【作者】 刘旭东；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【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。更多还原

【Abstract】 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.更多还原