产耐盐蛋白酶深海细菌的分离鉴定

The Isolation and Identification of Deep-sea Bacteria That Produce Salt-tolerant Proteases

摘要:

本文采用高盐富集、复筛发酵及耐盐稳定性实验的方式，从深海海泥中筛选产耐盐蛋白酶的深海海洋细菌。从深海海洋微生物中共筛选出25株耐盐菌，在这25株菌株中有14株在酪蛋白平板上能产生较大的水解圈；通过发酵复筛及耐盐稳定性实验，筛选得到一株酶活性能高且耐盐稳定性好的菌株，将其编号为SWJSS3；对菌株SWJSS3进行16S rDNA的鉴定并初步研究了盐浓度对产酶情况和酶稳定性的影响。菌株SWJSS3在0~10% (m/V）的盐浓度条件下均能生长，在0~10%的盐浓度下，菌液OD600的吸光值范围为0.08-1.98；通过发酵复筛其所产生的蛋白酶酶活在盐浓度为1%时达最高，为233.56±2.16 U/mL；所产蛋白酶在终浓度为15% (m/V)的NaCl溶液下，混匀4 ℃保存1 h，残余酶活为初始酶活的40.70±2.06%，继续存放一段时间到第9 h，酶活基本保持不变；通过16S rDNA鉴定其为铜绿假单胞菌，与Pseudomonas aeruginosa RP28 16S rDNA的相似性达到99%以上。

Abstract:

This study was aimed at screening deep-sea bacteria that produce salt-tolerant proteases from deep-sea mud samples based on high-salt accumulation, fermentation rescreening, and salt-tolerant stability tests. Twenty-five strains of deep-sea marine microorganisms were selected, 14 of which demonstrated an increase in hydrolysis cycles on casein plates. A strain with high enzyme activity and salt stability was obtained based on fermentation rescreening and a salt-tolerance test, and was labeled as SWJSS3. Strain SWJSS3 was identified using 16S rDNA and a preliminary study on the effects of salt concentration on its protease yield and stability was conducted. The strain was able to grow in medium with salt concentrations between 0% and 10% and an OD600 of the bacterial suspension ranging from 0.08 to 1.98. The activity of proteases produced by SWJSS3 was maximal for a 1% salt concentration in the fermentation rescreening (233.56 ± 2 U/mL). After treatment with 15% (final concentration) sodium chloride solution at 4 °C for 1 h, the enzyme activity decreased to 40.70 ± 2.06% of the initial enzyme activity, but remained stable for up to 9 h. strain SWJSS3 was identified as Pseudomonas aeruginosa, exhibiting 99% 16S rDNA sequence similarity to P. aeruginosa RP28.