应用比色法检测枯草芽孢杆菌变异菌株中1-脱氧野尻霉素含量

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.027

生物技术通报 ›› 2016, Vol. 32 ›› Issue (6): 187-192.doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.027

应用比色法检测枯草芽孢杆菌变异菌株中1-脱氧野尻霉素含量

龙玲, 杨艳, 朱乃硕

复旦大学生命科学学院遗传工程国家重点实验室微生物与分子免疫学研究室，上海 200438

收稿日期:2015-05-22 出版日期:2016-06-27 发布日期:2016-06-28
作者简介:龙玲，女，硕士研究生，研究方向：生物工程；E-mail：13210700143@fudan.edu.cn
基金资助:
国家科技重大专项(2012ZX10002006-002-003)，上海市科委科学基金项目(13431900602)

Colorimetric Method in the Determination of 1-Deoxynojirimycin Isolated from the Mutant Strain of Bacillus subtilis

LONG Ling, YANG Yan, ZHU Nai-shuo

Laboratory of Microbiology and Molecular Immunology，State Key Laboratory of Genetic Engineering，School of Life Sciences，Fudan University，Shanghai 200438

Received:2015-05-22 Published:2016-06-27 Online:2016-06-28

摘要/Abstract

摘要： 为寻找测定枯草芽孢杆菌黑色变种突变株Bacillus subtilis var. niger-231(B-231)的发酵液中1-脱氧野尻霉素(1-Deoxynojirimycin，DNJ)的新方法并提高其产发酵液中DNJ的含量。在有铜离子的存在下，DNJ与二硫化碳反应，用有机溶剂对产物萃取后在440 nm进行光度测定，得到DNJ含量。再采用紫外诱变方法处理突变株B-231。绘制了检测DNJ标准品的标准曲线，对测定DNJ的新方法做了精密度、灵敏度、准确度以及产物的稳定性的探讨，设计正交试验优化了反应条件，并将此方法运用到细菌发酵液中DNJ含量的测定中，通过遗传诱变手段处理原始细菌B-231，最终筛选到一株高产DNJ的菌株，命名为B. subtilis var. niger- 431(B-431)，DNJ含量达87.3 mg/L，其产生DNJ的量较原始菌株原突变株提高了4.2倍。首次提出了一种快速测定细菌发酵液中的1-脱氧野尻霉素的新方法，紫外诱变手段使细菌产生DNJ的能力有较大提高。

关键词: 枯草芽孢杆菌, 1-脱氧野尻霉素, α-糖苷酶抑制剂, 紫外诱变

Abstract: This work is to seek a novel method to detect 1-Deoxynojirimycin(DNJ)in the culture broth of Bacillus subtilis var. niger-231(B-231)and to improve DNJ production. DNJ reacts rapidly with carbon disulfide while Cu²⁺ existing，and the final product extracted by organic solvent was measured by spectrophotometer at 440 nm，thus DNJ content was determined. Then mutant strain B-231 was induced with ultraviolet. The standard curve for measuring standard DNJ was drawn，and the precision，sensitivity，accuracy and stability of the products while using the novel method of measuring DNJ were studied. Orthogonal test was designed to obtain the optimal reaction condition. The novel method was applied to measure the content of DNJ in the culture broth of bacteria. A high-yield DNJ strain was screened and named as B. subtilis var. niger- 431(B-431)by mutagenizing the B-231. The content of DNJ by B-431 was up to 87.3 mg/mL，and increased 4.2 times compared to B-231. This study established a novel method to determine 1-deoxynojirimycin in the culture broth of DNJ-producing strain for the first time. The induction by ultraviolet increased the capacity of this bacterium producing DNJ.

Key words: Bacillus subtilis, 1-deoxynojirimycin, α-glucosidase inhibitor, ultraviolet mutation

龙玲, 杨艳, 朱乃硕. 应用比色法检测枯草芽孢杆菌变异菌株中1-脱氧野尻霉素含量[J]. 生物技术通报, 2016, 32(6): 187-192.

LONG Ling, YANG Yan, ZHU Nai-shuo. Colorimetric Method in the Determination of 1-Deoxynojirimycin Isolated from the Mutant Strain of Bacillus subtilis[J]. Biotechnology Bulletin, 2016, 32(6): 187-192.

参考文献

[1] Kwon HJ, Chung JY, Kim JY, et al. Comparison of 1-Deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects：in vivo and in vitro studies. [J]. Journal of Agricultural and Food Chemistry, 2011, 59(7SI)：3014-3019.
[2] Lee S. 1-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes. [J]. Journal of Microbiology and Biotechnology, 2013, 23(5)：637-643.
[3] He H, Lu YH. Comparison of inhibitory activities and mechanisms of five mulberry plant bioactive components against alpha-glucosidase. [J]. J Agric Food Chem, 2013, 61(34)：8110-8119.
[4] Niwa T, Tsuruoka T, Goi H, et al. Novel glycosidase inhibitors, nojirimycin-b and d-mannonic-delta-lactam - isolation, structure determination and biological property[J]. Journal of Antibiotics, 1984, 37(12)：1579-1586.
[5] Onose S, Ikeda R, Nakagawa K, et al. Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillus species. [J]. Food Chemistry, 2013, 138(1)：516-523.
[6] Kim HS, Lee JY, Hwang KY, et al. Isolation and identification of a Bacillus sp producing alpha-glucosidase inhibitor 1-deoxynojirimycin. [J]. Korean Journal of Microbiology and Biotechnology, 2011, 39(1)：49-55.
[7] Nakagawa K, Kubota H, Kimura T, et al. Occurrence of orally administered mulberry 1-deoxynojirimycin in rat plasma[J]. Journal of Agricultural and Food Chemistry, 2007, 55(22)：8928-8933.
[8] Shibano M, Fujimoto Y, Kushino K, et al. Biosynthesis of 1-deoxynojirimycin in commelina communis：a difference between the microorganisms and plants[J]. Phytochemistry, 2004, 65(19)：2661-2665.
[9] Asano N, Oseki K, Tomioka E, et al. N-containing sugars from morus-alba and their glycosidase inhibitory activities[J]. Carbohydrate Research, 1994, 259(2)：243-255.
[10] Gruters RA, Neefjes JJ, Tersmette M, et al. Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase[J]. Nature, 1987, 330(6143)：74-77.
[11] Butters TD, Dwek RA, Platt FM. Therapeutic applications of imino sugars in lysosomal storage disorders[J]. Current Topics in Medicinal Chemistry, 2003, 3(5)：561-574.
[12] Kang KD, Cho YS, Song JH, et al. Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85[J]. Journal of Microbiology, 2011, 49(3)：431-440.
[13] Zhu YP, Yamaki K, Yoshihashi T, et al. Purification and identifica-tion of 1-Deoxynojirimycin(DNJ)in Okara fermented by Bacillus subtilis B2 from Chinese traditional food(Meitaoza)[J]. Journal of Agricultural and Food Fhemistry, 2010, 58(7)：4097-4103.
[14] Kimura T, Nakagawa K, Kubota H, et al. Food-grade Mulberry powder enriched with 1-Deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans[J]. Journal of Agricultural and Food Chemistry, 2007, 55(14)：5869-5874.
[15] 张志贤.实用有机定量分析[M]. 上海, 上海科技出版社：1965.
[16] 张志贤, 张瑞镐.有机官能团定量分析[M]. 北京, 化学工业出版社：1990.
[17] 周德庆.微生物学实验教程[M]. 北京, 高等教育出版社：2006.
[18] 白豆, 龙玲, 朱乃硕. 一株1-脱氧野尻霉素(DNJ)枯草芽孢杆菌黑色变种的诱变育种研究[J]. 复旦学报:自然科学版, 2006(1)：104-111.
[19] Yamaki K. and Mori Y. Evaluation of a-glucosidase inhibitory activity in colored foods：a trial using slope factors of regression curves[J]. Journal of the Japanese Society for Food Science and Technology-Nippon Shokuhin Kagaku Kogaku Kaishi, 2006, 53(4)：229-231.
[20] Kim JW, Kim SU, Lee HS, et al. Determination of 1-deoxynojirim-ycin in Morus alba L. leaves by derivatization with 9-fluorenylmethyl chloroformate followed by reversed-phase high-performance liquid chromatography[J]. Journal of Chromatography a, 2003, 1002(1-2)：93-99.
[21] Zhu YP, Li XT, Teng C, et al. Enhanced production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2 using response surface methodology[J]. Food and Bioproducts Processing, 2013, 91(3)：264-270.

应用比色法检测枯草芽孢杆菌变异菌株中1-脱氧野尻霉素含量

Colorimetric Method in the Determination of 1-Deoxynojirimycin Isolated from the Mutant Strain of Bacillus subtilis

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	赵志祥, 王殿东, 周亚林, 王培, 严婉荣, 严蓓, 罗路云, 张卓. 枯草芽孢杆菌Ya-1对辣椒枯萎病的防治及其对根际真菌群落的影响[J]. 生物技术通报, 2023, 39(9): 213-224.
[2]	杨冬, 唐璎. 枯草芽孢杆菌WTX1胞外酶降解AFB₁酶学特性及降解位点分析[J]. 生物技术通报, 2023, 39(4): 93-102.
[3]	祖雪, 周瑚, 朱华珺, 任佐华, 刘二明. 枯草芽孢杆菌K-268的分离鉴定及对水稻稻瘟病的防病效果[J]. 生物技术通报, 2022, 38(6): 136-146.
[4]	马艳琴, 邱益彬, 李莎, 徐虹. 透明质酸的生物合成及其代谢工程的研究进展[J]. 生物技术通报, 2022, 38(2): 252-262.
[5]	张倩, 徐春燕, 张铎, 王亚会, 梁新盈, 李慧. 黄褐土玉米秸秆腐解菌株筛选及其促腐能力研究[J]. 生物技术通报, 2022, 38(12): 233-243.
[6]	苗华彪, 曹艳, 杨梦瀚, 黄遵锡. 基于信号肽策略提高外源蛋白在枯草芽孢杆菌中的表达[J]. 生物技术通报, 2021, 37(6): 259-271.
[7]	唐璎, 黄佳, 邓展瑞, 杨晓楠. 一株枯草芽孢杆菌降解黄曲霉毒素B₁产物分析[J]. 生物技术通报, 2021, 37(12): 82-90.
[8]	张维娇, 金学荣, 徐雅晴, 李江华, 堵国成, 康振. 枯草芽孢杆菌表达与调控工具相关研究进展[J]. 生物技术通报, 2020, 36(4): 26-33.
[9]	付首颖, 夏苗苗, 张祎凝, 刘川, 涂然, 张大伟. 核黄素工业菌株高通量筛选方法的建立和应用[J]. 生物技术通报, 2020, 36(4): 47-53.
[10]	赵晓霞, 牛世全, 文娜, 苏锋锋. 黄芪根腐病生防芽孢杆菌的筛选鉴定与盆栽防效试验[J]. 生物技术通报, 2019, 35(9): 107-111.
[11]	邱锦, 黄火清, 姚斌, 罗会颖. 解淀粉芽孢杆菌淀粉酶催化活力改良及其在枯草芽孢杆菌中的高效表达[J]. 生物技术通报, 2019, 35(9): 134-143.
[12]	凌翓, 纪明华, 段海燕, 史吉平, 孙俊松. 木糖酸氧化途径在枯草芽孢杆菌的构建及转化乙醇酸的研究[J]. 生物技术通报, 2019, 35(6): 76-82.
[13]	戚家明, 杨娜, 孙杉杉, 明艳超, 郭亮, 张东旭, 徐志文. 一株具有噬菌体抗性的芽孢杆菌BS-2的鉴定及葡萄糖流加工艺优化[J]. 生物技术通报, 2019, 35(3): 210-216.
[14]	戚家明, 孙杉杉, 张东旭, 徐志文, 徐延平. 芽孢杆菌BS-6基于全基因组数据的分类鉴定及拮抗能力分析[J]. 生物技术通报, 2019, 35(10): 111-118.
[15]	杨何宝 ,胡美荣 ,郑翔 ,牟庆璇 ,高沛汝. 不同信号肽及分子伴侣对中性蛋白酶在枯草芽孢杆菌中分泌表达的影响[J]. 生物技术通报, 2018, 34(6): 134-140.