Bacillus sp. YX-1中温酸性α-淀粉酶的分离纯化及基因克隆和表达的研究
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摘要
α-淀粉酶(1,4-α-D-葡萄糖苷水解酶,EC3.2.1.1)从淀粉糖链内部水解1,4-α-D-葡萄糖苷键,开发筛选具有各种特性的α-淀粉酶成为目前酶制剂研究领域的一个重要方向。中温酸性α-淀粉酶以其中温和耐酸特性有广泛的应用领域,对于丰富淀粉酶品种具有重要实践价值。同时,认识和探寻微生物酶的耐酸特性和相关机理也有积极的理论意义。本文从筛选新酶的角度入手,从Bacillus sp. YX-1菌株中分离纯化得到一种具有新催化功能的α-淀粉酶,并对该酶的酶学特征和结构基因的克隆与表达等分子酶工程内容进行了研究。
针对实验室保藏的一株编号为YX-1的淀粉酶产酶菌株(该菌株在pH 4.5的固态培养基上表现出水解淀粉产透明圈的特性)进行菌体形态、生理生化以及分子生物学16S rDNA序列的鉴定,初步命名为Bacillus sp.YX-1。同时,通过比对分析发现该菌株16S rDNA序列与B. subtilis, B. licheniformis, B. stearothermophilus以及B. amyloliquefaciens等常见的α-淀粉酶生产菌株16S rDNA序列没有高同源性,而与之同源性较高的其它芽孢杆菌属菌株(Bacillus sp.)却没有相关产酶的报道。从物种进化分析结果来看,Bacillus sp. YX-1是一种与相关同源菌株存在分类差异的淀粉酶产酶新菌株。该菌株16S rDNA序列已在GenBank数据库登记,登记号为DQ883446。
采用紫外诱变的方法针对Bacillus sp.YX-1进行优良产酶菌株的选育,筛选得到一株突变菌株,产酶能力与原始菌株Bacillus sp.YX-1相比有显著提高。通过培养基优化,跟踪突变菌株产酶过程,发现44 h时达到产酶峰值162.24 U/mL,是出发菌株产酶能力的3倍。对其粗酶酶学特性分析表明,与原始菌株产酶酶学性质相似。
针对Bacillus sp.YX-1发酵粗酶进行了分离纯化,经硫酸铵分级沉淀、DEAE- Sepharose Fast Flow阴离子层析、Sephadex G-75凝胶过滤层析等步骤后获得电泳均一的淀粉酶,其酶蛋白的亚基分子量约为58 kDa。纯化后比活由粗酶的17.9 U/mg提高到607 U/mg,纯化倍数为34倍。采用得到的纯酶对可溶性淀粉底物进行水解,并与商品化中温α-淀粉酶比较发现,该酶是一个液化型水解内切酶。
采用纯化得到的Bacillus sp.YX-1α-淀粉酶,针对其主要酶学性质进行了研究。该酶最适反应pH为5.0,在pH 4.5-11.0范围内稳定,具有较好的耐酸特性和广泛的稳定性。该酶最适反应温度为40-50℃,在40-60℃范围内稳定。金属离子中,Ca2+对于酶活性没有显著的影响,Na+对酶活性有激活作用。Bacillus sp.YX-1α-淀粉酶与商品化中温α-淀粉酶比较而言,在pH 4.5-5.0的偏酸性条件下表现出较强的活性及稳定性;pH 5.0时该酶对于多种生淀粉底物也有良好的水解作用;同时,对于15-20%的高浓度玉米生淀粉底物也都能够进行有效的水解。经LC-MASS-MASS分析得到了酶蛋白中两个肽段的氨基酸序列,通过比对发现,该酶与NCBI中已报道的α-淀粉酶序列具有一定的同源性,同时,在氨基酸水平上也存在着差异。因此,该酶是一种具有研究价值和应用前景的中温酸性α-淀粉酶。
通过搜集NCBI数据库中芽孢杆菌属α-淀粉酶氨基酸序列,并根据序列的保守性和同源性设计引物,扩增获得Bacillus sp.YX-1α-淀粉酶的结构基因amy。该基因全长1545 bp,编码514个氨基酸残基。该基因已在GenBank数据库登记,登记号为EU159580。将结构基因全序列amy比对分析发现,Bacillus sp. YX-1中温α-淀粉酶与芽孢杆菌属α-淀粉酶表现出较高的同源性,然而,在表观酶学性质上Bacillus sp. YX-1中温α-淀粉酶表现出较好的耐酸特性和较强的生淀粉底物水解能力。在研究中分别从酶蛋白氨基酸组成、维系酶结构稳定的空间作用力以及酶蛋白结构等多角度来阐述Bacillus sp. YX-1中温α-淀粉酶基因序列与其催化功能(酸性条件下的活性和稳定性)之间的内在联系;并从基因序列角度初步分析了紫外诱变后突变菌株产酶能力提高的机理。
将Bacillus sp.YX-1中温α-淀粉酶结构基因插入表达载体pET28a中并转化相应表达宿主E. coli BL21 (DE3)中成功构建带有目的基因的重组菌株E. coli BL21 (DE3) (pET-28a-amy)。当诱导培养温度从37℃降低至30℃时,表达后形成的可溶性蛋白增加,并确定诱导培养条件为:诱导培养温度30℃,诱导物IPTG浓度0.5 mmol/L。在以上诱导培养条件下,该重组菌粗酶液催化活性为1.52 U/mg。利用His-tag亲和层析分离纯化了表达后的重组蛋白。
α-Amylases (EC3.2.1.1, 1,4-α-D-glucan-glucanohydrolase) are starch-degrading enzymes that catalyze the hydrolysis of internalα-1,4-O-glycosidic bonds in polysaccharides with the retention ofα-anomeric configuration in the products. It is one of the most important industrial enzymes, which can be used in wide number of industrial processes including biofuel, traditional brewing, food and textile.
Now most of the commercial bacterialα-amylases used in the liquefaction step have pH optima at around 6.5 and unstable at low pH, however, natural pH of starch slurry is usually around 4.5, therefore, it is necessary to improve the activity and stability of the enzymes at low pH values for omitting the pH adjustment step. Furthermore, it could supply the theoretical basis for elucidating the acid-stability mechanism of the microorganisms and its metabolites, developing and utilizing the new products by protein engineering. This study reports the purification, characterization and gene expression of a novelα-amylase from a newly isolated Bacillus sp. YX-1.
On the basis of the hydrolysis ability in the starch agar plate (pH 4.5), an isolate YX-1 was selected. The newly isolated strain was identified as a Bacillus species according to the characteristic of this strain and the 16S rDNA sequence. Moreover, a comparison of 16S rDNA sequence of this strain with other related Bacillus sp. shows that the 16S rDNA sequence of this strain has no high similarity with B. subtilis, B. licheniformis, B. stearothermophilus and B. amyloliquefaciens, which are the usualα-amylase-producing strains for industrial applications. The 16S rDNA sequence of the novel isolatedα-amylase-producing strain YX-1 has been deposited into the GenBank database under accession number DQ883446.
YX-1 was used as the parent strain to increase its enzyme production capacity by the method of UV mutation. After several screen procedures, one mutant strain with higher enzyme-producing ability was selected. The activity was about 200% higher than that of the parent strain. The crude enzyme of the mutant was similar with that of the parent strain which could hydrolyze starch substrate in acidic condition.
The amylase from Bacillus sp. YX-1 was purified reaching 34 folds of electrophoretic homogeneity by sequential ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. The enzyme had a molecular weight of 58kDa estimated by SDS-PAGE. The action mode of the enzyme was evaluated by studying the hydrolysis performance of soluble starch at pH 4.5 and 60℃. The purified amylase could convert starch substrate to low molecular oligosaccharides with rapid blue color reduction in iodine-starch reaction and appearance of reducing sugar, indicating an endo-type activity of this amylase.
The purifiedα-amylase exhibited the maximum activity at pH 5.0 and 40-50℃. Furthermore, the enzyme performed stable over the pH range of 4.5-11.0 and at 60℃. The enzyme preparation could hydrolyze various raw starches very well at pH 5.0, which was around the saccharification pH of industrial application. It is worth noting that the enzyme exhibits a high digestibility towards raw corn starch granule of concentrations ranging from 15 to 20%, omitting an energy intensive gelatinization step. These features are very important for industrial starch liquefaction. From the viewpoints of application, the optimum activity of the enzyme at low pH values and its suitable thermostability made it favorable in industrial operations for traditional fermentation and food processing. The amino acid sequences of two peptides from the purified enzyme were analyzed by LC-MASS-MASS, and the enzyme did not show strict identity to theα-amylases reported for some differences in amino acid residues.
To further investigate this enzyme, theα-amylase from Bacillus sp. YX-1 was subsequently cloned, sequenced and expressed in E. coli BL21. Theα-amylase gene (amy) was amplified using genomic DNA from Bacillus sp. YX-1 as a template and the specific primers based on theα-amylase genes of Bacillus species from the GenBank database. After cloning the gene encoding the enzyme, sequencing analysis revealed that the sequence contains one complete open reading frame with a length of 1545 base pairs and encodes a protein of 514 amino acid residues with a calculated molecular weight of 58 kDa plus a signal peptide of 31 amino acids. The nucleotide sequence of the Bacillus sp. YX-1α-amylase has been deposited in the GenBank database under accession number EU159580. The amino acid composition of the sequence and its relevant non-covalent action, the secondary structure analysis, the three dimensional structure prediction were utilized respectively to elucidate the enzymatic properties of this enzyme. The principle on the development of enzyme activity with UV mutation was also analyzed.
An expression vector, pET-28a-amy, containing theα-amylase gene (amy) inserted into pET-28a was constructed, and E. coli BL21 (DE3) was transformed with the ligation mixture to yield the transformant E. coli BL21 (DE3) (pET-28a-amy). SDS-PAGE analysis of expressed recombinant protein revealed that most of recombinant protein existed as inclusion body when expressed at 37℃with 0.5 mmol/L IPTG induction, while at 30℃, the amount of soluble protein increased, and thus the expression conditions were determined as cultivation temperature 30℃and IPTG 0.5 mmol/L. Under those conditions, the cell-free extract from recombinant E. coli showed a specificα-amylase activity of 1.52 U/mg.
The recombinant enzyme of N-terminal His6-tag protein was purified from the cell-free extract of E. coli transformant using a HisTrap HP affinity column to an apparent homogeneity on SDS-PAGE. The recombinant enzyme displayed physical-chemical characteristics similar to those observed for the native enzyme from Bacillus sp. YX-1.
针对实验室保藏的一株编号为YX-1的淀粉酶产酶菌株(该菌株在pH 4.5的固态培养基上表现出水解淀粉产透明圈的特性)进行菌体形态、生理生化以及分子生物学16S rDNA序列的鉴定,初步命名为Bacillus sp.YX-1。同时,通过比对分析发现该菌株16S rDNA序列与B. subtilis, B. licheniformis, B. stearothermophilus以及B. amyloliquefaciens等常见的α-淀粉酶生产菌株16S rDNA序列没有高同源性,而与之同源性较高的其它芽孢杆菌属菌株(Bacillus sp.)却没有相关产酶的报道。从物种进化分析结果来看,Bacillus sp. YX-1是一种与相关同源菌株存在分类差异的淀粉酶产酶新菌株。该菌株16S rDNA序列已在GenBank数据库登记,登记号为DQ883446。
采用紫外诱变的方法针对Bacillus sp.YX-1进行优良产酶菌株的选育,筛选得到一株突变菌株,产酶能力与原始菌株Bacillus sp.YX-1相比有显著提高。通过培养基优化,跟踪突变菌株产酶过程,发现44 h时达到产酶峰值162.24 U/mL,是出发菌株产酶能力的3倍。对其粗酶酶学特性分析表明,与原始菌株产酶酶学性质相似。
针对Bacillus sp.YX-1发酵粗酶进行了分离纯化,经硫酸铵分级沉淀、DEAE- Sepharose Fast Flow阴离子层析、Sephadex G-75凝胶过滤层析等步骤后获得电泳均一的淀粉酶,其酶蛋白的亚基分子量约为58 kDa。纯化后比活由粗酶的17.9 U/mg提高到607 U/mg,纯化倍数为34倍。采用得到的纯酶对可溶性淀粉底物进行水解,并与商品化中温α-淀粉酶比较发现,该酶是一个液化型水解内切酶。
采用纯化得到的Bacillus sp.YX-1α-淀粉酶,针对其主要酶学性质进行了研究。该酶最适反应pH为5.0,在pH 4.5-11.0范围内稳定,具有较好的耐酸特性和广泛的稳定性。该酶最适反应温度为40-50℃,在40-60℃范围内稳定。金属离子中,Ca2+对于酶活性没有显著的影响,Na+对酶活性有激活作用。Bacillus sp.YX-1α-淀粉酶与商品化中温α-淀粉酶比较而言,在pH 4.5-5.0的偏酸性条件下表现出较强的活性及稳定性;pH 5.0时该酶对于多种生淀粉底物也有良好的水解作用;同时,对于15-20%的高浓度玉米生淀粉底物也都能够进行有效的水解。经LC-MASS-MASS分析得到了酶蛋白中两个肽段的氨基酸序列,通过比对发现,该酶与NCBI中已报道的α-淀粉酶序列具有一定的同源性,同时,在氨基酸水平上也存在着差异。因此,该酶是一种具有研究价值和应用前景的中温酸性α-淀粉酶。
通过搜集NCBI数据库中芽孢杆菌属α-淀粉酶氨基酸序列,并根据序列的保守性和同源性设计引物,扩增获得Bacillus sp.YX-1α-淀粉酶的结构基因amy。该基因全长1545 bp,编码514个氨基酸残基。该基因已在GenBank数据库登记,登记号为EU159580。将结构基因全序列amy比对分析发现,Bacillus sp. YX-1中温α-淀粉酶与芽孢杆菌属α-淀粉酶表现出较高的同源性,然而,在表观酶学性质上Bacillus sp. YX-1中温α-淀粉酶表现出较好的耐酸特性和较强的生淀粉底物水解能力。在研究中分别从酶蛋白氨基酸组成、维系酶结构稳定的空间作用力以及酶蛋白结构等多角度来阐述Bacillus sp. YX-1中温α-淀粉酶基因序列与其催化功能(酸性条件下的活性和稳定性)之间的内在联系;并从基因序列角度初步分析了紫外诱变后突变菌株产酶能力提高的机理。
将Bacillus sp.YX-1中温α-淀粉酶结构基因插入表达载体pET28a中并转化相应表达宿主E. coli BL21 (DE3)中成功构建带有目的基因的重组菌株E. coli BL21 (DE3) (pET-28a-amy)。当诱导培养温度从37℃降低至30℃时,表达后形成的可溶性蛋白增加,并确定诱导培养条件为:诱导培养温度30℃,诱导物IPTG浓度0.5 mmol/L。在以上诱导培养条件下,该重组菌粗酶液催化活性为1.52 U/mg。利用His-tag亲和层析分离纯化了表达后的重组蛋白。
α-Amylases (EC3.2.1.1, 1,4-α-D-glucan-glucanohydrolase) are starch-degrading enzymes that catalyze the hydrolysis of internalα-1,4-O-glycosidic bonds in polysaccharides with the retention ofα-anomeric configuration in the products. It is one of the most important industrial enzymes, which can be used in wide number of industrial processes including biofuel, traditional brewing, food and textile.
Now most of the commercial bacterialα-amylases used in the liquefaction step have pH optima at around 6.5 and unstable at low pH, however, natural pH of starch slurry is usually around 4.5, therefore, it is necessary to improve the activity and stability of the enzymes at low pH values for omitting the pH adjustment step. Furthermore, it could supply the theoretical basis for elucidating the acid-stability mechanism of the microorganisms and its metabolites, developing and utilizing the new products by protein engineering. This study reports the purification, characterization and gene expression of a novelα-amylase from a newly isolated Bacillus sp. YX-1.
On the basis of the hydrolysis ability in the starch agar plate (pH 4.5), an isolate YX-1 was selected. The newly isolated strain was identified as a Bacillus species according to the characteristic of this strain and the 16S rDNA sequence. Moreover, a comparison of 16S rDNA sequence of this strain with other related Bacillus sp. shows that the 16S rDNA sequence of this strain has no high similarity with B. subtilis, B. licheniformis, B. stearothermophilus and B. amyloliquefaciens, which are the usualα-amylase-producing strains for industrial applications. The 16S rDNA sequence of the novel isolatedα-amylase-producing strain YX-1 has been deposited into the GenBank database under accession number DQ883446.
YX-1 was used as the parent strain to increase its enzyme production capacity by the method of UV mutation. After several screen procedures, one mutant strain with higher enzyme-producing ability was selected. The activity was about 200% higher than that of the parent strain. The crude enzyme of the mutant was similar with that of the parent strain which could hydrolyze starch substrate in acidic condition.
The amylase from Bacillus sp. YX-1 was purified reaching 34 folds of electrophoretic homogeneity by sequential ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. The enzyme had a molecular weight of 58kDa estimated by SDS-PAGE. The action mode of the enzyme was evaluated by studying the hydrolysis performance of soluble starch at pH 4.5 and 60℃. The purified amylase could convert starch substrate to low molecular oligosaccharides with rapid blue color reduction in iodine-starch reaction and appearance of reducing sugar, indicating an endo-type activity of this amylase.
The purifiedα-amylase exhibited the maximum activity at pH 5.0 and 40-50℃. Furthermore, the enzyme performed stable over the pH range of 4.5-11.0 and at 60℃. The enzyme preparation could hydrolyze various raw starches very well at pH 5.0, which was around the saccharification pH of industrial application. It is worth noting that the enzyme exhibits a high digestibility towards raw corn starch granule of concentrations ranging from 15 to 20%, omitting an energy intensive gelatinization step. These features are very important for industrial starch liquefaction. From the viewpoints of application, the optimum activity of the enzyme at low pH values and its suitable thermostability made it favorable in industrial operations for traditional fermentation and food processing. The amino acid sequences of two peptides from the purified enzyme were analyzed by LC-MASS-MASS, and the enzyme did not show strict identity to theα-amylases reported for some differences in amino acid residues.
To further investigate this enzyme, theα-amylase from Bacillus sp. YX-1 was subsequently cloned, sequenced and expressed in E. coli BL21. Theα-amylase gene (amy) was amplified using genomic DNA from Bacillus sp. YX-1 as a template and the specific primers based on theα-amylase genes of Bacillus species from the GenBank database. After cloning the gene encoding the enzyme, sequencing analysis revealed that the sequence contains one complete open reading frame with a length of 1545 base pairs and encodes a protein of 514 amino acid residues with a calculated molecular weight of 58 kDa plus a signal peptide of 31 amino acids. The nucleotide sequence of the Bacillus sp. YX-1α-amylase has been deposited in the GenBank database under accession number EU159580. The amino acid composition of the sequence and its relevant non-covalent action, the secondary structure analysis, the three dimensional structure prediction were utilized respectively to elucidate the enzymatic properties of this enzyme. The principle on the development of enzyme activity with UV mutation was also analyzed.
An expression vector, pET-28a-amy, containing theα-amylase gene (amy) inserted into pET-28a was constructed, and E. coli BL21 (DE3) was transformed with the ligation mixture to yield the transformant E. coli BL21 (DE3) (pET-28a-amy). SDS-PAGE analysis of expressed recombinant protein revealed that most of recombinant protein existed as inclusion body when expressed at 37℃with 0.5 mmol/L IPTG induction, while at 30℃, the amount of soluble protein increased, and thus the expression conditions were determined as cultivation temperature 30℃and IPTG 0.5 mmol/L. Under those conditions, the cell-free extract from recombinant E. coli showed a specificα-amylase activity of 1.52 U/mg.
The recombinant enzyme of N-terminal His6-tag protein was purified from the cell-free extract of E. coli transformant using a HisTrap HP affinity column to an apparent homogeneity on SDS-PAGE. The recombinant enzyme displayed physical-chemical characteristics similar to those observed for the native enzyme from Bacillus sp. YX-1.
引文
1. Gupta R, Gigras P, Mohapatra H, et al. Microbialα-amylases: a biotechnological perspective [J]. Process Biochem, 2003, 38: 1599-1616.
2. Pandey A, Nigam P, Soccol C R, et al. Advances in microbial amylases [J]. Biotechnol Appl Biochem, 2000, 31: 135-152.
3. van der Maarel M J E C, van der Veen B, Uitdehaag J C M, et al. Properties and applications of starch-converting enzymes of theα-amylase family [J]. J Biotechnol, 2002, 94: 137-155.
4.张树政主编.酶制剂工业[M].北京:科学出版社, 1998. 456-457
5. Sivaramakrishnan S, Gangadharan D, Nampoothiri K M, et al. alpha-amylases from microbial sources-an overview on recent developments [J]. Food Technol Biotechnol, 2006, 44(2): 173-184.
6. Sajedi R H, Naderi-Manesh H, Khajeh K, et al. A Ca-independent alpha-amylase that is active and stable at low pH from the Bacillus sp. KR-8104 [J]. Enzyme Microb Technol, 2005, 36(5-6): 666-671.
7. Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes: a review [J]. Biores Technol, 2003, 89(1): 17-34.
8. Markovitz A, Klein H P, Fischer E H. Purification, crystallization, and properties of the alpha-amylase of Pseudomonas saccharophila [J]. Biochim Biophys Acta, 1956, 19(2): 267-273.
9. Minoda Y, Yamada K. Acid-stableα-amylase of black Aspergilli I. Detection and purification of acid-stable dextrinizing amylase [J]. Agric Biol Chem, 1963, 27: 806-811.
10. Dong G Q, Vieille C, Savchenko A, et al. Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme [J]. Appl Environ Microbiol, 1997, 63(9): 3569-3576.
11. Dong G Q, Vieille C, Zeikus J G. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme [J]. Appl Environ Microbiol, 1997, 63(9): 3577-3584.
12. Jorgensen S, Vorgias C E, Antranikian G. Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis [J]. J Biol Chem, 1997, 272(26): 16335-16342.
13. Fiala G, Stetter K O. Pyrococcus furiosus sp. nov. represents a novel genus of marineheterotrophic archaebacteria growing optimally at 100℃[J]. Arch Microbiol, 1986, 145(1): 56-61.
14. Orapin B. Production of amyloglucosidase by submerged culture [J]. J Agric Sci, 1983, 16(3): 173-184.
15. Jodal I, Kandra L, Harangi J, et al. Hydrolysis of cyclodextrin by Aspergillus oryzae alpha-amylase [J]. Starch, 1984, 36(4): 140-143.
16. Bhella R S, Altosaar I. Purification and some properties of the extracellular alpha-amylase from Aspergillus awamori [J]. Can J Microbiol, 1985, 31(2): 149-153.
17. Mikami S, Iwano K, Shiinoki S, et al. Purification and some properties of acid-stableα-amylases from Shochu koji (Aspergillus kawachii) [J]. Agric Biol Chem, 1987, 51(9): 2495-2501.
18. Suganuma T, Tahara N, Kitahara K, et al. N-terminal sequence of amino acids and some properties of an acid-stable alpha-amylase from citric acid koji (Aspergillus usamii var) [J]. Biosci Biotechnol Biochem, 1996, 60(1): 177-179.
19. Moreira F G, de Lima F A, Pedrinho S R F, et al. Production of amylases by Aspergillus tamarii [J]. Rev Microbiol, 1999, 30(2): 157-162.
20. Schwermann B, Pfau K, Liliensiek B, et al. Purification, properties and structural aspects of a thermoacidophilicα-amylase from Alicyclobacillus acidocaldarius atcc 27009: Insight into acidostability of proteins [J]. Eur J Biochem, 1994, 226: 981-991.
21. Mamo G, Gessesse A. Purification and characterization of two raw-starch-digesting thermostableα-amylases from a thermophilic Bacillus [J]. Enzyme Microb Technol, 1999, 25(3-5): 433-438.
22. Ali M B, Mezghani M, Bejar S. A thermostableα-amylase producing maltohexaose from a new isolated Bacillus sp. US100: study of activity and molecular cloning of the corresponding gene [J]. Enzyme Microb Technol, 1999, 24: 584-589.
23. Hamilton L M, Kelly C T, Fogarty W M. Purification and properties of the raw starch-degrading alpha-amylase of Bacillus sp. IMD 434 [J]. Biotechnol Lett, 1999, 21(2): 111-115.
24. Wind R D, Buitelaar R M, Eggink G, et al. Characterization of a new Bacillus stearothermophilus Isolate: a highly thermostable alpha amylase producing strain [J]. Appl Microbiol Biotechnol, 1994, 41(2): 155-162.
25. Bolton D J, Kelly C T, Fogarty W M. Purification and characterization of the alpha-amylase of Bacillus flavothermus [J]. Enzyme Microb Technol, 1997, 20(5): 340-343.
26. Uguru G C, Robb D A, Akinyanju J A, et al. Purification, characterisation and mutagenicenhancement of a thermoactive alpha amylase from Bacillus subtilis [J]. J Ind Microbiol Biotechnol, 1997, 19(4): 273-279.
27. Salva T J G, Moraes I O. Effect of the carbon source on alpha amylase production by Bacillus subtilis BA-04 [J]. Rev Microbiol, 1995, 26(1): 46-51.
28. Marco J L, Bataus L A, Valencia F F, et al. Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli [J]. Appl Microbiol Biotechnol, 1996, 44(6): 746-752.
29. Ebisu S, Mori M, Takagi H, et al. Production of a fungal protein, Taka amylase A, by protein producing Bacillus brevis HPD31 [J]. J Ind Microbiol, 1993, 11(2): 83-88.
30. Sudo S, Ishikawa T, Takayasu sakamoto Y, et al. Characteristics of acid-stable alpha-amylase production by submerged culture of Aspergillus kawachii [J]. J Ferment Bioeng, 1993, 76(2): 105-110.
31. Sudo S, Ishikawa T, Sato K, et al. Comparison of acid-stable alpha-amylase production by Aspergillus kawachii in solid state and submerged cultures [J]. J Ferment Bioeng, 1994, 77(5): 483-489.
32. Nagamine K, Murashima K, Kato T, et al. Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii [J]. Biosci Biotechnol Biochem, 2003, 67(10): 2194-2202.
33. Khoo S L, Amirul A A, Kamaruzaman M, et al. Purification and characterization of alpha-amylase from Aspergillus flavus [J]. Folia Microbiol, 1994, 39(5): 392-398.
34. Iefuji H, Chino M, Kato M, et al. Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: Purification, characterization, cloning and sequencing [J]. Biochem J, 1996, 318: 989-996.
35. Prieto J A, Bort B R, Martinez J, et al. Purification and characterization of a new alpha amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae [J]. Biochem Cell Biol, 1995, 73(1-2): 41-49.
36. Mishra R S, Maheshwari R. Amylases of the thermophilic fungus Thermomyces lanuginosus: Their purification, properties, action on starch and response to heat [J]. J Biosci, 1996, 21(5): 653-672.
37. Hamilton L M, Kelly C T, Fogarty W M. Production and properties of the raw starch-digestingα-amylase of Bacillus sp. IMD 435 [J]. Process Biochem, 1999, 35: 27-31.
38. Mamo G, Gashe B A, Gessesse A. A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11 [J]. J Appl Microbiol, 1999, 86(4): 557-560.
39. Giraud E, Gosselin L, Marin B, et al. Purification and characterization of an extracellularamylase from Lactobacillus plantarum strain A6 [J]. J Appl Bacteriol, 1993, 75(3): 276-282.
40. Freer S N. Purification and characterization of the extracellular alpha amylase from Streptococcus bovis JB1 [J]. Appl Environ Microbiol, 1993, 59(5): 1398-1402.
41. Chung Y C, Kobayashi T, Kanai H, et al. Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococcus profundus DT5432 [J]. Appl Environ Microbiol, 1995, 61(4): 1502-1506.
42.李多川,杨依军,彭友良等.嗜热真菌Thermomyces lanuginosus热稳定α-淀粉酶的纯化及特性[J].微生物学报, 1997, 37(2): 107-114.
43.张文学,杨瑞,胡承等.白曲耐酸性α-淀粉酶的分离和纯化[J].四川大学学报, 2002, 34(2): 51-56.
44.陈波,李大力,杨树林.酸性α-淀粉酶生产菌株的筛选和酶的纯化及酶的性质研究[J].食品科学, 2005, 26(5): 119-122.
45. Demirkan E S, Mikami B, Adachi M, et al.α-Amylase from B. amyloliquefaciens: purification, characterization, raw starch degradation and expression in E. coli [J]. Process Biochem, 2005, 40: 2629-2636.
46. Mijts B N, Patel B K C. Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme [J]. Microbiology, 2002, 148: 2343-2349.
47. Feller G, Le Bussy O, Gerday C. Expression of psychrophilic genes in mesophilic hosts: Assessment of the folding state of a recombinant alpha-amylase [J]. Appl Environ Microbiol, 1998, 64(3): 1163-1165.
48. Kang H K, Lee J H, Kim D, et al. Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and determination of some of its properties [J]. Fems Microbiol Lett, 2004, 233(1): 53-64.
49. Leveque E, Haye B, Belarbi A. Cloning and expression of an alpha-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme [J]. Fems Microbiol Lett, 2000, 186(1): 67-71.
50. Kaneko A, Sudo S, Takayasu Sakamoto Y, et al. Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii [J]. J Ferment Bioeng, 1996, 81(4): 292-298.
51. Knox A M, du Preez J C, Kilian S G. Starch fermentation characteristics ofSaccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera [J]. Enzyme Microb Technol, 2004, 34(5): 453-460.
52. Morimura S, Zhang W X, Ichimura T, et al. Genetic engineering of white shochu-koji to achieve higher levels of acid-stable alpha-amylase and glucoamylase and other properties when used for shochu making on a laboratory scale [J]. J Inst Brew, 1999, 105(5): 309-314.
53.张文学,刘春莉,蒋宏.利用原生质体融合和诱变育种技术选育高酶活菌株[J].四川大学学报, 2003, 35(6): 66-70.
54.刘春莉,张文学,江孝明等.新型耐酸性液化糖化酶的分离提取及其特性[J].酿酒, 2003, 30(3): 73-76.
55. Janecek S.α-amylase family: molecular biology and evolution. [J]. Prog Biophys Mol Biol, 1997, 67(1): 67-97.
56. Nielsen J E, Borchert T V. Protein engineering of bacterial alpha-amylases [J]. Biochim Biophys Acta, 2000, 1543(2): 253-274.
57. Terada Y, Fujii K, Takaha T, et al. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: Production of cycloamylose [J]. Appl Environ Microbiol, 1999, 65(3): 910-915.
58. Potocki De Montalk G, Remaud-Simeon M, Willemot R W, et al. Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme [J]. J Bacteriol, 1999, 181(2): 375-381.
59. Lawson C L, Vanmontfort R, Strokopytov B, et al. Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form [J]. J Mol Biol, 1994, 236(2): 590-600.
60. Podkovyrov S M, Zeikus J G. Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli [J]. J Bacteriol, 1992, 174(16): 5400-5405.
61. Kiel J, Boels J M, Beldman G, et al. Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (Glgb) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis [J]. Mol Gen Genet, 1991, 230(1-2): 136-144.
62. Amemura A, Chakraborty R, Fujita M, et al. Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15 [J]. J Biol Chem, 1988, 263(19): 9271-9275.
63. Cha H J, Yoon H G, Kim Y W, et al. Molecular and enzymatic characterization of amaltogenic amylase that hydrolyzes and transglycosylates acarbose [J]. Eur J Biochem, 1998, 253(1): 251-262.
64. Kashiwabara S, Ogawa S, Miyoshi N, et al. Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228 [J]. Biosci Biotechnol Biochem, 1999, 63(10): 1736-1748.
65. Aiba H, Baba T, Hayashi K, et al. A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map [J]. DNA Res, 1996, 3(6): 363-77.
66. Kim I C, Cha J H, Kim J R, et al. Catalytic properties of the cloned amylase from Bacillus licheniformis [J]. J Biol Chem, 1992, 267(31): 22108-22114.
67. Reddy N S, Nimmagadda A, Rao K R S S. An overview of the microbialα-amylase family [J]. Afri J Biotechnol, 2003, 2(12): 645-648.
68. Swift H J, Brady L, Derewenda Z S, et al. Structure and molecular model refinement of Aspergillus oryzae (Taka) alpha-amylase: an application of the simulated annealing method [J]. Acta Crystallogr B, 1991, 47: 535-544.
69. Boel E, Brady L, Brzozowski A M, et al. Calcium binding in alpha-amylases: an X-ray diffraction study at 2.1A resolution of two enzymes from Aspergillus [J]. Biochemistry, 1990, 29(26): 6244-6249.
70. Brady R L, Brzozowski A M, Derewenda Z S, et al. Solution of the structure of Aspergillus niger acid alpha-amylase by combined molecular replacement and multiple isomorphous replacement methods [J]. Acta Crystallogr B, 1991, 47: 527-535.
71. Machius M, Wiegand G, Huber R. Crystal structure of calcium depleted Bacillus licheniformis alpha amylase at 2.2-angstrom resolution [J]. J Mol Biol, 1995, 246(4): 545-559.
72. Lee S Y, Kim S, Sweet R, et al. Crystallization and a preliminary X-ray crystallographic study of alpha-amylase from Bacillus licheniformis. [J]. Arch Biochem Biophys, 1991, 291(2): 255-257.
73. Hwang K Y, Song H K, Chang C, et al. Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 angstrom resolution [J]. Mol Cells, 1997, 7(2): 251-258.
74. Chang C S, Kim K K, Hwang K Y, et al. Crystallization and preliminary-X-ray crystallographic analysis of alpha-amylase from Bacillus subtilis [J]. J Mol Biol, 1993, 229(1): 235-238.
75. Mizuno H, Morimoto Y, Tsukihara T, et al. Crystallization and preliminary-X-ray studies of wild-type and catalytic-site mutant alpha-amylase from Bacillus subtilis [J]. J Mol Biol,1993, 234(4): 1282-1283.
76. Fujimoto Z, Takase K, Doui N, et al. Crystal structure of a catalytic-site mutant alpha-amylase from Bacillus subtilis complexed with maltopentaose [J]. J Mol Biol, 1998, 277(2): 393-407.
77. Dauter Z, Dauter M, Brzozowski A M, et al. X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 angstrom resolution [J]. Biochemistry, 1999, 38(26): 8385-8392.
78. Suvd D, Takase K, Fujimoto Z, et al. Purification, crystallization and preliminary X-ray crystallographic study of alpha-amylase from Bacillus stearothermophilus [J]. Acta Crystallographica Section D-Biological Crystallography, 2000, 56: 200-202.
79. Brzozowski A M, Lawson D M, Turkenburg J P, et al. Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes [J]. Biochemistry, 2000, 39(31): 9099-9107.
80. Eijsink V G H, Bj?rk A, Gaseidnes S, et al. Rational engineering of enzyme stability [J]. J Biotechnol, 2004, 113: 105-120.
81. Declerck N, Machius M, Joyet P, et al. Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50℃temperature range [J]. Protein Eng, 2003, 16(4): 287-293.
82. Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformisα-Amylase through Introduction of hydrophobic residues at the surface [J]. J Biol Chem, 2003, 278(13): 11546-11553.
83. Verhaert R M D, Beekwilder J, Olsthoorn R, et al. Phage display selects for amylases with improved low pH starch-binding [J]. J Biotechnol, 2002, 96: 103-118.
84. Matzke J, Schwermann B, Bakker E P. Acidostable and acidophilic proteins: the example of theα-amylase from Alicyclobacillus acidocaldarius [J]. Comp Biochem Physiol, 1997, 118A: 475-479.
85. Schafer K, Magnusson U, Schiefner F S A, et al. X-ray structures of the maltose–maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins [J]. J Mol Biol, 2004, 335: 261-274.
86. Nielsen J E, Beier L, Otzen D, et al. Electrostatics in the active site of an alpha-amylase [J]. Eur J Biochem, 1999, 264(3): 816-824.
87. Nielsen J E, Borchert T V, Vriend G. The determinants of alpha-amylase pH-activity profiles [J]. Protein Eng, 2001, 14(7): 505-512.
88. Nielsen J E, McCammon J A. Calculating pKa values in enzyme active sites [J]. ProteinSci, 2003, 12(9): 1894-1901.
89.周亚凤,张先恩, Cass A E G.分子酶工程学研究进展[J].生物工程学报, 2002, 18(4): 401-406.
90. Warshel A, Naray szabo G, Sussman F, et al. How do serine proteases really work [J]. Biochemistry, 1989, 28(9): 3629-3637.
91. McCarter J D, Withers S G. Mechanisms of enzymatic glycoside hydrolysis [J]. Curr Opin Struct Biol, 1994, 4(6): 885-892.
92. Hashida M, Bisgaard-Frantzen H. Protein engineering of new industrial amylases [J]. Trends Glycosci Glycotechnol, 2000, 12(68): 389-401.
93. Shaw A, Bott R, Day A G. Protein engineering of alpha-amylase for low pH performance [J]. Curr Opin Biotechnol, 1999, 10(4): 349-352.
94. Suzuki Y, Ito N, Yuuki T, et al. Amino-acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation [J]. J Biol Chem, 1989, 264(32): 18933-18938.
95. Conrad B, Hoang V, Polley A, et al. Hybrid Bacillus amyloliquefaciens X Bacillus licheniformis alpha-amylases. construction, properties and sequence determinants [J]. Eur J Biochem, 1995, 230(2): 481-490.
96. Vihinen M, Mantsala P. Microbial amylolytic enzymes [J]. Crit Rev Biochem Mol, 1989, 24(4): 329-418.
97.诸葛斌,姚惠源,姚卫蓉.生淀粉糖化酶的结构和作用机理[J].工业微生物, 2001, 31(1): 49-51.
98. Goyal N, Gupta J K, Soni S K. A novel raw starch digesting thermostableα-amylase from Bacillus sp.I-3 and its use in the direct hydrolysis of raw potato starch [J]. Enzyme Microb Technol, 2005, 37: 723-734.
99. Mitsuiki S, Mukae K, Sakai M, et al. Comparative characterization of raw starch hydrolyzingα-amylases from various Bacillus strains [J]. Enzyme Microb Technol, 2005, 37: 410-416.
100.Morita H, Fujio Y. Effect of organic nitrogen sources on raw starch-digesting glucoamylase production of Rhizopus sp. MKU 40 [J]. Starch, 2000, 52(1): 18-21.
101.Stoffer B, Frandsen T P, Busk P K, et al. Production, purification and characterization of the catalytic domain of glucoamylase from Aspergillus niger [J]. Biochem J, 1993, 292: 197-202.
102.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 429-38.
103.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Degradation of raw starch granules byalpha-amylase purified from culture of Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 422-428.
104.Anindyawati T, Melliawati R, Ito K, et al. Three different types of alpha-amylases from Aspergillus awamori KT-11: Their purifications, properties, and specificities [J]. Biosci Biotechnol Biochem, 1998, 62(7): 1351-1357.
105.Iefuji H, Iimura Y, Obata T. Isolation and characterization of a yeast Cryptococcus sp. S-2 that produces raw starch-digesting alpha-amylase, xylanase, and polygalacturonase [J]. Biosci Biotechnol Biochem, 1994, 58(12): 2261-2262.
106.Belshaw N J, Williamson G. Specificity of the binding domain of glucoamylase-1 [J]. Eur J Biochem, 1993, 211(3): 717-724.
107.Morita H, Fujio Y. High specific activity of raw-starch digesting glucoamylase producing Rhizopus sp. A-11 in liquid culture [J]. Starch, 1997, 49(7-8): 293-296.
108.Morita H, Matsunaga M, Mizuno K, et al. A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains [J]. J Gen Appl Microbiol, 1998, 44(3): 211-216.
109.Hayashida S, Nakahara K, Kanlayakrit W, et al. Characteristics and function of raw-starch-affinity site on Aspergillus awamori Var. kawachi glucoamylaseⅠMolecule [J]. Agric Biol Chem, 1989, 53(1): 143-149.
110.Hamilton L M, Kelly C T, Fogarty W M. Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD 434 [J]. Carbohyd Res, 1998, 314(3-4): 251-257.
111.Haifeng L, Zhenming C, Xiaohong W, et al. Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion [J]. Enzyme Microb Technol, 2007, 40(5): 1006-1012.
112.Haifeng L, Zhenming C, Xiaohui D, et al. Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme [J]. Process Biochem, 2007, 42(3): 462-465.
113.Kaneko T, Ohno T, Ohisa N. Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory [J]. Biosci Biotechnol Biochem, 2005, 69(6): 1073-81.
114.Declerck N, Machius M, Chambert R, et al. Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: Thermodynamic studies and structural interpretation [J]. Protein Eng, 1997, 10(5): 541-549.
115.Jeang C L, Lee Y H, Chang L W. Purification and characterization of a raw-starch digesting amylase from a soil bacterium--Cytophaga sp. [J]. Biochem Mol Biol Int, 1995,35(3): 549-557.
116.Jeang C L, Chen L S, Chen M Y, et al. Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli [J]. Appl Environ Microbiol, 2002, 68(7): 3651-3654.
117.Hamilton L M, Kelly C T, Fogarty W M. Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD 434 [J]. Carbohyd Res, 1998, 314: 251-257.
118.Kim J, Nanmori T, Shinke R. Thermostable, raw-starch-digesting amylase from Bacillus stearothermophilus [J]. Appl Environ Microbiol, 1989, 55(6): 1638-1639.
119.Richardson T H, Tan X Q, Frey G, et al. A novel, high performance enzyme for starch liquefaction - Discovery and optimization of a low pH, thermostable alpha-amylase [J]. J Biol Chem, 2002, 277(29): 26501-26507.
120.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001.
121.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual 2nd ed [M]. New York: 1989.
122.Liu X D, Xu Y. A novel raw starch digestingα-amylase from a newly isolated Bacillus sp. YX-1: Purification and characterization [J]. Biores Technol, 2008, 99: 4315-4320.
123.Guan Y, Zheng B J, He Y Q, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China [J]. Science, 2003, 302(5643): 276-278.
124.Delong E F, Wickham G S, Pace N R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells [J]. Science, 1989, 243(4896): 1360-1363.
125.Peiris J S M, Lai S T, Poon L L M, et al. Coronavirus as a possible cause of severe acute respiratory syndrome [J]. Lancet, 2003, 361(9366): 1319-1325.
126.Ksiazek T G, Erdman D, Goldsmith C S, et al. A novel coronavirus associated with severe acute respiratory syndrome [J]. N Engl J Med, 2003, 348(20): 1953-1966.
127.Alikhajeh J, Khajeh K, Naderi-Manesh M, et al. Kinetic analysis, structural studies and prediction of pKa values of Bacillus sp. KR-8104α-amylase: The determinants of pH-activity profile [J]. Enzyme Microb Technol, 2007, 41: 337-345.
128.Sajedi R H, Taghdir M, Naderi-Manesh H, et al. Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+-independent alpha-amylase with acidic pH-profile [J]. J Biochem Mol Biol, 2007, 40(3): 315-324.
129.Liu W H, Yang C H. Isolation and identification of the lignocellulolytic and thermophilic actinomycetes [J]. Food Sci Agric Chem, 2002, 4: 89-94.
130.Yang C, Cheng K, Liu W. Optimization of medium composition for the production ofextracellular amylase by Thermobifida fusca using a response surface methodology [J]. Food Sci Agric Chem, 2003, 5(1): 35-40.
131.Yang C H, Liu W H. Purification and properties of a maltotriose-producing alpha-amylase from Thermobifida fusca [J]. Enzyme Microb Technol, 2004, 35(2-3): 254-260.
132.Yang C H, Liu W H. Cloning and characterization of a maltotriose-producing alpha-amylase gene from Thermobifida fusca [J]. J Ind Microbiol Biotechnol, 2007, 34(4): 325-330.
133.Schallmey M, Singh A, Ward O P. Developments in the use of Bacillus species for industrial production [J]. Can J Microbiol, 2004, 50: 1-17.
134.Hillier P, Wase D A J, Emery A N. Production ofα-amylase by Bacillus amyloliquefaciens in batch and continuous culture using a defined synthetic medium [J]. Biotechnol Lett, 1996, 18: 795-800.
135.Safarikova M, Roy I, Gupta M N, et al. Magnetic alginate microparticles for purification of alpha-amylases [J]. J Biotechnol, 2003, 105(3): 255-260.
136.Amritkar N, Kamat M, Lali A. Expanded bed affinity purification of bacterial alpha-amylase and cellulase on composite substrate analogue-cellulose matrices [J]. Process Biochem, 2004, 39(5): 565-570.
137.Zhi W B, Deng Q Y, Song J G, et al. One-step purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems [J]. J Chromatogr A, 2005, 1070(1-2): 215-219.
138.Najafi M F, Kembhavi A. One step purification and characterization of an extracellular alpha-amylase from marine Vibrio sp. [J]. Enzyme Microb Technol, 2005, 36(4): 535-539.
139.Liao Y C, Syu M J. Novel immobilized metal ion affinity adsorbent based on cross-linked beta-cyclodextrin matrix for repeated adsorption of alpha-amylase [J]. Biochem Eng J, 2005, 23(1): 17-24.
140.Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities [J]. Biochem J, 1991, 280: 309-316.
141.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970, 227: 680-685.
142.Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the folin-phenol reagent [J]. J Biol Chem, 1951, 193: 265-275.
143.Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J].Anal Chem, 1959, 31: 426-428.
144.Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective [J]. Process Biochem, 2003, 38(11): 1599-1616.
145.Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes:a review [J]. Biores Technol, 2003, 89: 17-34.
146.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 429-438.
147.Najafi M F, Deobagkar D, Deobagkar D. Purification and characterization of an extracellularα-amylase from Bacillus subtilis AX20 [J]. Protein Expres Purif, 2005, 41: 349-354.
148.Declerck N, Machius M, Wiegand G, et al. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase [J]. J Mol Biol, 2000, 301(4): 1041-1057.
149.Nielsen H, Engelbrecht J, Brunak S, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites [J]. Protein Eng, 1997, 10(1): 1-6.
150.Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0 [J]. J Mol Biol, 2004, 340(4): 783-795.
151.Granum P E. Purification and physicochemical properties of an extra-cellular amylase from a strain of Bacillus amyloliquefaciens isolated from a strain of Bacillus amyliquefaciens isolated from dry onion powder [J]. J Food Biochem, 1979, 3: 1~12.
152.Bessler C, Schmitt J, Maurer K H, et al. Directed evolution of a bacterialα-amylase: Toward enhanced pH-performance and higher specific activity [J]. Protein Sci, 2003, 12: 2141-2149.
153.Ben Messaoud E, Ben Ali M, Elleuch N, et al. Purification and properties of a maltoheptaose and maltohexaose-forming amylase produced by Bacillus subtilis US116 [J]. Enzyme Microb Technol, 2004, 34(7): 662-666.
154.Jones D T. Protein secondary structure prediction based on position-specific scoring matrices [J]. J Mol Biol, 1999, 292(2): 195-202.
155.Bryson K, McGuffin L J, Marsden R L, et al. Protein structure prediction servers at University College London [J]. Nucl Acids Res, 2005, 33: W36-W38.
156.Schwede T, Diemand A, Guex N, et al. Protein structure computing in the genomic era [J]. Res Microbiol, 2000, 151(2): 107-112.
157.Schwede T, Kopp J, Guex N, et al. SWISS-MODEL: an automated protein homology-modeling server [J]. Nucleic Acids Res, 2003, 31(13): 3381-3385.
158.Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling [J]. Bioinformatics, 2006, 22(2): 195-201.
159.Holm L, Koivula A K, Lehtovaara P M, et al. Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase [J]. Protein Eng, 1990, 3(3): 181-191.
160.Hockney R C. Recent developments in heterologous protein production in Escherichia coli [J]. Trends Biotechnol, 1994, 12(11): 456-463.
161.Donovan R S, Robinson C W, Glick B R. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter [J]. J Ind Microbiol, 1996, 16(3): 145-154.
162.Chalmers J J, Kim E, Telford J N, et al. Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor [J]. Appl Environ Microbiol, 1990, 56(1): 104-111.
163.朱红裕,李强.外源蛋白在大肠杆菌中的可溶性表达策略[J].过程工程学报, 2006, 6(1): 150-155.
164.Zamost B L, Nielsen H K, Starnes R L. Thermostable enzymes for industrial applications [J]. J Ind Microbiol, 1991, 8(2): 71-81.
165.Schenk P M, Baumann S, Mattes R, et al. Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare (Arg) tRNAs [J]. Biotechniques, 1995, 19(2): 196-198.
166.Wakagi T, Oshima T, Imamura H, et al. Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon [J]. Biosci Biotechnol Biochem, 1998, 62(12): 2408-2414.
167.袁铁铮,姚斌,罗会颖等.一种高温酸性α-淀粉酶基因的高效表达和表达产物分析[J].高技术通讯, 2005, 15(11): 63-68.
168.郭建强,李运敏,岳丽丽等.超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达[J].生物工程学报, 2006, 22(2): 237-242.
169.Westers L, Westers H, Quax W J. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism [J]. BBA-Mol Cell Res, 2004, 1694(1-3): 299-310.
170.Heng C, Chen Z, Du L, et al. Expression and secretion of an acid-stable alpha-amylase gene in Bacillus subtilis by SacB promoter and signal peptide [J]. Biotechnol Lett, 2005,27(21): 1731-1736.
171.Miyata S, Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds .17. alpha-amylase biosynthesis: signal sequence prevents normal conversion of the unprocessed precursor molecule to the biologically-active form [J]. Proc Natl Acad Sci USA, 1982, 79(24): 7792-7795.
172.Miyata S, Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds .18. biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes [J]. J Cell Biol, 1983, 96(3): 802-806.
173.Janecek S. Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases [J]. Eur J Biochem, 1994, 224(2): 519-524.
174.李辉,郭建强,岳丽丽等.重组超耐热酸性α-淀粉酶的分离纯化及其性质研究[J].微生物学报, 2005, 45(4): 547-550.
2. Pandey A, Nigam P, Soccol C R, et al. Advances in microbial amylases [J]. Biotechnol Appl Biochem, 2000, 31: 135-152.
3. van der Maarel M J E C, van der Veen B, Uitdehaag J C M, et al. Properties and applications of starch-converting enzymes of theα-amylase family [J]. J Biotechnol, 2002, 94: 137-155.
4.张树政主编.酶制剂工业[M].北京:科学出版社, 1998. 456-457
5. Sivaramakrishnan S, Gangadharan D, Nampoothiri K M, et al. alpha-amylases from microbial sources-an overview on recent developments [J]. Food Technol Biotechnol, 2006, 44(2): 173-184.
6. Sajedi R H, Naderi-Manesh H, Khajeh K, et al. A Ca-independent alpha-amylase that is active and stable at low pH from the Bacillus sp. KR-8104 [J]. Enzyme Microb Technol, 2005, 36(5-6): 666-671.
7. Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes: a review [J]. Biores Technol, 2003, 89(1): 17-34.
8. Markovitz A, Klein H P, Fischer E H. Purification, crystallization, and properties of the alpha-amylase of Pseudomonas saccharophila [J]. Biochim Biophys Acta, 1956, 19(2): 267-273.
9. Minoda Y, Yamada K. Acid-stableα-amylase of black Aspergilli I. Detection and purification of acid-stable dextrinizing amylase [J]. Agric Biol Chem, 1963, 27: 806-811.
10. Dong G Q, Vieille C, Savchenko A, et al. Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme [J]. Appl Environ Microbiol, 1997, 63(9): 3569-3576.
11. Dong G Q, Vieille C, Zeikus J G. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme [J]. Appl Environ Microbiol, 1997, 63(9): 3577-3584.
12. Jorgensen S, Vorgias C E, Antranikian G. Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis [J]. J Biol Chem, 1997, 272(26): 16335-16342.
13. Fiala G, Stetter K O. Pyrococcus furiosus sp. nov. represents a novel genus of marineheterotrophic archaebacteria growing optimally at 100℃[J]. Arch Microbiol, 1986, 145(1): 56-61.
14. Orapin B. Production of amyloglucosidase by submerged culture [J]. J Agric Sci, 1983, 16(3): 173-184.
15. Jodal I, Kandra L, Harangi J, et al. Hydrolysis of cyclodextrin by Aspergillus oryzae alpha-amylase [J]. Starch, 1984, 36(4): 140-143.
16. Bhella R S, Altosaar I. Purification and some properties of the extracellular alpha-amylase from Aspergillus awamori [J]. Can J Microbiol, 1985, 31(2): 149-153.
17. Mikami S, Iwano K, Shiinoki S, et al. Purification and some properties of acid-stableα-amylases from Shochu koji (Aspergillus kawachii) [J]. Agric Biol Chem, 1987, 51(9): 2495-2501.
18. Suganuma T, Tahara N, Kitahara K, et al. N-terminal sequence of amino acids and some properties of an acid-stable alpha-amylase from citric acid koji (Aspergillus usamii var) [J]. Biosci Biotechnol Biochem, 1996, 60(1): 177-179.
19. Moreira F G, de Lima F A, Pedrinho S R F, et al. Production of amylases by Aspergillus tamarii [J]. Rev Microbiol, 1999, 30(2): 157-162.
20. Schwermann B, Pfau K, Liliensiek B, et al. Purification, properties and structural aspects of a thermoacidophilicα-amylase from Alicyclobacillus acidocaldarius atcc 27009: Insight into acidostability of proteins [J]. Eur J Biochem, 1994, 226: 981-991.
21. Mamo G, Gessesse A. Purification and characterization of two raw-starch-digesting thermostableα-amylases from a thermophilic Bacillus [J]. Enzyme Microb Technol, 1999, 25(3-5): 433-438.
22. Ali M B, Mezghani M, Bejar S. A thermostableα-amylase producing maltohexaose from a new isolated Bacillus sp. US100: study of activity and molecular cloning of the corresponding gene [J]. Enzyme Microb Technol, 1999, 24: 584-589.
23. Hamilton L M, Kelly C T, Fogarty W M. Purification and properties of the raw starch-degrading alpha-amylase of Bacillus sp. IMD 434 [J]. Biotechnol Lett, 1999, 21(2): 111-115.
24. Wind R D, Buitelaar R M, Eggink G, et al. Characterization of a new Bacillus stearothermophilus Isolate: a highly thermostable alpha amylase producing strain [J]. Appl Microbiol Biotechnol, 1994, 41(2): 155-162.
25. Bolton D J, Kelly C T, Fogarty W M. Purification and characterization of the alpha-amylase of Bacillus flavothermus [J]. Enzyme Microb Technol, 1997, 20(5): 340-343.
26. Uguru G C, Robb D A, Akinyanju J A, et al. Purification, characterisation and mutagenicenhancement of a thermoactive alpha amylase from Bacillus subtilis [J]. J Ind Microbiol Biotechnol, 1997, 19(4): 273-279.
27. Salva T J G, Moraes I O. Effect of the carbon source on alpha amylase production by Bacillus subtilis BA-04 [J]. Rev Microbiol, 1995, 26(1): 46-51.
28. Marco J L, Bataus L A, Valencia F F, et al. Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli [J]. Appl Microbiol Biotechnol, 1996, 44(6): 746-752.
29. Ebisu S, Mori M, Takagi H, et al. Production of a fungal protein, Taka amylase A, by protein producing Bacillus brevis HPD31 [J]. J Ind Microbiol, 1993, 11(2): 83-88.
30. Sudo S, Ishikawa T, Takayasu sakamoto Y, et al. Characteristics of acid-stable alpha-amylase production by submerged culture of Aspergillus kawachii [J]. J Ferment Bioeng, 1993, 76(2): 105-110.
31. Sudo S, Ishikawa T, Sato K, et al. Comparison of acid-stable alpha-amylase production by Aspergillus kawachii in solid state and submerged cultures [J]. J Ferment Bioeng, 1994, 77(5): 483-489.
32. Nagamine K, Murashima K, Kato T, et al. Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii [J]. Biosci Biotechnol Biochem, 2003, 67(10): 2194-2202.
33. Khoo S L, Amirul A A, Kamaruzaman M, et al. Purification and characterization of alpha-amylase from Aspergillus flavus [J]. Folia Microbiol, 1994, 39(5): 392-398.
34. Iefuji H, Chino M, Kato M, et al. Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: Purification, characterization, cloning and sequencing [J]. Biochem J, 1996, 318: 989-996.
35. Prieto J A, Bort B R, Martinez J, et al. Purification and characterization of a new alpha amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae [J]. Biochem Cell Biol, 1995, 73(1-2): 41-49.
36. Mishra R S, Maheshwari R. Amylases of the thermophilic fungus Thermomyces lanuginosus: Their purification, properties, action on starch and response to heat [J]. J Biosci, 1996, 21(5): 653-672.
37. Hamilton L M, Kelly C T, Fogarty W M. Production and properties of the raw starch-digestingα-amylase of Bacillus sp. IMD 435 [J]. Process Biochem, 1999, 35: 27-31.
38. Mamo G, Gashe B A, Gessesse A. A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11 [J]. J Appl Microbiol, 1999, 86(4): 557-560.
39. Giraud E, Gosselin L, Marin B, et al. Purification and characterization of an extracellularamylase from Lactobacillus plantarum strain A6 [J]. J Appl Bacteriol, 1993, 75(3): 276-282.
40. Freer S N. Purification and characterization of the extracellular alpha amylase from Streptococcus bovis JB1 [J]. Appl Environ Microbiol, 1993, 59(5): 1398-1402.
41. Chung Y C, Kobayashi T, Kanai H, et al. Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococcus profundus DT5432 [J]. Appl Environ Microbiol, 1995, 61(4): 1502-1506.
42.李多川,杨依军,彭友良等.嗜热真菌Thermomyces lanuginosus热稳定α-淀粉酶的纯化及特性[J].微生物学报, 1997, 37(2): 107-114.
43.张文学,杨瑞,胡承等.白曲耐酸性α-淀粉酶的分离和纯化[J].四川大学学报, 2002, 34(2): 51-56.
44.陈波,李大力,杨树林.酸性α-淀粉酶生产菌株的筛选和酶的纯化及酶的性质研究[J].食品科学, 2005, 26(5): 119-122.
45. Demirkan E S, Mikami B, Adachi M, et al.α-Amylase from B. amyloliquefaciens: purification, characterization, raw starch degradation and expression in E. coli [J]. Process Biochem, 2005, 40: 2629-2636.
46. Mijts B N, Patel B K C. Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme [J]. Microbiology, 2002, 148: 2343-2349.
47. Feller G, Le Bussy O, Gerday C. Expression of psychrophilic genes in mesophilic hosts: Assessment of the folding state of a recombinant alpha-amylase [J]. Appl Environ Microbiol, 1998, 64(3): 1163-1165.
48. Kang H K, Lee J H, Kim D, et al. Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and determination of some of its properties [J]. Fems Microbiol Lett, 2004, 233(1): 53-64.
49. Leveque E, Haye B, Belarbi A. Cloning and expression of an alpha-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme [J]. Fems Microbiol Lett, 2000, 186(1): 67-71.
50. Kaneko A, Sudo S, Takayasu Sakamoto Y, et al. Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii [J]. J Ferment Bioeng, 1996, 81(4): 292-298.
51. Knox A M, du Preez J C, Kilian S G. Starch fermentation characteristics ofSaccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera [J]. Enzyme Microb Technol, 2004, 34(5): 453-460.
52. Morimura S, Zhang W X, Ichimura T, et al. Genetic engineering of white shochu-koji to achieve higher levels of acid-stable alpha-amylase and glucoamylase and other properties when used for shochu making on a laboratory scale [J]. J Inst Brew, 1999, 105(5): 309-314.
53.张文学,刘春莉,蒋宏.利用原生质体融合和诱变育种技术选育高酶活菌株[J].四川大学学报, 2003, 35(6): 66-70.
54.刘春莉,张文学,江孝明等.新型耐酸性液化糖化酶的分离提取及其特性[J].酿酒, 2003, 30(3): 73-76.
55. Janecek S.α-amylase family: molecular biology and evolution. [J]. Prog Biophys Mol Biol, 1997, 67(1): 67-97.
56. Nielsen J E, Borchert T V. Protein engineering of bacterial alpha-amylases [J]. Biochim Biophys Acta, 2000, 1543(2): 253-274.
57. Terada Y, Fujii K, Takaha T, et al. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: Production of cycloamylose [J]. Appl Environ Microbiol, 1999, 65(3): 910-915.
58. Potocki De Montalk G, Remaud-Simeon M, Willemot R W, et al. Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme [J]. J Bacteriol, 1999, 181(2): 375-381.
59. Lawson C L, Vanmontfort R, Strokopytov B, et al. Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form [J]. J Mol Biol, 1994, 236(2): 590-600.
60. Podkovyrov S M, Zeikus J G. Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli [J]. J Bacteriol, 1992, 174(16): 5400-5405.
61. Kiel J, Boels J M, Beldman G, et al. Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (Glgb) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis [J]. Mol Gen Genet, 1991, 230(1-2): 136-144.
62. Amemura A, Chakraborty R, Fujita M, et al. Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15 [J]. J Biol Chem, 1988, 263(19): 9271-9275.
63. Cha H J, Yoon H G, Kim Y W, et al. Molecular and enzymatic characterization of amaltogenic amylase that hydrolyzes and transglycosylates acarbose [J]. Eur J Biochem, 1998, 253(1): 251-262.
64. Kashiwabara S, Ogawa S, Miyoshi N, et al. Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228 [J]. Biosci Biotechnol Biochem, 1999, 63(10): 1736-1748.
65. Aiba H, Baba T, Hayashi K, et al. A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map [J]. DNA Res, 1996, 3(6): 363-77.
66. Kim I C, Cha J H, Kim J R, et al. Catalytic properties of the cloned amylase from Bacillus licheniformis [J]. J Biol Chem, 1992, 267(31): 22108-22114.
67. Reddy N S, Nimmagadda A, Rao K R S S. An overview of the microbialα-amylase family [J]. Afri J Biotechnol, 2003, 2(12): 645-648.
68. Swift H J, Brady L, Derewenda Z S, et al. Structure and molecular model refinement of Aspergillus oryzae (Taka) alpha-amylase: an application of the simulated annealing method [J]. Acta Crystallogr B, 1991, 47: 535-544.
69. Boel E, Brady L, Brzozowski A M, et al. Calcium binding in alpha-amylases: an X-ray diffraction study at 2.1A resolution of two enzymes from Aspergillus [J]. Biochemistry, 1990, 29(26): 6244-6249.
70. Brady R L, Brzozowski A M, Derewenda Z S, et al. Solution of the structure of Aspergillus niger acid alpha-amylase by combined molecular replacement and multiple isomorphous replacement methods [J]. Acta Crystallogr B, 1991, 47: 527-535.
71. Machius M, Wiegand G, Huber R. Crystal structure of calcium depleted Bacillus licheniformis alpha amylase at 2.2-angstrom resolution [J]. J Mol Biol, 1995, 246(4): 545-559.
72. Lee S Y, Kim S, Sweet R, et al. Crystallization and a preliminary X-ray crystallographic study of alpha-amylase from Bacillus licheniformis. [J]. Arch Biochem Biophys, 1991, 291(2): 255-257.
73. Hwang K Y, Song H K, Chang C, et al. Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 angstrom resolution [J]. Mol Cells, 1997, 7(2): 251-258.
74. Chang C S, Kim K K, Hwang K Y, et al. Crystallization and preliminary-X-ray crystallographic analysis of alpha-amylase from Bacillus subtilis [J]. J Mol Biol, 1993, 229(1): 235-238.
75. Mizuno H, Morimoto Y, Tsukihara T, et al. Crystallization and preliminary-X-ray studies of wild-type and catalytic-site mutant alpha-amylase from Bacillus subtilis [J]. J Mol Biol,1993, 234(4): 1282-1283.
76. Fujimoto Z, Takase K, Doui N, et al. Crystal structure of a catalytic-site mutant alpha-amylase from Bacillus subtilis complexed with maltopentaose [J]. J Mol Biol, 1998, 277(2): 393-407.
77. Dauter Z, Dauter M, Brzozowski A M, et al. X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 angstrom resolution [J]. Biochemistry, 1999, 38(26): 8385-8392.
78. Suvd D, Takase K, Fujimoto Z, et al. Purification, crystallization and preliminary X-ray crystallographic study of alpha-amylase from Bacillus stearothermophilus [J]. Acta Crystallographica Section D-Biological Crystallography, 2000, 56: 200-202.
79. Brzozowski A M, Lawson D M, Turkenburg J P, et al. Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes [J]. Biochemistry, 2000, 39(31): 9099-9107.
80. Eijsink V G H, Bj?rk A, Gaseidnes S, et al. Rational engineering of enzyme stability [J]. J Biotechnol, 2004, 113: 105-120.
81. Declerck N, Machius M, Joyet P, et al. Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50℃temperature range [J]. Protein Eng, 2003, 16(4): 287-293.
82. Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformisα-Amylase through Introduction of hydrophobic residues at the surface [J]. J Biol Chem, 2003, 278(13): 11546-11553.
83. Verhaert R M D, Beekwilder J, Olsthoorn R, et al. Phage display selects for amylases with improved low pH starch-binding [J]. J Biotechnol, 2002, 96: 103-118.
84. Matzke J, Schwermann B, Bakker E P. Acidostable and acidophilic proteins: the example of theα-amylase from Alicyclobacillus acidocaldarius [J]. Comp Biochem Physiol, 1997, 118A: 475-479.
85. Schafer K, Magnusson U, Schiefner F S A, et al. X-ray structures of the maltose–maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins [J]. J Mol Biol, 2004, 335: 261-274.
86. Nielsen J E, Beier L, Otzen D, et al. Electrostatics in the active site of an alpha-amylase [J]. Eur J Biochem, 1999, 264(3): 816-824.
87. Nielsen J E, Borchert T V, Vriend G. The determinants of alpha-amylase pH-activity profiles [J]. Protein Eng, 2001, 14(7): 505-512.
88. Nielsen J E, McCammon J A. Calculating pKa values in enzyme active sites [J]. ProteinSci, 2003, 12(9): 1894-1901.
89.周亚凤,张先恩, Cass A E G.分子酶工程学研究进展[J].生物工程学报, 2002, 18(4): 401-406.
90. Warshel A, Naray szabo G, Sussman F, et al. How do serine proteases really work [J]. Biochemistry, 1989, 28(9): 3629-3637.
91. McCarter J D, Withers S G. Mechanisms of enzymatic glycoside hydrolysis [J]. Curr Opin Struct Biol, 1994, 4(6): 885-892.
92. Hashida M, Bisgaard-Frantzen H. Protein engineering of new industrial amylases [J]. Trends Glycosci Glycotechnol, 2000, 12(68): 389-401.
93. Shaw A, Bott R, Day A G. Protein engineering of alpha-amylase for low pH performance [J]. Curr Opin Biotechnol, 1999, 10(4): 349-352.
94. Suzuki Y, Ito N, Yuuki T, et al. Amino-acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation [J]. J Biol Chem, 1989, 264(32): 18933-18938.
95. Conrad B, Hoang V, Polley A, et al. Hybrid Bacillus amyloliquefaciens X Bacillus licheniformis alpha-amylases. construction, properties and sequence determinants [J]. Eur J Biochem, 1995, 230(2): 481-490.
96. Vihinen M, Mantsala P. Microbial amylolytic enzymes [J]. Crit Rev Biochem Mol, 1989, 24(4): 329-418.
97.诸葛斌,姚惠源,姚卫蓉.生淀粉糖化酶的结构和作用机理[J].工业微生物, 2001, 31(1): 49-51.
98. Goyal N, Gupta J K, Soni S K. A novel raw starch digesting thermostableα-amylase from Bacillus sp.I-3 and its use in the direct hydrolysis of raw potato starch [J]. Enzyme Microb Technol, 2005, 37: 723-734.
99. Mitsuiki S, Mukae K, Sakai M, et al. Comparative characterization of raw starch hydrolyzingα-amylases from various Bacillus strains [J]. Enzyme Microb Technol, 2005, 37: 410-416.
100.Morita H, Fujio Y. Effect of organic nitrogen sources on raw starch-digesting glucoamylase production of Rhizopus sp. MKU 40 [J]. Starch, 2000, 52(1): 18-21.
101.Stoffer B, Frandsen T P, Busk P K, et al. Production, purification and characterization of the catalytic domain of glucoamylase from Aspergillus niger [J]. Biochem J, 1993, 292: 197-202.
102.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 429-38.
103.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Degradation of raw starch granules byalpha-amylase purified from culture of Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 422-428.
104.Anindyawati T, Melliawati R, Ito K, et al. Three different types of alpha-amylases from Aspergillus awamori KT-11: Their purifications, properties, and specificities [J]. Biosci Biotechnol Biochem, 1998, 62(7): 1351-1357.
105.Iefuji H, Iimura Y, Obata T. Isolation and characterization of a yeast Cryptococcus sp. S-2 that produces raw starch-digesting alpha-amylase, xylanase, and polygalacturonase [J]. Biosci Biotechnol Biochem, 1994, 58(12): 2261-2262.
106.Belshaw N J, Williamson G. Specificity of the binding domain of glucoamylase-1 [J]. Eur J Biochem, 1993, 211(3): 717-724.
107.Morita H, Fujio Y. High specific activity of raw-starch digesting glucoamylase producing Rhizopus sp. A-11 in liquid culture [J]. Starch, 1997, 49(7-8): 293-296.
108.Morita H, Matsunaga M, Mizuno K, et al. A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains [J]. J Gen Appl Microbiol, 1998, 44(3): 211-216.
109.Hayashida S, Nakahara K, Kanlayakrit W, et al. Characteristics and function of raw-starch-affinity site on Aspergillus awamori Var. kawachi glucoamylaseⅠMolecule [J]. Agric Biol Chem, 1989, 53(1): 143-149.
110.Hamilton L M, Kelly C T, Fogarty W M. Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD 434 [J]. Carbohyd Res, 1998, 314(3-4): 251-257.
111.Haifeng L, Zhenming C, Xiaohong W, et al. Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion [J]. Enzyme Microb Technol, 2007, 40(5): 1006-1012.
112.Haifeng L, Zhenming C, Xiaohui D, et al. Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme [J]. Process Biochem, 2007, 42(3): 462-465.
113.Kaneko T, Ohno T, Ohisa N. Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory [J]. Biosci Biotechnol Biochem, 2005, 69(6): 1073-81.
114.Declerck N, Machius M, Chambert R, et al. Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: Thermodynamic studies and structural interpretation [J]. Protein Eng, 1997, 10(5): 541-549.
115.Jeang C L, Lee Y H, Chang L W. Purification and characterization of a raw-starch digesting amylase from a soil bacterium--Cytophaga sp. [J]. Biochem Mol Biol Int, 1995,35(3): 549-557.
116.Jeang C L, Chen L S, Chen M Y, et al. Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli [J]. Appl Environ Microbiol, 2002, 68(7): 3651-3654.
117.Hamilton L M, Kelly C T, Fogarty W M. Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD 434 [J]. Carbohyd Res, 1998, 314: 251-257.
118.Kim J, Nanmori T, Shinke R. Thermostable, raw-starch-digesting amylase from Bacillus stearothermophilus [J]. Appl Environ Microbiol, 1989, 55(6): 1638-1639.
119.Richardson T H, Tan X Q, Frey G, et al. A novel, high performance enzyme for starch liquefaction - Discovery and optimization of a low pH, thermostable alpha-amylase [J]. J Biol Chem, 2002, 277(29): 26501-26507.
120.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001.
121.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual 2nd ed [M]. New York: 1989.
122.Liu X D, Xu Y. A novel raw starch digestingα-amylase from a newly isolated Bacillus sp. YX-1: Purification and characterization [J]. Biores Technol, 2008, 99: 4315-4320.
123.Guan Y, Zheng B J, He Y Q, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China [J]. Science, 2003, 302(5643): 276-278.
124.Delong E F, Wickham G S, Pace N R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells [J]. Science, 1989, 243(4896): 1360-1363.
125.Peiris J S M, Lai S T, Poon L L M, et al. Coronavirus as a possible cause of severe acute respiratory syndrome [J]. Lancet, 2003, 361(9366): 1319-1325.
126.Ksiazek T G, Erdman D, Goldsmith C S, et al. A novel coronavirus associated with severe acute respiratory syndrome [J]. N Engl J Med, 2003, 348(20): 1953-1966.
127.Alikhajeh J, Khajeh K, Naderi-Manesh M, et al. Kinetic analysis, structural studies and prediction of pKa values of Bacillus sp. KR-8104α-amylase: The determinants of pH-activity profile [J]. Enzyme Microb Technol, 2007, 41: 337-345.
128.Sajedi R H, Taghdir M, Naderi-Manesh H, et al. Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+-independent alpha-amylase with acidic pH-profile [J]. J Biochem Mol Biol, 2007, 40(3): 315-324.
129.Liu W H, Yang C H. Isolation and identification of the lignocellulolytic and thermophilic actinomycetes [J]. Food Sci Agric Chem, 2002, 4: 89-94.
130.Yang C, Cheng K, Liu W. Optimization of medium composition for the production ofextracellular amylase by Thermobifida fusca using a response surface methodology [J]. Food Sci Agric Chem, 2003, 5(1): 35-40.
131.Yang C H, Liu W H. Purification and properties of a maltotriose-producing alpha-amylase from Thermobifida fusca [J]. Enzyme Microb Technol, 2004, 35(2-3): 254-260.
132.Yang C H, Liu W H. Cloning and characterization of a maltotriose-producing alpha-amylase gene from Thermobifida fusca [J]. J Ind Microbiol Biotechnol, 2007, 34(4): 325-330.
133.Schallmey M, Singh A, Ward O P. Developments in the use of Bacillus species for industrial production [J]. Can J Microbiol, 2004, 50: 1-17.
134.Hillier P, Wase D A J, Emery A N. Production ofα-amylase by Bacillus amyloliquefaciens in batch and continuous culture using a defined synthetic medium [J]. Biotechnol Lett, 1996, 18: 795-800.
135.Safarikova M, Roy I, Gupta M N, et al. Magnetic alginate microparticles for purification of alpha-amylases [J]. J Biotechnol, 2003, 105(3): 255-260.
136.Amritkar N, Kamat M, Lali A. Expanded bed affinity purification of bacterial alpha-amylase and cellulase on composite substrate analogue-cellulose matrices [J]. Process Biochem, 2004, 39(5): 565-570.
137.Zhi W B, Deng Q Y, Song J G, et al. One-step purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems [J]. J Chromatogr A, 2005, 1070(1-2): 215-219.
138.Najafi M F, Kembhavi A. One step purification and characterization of an extracellular alpha-amylase from marine Vibrio sp. [J]. Enzyme Microb Technol, 2005, 36(4): 535-539.
139.Liao Y C, Syu M J. Novel immobilized metal ion affinity adsorbent based on cross-linked beta-cyclodextrin matrix for repeated adsorption of alpha-amylase [J]. Biochem Eng J, 2005, 23(1): 17-24.
140.Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities [J]. Biochem J, 1991, 280: 309-316.
141.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970, 227: 680-685.
142.Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the folin-phenol reagent [J]. J Biol Chem, 1951, 193: 265-275.
143.Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J].Anal Chem, 1959, 31: 426-428.
144.Gupta R, Gigras P, Mohapatra H, et al. Microbial alpha-amylases: a biotechnological perspective [J]. Process Biochem, 2003, 38(11): 1599-1616.
145.Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes:a review [J]. Biores Technol, 2003, 89: 17-34.
146.Matsubara T, Ben Ammar Y, Anindyawati T, et al. Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11 [J]. J Biochem Mol Biol, 2004, 37(4): 429-438.
147.Najafi M F, Deobagkar D, Deobagkar D. Purification and characterization of an extracellularα-amylase from Bacillus subtilis AX20 [J]. Protein Expres Purif, 2005, 41: 349-354.
148.Declerck N, Machius M, Wiegand G, et al. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase [J]. J Mol Biol, 2000, 301(4): 1041-1057.
149.Nielsen H, Engelbrecht J, Brunak S, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites [J]. Protein Eng, 1997, 10(1): 1-6.
150.Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0 [J]. J Mol Biol, 2004, 340(4): 783-795.
151.Granum P E. Purification and physicochemical properties of an extra-cellular amylase from a strain of Bacillus amyloliquefaciens isolated from a strain of Bacillus amyliquefaciens isolated from dry onion powder [J]. J Food Biochem, 1979, 3: 1~12.
152.Bessler C, Schmitt J, Maurer K H, et al. Directed evolution of a bacterialα-amylase: Toward enhanced pH-performance and higher specific activity [J]. Protein Sci, 2003, 12: 2141-2149.
153.Ben Messaoud E, Ben Ali M, Elleuch N, et al. Purification and properties of a maltoheptaose and maltohexaose-forming amylase produced by Bacillus subtilis US116 [J]. Enzyme Microb Technol, 2004, 34(7): 662-666.
154.Jones D T. Protein secondary structure prediction based on position-specific scoring matrices [J]. J Mol Biol, 1999, 292(2): 195-202.
155.Bryson K, McGuffin L J, Marsden R L, et al. Protein structure prediction servers at University College London [J]. Nucl Acids Res, 2005, 33: W36-W38.
156.Schwede T, Diemand A, Guex N, et al. Protein structure computing in the genomic era [J]. Res Microbiol, 2000, 151(2): 107-112.
157.Schwede T, Kopp J, Guex N, et al. SWISS-MODEL: an automated protein homology-modeling server [J]. Nucleic Acids Res, 2003, 31(13): 3381-3385.
158.Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling [J]. Bioinformatics, 2006, 22(2): 195-201.
159.Holm L, Koivula A K, Lehtovaara P M, et al. Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase [J]. Protein Eng, 1990, 3(3): 181-191.
160.Hockney R C. Recent developments in heterologous protein production in Escherichia coli [J]. Trends Biotechnol, 1994, 12(11): 456-463.
161.Donovan R S, Robinson C W, Glick B R. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter [J]. J Ind Microbiol, 1996, 16(3): 145-154.
162.Chalmers J J, Kim E, Telford J N, et al. Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor [J]. Appl Environ Microbiol, 1990, 56(1): 104-111.
163.朱红裕,李强.外源蛋白在大肠杆菌中的可溶性表达策略[J].过程工程学报, 2006, 6(1): 150-155.
164.Zamost B L, Nielsen H K, Starnes R L. Thermostable enzymes for industrial applications [J]. J Ind Microbiol, 1991, 8(2): 71-81.
165.Schenk P M, Baumann S, Mattes R, et al. Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare (Arg) tRNAs [J]. Biotechniques, 1995, 19(2): 196-198.
166.Wakagi T, Oshima T, Imamura H, et al. Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon [J]. Biosci Biotechnol Biochem, 1998, 62(12): 2408-2414.
167.袁铁铮,姚斌,罗会颖等.一种高温酸性α-淀粉酶基因的高效表达和表达产物分析[J].高技术通讯, 2005, 15(11): 63-68.
168.郭建强,李运敏,岳丽丽等.超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达[J].生物工程学报, 2006, 22(2): 237-242.
169.Westers L, Westers H, Quax W J. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism [J]. BBA-Mol Cell Res, 2004, 1694(1-3): 299-310.
170.Heng C, Chen Z, Du L, et al. Expression and secretion of an acid-stable alpha-amylase gene in Bacillus subtilis by SacB promoter and signal peptide [J]. Biotechnol Lett, 2005,27(21): 1731-1736.
171.Miyata S, Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds .17. alpha-amylase biosynthesis: signal sequence prevents normal conversion of the unprocessed precursor molecule to the biologically-active form [J]. Proc Natl Acad Sci USA, 1982, 79(24): 7792-7795.
172.Miyata S, Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds .18. biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes [J]. J Cell Biol, 1983, 96(3): 802-806.
173.Janecek S. Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases [J]. Eur J Biochem, 1994, 224(2): 519-524.
174.李辉,郭建强,岳丽丽等.重组超耐热酸性α-淀粉酶的分离纯化及其性质研究[J].微生物学报, 2005, 45(4): 547-550.