产耐酸性α-淀粉酶菌株的分离、鉴定、酶学特性研究及发酵培养基的优化

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 159-163.

产耐酸性α-淀粉酶菌株的分离、鉴定、酶学特性研究及发酵培养基的优化

王建玲, 陈志鑫, 刘逸寒, 路福平

(天津科技大学生物工程学院工业发酵微生物教育部重点实验室，天津 300457)

收稿日期:2013-10-23 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:王建玲，女，研究员，研究方向：酶与应用微生物；E-mail：wjl01@tust.edu.cn
基金资助:
天津科技大学实验室开放基金项目(1204A211)，国家自然科学基金资助项目(31101219)，国家高技术研究发展计划(“863”计划)项目(2013AA102106-07)

Screening，Identification and Characterization of an Acid q-amylase Producing Strain and Optimization of Its Fermentable Condition

Wang Jianling, Chen Zhixin, Liu Yihan, Lu Fuping

(Key Laboratory of Industrial Fermentation Microbiology，Ministry of Education，College of Biotechnology，Tianjin University of Science& Technology，Tianjin 300457)

Received:2013-10-23 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 从富含淀粉的土壤中筛选获得多株产耐酸性α-淀粉酶的菌株，将酶活最高的一株经16S rDNA鉴定为枯草芽孢杆菌(Bacillus subtilis)，命名为Bacillus subtilis A-7，其所产的耐酸性α-淀粉酶最适温度和pH分别为60℃和4.5。通过单因素筛选及正交试验优化发酵培养基，得到最佳配方为可溶性淀粉 2%，蛋白胨 2%，CaCl₂ 0.07%，Na₂HPO₄ 0.8%，在此条件下耐酸性α-淀粉酶活力达到221 U/mL，是未优化条件下的2.3倍。

关键词: 耐酸性α-淀粉酶, 16S, rDNA鉴定, 枯草芽孢杆菌, 最适温度, 最适pH, 正交试验

Abstract: Multiple new acid α-amylase producing strains were isolated from soil which is rich in starch. A strain which has the highest enzyme activity was identified as Bacillus subtilis A-7 through analysis of 16S rDNA gene. The optimum temperature and pH was 60℃ and 4.5. The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium. The composition of the best medium was 2% soluble starch, 2% peptone, 0.07% CaCl₂, 0.8% Na₂HPO₄. Under this optimal condition, the activity of acid α-amylase was 221 U/mL and 2.3-fold more than that of no optimization.

Key words: Acid α-amylase 16S, rDNA, Bacillus subtilis, Optimum temperature, Optimum pH, Orthogonal experiment

王建玲, 陈志鑫, 刘逸寒, 路福平. 产耐酸性α-淀粉酶菌株的分离、鉴定、酶学特性研究及发酵培养基的优化[J]. 生物技术通报, 2014, 0(4): 159-163.

Wang Jianling, Chen Zhixin, Liu Yihan, Lu Fuping. Screening，Identification and Characterization of an Acid q-amylase Producing Strain and Optimization of Its Fermentable Condition[J]. Biotechnology Bulletin, 2014, 0(4): 159-163.

参考文献

[1] 张树政.酶工业制剂(下册)［M].北京：科学出版社, 1998：458-489.
[2] Pandey A, Nigam P, Soccol C, et al. Advances in microbial amylases[J]. Biotechnol Appl Biochem, 2000, 31：135-152.
[3] Gupta R, Gigras P, Mohapatra H, et al. Microbial α-amylases：a biotechnological perspective[J]. Process Biochemistry, 2003, 38(11)：1599-1616.
[4] Kandra L. α-Amylases of medical and industrial importance[J]. J Mol Struct, 2003, 666：487-498.
[5] 高崎义幸.日本开发耐热耐酸性α-淀粉酶[J]. 日本食品工业, 1994, 6(30)：44-50.
[6] Sajedi RH, Naderi-Manesh H, Khajeh K, et al. A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104[J]. Enzyme Microb Technol, 2005, 36(5)：666-671.
[7] Richardson TH, Tan X, Frey G, et al. A novel, high performance enzyme for starch liquefaction discovery and optimization of a low pH, thermostable α-amylase[J]. Journal of Biological Chemistry, 2002, 277(29)：26501-26507.
[8] Minoda Y, ARAI M, TORIGOE Y, et al. Acid-stable α-amylase of black Aspergilli[J]. Agricultural and Biological Chemistry, 1968, 32(1)：104-109.
[9] Morimura S, Zhang WX, Ichimura T, et al. Genetic engineering of white Shochu-koji to achieve higher levels of acid-stable α-amylase and glucoamylase and other properties when used for Shochu making on a laboratory scale[J]. Journal of the Institute of Brewing, 1999, 105(3)：309-314.
[10] 刘雅琴, 乌日娜, 段金华.耐酸性α-淀粉酶产生菌的发酵条件优化[J].安徽农业科学, 2010, 38(35)：19888-19890.
[11] 张丽靖, 沈江锋, 金庆超, 等.一株酸性淀粉酶产生菌的分离, 鉴定及酶学特性初步研究[J].生物技术通报, 2011(5)：143-145.
[12] Buchanan RE, Gibbons NE. 伯杰细菌鉴定手册［M].第八版. 北京：科学出版社, 1984：353-357.
[13] Sambrook J, Russell DW. 分子克隆实验指南精编版［M]. 北京：化学工业出版社, 2008.
[14] Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformis α-amylase through introduction of hydrophobic residues at the surface[J]. Journal of Biological Chemistry, 2003, 278(13)：11546-11553.
[15] Sanchez S, Demain AL. Enzymes and bioconversions of industrial, pharmaceutical, and biotechnological significance[J]. Organic Process Research & Development, 2010, 15(1)：224-230.
[16] 胡元森, 潘涛, 李翠香.酸性淀粉酶的分离纯化与酶学性质研究[J].生物技术通报, 2010(3)：199-202.
[17] Sharma A, Satyanarayana T. Microbial acid-stable α-amylases：Characteristics, genetic engineering and applications[J]. Process Biochemistry, 2013, 48(2)：201-211.