竹鼠肠道纤维素降解菌的分离鉴定及其产酶特性研究

Isolation,Identification and Enzyme Production Characteristics of Cellulolytic Bacterium from Intestine of Bamboo Rats

本试验旨在从竹鼠肠道内容物中分离筛选易培养、具有高纤维素酶活性的菌株。称取竹鼠肠道内容物1.0 g,以刚果红平板进行纤维素降解菌的初筛,再分别以羧甲基纤维素钠(CMC⁃Na)、微晶纤维素、D-水杨苷和滤纸为碳源,采用3,5-二硝基水杨酸(DNS)法测定菌株内切葡聚糖酶(Cen)、外切葡聚糖酶(Cex)、β-葡萄糖苷酶(BG)和滤纸酶(FPA)活性。采用生理生化试验和16S rDNA PCR方法进行菌株种属鉴定。结果显示:从竹鼠肠道内容物中分离获得4株产纤维素酶的需氧菌(GL⁃4、GL⁃5、GL⁃6和GL⁃8),其中GL⁃4的Cen和FPA活性最高,分别为49.32和25.13 U/mL;GL⁃8的Cex活性最高,为30.59 U/mL;GL⁃5的BG活性最高,为35.08 U/mL。对纤维素酶总活性最高的GL⁃4进行了培养条件优化,其在37℃、pH 8.0条件下培养24 h时,FPA活性最高。对产纤维素酶活性较高的3株菌(GL⁃4、GL⁃5和GL⁃8)进行种属鉴定,显示3株菌均为枯草芽孢杆菌。综上可知,本试验从竹鼠肠道内容物中分离到3株需氧枯草芽孢杆菌,这3株菌均具有较高的产酶能力;对菌株GL⁃4进行培养条件优化,得出其产酶最适条件为发酵时间24 h、发酵培养基pH 8和发酵温度37℃,可作为高粗纤维含量非粮型饲料开发利用的候选菌株。

This experiment aimed to isolate and screen bacterial strains with high cellulase activity from the in⁃testinal contents of bamboo rats.Cellulose degrading bacteria in the 1.0 g intestinal contents of bamboo rats were preliminary screened by Congo red plate,and then used sodium carboxymethyl cellulose(CMC⁃Na),microcrystalline cellulose,D⁃salicylic acid and filter paper as carbon sources,to determine the activities of en⁃doglucanase(Cen),exoglucanase(Cex),β⁃glucosidase(BG)and filter paper enzyme(FPA)of bacterial strains with 3,5⁃dinitrosalicylic acid(DNS)method.The species identification of bacterial strains through physiological and biochemical tests and 16S rDNA PCR method.The results showed that 4 cellulase⁃producing aerobic bacteria(GL⁃4,GL⁃5,GL⁃6 and GL⁃8)were isolated from the intestinal contents of bamboo rats.A⁃mong them,GL⁃4 had the highest Cen and FPA activities which were 49.32 and 25.13 U/mL,respectively;GL⁃8 had the highest Cex activity which was 30.59 U/mL;GL⁃5 had the highest BG activity which was 35.08 U/mL.The culture conditions of GL⁃4 which had the highest total cellulase activity were optimized.When the temperature was 37℃,the culture time was 24 h and the pH of the medium was 8.0,the GL⁃4 had the highest FPA activity.The species identification of 3 bacterial strains(GL⁃4,GL⁃5 and GL⁃8)with high cellulase activity showed that all bacterial strains were Bacillus subtilis.In conclusion,three strains of aerobic Bacillus subtilis are isolated from the intestinal contents of bamboo rats,and all the strains have high cellulase⁃producing capacity and are easy to cultivate.The optimal conditions for cellulase⁃producing of GL⁃4 which had the highest total cellulase activity are 24 h of culture time,37℃of culture temperature and pH 8 of medium.The GL⁃4 can be used as a candidate strain for the development and utilization of non⁃grain feeds with high crude fiber content.