一株高产纳豆激酶菌株的鉴定与分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.01.030

生物技术通报 ›› 2016, Vol. 32 ›› Issue (1): 187-194.doi: 10.13560/j.cnki.biotech.bull.1985.2016.01.030

一株高产纳豆激酶菌株的鉴定与分析

王雪妍, 孙玉飞, 冯菲, 陈晓飞, 李锋, 周伏忠

河南省科学院生物研究所有限责任公司河南省微生物工程重点实验室, 郑州 450008

收稿日期:2015-05-06 出版日期:2016-01-09 发布日期:2016-01-22
作者简介:王雪妍, 女, 研究方向：微生物发酵工程;E-mail：1060877396@qq.com
基金资助:
河南省重点实验室建设专项项目(122300413214), 河南省重点科技攻关项目(142102210087), 郑州市攻关计划项目(141PPTGG415)资助项目。

Identification and Analysis of a Nattokinase-producing Strain

WANG Xue-yan, SUN Yu-fei, FENG Fei, CHEN Xiao-fei, LI Feng, ZHOU Fu-zhong

Institute of Biology Co., Ltd., Henan Academy of Sciences, Key Laboratory of Microbial Engineering of Henan Province, Zhengzhou 450008

Received:2015-05-06 Published:2016-01-09 Online:2016-01-22

摘要/Abstract

摘要： 鉴定高产纳豆激酶菌株Td, 分析纳豆激酶的分子特征。利用菌体形态、生理生化特征、以及分子生物学方法对菌株Td进行鉴定;并采用MALDI-TOF质谱测定与分析、SDS-PAGE和纤溶活性测定等方法检测纳豆激酶特性, 利用PCR方法扩增纳豆激酶的基因全长。结合菌体形态、生理生化特征和16S rDNA、gyrA基因序列、DNA-DNA杂交率等实验结果, 鉴定菌株Td为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp. subtilis);菌株Td发酵产生的纳豆激酶产量可达300 mg/L以上, 占发酵液总蛋白的40%以上;纤溶活性达230 U/mL以上;氨基酸序列与subtilisin E的序列相似性最高;基因全长序列为1 143 bp。枯草芽孢杆菌枯草亚种Td是一株高产、高活性纳豆激酶的产生菌, 具有优良的工业化开发价值。

关键词: 枯草芽孢杆菌枯草亚种, 纳豆激酶, 菌株鉴定

Abstract: The objective of this work is to identify an isolate Td of yielding high nattokinase, and to analyze the molecular characteristics of its nattokinase. The morphologic, biochemical and physiological characterizations, and molecular biological methods were used to identify the taxonomic position of isolate Td;mass spectrometric determination and analysis of MALDI-TOF, SDS-PAGE and fibrinolytic activity were employed to measure the characteristics of nattokinase, and PCR method was adopted to clone the full length of nattokinase gene. The bacterial strain Td was identified as Bacillus subtilis subsp. subtilis according to the results of morphologic, biochemical and physiological characterizations, as well as 16S rDNA, the sequences of 16S rDNA gyrA, hybrid rate of DNA-DNA. The yield of nattokinase produced by strain Td reached 300 mg/L, accounted for over 40% of the total protein. Fibrinolytic activity was over 230 U/mL. Amino acid sequence of nattokinase produced by Td showed the highest similarity to the subtilisin E. and the full length of the gene was 1143 bp. Conclusively, as a high-yield and high-activity strain for nattokinase, B. subtilis subsp. subtilis Td is a promising candidate for industrial development and utilization.

Key words: Bacillus subtilis subsp. subtilis, nattokinase, strain identification

王雪妍, 孙玉飞, 冯菲, 陈晓飞, 李锋, 周伏忠. 一株高产纳豆激酶菌株的鉴定与分析[J]. 生物技术通报, 2016, 32(1): 187-194.

WANG Xue-yan, SUN Yu-fei, FENG Fei, CHEN Xiao-fei, LI Feng, ZHOU Fu-zhong. Identification and Analysis of a Nattokinase-producing Strain[J]. Biotechnology Bulletin, 2016, 32(1): 187-194.

参考文献

［1］Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme(nattokinase)in the vegetable cheese Natto：a typical and popular soybean food in the Japanese diet［J］. Experientia, 1987, 43(10)：1110-1111.
［2］Ku TW, Tsai RL, Pan TM. A simple and cost-saving approach to optimize the production of subtilisin NAT by submerged cultivation of Bacillus subtilis Natto［J］. Journal of Agricultural and Food Chemistry, 2009, 57(1), 292-296.
［3］Choi NS, Chang KT, Jae MP, et al. Cloning, expression, and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4［J］. FEMS Microbiology Letters, 2004, 236(2)：325-331.
［4］Peng Y, Yang XJ, Xiao L, et al. Cloning and expression of a fibrinolytic enzyme(subtilisin DFE)gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis［J］. Research in Microbiology, 2004, 155(3)：167-173.
［5］Agrebi R, Haddar A, Hmidet N, et al. BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26：purification, biochemical and molecular characterization［J］. Process Biochemistry, 2009, 44(11)：1252-1259.
［6］Nakamura T, Yamagata Y, Ichishima E. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis(natto)［J］. Bioscience, Biotechnology, and Biochemistry, 1992, 56(11)：1869-1871.
［7］Nishito Y, Osana Y, Hachiya T, et al. Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data［J］. BMC Genomics, 2010, 11：243-254.
［8］周伏忠, 贾蕴莉, 陈国参, 等. 纤溶酶高产菌株筛选及其液体发酵条件研究［J］. 河南科学, 2008, 26(1)：125-130.
［9］熊迎新, 尹宗宁, 李婉宜. 纳豆激酶固体浅盘发酵工艺的优化［J］. 中国生化药物杂志, 2005(5)：15-17.
［10］Cho YH, Song JY, Kim KM, et al. Production of nattokinase by batch and fed-batch culture of Bacillus subtilis［J］. New Biotechnology, 2010, 27(4)：341-346.
［11］Wang C, Du M, Zheng D, et al. Purification and characterization of nattokinase from Bacillus subtilis natto B-12［J］. Journal of Agricultural and Food Chemistry, 2009, 57(20)：9722-9729.
［12］ Chen PT, Chao YP. Enhanced production of recombinant nattokinase in Bacillus subtilis by the elimination of limiting factors［J］. Biotechnology Letters, 2006, 28(19)：1595-1600.
［13］ Kim JY, Gum SN, Paik JK, et al. Effects of nattokinase on blood pressure：a randomized, controlled trial［J］. Hypertension Research, 2008, 31(8)：1583-1588.
［14］ Yang NC, Chou CW, Chen CY, et al. Combined nattokinase with red yeast rice but not nattokinase alone has potent effects on blood lipids in human subjects with hyperlipidemia［J］. Asia Pacific Journal of Clinical Nutrition, 2009, 18(3)：310-317.
［15］陈晓飞, 杜迅, 刘安邦, 等. 纳豆激酶酶学性质的初步研究［J］. 河南科学, 2008(10)：1212-1214.
［16］陈晓飞, 周伏忠, 陈国参, 等. 纳豆激酶的分离纯化及体外溶栓特性研究［J］. 大豆科学, 2010, 29(2)：295-298.
［17］陈晓飞, 周伏忠, 陈国参, 等. 纳豆激酶酶学性质研究［J］. 河南科学, 2010, 28(1)：41-43.
［18］Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity［J］. Archive of Biochemistry and Biophysics. 1952, 40(2)：346-351.
［19］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.
［20］萨姆布鲁克, 拉塞尔著;黄培堂, 等译. 分子克隆实验指南［M］.第3版. 北京：科学出版社, 2008.
［21］Rooney AP, Price NPJ, Ehrhardt C, et al. Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. ［J］. International Journal of Systematic and Evolutionary Microbiology, 2009, 59：2429-2436.
［22］曹凤明, 杨小红, 马鸣超, 等. 枯草芽孢杆菌近缘种群鉴定方法研究进展［J］. 微生物学通报, 2014, 41(5)：968-974.
［23］Chun J, Bae KS. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence［J］. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2000, 78(2)：123-127.
［24］Yamamoto S, Harayama S. Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes［J］. International Journal of Systematic Bacteriology, 1998, 48：813-819.
［25］Kamata H, Yamagata Y, Nakamura, et al. Characterization of the complex between a macroglobulin and a serine proteinase from Bacillus natto［J］. Agricultural and Biological Chemistry, 1989, 53：2695-2702.

[1]	刘宇程, 邱恋, 梁晶晶, 李玲丽, 马丽丽. 一株聚丙烯酰胺降解菌的筛选及降解特性研究[J]. 生物技术通报, 2019, 35(9): 178-183.
[2]	倪楠, 辛志宏, 许斌, 张丽静, 王艳, 江均平, 王凤忠, 李淑英. 纳豆激酶豆乳液体发酵条件优化[J]. 生物技术通报, 2019, 35(10): 212-219.
[3]	赵仲麟, 李淑英, 聂莹, 李燕, 袁超, 唐选明. 纳豆激酶生产菌的筛选及发酵纳豆特性研究[J]. 生物技术通报, 2015, 31(3): 161-164.
[4]	刘靓蕾, 张妍, 孙晓蕾, 韩岚, 满达呼斯琴, 哈斯阿古拉. 人工合成的纳豆激酶基因转化烟草的研究[J]. 生物技术通报, 2014, 0(10): 94-100.
[5]	李淑英, 聂莹, 杜欢, 赵仲麟, 袁超, 李燕, 宋晓燕, 唐选明. 纳豆激酶生产菌BSNK-5 鉴定及发酵条件优化[J]. 生物技术通报, 2013, 0(2): 184-189.
[6]	叶胜蓝;汪江峰;黄剑;. 土壤放线菌P3-2的分类鉴定及抗菌活性研究[J]. , 2012, 0(01): 156-161.
[7]	姜嵛;梁晨;姜国勇;. 植物内生枯草芽孢杆菌纳豆激酶同源基因的序列分析及其在大肠杆菌中的表达[J]. , 2011, 0(03): 97-99.
[8]	逯京华;孙智杰;. 纳豆激酶的研究现状与展望[J]. , 2009, 0(08): 41-45.

一株高产纳豆激酶菌株的鉴定与分析

Identification and Analysis of a Nattokinase-producing Strain

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 8

编辑推荐

Metrics

本文评价