纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究

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。

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

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 16S rRNA 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 prope-rties 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 condi-tions 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.