产电海洋脂肪酶生产菌的分离筛选鉴定及培养条件研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0833

摘要/Abstract

摘要：

旨在为研究海洋产脂肪酶细菌的产酶特征,并初步研究其产电性能。从舟山海域潮间带海泥中筛选具有产脂肪酶性能的海洋细菌,以橄榄油为唯一碳源进行平板筛选,采用NaOH滴定分析法测定脂肪酶活性,并初步研究了发酵条件对酶活的影响。为了确定菌株的分类地位,利用16S rRNA基因分析进行鉴定,构建系统发育进化树,并通过单因素实验初步研究了各培养条件对产酶活性的影响,同时初步探究其微生物产电性能。结果显示：从潮间带海泥中筛选到具有产脂肪酶活性的菌株2株,分别标记为S11和S22,分子鉴定结果显示,S11与假单胞属Pseudomonas putida WAB1947,S22与芽孢杆菌属Bacillus substills LSRB在进化地位上最为接近。单因素实验结果显示,培养时间48 h、初始pH值7.0、尿素含量为0.35 g/L时两株菌株对应的产酶活性最好。构建微生物燃料电池（MFC）进行电压采集,结果显示S11、S22的最大输出电压分别为0.055 V和0.031 V。菌株S11和S22分别为假单胞属Pseudomonas putida和芽孢杆菌属Bacillus substills,并表现出一定的产电性能。

关键词: 脂肪酶, 16S rRNA, 进化树, 微生物燃料电池

Abstract:

The objective of this work is to study the characteristics and electricity-producing of lipase-producing bacteria in the sea. The strains with lipase-producing feature were screened from the intertidal marine mud in Zhoushan sea area. Olive oil was used as the sole carbon source for plate screening,NaOH titration was used to determine the lipase activity,and the effect of fermentation condition on the lipase activity was preliminarily studied. To determine the taxonomic status of strains,16S rRNA gene analysis was used for identification,phylogenetic tree was constructed,and the effects of various culture conditions on the enzyme production activity were preliminarily studied through single-factor experiment. The microbial electric performance was preliminarily explored. The results showed that two strains with lipase-producing activity were selected from the intertidal mud,labeled S11 and S22,respectively. Molecular identification results revealed that S11 and Pseudomonas putidawa WAB1947,S22 and Bacillus substills LSRB were the closest in phylogenetic status respectively. The results of single-factor experiment demonstrated that the corresponding enzyme activity of the two strains was the best when the culture time was 48 h,the initial pH value was 7.0,and the urea content was 0.35 g/L. Microbial fuel cell（MFC）was constructed for voltage acquisition,and the results showed that the maximum output voltage of S11 and S22 was 0.055 V and 0.031 V,respectively. In conclusion,strain S11 and S22 is Pseudomonas putida and Bacillus substills,respectively,and they present certain electricity-producing capacity.

Key words: lipase, 16S rRNA, phylogenetic tree, microbial fuel cell

黄阳天, 陆育彪, 黄益帖, 孟繁龙, 徐凯文, 李鹏. 产电海洋脂肪酶生产菌的分离筛选鉴定及培养条件研究[J]. 生物技术通报, 2020, 36(12): 91-97.

HUANG Yang-tian, LU Yu-biao, HUANG Yi-tie, MENG Fan-long, XU Kai-wen, LI Peng. Screening and Identification of Marine Electricity-producing and Lipase-producing Bacteria and Preliminary Study on Its Culture Conditions[J]. Biotechnology Bulletin, 2020, 36(12): 91-97.

图/表 10

表1 脂肪酶活性测定反应体系

操作步骤	空白对照/mL	样品/mL（3个重复）
0.05 mol/L pH8.0 Tris-HCl缓冲溶液	5	5
橄榄油乳化液	4	4
	60℃水浴预热5-10 min
95%无水乙醇	15	0
发酵液	1.0（灭活）	1.0
	60℃水浴、震荡10 min
95%无水乙醇	0	15
1%酚酞	0.025	0.025
0.05 mol/L NaOH溶液滴定至终点	V_空	V_样

图1 菌株S11和S22的系统发育树

图2 菌株S11和S22的SEM扫描图

图3 培养时间对脂肪酶酶活的影响

图4 初始pH值对脂肪酶酶活的影响

图5 尿素含量对脂肪酶酶活的影响

图6 培养温度对脂肪酶酶活的影响

图7 橄榄油含量对脂肪酶酶活的影响

图8 甘油含量对脂肪酶酶活的影响

图9 菌株输出电压采集图

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