纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究  

Isolation, Identification, Mutagenesis of Cellulose-degrading Bacterium CMC-4 and Its Enzymatic Properties

作　　者：王霞[1] 华琳 张海龙 朱安宁[3] 曹慧[2] 

机构地区：[1]环境保护部南京环境科学研究所,南京210042 [2]南京农业大学生命科学学院/农业部农业环境微生物重点实验室,南京210095 [3]中国科学院南京土壤研究所,南京210008

出　　处：《土壤》2017年第5期919-925,共7页Soils

基　　金：国家重点基础研究发展计划项目(2011CB100504);江苏省环境监测科研基金项目(0713)资助

摘　　要：从秸秆还田土壤中初筛获得一株高效纤维素降解菌CMC-4,根据菌株形态、理化性质及16S rDNA序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以此为出发菌株,经亚硝酸钠诱变获得一株稳定高产纤维素酶突变株CMC-4-3。对CMC-4和CMC-4-3的产酶条件和酶学性质进行比较分析,结果表明:CMC-4-3纤维素酶活力较CMC-4提高67.5%;其最适产酶条件是:37℃、pH 6.0、葡萄糖为碳源、蛋白胨为氮源、接种量2.0%、装液量60 ml/250 ml。该菌株所产纤维素酶的最适反应pH为6.0,且在4.0~7.0酶活力较稳定;最适反应温度为50℃,在20~80℃范围内均保持稳定;金属离子Fe^(2+)、Mg^(2+)、Co^(2+)、K^+对酶有激活作用,其余离子均有不同程度抑制作用,而Cu^(2+)和Ca^(2+)抑制作用最强,酶活力减弱近50%,是该酶的强效抑制因子。诱变前后菌株产酶条件和酶学性质等部分表型发生了变化,而突变菌株显示出了更宽泛的环境适应范围。据此,获得一株高效产纤维素酶、耐受范围广的具纤维素降解能力的地衣芽孢杆菌,而CMC-4-3和CMC-4的表型可作为深入探讨基因型变化的线索。A strain with highly efficient to degrade cellulose was screened from straw returning soil, termed CMC-4. This strain was identified as Bacillus licheniformis by 16 S r RNA gene sequencing, physiological and biochemical properties. Using sodium nitrite as mutagene, a mutant strain CMC-4-3 with stable high-cellulase producing was obtained. The enzymatic properties and enzyme producing conditions of CMC-4 and CMC-4-3 were analyzed. The results showed that cellulase activity of CMC-4-3 was two times as that of CMC-4. And the optimal conditions for enzyme producing of CMC-4-3 were as follows： medium Volume 60 ml/250 ml, inoculation size 2.0%, source of carbon glucose, nitrogen source peptone, the optimum temperature 37 ℃, optimum pH 6.0. The optimal pH and temperature of reaction for the cellulase were 6.0 and 50 ℃, and the enzyme activity was stable at the ranges of pH from 4.0 to 7.0 and temperature from 20 ℃to 80 ℃. Fe2＋, Mg2＋, Co2＋ and K＋ could activate this cellulase, while other metal ions had some inhibiting effects on enzyme activity. Among inhibitory ions, the inhibition levels of Cu2＋ and Ca2＋ were strongest, with a 50% decrease in enzyme activity, thus proved to be the strongest inhibitors for this cellulase. Compared with wild strain CMC-4, the enzymatic properties and enzyme producing conditions for the mutant strain CMC4-3 changed partly, and showed more wide range of environment adaption. Thus a strain of Bacillus licheniformis with stable high-cellulase producing and wide adaption range was obtained. Moreover, the phenotypes of CMC-4 and CMC-4-3 could be the clue for studying genotype changes and the underlying molecular mechanism of mutation breeding.

关 键 词：地衣芽孢杆菌 纤维素酶 诱变 酶学特性 产酶条件 

分 类 号：S154.39[农业科学—土壤学]