et al. The Vienna RNA websuite[J]. Nucleic Acids Res,2008,36(suppl2): W70-W74.
[146] Marchler-Bauer A, Lu S, Anderson JB, et al. CDD: a Conserved Domain Database for the functionalannotation of proteins[J]. Nucleic Acids Res,2011,39(suppl1): D225-D229.
[147] Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on aminoacid sequence similarities[J]. Biochem J,1993,293(Pt3):781-788.
[148] Wiatrowski HA, van Denderen BJW, Berkey CD, et al. Mutations in the Gal83glycogen-bindingdomain activate the Snf1/Gal83kinase pathway by a glycogen-independent mechanism[J]. Mol CelBiol,2004,24(1):352-361.
[149] Polekhina G, Gupta A, Michell BJ, et al. AMPK beta subunit targets metabolic stress sensing toglycogen[J]. Curr Biol,2003,13(10):867-871.
[150] Hudson ER, Pan DA, James J, et al. A novel domain in AMP-activated protein kinase causesglycogen storage bodies similar to those seen in hereditary cardiac arrhythmias[J]. Curr Biol,2003,13(10):861-866.
[151] Rodríguez-Sanoja R, Oviedo N, Sánchez S. Microbial starch-binding domain[J]. Curr OpinMicrobiol,2005,8(3):260-267.
[152] Hatada Y, Igarashi K, Ozaki K, et al. Amino acid sequence and molecular structure of an alkalineamylopullulanase from Bacillus that hydrolyzes α-1,4and α-1,6linkages in polysaccharides at differentactive sites[J]. J Biol Chem,1996,271(39):24075-24083.
[153] Lévêque E, Jane ek, Haye B, et al. Thermophilic archaeal amylolytic enzymes[J]. Enzye MicrobTechnol,2000,26(1):3-14.
[154] Nakajima R, Imanaka T, Aiba S. Comparison of amino acid sequences of eleven differentα-amylases[J].Appl Microbiol Biotechnol,1986,23(5):355-360.
[155] Mathupala SP, Lowe SE, Podkovyrov SM, et al. Sequencing of the amylopullulanase (apu) gene ofThermoanaerobacter ethanolicus39E, and identification of the active site by site-directedmutagenesis[J]. J Biol Chem,1993,268(22):16332-16344.
[156] Yamashita M, Matsumoto D, Murooka Y. Amino acid residues specific for the catalytic actiontowards α-1,6-glucosidic linkages in Klebsiella pullulanase[J]. J Ferment Bioeng,1997,84(4):283-290.
[157] Fang TY, Tseng WC, Yu CJ, et al. Characterization of the thermophilic isoamylase from thethermophilic archaeon Sulfolobus solfataricus ATCC35092[J]. J Mol Catal B-Enzymatic,2005,33(3-6):99-107.
[158] Saha BC, Zeikus JG. Novel highly thermostable pullulanase from thermophiles[J]. Trends Biotechnol,1989,7(9):234-239.
[159] Chica R A, Doucet N, Pelletier J N. Semi-rational approaches to engineering enzyme activity:combining the benefits of directed evolution and rational design [J]. Curr Opin Biotechnol,2005,16(4):378-84.
[160] Uitdehaag J C, Mosi R, Kalk K H, et al. X-ray structures along the reaction pathway of cyclodextringlycosyltransferase elucidate catalysis in the α-amylase family [J]. Nat Struc Mol Biol,1999,6(5):432-6.
[161] Knegtel R M, Strokopytov B, Penninga D, et al. Crystallographic studies of the interaction ofcyclodextrin glycosyltransferase from Bacillus circulans strain251with natural substrates andproducts [J]. J Biol Chem,1995,270(49):29256-64.
[162] van der Veen BA, Leemhuis H, Kralj S, et al. Hydrophobic amino acid residues in the acceptorbinding site are main determinants for reaction mechanism and specificity ofcyclodextrin-glycosyltransferase[J]. J Biol Chem,2001,276(48):44557-44562.
[163] Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformis α-amylasethrough introduction of hydrophobic residues at the surface[J]. J Biol Chem,2003,278(13):11546-11553.
[164] Suzuki Y, Hatagaki K, Oda H. A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP1228, and evidence for the proline theory of increasing proteinthermostability[J]. Appl Microbiol Biotechnol,1991,34(6):707-714.
[165] Yang A-S, Honig B. On the pH dependence of protein stability[J]. J Mol Biol,1993,231(2):459-474.
[166] Gaidenko TA, Price CW. General stress transcription factor B and sporulation transcription factor Heach contribute to survival of Bacillus subtilis under extreme growth conditions[J]. J Bacteriol,1998,180(14):3730-3733.
[167] Darland G, BrocK TD. Bacillus acidocaldarius sp.nov., an acidophilic thermophilic spore-formingbacterium[J]. J Gen Microbiol,1971,67(1):9-15.
[168] Kashima Y, Udaka S. High-level production of hyperthermophilic cellulase in the Bacillus brevisexpression and secretion system[J]. Biosci Biotechnol Biochem,2004,68(1):235-237.
[169] Al-Quadan F, Akel H, Natshi R. Characteristics of a novel highly thermostable and extremelythermophilic alkalitolerant amylase from hyperthermophilic Bacillus Strain HUTBS71[J]. Online J BiolSci,9(3):67-74.
[170] Davail S, Feller G, Narinx E, et al. Cold adaptation of proteins. Purification, characterization, andsequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41[J]. J Biol Chem,1994,269(26):17448-17453.
[171] Forsyth G, Logan NA. Isolation of Bacillus thuringiensis from northern victoria land, antarctica[J].Lett Appl Microbiol,2000,30(3):263-266.
[172] Boer A, Priest F, Diderichsen B. On the industrial use of Bacillus licheniformis: a review[J]. ApplMicrobiol Biotechnol,1994,40(5):595-598.
[173] Zegers ND, Kluter E, Van Der Stap H, et al. Expression of the protective antigen of Bacillusanthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax[J]. J ApplMicrobiol,1999,87(2):309-314.
[174] Green S, Zeiger E, Palmer KA, et al. Protocols for the dominant lethal test, host‐mediated assay,and in vivo cytogenetic test used in the food and drug administration's review of substances in theGRAS (generally recognized as safe) list[J]. J Tox Environ Health,1976,1(6):921-928.
[175] Imanaka T, Tanaka T, Tsunekawa H, et al. Cloning of the genes for penicillinase, penP and penI, ofBacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillussubtilis, and Bacillus licheniformis[J]. J Bacteriol,1981,147(3):776-786.
[176] Fujii M, Takagi M, Imanaka T, et al. Molecular cloning of a thermostable neutral protease gene fromBacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus andBacillus subtilis[J]. J Bacteriol,1983,154(2):831-837.
[177] Jacobs MF. Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis andBacillus subtilis[J]. Gene,1995,152(1):69-74.
[178] Ishiwa H, Tsuchida N. New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Constructionand characterization of plasmid pHY460with twelve unique cloning sites[J]. Gene,1984,32(1-2):129-134.
[179] Suter-Crazzolara C, Unsicker K. Improved expression of toxic proteins in E. coli[J]. BioTechniques,1995,19(2):202-204.
[180] Tabor S. Expression using the T7RNA polymerase/promoter system. In: Current Protocols inMolecular Biology: John Wiley&Sons, Inc.;2001.
[181] S rensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm ofEscherichia coli[J]. Microb Cell Fact,2005,4(1):1.
[182] Palva I. Molecular cloning of α-amylase gene from Bacillus amyloliquefaciens and its expression inB. subtilis[J]. Gene,1982,19(1):81-87.
[183] Osborn MJ. Structure and biosynthesis of the bacterial cell Wall[J]. Annu Rev Biochem,1969,38(1):501-538.
[184] Brigidi P, Rossi E, Riccardi G, et al. A highly efficient electroporation system for transformation ofBacillus licheniformis[J]. Biotechnol Tech,1991,5(1):5-8.
[185] Xue G-P, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiencyof the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis[J]. J Microbiol Method,1999,34(3):183-191.
[186] Feng Y, Yang W, Ong S, et al. Fermentation of starch for enhanced alkaline protease production byconstructing an alkalophilic Bacillus pumilus strain[J]. Appl Microbiol Biotechnol,2001,57(1-2):153-160.
[187] Gessesse A, Gashe B. Production of alkaline protease by an alkaliphilic bacteria isolated from analkaline soda lake[J]. Biotechnol Lett,1997,19(5):479-481.
[188] Guzmán-Maldonado H, Paredes-López O, Biliaderis CG. Amylolytic enzymes and products derivedfrom starch: A review[J]. Crit Rev Food Sci Nutr,1995,35(5):373-403.
[189] Shaw Jei-Fu SJ-R. Production of high-maltose syrup and high-protein flour from rice by anenzymatic method[J]. J Biosci Biotechnol Biochem,1992,56(7):1071-1073.
[190] Volz GW, Caldwell ML. A study of the action of purified amylase from Aspergillus oryzae,taka-amylase[J]. J Biol Chem,1947,171(2):667-674.
[191] Yuan X-L, Kaaij R, Hondel CMJJ, et al. Aspergillus niger genome-wide analysis reveals a largenumber of novel alpha-glucan acting enzymes with unexpected expression profiles[J]. Mol Gen Genom,2008,279(6):545-561.
[2] Manners DJ. Recent developments in our understanding of amylopectin structure[J]. Carbohydr Polym,1989,11(2):87-112.
[3] Moore R, Storey R, Uno G. Principles of Botany. In: New York: McGraw Hill;2001.
[4] Gupta R, Gigras P, Mohapatra H, et al. Microbial α-amylases: a biotechnological perspective[J].Process Biochem,2003,38(11):1599-1616.
[5] Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, et al. a-Amylases from microbial sources–anoverview on recent developments[J]. Food Technol Biotechnol,2006,44(2):173-184.
[6] Miguel A N S M, Martins-Meyer T S, Da Costa Figueiredo E R V S, et al. Enzymes in Bakery: Currentand Future Trends [J].2013,
[7] kerberg A, Liljeberg H, Bj rck I. Effects of amylose/amylopectin ratio and baking conditions onresistant starch formation and glycaemic indices[J]. J Cereal Sci,1998,28(1):71-80.
[8] Margareta Leeman A, Karlsson ME, Eliasson A-C, et al. Resistant starch formation in temperaturetreated potato starches varying in amylose/amylopectin ratio[J]. Carbohydr Polym,2006,65(3):306-313.
[9] Lynd LR, Cushman JH, Nichols RJ, et al. Fuel ethanol from cellulosic biomass[J].Science(Washington),1991,251(4999):1318-1323.
[10] Gunja ZH, Manners D, Maung K. Studies on carbohydrate-metabolizing enzymes.7. Yeastisoamylase[J]. Biochem J,1961,81(2):392.
[11] Ishizaki Y, Taniguchi H, Maruyama Y, et al. Debranching enzymes of potato tubers (Solanumtuberosum L.). I. Purification and some properties of potato isoamylase[J]. Agr Biol Chem,1983,47(4):771.
[12] Nakamura Y, Umemoto T, Ogata N, et al. Starch debranching enzyme (R-enzyme or pullulanase) fromdeveloping rice endosperm: purification, cDNA and chromosomal localization of the gene[J]. Planta,1996,199(2):209-218.
[13] Doman-Pytka M, Bardowski J. Pullulan degrading enzymes of bacterial origin[J]. Crit Rev Microbiol,2004,30(2):107-121.
[14] Hatada Y, Igarashi K, Ozaki K, et al. Amino acid sequence and molecular structure of an alkalineamylopullulanase from Bacillus that hydrolyzes α-1,4and α-1,6linkages in polysaccharides atdifferent active sites[J]. J Biol Chem,1996,271(39):24075-24083.
[15] Katsuya Y, Mezaki Y, Kubota M, et al. Three-dimensional structure of Pseudomonas isoamylase at2.2A resolution[J]. J Mol Biol,1998,281(5):885-897.
[16] Akai H, Yokobayashi K, Misaki A, et al. Complete hydrolysis of branching linkages in glycogen byPseudomonas isoamylase: distribution of linear chains[J]. Biochim Biophys Acta (BBA)-GeneralSubjects,1971,237(3):422-429.
[17] Yokobayashi K, Misaki A, Harada T. Purification and properties of pseudomonas isoamylase[J].Biochim Biophys Acta (BBA)-Enzymology,1970,212(3):458-469.
[18] Gunja-Smith Z, Marshall J, Mercier C, et al. A revision of the Meyer-Bernfeld model of glycogen andamylopectin[J]. FEBS letters,1970,12(2):101.
[19] Kobayashi S, Kainuma K, Suzuki S. Purification and some properties of Bacillus maceranscycloamylose (cyclodextrin) glucanotransferase[J]. Carbohydr Research,1978,61(1):229-238.
[20] Horwath RO, Lally JA, Rotheim P. Process for producing. alpha.-1,6glucosidases using thermophilicmicroorganisms. In: Google Patents;1977.
[21] Maruo B, Kobayashi T. Enzymic scission of the branch links in amylopectin[J]. Nature,1951,167,606-607.
[22] Lee EY, Carter J, Nielsen L, et al. Purification and properties of yeast amylo-1,6-glucosidase-oligo-1,4. far.1,4-glucantransferase[J]. Biochemistry,1970,9(11):2347-2355.
[23] Spencer-Martins I. Extracellular isoamylase produced by the yeast Lipomyces kononenkoae[J]. ApplEnviron Microbiol,1982,44(6):1253-1257.
[24] Odibo F, Okafor N, Tom M, et al. Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea[J]. World J Microbiol Biotechnol,1992,8(2):102-105.
[25] Harada T, Yokobayashi K, Misaki A. Formation of isoamylase by Pseudomonas[J]. Appl Microbiol,1968,16(10):1439-1444.
[26] Harada T, Misaki A, Akai H, et al. Characterization of Pseudomonas isoamylase by its actions onamylopectin and glycogen: Comparison with Aerobacter pullulanase[J]. Biochim Biophys Acta (BBA)-Enzymology,1972,268(2):497-505.
[27] Fujita M, Futai M, Amemura A. In vivo expression of the Pseudomonas stutzeri maltotetraose-formingamylase gene (amyP)[J]. J Bacteriol,1990,172(3):1595-1599.
[28] Nielsen GC, Diers IV, Outtrup H, et al. Debranching enzyme product, preparation and use thereof. In:Google Patents;1985.
[29] Van Der Maarel M J, Van Der Veen B, Uitdehaag J, et al. Properties and applications ofstarch-converting enzymes of the α-amylase family [J]. J Biotechnol,2002,94(2):137-55.
[30] Urlaub H, Wober G. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillusamyloliquefaciens[J]. Febs Letters,1975,57(1):1-4.
[31] Yusaku F, Shiosaka S, Ueda S. Isoamylase production by aerobacter aerogenes:(I) Effect of nitrogensource[J]. Soc Biosci Bioeng, Japan.1970,48:8-13.
[32] Spencer-Martins I. Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae[J]. ApplEnviron Microbiol,1982,44(6):1253-1257.
[33] Ara K, Saeki K, Ito S. Purification and characterization of an alkaline isoamylase from an alkalophilicstrain of Bacillus[J]. J Gen Microbiol,1993,139(4):781-786.
[34] Yamada Y, Sato T, Ohya T. Isoamylase and process for producing the same. In: Google Patents;1994.
[35] Castro G, Garcia G, Sineriz F. Extracellular isoamylase produced by Bacillus circulans MIR‐137[J]. JAppl Microbiol,1992,73(6):520-523.
[36] Ma YJ, Lin LL, Chien HR, et al. Efficient utilization of starch by a recombinant strain ofSaccharomyces cerevisiae producing glucoamylase and isoamylase[J]. Biotechnol Appl Biochem.2000,31(1):55-59.
[37] Prayitno N, Archibald A. Distribution of muropeptides in walls of Bacillus subtilis and atemperature-sensitive mutant[J]. World J Microbiol Biotechnol,1996,12(6):579-584.
[38] Ghosh B, Ray R. Saccharification of raw native starches by extracellular isoamylase of Rhizopusoryzae[J]. Biotechnology,2010,9(2):224-228.
[39] Krohn B, Barry G, Kishore G. An isoamylase with neutral pH optimum from a Flavobacterium species:cloning, characterization and expression of the iam gene[J]. Mol Gen Gen,1997,254(5):469-478.
[40] Gomes I, Gomes J, Steiner W. Highly thermostable amylase and pullulanase of the extremethermophilic eubacterium Rhodothermus marinus: production and partial characterization[J]. BioresourTechnol,2003,90(2):207-214.
[41] Woo E-J, Lee S, Cha H, et al. Structural insight into the bifunctional mechanism of theglycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus[J]. J Biol Chem,2008,283(42):28641-28648.
[42] Ueda S, Nanri N. Production of isoamylase by Escherichia intermedia[J]. Appl Microbiol,1967,15(3):492-496.
[43] Jeanningros R, Creuzet-Sigal N, Frixon C, et al. Purification and properties of a debranching enzymefrom Escherichia coli[J]. Biochim Biophys Acta,1976,438(1):186-199.
[44] Kato M. Trehalose production with a new enzymatic system from Sulfolobus solfataricus KM1[J]. JMol Catal B: Enzymatic,1999,6(3):223-233.
[45] Chen JH, Chen ZY, Chow TY, et al. Nucleotide sequence and expression of the isoamylase gene froman isoamylase-hyperproducing mutant, Pseudomonas amyloderamosa JD210[J]. Biochim Biophys Acta,1990,1087(3):309-315.
[46] Wu XC, Lee W, Tran L, et al. Engineering a Bacillus subtilis expression-secretion system with a straindeficient in six extracellular proteases[J]. J Bacteriol,1991,173(16):4952-4958.
[47] Amemura A, Chakraborty R, Fujita M, et al. Cloning and nucleotide sequence of the isoamylase genefrom Pseudomonas amyloderamosa SB-15[J]. J Biol Chem,1988,263(19):9271-9275.
[48] Chen P-H, Lin L-L, Hsu W-H. Expression of Pseudomonas amyloderamosa isoamylase gene inSaccharomyces cerevisiae[J]. Biotechnol Lett,1998,20(8):735-739.
[49] Tognoni A, Carrera P, Galli G, et al. Cloning and nucleotide sequence of the isoamylase gene from astrain of Pseudomonas sp[J]. J Gen Microbiol,1989,135(1):37-45.
[50] Abe J, Ushijima C, Hizukuri S. Expression of the isoamylase gene of Flavobacterium odoratum KU inEscherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis[J].Appl Environ Microbiol,1999,65(9):4163-4170.
[51] Barry G, Kishore G, Krohn B. Isoamylase gene from Flavobacterium sp. compositions containing itand methods using it. Patent CA2195786WO1996/003513. In;1996.
[52] Krohn BM, Barry GF, Kishore GM. An isoamylase with neutral pH optimum from a Flavobacteriumspecies: cloning, characterization and expression of the iam gene[J]. Mol Gen Genet,1997,254(5):469-478.
[53] Cho KM, Kim EJ, Math RK, et al. Cloning of isoamylase gene of Pectobacterium carotovorum subsp.carotovorum LY34and identification of essential residues of enzyme[J]. J Life Sci,2007,17:1182-1190.
[54] Haki G, Rakshit S. Developments in industrially important thermostable enzymes: a review [J].Bioresour Technol,2003,89(1):17-34.
[55] Wu X-C, Lee W, Tran L, et al. Engineering a Bacillus subtilis expression-secretion system with a straindeficient in six extracellular proteases [J]. J Bacteriol,1991,173(16):4952-8.
[56] Gu Rout-Fleury A-M, Frandsen N, Stragier P. Plasmids for ectopic integration in Bacillus subtilis [J].Gene,1996,180(1):57-61.
[57] Wells J A, Ferrari E, Henner D J, et al. Cloning, sequencing, and secretion of Bacillusamyloliquefaciens subtillisin in Bacillus subtilis [J]. Nucleic Acids Research,1983,11(22):7911-25.
[58] Jacobs M, Eliasson M, Uhl N M, et al. Cloning, sequencing and expression of subtilisin Carlsbergfrom Bacillus licheniformis [J]. Nucleic Acids Res,1985,13(24):8913-26.
[59] Kobayashi K, Ehrlich SD, Albertini A, et al. Essential Bacillus subtilis genes[J]. P Natl A Sci,2003,100(8):4678-4683.
[60] Wong S-L. Advances in the use of Bacillus subtilis for the expression and secretion of heterologousproteins[J]. Curr Opin Biotechnol,1995,6(5):517-522.
[61] Zhang X-Z, Cui Z-L, Hong Q, et al. High-Level expression and secretion of methyl parathionhydrolase in Bacillus subtilis WB800[J]. Appl Environ Microbiol,2005,71(7):4101-4103.
[62] Cho H-Y, Yukawa H, Inui M, et al. Production of minicellulosomes from Clostridium cellulovorans inBacillus subtilis WB800[J]. Appl Environ Microbiol,2004,70(9):5704-5707.
[63] Vagner V, Dervyn E, Ehrlich SD. A vector for systematic gene inactivation in Bacillus subtilis[J].Microbiology,1998,144(11):3097-3104.
[64] Koehler TM. Bacillus anthracis genetics and virulence gene regulation[J]. Curr Top MicrobiolImmunol,2002,271:143-164.
[65] Dai Z, Sirard JC, Mock M, et al. The atxA gene product activates transcription of the anthrax toxingenes and is essential for virulence[J]. Mol Microbiol,2006,16(6):1171-1181.
[66] Brown DP, Ganova‐Raeva L, Green BD, et al. Characterization of spo0A homologues in diverseBacillus and Clostridium species identifies a probable DNA‐binding domain[J]. Mol Microbiol,2006,14(3):411-426.
[67] Cendrowski S, Macarthur W, Hanna P. Bacillus anthracis requires siderophore biosynthesis for growthin macrophages and mouse virulence [J]. Mol Microbiol,2004,51(2):407-17.
[68] Biswas I, Gruss A, Ehrlich SD, et al. High-efficiency gene inactivation and replacement system forgram-positive bacteria[J]. J Bacteriol,1993,175(11):3628-3635.
[69] Weisblum B, Graham MY, Gryczan T, et al. Plasmid copy number control: isolation andcharacterization of high-copy-number mutants of plasmid pE194[J]. J Bacteriol,1979,137(1):635-643.
[70] Thorne CB, Stull HB. Factors affecting transformation of Bacillus licheniformis[J]. J Bacteriol,1966,91(3):1012-1020.
[71] Hanahan D. Studies on transformation of Escherichia coli with plasmids [J]. J Mol Biol,1983,166(4):557-80.
[72] Bott KF, Wilson GA. Development of competence in the Bacillus subtilis transformation system[J]. Jof Bacteriol,1967,94(3):562-570.
[73] Vehmaanper J. Transformation of Bacillus amyloliquefaciens by electroporation[J]. FEMS MicrobiolLett,1989,61(1–2):165-169.
[74] Waschkau B, Waldeck J, Wieland S, et al. Generation of readily transformable Bacillus licheniformismutants[J]. Appl Microbiol Biotechnol,2008,78(1):181-188.
[75] Chang S, Cohen S. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA[J].Mol Gen Gen,1979,168(1):111-115.
[76] Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Anal Chem,1959,31(3):426-8.
[77] Kuzminov A. DNA replication meets genetic exchange: Chromosomal damage and its repair byhomologous recombination[J]. P Natl A Sci,2001,98(15):8461-8468.
[78] Kogoma T. Stable DNA replication: interplay between DNA replication, homologous recombination,and transcription[J]. Microbiol Mol Biol Rev,1997,61(2):212-238.
[79] Maguin E, Prévost H, Ehrlich SD, et al. Efficient insertional mutagenesis in lactococci and othergram-positive bacteria[J]. J Bacteriol,1996,178(3):931-935.
[80] Zakataeva N, Nikitina O, Gronskiy S, et al. A simple method to introduce marker-free geneticmodifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains[J].Appl Microbiol Biotechnol,2010,85(4):1201-1209.
[81] Maguin E, Duwat P, Hege T, et al. New thermosensitive plasmid for gram-positive bacteria[J]. JBacteriol,1992,174(17):5633-5638.
[82] Framson PE, Nittayajarn A, Merry J, et al. New genetic techniques for group B streptococci:high-efficiency transformation, maintenance of temperature-sensitive pWV01plasmids, andmutagenesis with Tn917[J]. Appl Environ Microbiol,1997,63(9):3539-3547.
[83] Gutierrez JA, Crowley PJ, Brown DP, et al. Insertional mutagenesis and recovery of interrupted genesof Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displayingacid sensitivity and nutritional requirements[J]. J Bacteriol,1996,178(14):4166-4175.
[84] McCleary BV, Codd R. Measurement of β-amylase in cereal flours and commercial enzymepreparations[J]. J Cereal Sci,1989,9(1):17-33.
[85] Lizotte PA, Henson CA, Duke SH. Purification and characterization of pea epicotyl β-amylase[J].Plant Physiol,1990,92(3):615-621.
[86] Matsuura Y, Kusunoki M, Harada W, et al. Structure and possible catalytic residues of taka-amylaseA[J]. J Biochem,1984,95(3):697-702.
[87] Suganuma T, Matsuno R, Ohnishi M, et al. A study of the mechanism of action of taka-amylase A onlinear oligosaccharides by product analysis and computer simulation[J]. J Biochem,1978,84(2):293-316.
[88] Hyun HH, Zeikus JG. General biochemical characterization of thermostable extracellular β-amylasefrom Clostridium thermosulfurogenes[J]. Appl Environ Microbiol,1985,49(5):1162-1167.
[89] Mikami B, Adachi M, Kage T, et al. Structure of raw starch-digesting Bacillus cereus β-amylasecomplexed with maltose[J]. Biochemistry,1999,38(22):7050-7061.
[90] Kim IC, Cha JH, Kim JR, et al. Catalytic properties of the cloned amylase from Bacilluslicheniformis[J]. J Biol Chem,1992,267(31):22108-22114.
[91] Sauer S, Kliem M. Mass spectrometry tools for the classification and identification of bacteria[J]. NatRev Microbiol,2010,8(1):74-82.
[92] Facklam R, Elliott J. Identification, classification, and clinical relevance of catalase-negative,gram-positive cocci, excluding the streptococci and enterococci[J]. Clin Microbiol Rev,1995,8(4):479-495.
[93] Petti C, Polage C, Schreckenberger P. The role of16S rRNA gene sequencing in identification ofmicroorganisms misidentified by conventional methods[J]. J Clin Microbiol,2005,43(12):6123-6125.
[94] Klijn N, Weerkamp AH, de Vos WM. Identification of mesophilic lactic acid bacteria by usingpolymerase chain reaction-amplified variable regions of16S rRNA and specific DNA probes[J]. ApplEnviron Microbiol,1991,57(11):3390-3393.
[95] Laguerre G, Allard M-R, Revoy F, et al. Rapid identification of rhizobia by restriction fragment lengthpolymorphism analysis of PCR-amplified16S rRNA genes[J]. Appl Environ Microbiol,1994,60(1):56-63.
[96] Xu D, C té J-C. Phylogenetic relationships between Bacillus species and related genera inferred fromcomparison of3′end16S rDNA and5′end16S–23S ITS nucleotide sequences[J]. Int J Sys EvolMicrobiol,2003,53(3):695-704.
[97] Relman D, Schmidt T, MacDermott R, et al. Identification of the uncultured bacillus of Whipple'sdisease[J]. New Eng J Med,1992,327(5):293.
[98] Sachidanandam R, Weissman D, Schmidt SC, et al. A map of human genome sequence variationcontaining1.42million single nucleotide polymorphisms[J]. Nature,2001,409(6822):928-933.
[99] Abbott A. And now for the proteome[J]. Nature,2001,409(6822):747-747.
[100] Miranker A, Robinson CV, Radford SE, et al. Detection of transient protein folding populations bymass spectrometry[J]. Science (New York, NY),1993,262(5135):896.
[101] Kussmann M, Nordhoff E, Rahbek‐Nielsen H, et al. Matrix‐assisted laser desorption/ionizationmass spectrometry sample preparation techniques designed for various peptide and protein analytes[J].J Mass Spectr,1998,32(6):593-601.
[102] Dan S, Marton I, Dekel M, et al. Cloning, expression, characterization, and nucleophile identificationof family3, Aspergillus niger β-Glucosidase[J]. J Biol Chem,2000,275(7):4973-4980.
[103] Wilson GA, Young FE. Isolation of a sequence-specific endonuclease (BamI) from Bacillusamyloliquefaciens H[J]. J. Mol Biol,1975,97:123-125.
[104] Weisburg WG, Barns SM, Pelletier DA, et al.16S ribosomal DNA amplification for phylogeneticstudy[J]. J Bacteriol,1991,173(2):697-703.
[105] Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patternsproduced by pulsed-field gel electrophoresis: criteria for bacterial strain typing[J]. J Clin Microbiol,1995,33(9):2233.
[106] Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutationdetection by pyrosequencing[J]. J Mol Diag,2005,7(3):413-421.
[107] Sch gger H, Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for theseparation of proteins in the range from1to100kDa[J]. Anal Biochem,1987,166(2):368.
[108] Rosenfeld J, Capdevielle Jl, Guillemot JC, et al. In-gel digestion of proteins for internal sequenceanalysis after one-or two-dimensional gel electrophoresis[J]. Anal Biochem,1992,203(1):173.
[109] Wen B, Doneanu CE, Gartner CA, et al. Fluorescent photoaffinity labeling of cytochrome P4503A4by lapachenole: identification of modification sites by mass spectrometry[J]. Biochemistry.2005,44(6):1833-1845.
[110] Ma M, Wang J, Chen R, et al. Expanding the crustacean neuropeptidome using a multi-faceted massspectrometric approach[J]. J Proteome Res,2009,8(5):2426.
[111] Nielsen P, Rainey F A, Outtrup H, et al. Comparative16S rDNA sequence analysis of somealkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus [J]. FemsMicrobiol Lett,1994,117(1):61-5.
[112] Kuhn P, Knapp M, Soltis SM, et al. The0.78structure of a serine protease: Bacillus lentussubtilisin[J]. Biochemistry,1998,37(39):13446-13452.
[113] Betzel C, Klupsch S, Papendorf G, et al. Crystal structure of the alkaline proteinase Savinase fromBacillus lentus at1.4resolution[J]. J Mol Biol,.1992,223(2):427-445.
[114] Sabioni JG, Park YK. Production and characterization of cyclodextrin glycosyltransferase fromBacillus lentus[J]. Starch‐St rke,2006,44(6):225-229.
[115] Zhang X, Huang C, Tang X, et al. Identification of structural proteins from shrimp white spotsyndrome virus (WSSV) by2DE-MS[J]. Proteins: Struc Func Bioinform,2004,55(2):229-235.
[116] Ou K, Kesuma D, Ganesan K, et al. Quantitative profiling of drug-associated proteomic alterationsby combined2-nitrobenzenesulfenyl chloride (nbs) isotope labeling and2DE/MS identification[J]. JProteome Res,2006,5(9):2194-2206.
[117] Peng J, Elias JE, Thoreen CC, et al. evaluation of multidimensional chromatography coupled withtandem mass spectrometry (LC/LC MS/MS) for large-scale protein analysis: the yeast proteome[J]. JProteome Res,2002,2(1):43-50.
[118] Jemal M. High-throughput quantitative bioanalysis by LC/MS/MS[J]. Biomed Chromatog,2000,14(6):422-429.
[119] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Matrix effect in quantitative LC/MS/MSanalyses of biological fluids: a method for determination of finasteride in human plasma at picogramper milliliter concentrations[J]. Anal Chem,1998,70(5):882-889.
[120] Washburn MP, Ulaszek R, Deciu C, et al. Analysis of quantitative proteomic data generated viamultidimensional protein identification technology[J]. Anal Chem,2002,74(7):1650-1657.
[121] Wolters DA, Washburn MP, Yates JR. An automated multidimensional protein identificationtechnology for shotgun proteomics[J]. Anal Chem,2001,73(23):5683-5690.
[122] Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome by multidimensionalprotein identification technology[J]. Nature Biotechnol,2001,19(3):242-247.
[123] Keller A, Nesvizhskii AI, Kolker E, et al. Empirical statistical model to estimate the accuracy ofpeptide identifications made by MS/MS and database search[J]. Anal Chem,2002,74(20):5383-5392.
[124] Korfmacher WA, Cox KA, Bryant MS, et al. HPLC-API/MS/MS: a powerful tool for integratingdrug metabolism into the drug discovery process[J]. Drug Discovery Today,1997,2(12):532-537.
[125] Bairoch A, Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in2000[J]. Nucleic Acids Res,2000,28(1):45-48.
[126] Bairoch A, Boeckmann B. The SWISS-PROT protein sequence data bank[J]. Nucleic Acids Res,1992,20(suppl):2019-2022.
[127] Wong JH, Balmer Y, Cai N, et al. Unraveling thioredoxin-linked metabolic processes of cerealstarchy endosperm using proteomics[J]. FEBS Lett,2003,547(1–3):151-156.
[128] Severin A, Nickbarg E, Wooters J, et al. Proteomic analysis and identification of streptococcuspyogenes surface-associated proteins[J]. J Bacteriol,2007,189(5):1514-1522.
[129] Xu SB, Li T, Deng ZY, et al. Dynamic proteomic analysis reveals a switch between central carbonmetabolism and alcoholic fermentation in rice filling grains[J]. Plant Physiol,2008,148(2):908-925.
[130] Dahiyat B I, Mayo S L. De novo protein design: fully automated sequence selection [J]. Science,1997,278(5335):82-7.
[131] Dancik V, Addona T A, Clauser K R, et al. De novo peptide sequencing via tandem massspectrometry [J]. J Comput Biol,1999,6(3-4):327-42.
[132] Ochman H, Gerber A S, Hartl D L. Genetic applications of an inverse polymerase chain reaction [J].Genetics,1988,120(3):621-3.
[133] Shobha MS, Tharanathan RN. Nonspecific activity of Bacillus acidopullulyticus pullulanase ondebranching of guar galactomannan[J]. J Agr Food Chem,2008,56(22):10858-10864.
[134] Matuschek M, Burchhardt G, Sahm K, et al. Pullulanase of Thermoanaerobacteriumthermosulfurigenes EM1(Clostridium thermosulfurogenes): molecular analysis of the gene, compositestructure of the enzyme, and a common model for its attachment to the cell surface[J]. J Bacteriol,1994,176(11):3295-3302.
[135] Albertson GD, McHale RH, Gibbs MD, et al. Cloning and sequence of a type I pullulanase from anextremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus[J]. Biochimica etBiophysica Acta (BBA)-Gene Struc Expre,1997,1354(1):35-39.
[136] Bertoldo C, Duffner F, Jorgensen PL, et al. Pullulanase Type I from Fervidobacterium pennavoransVen5: Cloning, sequencing, and expression of the gene and biochemical characterization of therecombinant enzyme[J]. Appl Environ Microbiol,1999,65(5):2084-2091.
[137] Ben Messaoud E, Ben Ammar Y, Mellouli L, et al. Thermostable pullulanase type I from newisolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli[J].Enzym Microb Technol,2002,31(6):827-832.
[138] Zouari Ayadi D, Ben Ali M, Jemli S, et al. Heterologous expression, secretion and characterization ofthe Geobacillus thermoleovorans US105type I pullulanase[J]. Appl Microbiol Biotechnol,2008,78(3):473-481.
[139] Kuriki T, Park J-H, Okada S, et al. Purification and characterization of thermostable pullulanase fromBacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis[J].Appl Environ Microbiol,1988,54(11):2881-2883.
[140] Urlaub H, Wober G. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillusamyloliquefaciens[J]. FEBS Lett,1975,57(1):1-4.
[141] Gunja ZH, Manners DJ, Khin M. Studies on carbohydrate-metabolizing enzymes.3. Yeast branchingenzyme[J]. Biochem J,1960,75:441-450.
[142] Kitagawa H, Amemura A, Harada T. Studies on the inhibition and molecular properties of crystallinePseudomonas isoamylase[J]. Agr Biol Chem,1975,39.
[143] Marshall JJ. Inhibition of pullulanase by Schardinger dextrins[J]. FEBS Lett,1973,37(2):269-273.
[144] Jeon JH, Kim S-J, Lee HS, et al. Novel metagenome-derived carboxylesterase that hydrolyzesβ-lactam antibiotics[J].Appl Environ Microbiol,2011,77(21):7830-7836.
[145] Gruber AR, Lorenz R, Bernhart SH, et al. The Vienna RNA websuite[J]. Nucleic Acids Res,2008,36(suppl2): W70-W74.
[146] Marchler-Bauer A, Lu S, Anderson JB, et al. CDD: a Conserved Domain Database for the functionalannotation of proteins[J]. Nucleic Acids Res,2011,39(suppl1): D225-D229.
[147] Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on aminoacid sequence similarities[J]. Biochem J,1993,293(Pt3):781-788.
[148] Wiatrowski HA, van Denderen BJW, Berkey CD, et al. Mutations in the Gal83glycogen-bindingdomain activate the Snf1/Gal83kinase pathway by a glycogen-independent mechanism[J]. Mol CelBiol,2004,24(1):352-361.
[149] Polekhina G, Gupta A, Michell BJ, et al. AMPK beta subunit targets metabolic stress sensing toglycogen[J]. Curr Biol,2003,13(10):867-871.
[150] Hudson ER, Pan DA, James J, et al. A novel domain in AMP-activated protein kinase causesglycogen storage bodies similar to those seen in hereditary cardiac arrhythmias[J]. Curr Biol,2003,13(10):861-866.
[151] Rodríguez-Sanoja R, Oviedo N, Sánchez S. Microbial starch-binding domain[J]. Curr OpinMicrobiol,2005,8(3):260-267.
[152] Hatada Y, Igarashi K, Ozaki K, et al. Amino acid sequence and molecular structure of an alkalineamylopullulanase from Bacillus that hydrolyzes α-1,4and α-1,6linkages in polysaccharides at differentactive sites[J]. J Biol Chem,1996,271(39):24075-24083.
[153] Lévêque E, Jane ek, Haye B, et al. Thermophilic archaeal amylolytic enzymes[J]. Enzye MicrobTechnol,2000,26(1):3-14.
[154] Nakajima R, Imanaka T, Aiba S. Comparison of amino acid sequences of eleven differentα-amylases[J].Appl Microbiol Biotechnol,1986,23(5):355-360.
[155] Mathupala SP, Lowe SE, Podkovyrov SM, et al. Sequencing of the amylopullulanase (apu) gene ofThermoanaerobacter ethanolicus39E, and identification of the active site by site-directedmutagenesis[J]. J Biol Chem,1993,268(22):16332-16344.
[156] Yamashita M, Matsumoto D, Murooka Y. Amino acid residues specific for the catalytic actiontowards α-1,6-glucosidic linkages in Klebsiella pullulanase[J]. J Ferment Bioeng,1997,84(4):283-290.
[157] Fang TY, Tseng WC, Yu CJ, et al. Characterization of the thermophilic isoamylase from thethermophilic archaeon Sulfolobus solfataricus ATCC35092[J]. J Mol Catal B-Enzymatic,2005,33(3-6):99-107.
[158] Saha BC, Zeikus JG. Novel highly thermostable pullulanase from thermophiles[J]. Trends Biotechnol,1989,7(9):234-239.
[159] Chica R A, Doucet N, Pelletier J N. Semi-rational approaches to engineering enzyme activity:combining the benefits of directed evolution and rational design [J]. Curr Opin Biotechnol,2005,16(4):378-84.
[160] Uitdehaag J C, Mosi R, Kalk K H, et al. X-ray structures along the reaction pathway of cyclodextringlycosyltransferase elucidate catalysis in the α-amylase family [J]. Nat Struc Mol Biol,1999,6(5):432-6.
[161] Knegtel R M, Strokopytov B, Penninga D, et al. Crystallographic studies of the interaction ofcyclodextrin glycosyltransferase from Bacillus circulans strain251with natural substrates andproducts [J]. J Biol Chem,1995,270(49):29256-64.
[162] van der Veen BA, Leemhuis H, Kralj S, et al. Hydrophobic amino acid residues in the acceptorbinding site are main determinants for reaction mechanism and specificity ofcyclodextrin-glycosyltransferase[J]. J Biol Chem,2001,276(48):44557-44562.
[163] Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformis α-amylasethrough introduction of hydrophobic residues at the surface[J]. J Biol Chem,2003,278(13):11546-11553.
[164] Suzuki Y, Hatagaki K, Oda H. A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP1228, and evidence for the proline theory of increasing proteinthermostability[J]. Appl Microbiol Biotechnol,1991,34(6):707-714.
[165] Yang A-S, Honig B. On the pH dependence of protein stability[J]. J Mol Biol,1993,231(2):459-474.
[166] Gaidenko TA, Price CW. General stress transcription factor B and sporulation transcription factor Heach contribute to survival of Bacillus subtilis under extreme growth conditions[J]. J Bacteriol,1998,180(14):3730-3733.
[167] Darland G, BrocK TD. Bacillus acidocaldarius sp.nov., an acidophilic thermophilic spore-formingbacterium[J]. J Gen Microbiol,1971,67(1):9-15.
[168] Kashima Y, Udaka S. High-level production of hyperthermophilic cellulase in the Bacillus brevisexpression and secretion system[J]. Biosci Biotechnol Biochem,2004,68(1):235-237.
[169] Al-Quadan F, Akel H, Natshi R. Characteristics of a novel highly thermostable and extremelythermophilic alkalitolerant amylase from hyperthermophilic Bacillus Strain HUTBS71[J]. Online J BiolSci,9(3):67-74.
[170] Davail S, Feller G, Narinx E, et al. Cold adaptation of proteins. Purification, characterization, andsequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41[J]. J Biol Chem,1994,269(26):17448-17453.
[171] Forsyth G, Logan NA. Isolation of Bacillus thuringiensis from northern victoria land, antarctica[J].Lett Appl Microbiol,2000,30(3):263-266.
[172] Boer A, Priest F, Diderichsen B. On the industrial use of Bacillus licheniformis: a review[J]. ApplMicrobiol Biotechnol,1994,40(5):595-598.
[173] Zegers ND, Kluter E, Van Der Stap H, et al. Expression of the protective antigen of Bacillusanthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax[J]. J ApplMicrobiol,1999,87(2):309-314.
[174] Green S, Zeiger E, Palmer KA, et al. Protocols for the dominant lethal test, host‐mediated assay,and in vivo cytogenetic test used in the food and drug administration's review of substances in theGRAS (generally recognized as safe) list[J]. J Tox Environ Health,1976,1(6):921-928.
[175] Imanaka T, Tanaka T, Tsunekawa H, et al. Cloning of the genes for penicillinase, penP and penI, ofBacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillussubtilis, and Bacillus licheniformis[J]. J Bacteriol,1981,147(3):776-786.
[176] Fujii M, Takagi M, Imanaka T, et al. Molecular cloning of a thermostable neutral protease gene fromBacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus andBacillus subtilis[J]. J Bacteriol,1983,154(2):831-837.
[177] Jacobs MF. Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis andBacillus subtilis[J]. Gene,1995,152(1):69-74.
[178] Ishiwa H, Tsuchida N. New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Constructionand characterization of plasmid pHY460with twelve unique cloning sites[J]. Gene,1984,32(1-2):129-134.
[179] Suter-Crazzolara C, Unsicker K. Improved expression of toxic proteins in E. coli[J]. BioTechniques,1995,19(2):202-204.
[180] Tabor S. Expression using the T7RNA polymerase/promoter system. In: Current Protocols inMolecular Biology: John Wiley&Sons, Inc.;2001.
[181] S rensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm ofEscherichia coli[J]. Microb Cell Fact,2005,4(1):1.
[182] Palva I. Molecular cloning of α-amylase gene from Bacillus amyloliquefaciens and its expression inB. subtilis[J]. Gene,1982,19(1):81-87.
[183] Osborn MJ. Structure and biosynthesis of the bacterial cell Wall[J]. Annu Rev Biochem,1969,38(1):501-538.
[184] Brigidi P, Rossi E, Riccardi G, et al. A highly efficient electroporation system for transformation ofBacillus licheniformis[J]. Biotechnol Tech,1991,5(1):5-8.
[185] Xue G-P, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiencyof the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis[J]. J Microbiol Method,1999,34(3):183-191.
[186] Feng Y, Yang W, Ong S, et al. Fermentation of starch for enhanced alkaline protease production byconstructing an alkalophilic Bacillus pumilus strain[J]. Appl Microbiol Biotechnol,2001,57(1-2):153-160.
[187] Gessesse A, Gashe B. Production of alkaline protease by an alkaliphilic bacteria isolated from analkaline soda lake[J]. Biotechnol Lett,1997,19(5):479-481.
[188] Guzmán-Maldonado H, Paredes-López O, Biliaderis CG. Amylolytic enzymes and products derivedfrom starch: A review[J]. Crit Rev Food Sci Nutr,1995,35(5):373-403.
[189] Shaw Jei-Fu SJ-R. Production of high-maltose syrup and high-protein flour from rice by anenzymatic method[J]. J Biosci Biotechnol Biochem,1992,56(7):1071-1073.
[190] Volz GW, Caldwell ML. A study of the action of purified amylase from Aspergillus oryzae,taka-amylase[J]. J Biol Chem,1947,171(2):667-674.
[191] Yuan X-L, Kaaij R, Hondel CMJJ, et al. Aspergillus niger genome-wide analysis reveals a largenumber of novel alpha-glucan acting enzymes with unexpected expression profiles[J]. Mol Gen Genom,2008,279(6):545-561.