芽孢杆菌种间原生质体融合选育高产Surfactin新菌株

doi:10.13560/j.cnki.biotech.bull.1985.2019-0235

生物技术通报 ›› 2019, Vol. 35 ›› Issue (8): 238-245.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0235

芽孢杆菌种间原生质体融合选育高产Surfactin新菌株

潘旭耀, 魏韬, 谭柱豪, 林龙镇, 郭丽琼, 林俊芳

1. 华南农业大学食品学院生物工程系,广州 510640;
2. 广东省微生态制剂工程技术研究中心, 广州 510640

收稿日期:2019-03-21 出版日期:2019-08-26 发布日期:2019-08-05
作者简介:潘旭耀,男,硕士,研究方向：食品科学;E-mail：401693266@qq.com
基金资助:
国家自然科学基金项目（31572178,31372116）,广州市科技项目（201607010197）,广东省科技项目（2015A020209121）

Breeding of a New Strain of High-yield Surfactin by Bacillus Interspecies Protoplast Fusion

PAN Xu-yao, WEI Tao, TAN Zhu-hao, LIN Long-zhen, GUO Li-qiong, LIN Jun-fang

1. Department of Bioengineering,College of Food Science,South China Agricultural University,Guangzhou 510640;
2. Guangdong Microecological Preparation Engineering Technology Research Center,Guangzhou 510640

Received:2019-03-21 Published:2019-08-26 Online:2019-08-05

摘要/Abstract

摘要： 旨在获得纳豆芽孢杆菌和暹罗芽孢杆菌种间融合高产Surfactin的新菌株。以纳豆芽孢杆菌和暹罗芽孢杆菌为亲本菌株,通过制备纳豆芽孢杆菌和暹罗芽孢杆菌的原生质体,采用PEG介导的双亲灭活标记法融合原生质体,拟融合子通过PCR鉴定、CPC-BTB法高通量筛选、HPLC复筛以及融合菌株的稳定性验证获得高产Surfactin的新菌株。研究结果表明纳豆芽孢杆菌和暹罗芽孢杆菌的原生质体制备最佳酶解浓度分别为0.25 mg/mL和0.2 mg/mL,最佳酶解时间分别为25 min和20 min,其原生质体得率分别为83.1%和84.3%。两者的原生质体融合条件为：纳豆芽孢杆菌紫外灭活80 min,暹罗芽孢杆菌85℃热灭活40 min,融合体系为50%的PEG6000在38℃下融合时间15 min。获得了遗传稳定的融合菌株F8,该菌株Surfactin产量相对于纳豆芽杆菌提高了33.3%,暹罗芽孢杆菌提高了60%。

关键词: 原生质体融合, Surfactin, 双亲灭活, CPC-BTB筛选

Abstract: This work aims to obtain a new strain of high-yield Surfactin via Bacillus natto and Bacillus siamansi interspecies protoplast fusion. The protoplasts of B. natto and B. siamansi were prepared using B. natto and B. siamansi as parent strains,and the protoplasts were fused by PEG-mediated parental inactivation labeling. A new strain producing high yield Surfactin was obtained after the potential fusions identified by PCR,high-throughput screening by CPC-BTB method,HPLC rescreening and stability verification of fusion strains. The results showed as such：the optimal enzymatic hydrolysis concentration of protoplasts of B. natto and B. siamansi were 0.25 mg/mL and 0.2 mg/mL,respectively;the optimal enzymatic hydrolysis time was 25 min and 20 min,respectively;and protoplast yield were 83.1% and 84.3%,respectively. The conditions of protoplast fusion were as follows：B. natto was inactivated by UV for 80 min,B. siamansi was inactivated at 85℃ for 40 min,and the fusion system was 50% PEG6000 at 38℃ for 15 min. A stable fusion strain F8 was obtained,and it produced a 33.3% increase in Surfactin production compared to sole B. natto and a 60% increase by sole B. siamansi.

Key words: protoplast fusion, surfactin, parental inactivation, CPC-BTB reaction

潘旭耀, 魏韬, 谭柱豪, 林龙镇, 郭丽琼, 林俊芳. 芽孢杆菌种间原生质体融合选育高产Surfactin新菌株[J]. 生物技术通报, 2019, 35(8): 238-245.

PAN Xu-yao, WEI Tao, TAN Zhu-hao, LIN Long-zhen, GUO Li-qiong, LIN Jun-fang. Breeding of a New Strain of High-yield Surfactin by Bacillus Interspecies Protoplast Fusion[J]. Biotechnology Bulletin, 2019, 35(8): 238-245.

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芽孢杆菌种间原生质体融合选育高产Surfactin新菌株

Breeding of a New Strain of High-yield Surfactin by Bacillus Interspecies Protoplast Fusion

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