嗜盐淀粉酶产生菌的筛选鉴定及其酶学性质研究

Isolation,Identification and Enzymatic Characteristics of a Halophilic-amylase-producing Strain

关键词:  阴沟肠杆菌  嗜盐淀粉酶  酶学性质

Key words:  Enterobacter cloacae  halophilic amylase  enzymatic property

摘要:

[目的]筛选分离新的嗜盐淀粉酶产生菌,并对其进行酶学性质研究。[方法]从广西大学奶牛厂的奶牛粪便土壤中分离得到一株能产耐盐淀粉酶的菌株,利用16S rRNA序列对比进行鉴定,经破胞提取胞内总蛋白测定嗜盐淀粉酶酶学性质。[结果]经16S rRNA初步鉴定该菌株为阴沟肠杆菌属(Enterobacter cloacae)。该菌株是非嗜盐菌株,却能产生一种胞内嗜盐淀粉酶。酶学性质研究表明该酶在NaCl终浓度为4mol/L时,酶活力最强,当盐浓度为5.5mol/L时,仍能保持最高酶活的60%;以MOPS-NaCl配制的1%可溶性淀粉溶液为底物时,其最适反应温度为50℃,最适pH值为6.5,pH值在4.5~8.5时均能保持60%以上的相对活力;在35~45℃的温度范围内显示较好的稳定性,相对酶活保持在80%以上,但当温度达到50℃时,酶活力急剧下降;Ca²⁺浓度对酶活力几乎没有影响;EDTA浓度在10~140mmol/L时酶活力变化不大,大于140mmol/L之后,酶活力逐渐下降。[结论]该菌株是在非嗜盐菌株中发现的具有嗜盐淀粉酶基因的菌株,其嗜盐淀粉酶在高盐环境下具有很高的酶活力。

Abstract:

[Objective] Screening and isolating halophilic-amylase-producing strains, and studying on their enzymology properties.[Methods] A bacterium which produced salt tolerance amylase was isolated from the cow manure soil in the dairy plant of Guangxi University. The strain was identified by 16S rRNA alignment, and extracted total intracellular protein via cracking the strains' cell to study on the enzymology properties.[Results] By analyzing of 16S rRNA sequence, the strain was initially identified as genus of Enterobacter cloacae. It is a non-halophilic strain, but can produce a kind of intracellular halophilic amylase. Studies on enzymology properties had shown that it had the highest enzyme activity when the final NaCl concentration reached 4mol/L.It had still maintained 60% relative activity at the 5.5mol/L salt concentra-tion. When MOPS-NaCl soluble starch was used as substrate, its optimal activity was at 50℃ and pH 6.5, while more than 60% relative enzyme activity maintained from pH4.5 to pH8.5;the thermostability holded high stability from 35℃ to 45℃ and the relative activity is more than 80%, however, when the temperature reached 50℃, the activity dropped sharply;Ca²⁺ concentration had almost no effect on enzyme activity; the enzyme activity had hardly changed when the EDTA concentration was among 10~140mmol/L, but decreased gradually above 140mmol/L.[Conclusion] The experimental strains were found in the non-halophilic strain with halophilic amylase gene, and the halophilic amylase has the very high enzyme activity in high salt environment.