海洋低温甾醇酯酶菌株的筛选鉴定及其酶学特性研究
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摘要
从大连渤海湾海泥海水样品中分离出一株高产海洋甾醇酯酶的菌株Q-06,对其进行形态观察、生理生化试验和分子生物学(16S r DNA)鉴定,并对其产海洋甾醇酯酶进行酶学特性研究。结果表明,菌株Q-06被鉴定为莓实假单胞菌(Pseudomonas fragi),其最适作用温度和pH值分别为30℃和7.0,该酶在低温下酶活性较高,有一定的耐弱碱性。Ag~+对酶的抑制性较强,Mg~(2+) 、Ni~(2+) 、Ca~(2+) 等对甾醇酯酶的活性有微弱的激活作用,乙二胺四乙酸(EDTA)和十二烷基硫酸钠(SDS)均可以有效地抑制甾醇酯酶活性。
A strain Q-06 with high production of marine sterol esterase was isolated from marine mud and water samples in the Bohai Bay sea area of Dalian, and was identified by morphological observation, physiological and biochemical experiments and molecular biology(16S r DNA) identification.Results showed that the strain Q-06 was identified as Pseudomonas fragi. The enzymatic properties of the sterol esterase produced by strain Q-06 were researched. The results showed that the optimum temperature and pH value of the sterol esterase were 30 ℃ and 7.0, respectively. The enzyme activity of sterol esterase was higher at low temperature, and there was a certain resistance to alkalescence. The inhibitory effect of Ag~+ on the sterol esterase was significant, the activation effect of Mg~(2+) , Ni~(2+) and Ca~(2+) on sterol esterase was weaker, and the ethylene diamine tetraacetic acid(EDTA)and sodium dodecyl sulfate(SDS) could effectively inhibit the activity of sterol esterase.
A strain Q-06 with high production of marine sterol esterase was isolated from marine mud and water samples in the Bohai Bay sea area of Dalian, and was identified by morphological observation, physiological and biochemical experiments and molecular biology(16S r DNA) identification.Results showed that the strain Q-06 was identified as Pseudomonas fragi. The enzymatic properties of the sterol esterase produced by strain Q-06 were researched. The results showed that the optimum temperature and pH value of the sterol esterase were 30 ℃ and 7.0, respectively. The enzyme activity of sterol esterase was higher at low temperature, and there was a certain resistance to alkalescence. The inhibitory effect of Ag~+ on the sterol esterase was significant, the activation effect of Mg~(2+) , Ni~(2+) and Ca~(2+) on sterol esterase was weaker, and the ethylene diamine tetraacetic acid(EDTA)and sodium dodecyl sulfate(SDS) could effectively inhibit the activity of sterol esterase.
引文
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[17]王强,李旭,窦少华,等.海洋葡甘聚糖酶菌株的分离鉴定及酶学性质研究[J].中国酿造,2016,35(6):65-69.
[18]任楠楠,王晓辉,迟乃玉,等.微生物甾醇酯酶的研究进展[J].中国酿造,2017,36(6):9-13.
[19]曾诚.甾醇酯酶产生菌的筛选、鉴定及酶学性质研究[D].南京:南京林业大学,2013.
[20] VAQUERO M E, PRIETO A, BARRIUSO J, et al. Expression and properties of three novel fungal lipases/sterol esterases predicted in silico:comparison with other enzymes of the Candida rugosa-like family[J].Appl Microbiol Biot, 2015, 99(23):10057-10067.
[2] VAQUERO M E, BARRIUSO J, MARTíNEZ M J, et al. Properties,structure, and applications of microbial sterol esterases[J]. Appl Microbiol Biot, 2016, 100(5):2047-2061.
[3]罗日明.脂肪酶催化合成植物甾醇酯的研究[D].广州:华南理工大学,2013.
[4]朱婧,史云洁,王颜,等.含植物甾醇酯牛奶对高胆固醇血症患者血脂水平影响的随机对照研究[J].卫生研究,2016,49(5):718-724.
[5]张蕾.植物甾醇及制品降心脑血管疾病风险的功能性评价[D].天津:天津商业大学,2014.
[6]郭玮,陈方俊,潘柏申.胆固醇检测方法的发展历史及标准化[J].检验医学,2013,28(11):970-974.
[7] BROWN A W, HANG J L, DUSSAULT P H, et al. Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase[J]. J Nutr Biochem, 2010, 21(8):736-740.
[8]叶聿程,李祖巍,张向明,等.一株降解树脂细菌的鉴定及发酵条件的优化[J].华东纸业,2013,44(1):36-44.
[9] ZENG C, FAN Y X, DING S J. Screening of sterol esterase-producing strain Chryseobacterium sp. and its emzymatic properties[J]. Chem Ind Forest Prod, 2013, 33(3):43-49.
[10] PETERS J, ONGURI V, NISHIMOTO S K, et al. The Chlamydia trachomatis CT149 protein exhibits esterase activity in vitro and catalyzes cholesteryl ester hydrolysis when expressed in He La cells[J]. Microb Infect, 2012, 14(13):1196-1204.
[11] S?E J B, J?RGENSEN T L. Method for producing phytosterol/phytostanol phospholipid esters:EP2010071352[P]. 2012-02-01.
[12]杨丽娜,迟乃玉,石群,等.海洋氨肽酶菌株的筛选鉴定及其酶学特性研究[J].中国酿造,2017,36(3):66-70.
[13]谭晓晶. Rhodococcus sp.胆固醇酯酶分离纯化与基本性质研究[D].成都:四川大学,2006
[14]曾诚,范远鑫,丁少军.甾醇酯酶菌株Chryseobacterium sp.的筛选及酶学性质研究[J].林产化学与工业,2013,33(3):43-49.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:91-100.
[16]张庆芳,迟乃玉.微生物实验技术[M].沈阳:辽宁科学技术出版社,2001:119-141.
[17]王强,李旭,窦少华,等.海洋葡甘聚糖酶菌株的分离鉴定及酶学性质研究[J].中国酿造,2016,35(6):65-69.
[18]任楠楠,王晓辉,迟乃玉,等.微生物甾醇酯酶的研究进展[J].中国酿造,2017,36(6):9-13.
[19]曾诚.甾醇酯酶产生菌的筛选、鉴定及酶学性质研究[D].南京:南京林业大学,2013.
[20] VAQUERO M E, PRIETO A, BARRIUSO J, et al. Expression and properties of three novel fungal lipases/sterol esterases predicted in silico:comparison with other enzymes of the Candida rugosa-like family[J].Appl Microbiol Biot, 2015, 99(23):10057-10067.
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