食品与发酵工业 >

2024 , Vol. 50 >Issue 16: 30 - 39

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.039631

研究报告

产碱性蛋白酶地衣芽孢杆菌合成培养基筛选与优化

  • 姚巧儿 ,
  • 张英 ,
  • 王永红
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  • (华东理工大学,生物反应器工程国家重点实验室,上海,200237)
第一作者：硕士研究生(王永红教授为通信作者,E-mail:yhwang@ecust.edu.cn)

收稿日期: 2024-04-19

  修回日期: 2024-05-09

  网络出版日期: 2024-09-19

基金资助

国家重点研发计划(2021YFC2100205)

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Screening and optimization of chemically defined medium for alkaline protease production by Bacillus licheniformis

  • YAO Qiaoer ,
  • ZHANG Ying ,
  • WANG Yonghong
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  • (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China)

Received date: 2024-04-19

  Revised date: 2024-05-09

  Online published: 2024-09-19

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摘要

该研究对于产碱性蛋白酶地衣芽孢杆菌(Bacillus licheniformis)的合成培养基进行了系统的筛选与优化。基于豆粕成分分析及文献报道构建初始化学合成培养基CDM0,并通过单一组分移除实验,初步确定对菌体生长有显著影响(生长降低20%以上)的组分,包括3种碱基、4种维生素、11种氨基酸以及一些金属离子和无机盐。在此基础上进行了组分添加实验,分别添加单一组分移除实验从CDM0中剔除的组分,进而筛选出对菌体生长具有20%以上促进作用的5种关键营养物质,并用全排列方法得到了26种组合添加方式,经实验确定了添加的最佳组合为(NH4)2SO4、腺嘌呤和天冬酰胺。此外,还研究了甘油、5种单糖和3种双糖对菌体生长及碱性蛋白酶合成的影响,最终以半乳糖为碳源确定了一种优化后的化学全合成培养基CDM3。实验结果表明,与CDM0相比,CDM3显著提高了菌体的生长速率和碱性蛋白酶的产量,其中酶活力提升至10 300.7 U/mL,提高了约2.5倍。该实验结果为今后研究该菌株的代谢特性奠定了基础。

关键词： 地衣芽孢杆菌; 碱性蛋白酶; 化学合成培养基; 筛选优化; 碳源

本文引用格式

姚巧儿 , 张英 , 王永红 . 产碱性蛋白酶地衣芽孢杆菌合成培养基筛选与优化[J]. 食品与发酵工业, 2024 , 50(16) : 30 -39 . DOI: 10.13995/j.cnki.11-1802/ts.039631

Abstract

In this study, a systematic screening and optimization approach was employed to identify and characterize the optimal chemically defined medium for cultivating Bacillus licheniformis to produce alkaline protease.The initial chemically defined medium CDM0 was constructed based on the analysis of the composition of soybean meal and relevant literature reviews.The components that significantly affected the growth of the bacterium (growth reduced by more than 20%) were preliminarily identified through single component removal experiments, including three bases, four vitamins, 11 amino acids, as well as various metal ions and inorganic salts.Based on this, the components that were taken out of CDM0 were added respectively and then five key nutrients with a more than 20% enhancement in cell growth were screened out.Then the optimal combination of (NH4)2SO4, adenine, and asparagine was obtained from a total of 26 combinations of the five key nutrients.Furthermore, the effects of glycerol, five monosaccharides, and three disaccharides on bacterial growth and alkaline protease chemically defined were also investigated.Ultimately, an optimized chemical complete synthesis medium, CDM3, was developed with galactose as the carbon source.The experimental results demonstrated that the cell growth and alkaline protease production under CDM3 were found to be significantly increased compared to the initial medium CDM0, where the enzyme viability was elevated by 2.5-fold to 10 300.7 U/mL.The findings of this experiment provide a foundation for future research on the metabolic characteristics of this strain.

Key words： Bacillus licheniformis; alkaline protease; chemically defined medium; screening and optimization; carbon source

参考文献

[1] 程晓芳, 袁丹丹, 张余慧, 等.蛋白酶在食品工业中的应用研究进展[J].食品研究与开发, 2018, 39(7):221-224.
CHENG X F,YUAN D D,ZHANG Y H,et al.Research progress of proteases in the food industry[J].Food Research and Development,2018, 39(7):221-224.
[2] KHAMBHATY Y.Applications of enzymes in leather processing[J].Environmental Chemistry Letters, 2020, 18(3):747-769.
[3] ZHANG G Q, CHEN Y K, LI Q H, et al.Growth-coupled evolution and high-throughput screening assisted rapid enhancement for amylase-producing Bacillus licheniformis[J].Bioresource Technology, 2021, 337:125467.
[4] 艾雨晴, 陈松骏, 秦娟, 等.微生物产蛋白酶的研究进展[J].食品工业科技, 2021, 42(19):451-458.
AI Y Q, CHEN S J, QIN J, et al.Advances in proteases producted by microorganisms[J].Science and Technology of Food Industry, 2021, 42(19):451-458.
[5] SONI S, CHHOKAR V, BENIWAL V, et al.Cost effective media optimization for PHB production by Bacillus badius MTCC 13004 using the statistical approach[J].International Journal of Biological Macromolecules, 2023, 233:123575.
[6] ZHAO F, ZHENG M Y, XU X M.Microbial conversion of agro-processing waste (peanut meal) to rhamnolipid by Pseudomonas aeruginosa:Solid-state fermentation, water extraction, medium optimization and potential applications[J].Bioresource Technology, 2023, 369:128426.
[7] 周魏, 曾嵩玉, 余金凤, 等.一株地衣芽胞杆菌产碱性蛋白酶条件优化[J].微生物学通报, 2022, 49(7):2753-2766.
ZHOU W, ZENG S Y, YU J F, et al.Optimization of alkaline protease production by a strain of Bacillus licheniformis[J].Microbiology China,2022,49(7):2753-2766.
[8] 何冬萍, 周康熙, 朱晓萍, 等.葛根淀粉为基质液态发酵生产红曲色素的培养基成分优化[J].中国食品学报, 2019, 19(2):108-115.
HE D P, ZHOU K X, ZHU X P, et al.Optimization of medium components producing Hongqu pigments in liquid-state fermentation using kudzu root starch as culture media[J].Journal of Chinese Institute of Food Science and Technology, 2019, 19(2):108-115.
[9] 胡栋, 柯灵超, 张敬宇, 等.响应面法设计优化阿维菌素化学合成发酵培养基[J].中国抗生素杂志, 2018, 43(8):1055-1061.
HU D, KE L C, ZHANG J Y, et al.Development and optimization of a fermentation chemically defined medium composition for the production of avermectins by response surface methodology[J].Chinese Journal of Antibiotics, 2018, 43(8):1055-1061.
[10] 汤宝, 王金子, 赵延存, 等.抗真菌物质HSAF发酵全合成培养基的筛选[J].中国生物防治学报, 2022, 38(3):645-652.
TANG B, WANG J Z, ZHAO Y C, et al.Screening of chemically defined medium for antifungal substance HSAF[J].Chinese Journal of Biological Control, 2022, 38(3):645-652.
[11] 施盛超, 刘志豪, 王永红.拟干酪乳杆菌全合成培养基氨基酸及维生素组分优化研究[J].食品与发酵工业, 2023, 49(18):24-30.
SHI S C, LIU Z H, WANG Y H.Optimization of amino acids and vitamins components in complete chemical defined medium of Lactobacillus paracasei[J].Food and Fermentation Industries, 2023, 49(18):24-30.
[12] 宋萍, 戚小灵, 胡燚, 等.响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基[J].中国生物工程杂志, 2010, 30(8):100-105.
SONG P, QI X L, HU Y, et al.Optimization of lipase production conditions by Bacillus subtilis using surface methodology[J].China Biotechnology, 2010, 30(8):100-105.
[13] 廖建国, 洪铭, 储炬.运用高通量筛选技术优化红霉素A发酵的合成培养基[J].中国抗生素杂志, 2018, 43(1):51-58.
LIAO J G, HONG M, CHU J.Optimizition of synthetic medium for erythromycin A production by highthroughput screening technology[J].Chinese Journal of Antibiotics, 2018, 43(1):51-58.
[14] 胡悦, 李汉文, 喻晨, 等.LiCl-ARTP复合诱变选育高产碱性蛋白酶菌株及其发酵条件优化[J].中国酿造, 2021, 40(2):59-65.
HU Y, LI H W, YU C, et al.Breeding of high yield alkaline protease strain by LiCl-ARTP compound mutation and fermentation condition optimization[J].China Brewing, 2021, 40(2):59-65
[15] ZHANG J, ZHOU J W, LIU J, et al.Development of chemically defined media supporting high cell density growth of Ketogulonicigenium vulgare and Bacillus megaterium[J].Bioresource Technology, 2011, 102(7):4807-4814.
[16] KUMARI R, SHARMA N, SHARMA S, et al.Production and characterization of bioactive peptides in fermented soybean meal produced using proteolytic Bacillus species isolated from kinema[J].Food Chemistry, 2023, 421:136130.
[17] IBÁÑEZ M A, DE BLAS C, CÁMARA L, et al.Chemical composition, protein quality and nutritive value of commercial soybean meals produced from beans from different countries:A meta-analytical study[J].Animal Feed Science and Technology, 2020, 267:114531.
[18] MÜLLER J, BECKERS M, MUßMANN N, et al.Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium[J].Microbial Cell Factories, 2018, 17(1):106.
[19] CHAMBI D, LUNDQVIST J, NYGREN E, et al.Production of exopolysaccharides by cultivation of halotolerant Bacillus atrophaeus BU4 in glucose- and xylose-based synthetic media and in hydrolysates of quinoa stalks[J].Fermentation, 2022, 8(2):79.
[20] XIAO F, GUO F F.Impacts of essential amino acids on energy balance[J].Molecular Metabolism, 2022, 57:101393.
[21] TOMITA T.Structure, function, and regulation of enzymes involved in amino acid metabolism of bacteria and archaea[J].Bioscience, Biotechnology, and Biochemistry, 2017, 81(11):2050-2061.
[22] WANG X, XIA K, YANG X J, et al.Growth strategy of microbes on mixed carbon sources[J].Nature Communications, 2019, 10(1):1279.
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