一株烟用产纤维素酶菌株的筛选、鉴定及酶学特性研究

A High Cellulase-producing Bacterium from Tobacco Lamina

【目的】从不同品种烟叶原料中分离筛选高产纤维素酶细菌菌株,为提高烟叶原料使用价值提供优秀菌种资源。【方法】采用稀释涂布法从37份片烟样品中分离纯化菌株,利用刚果红染色法结合摇瓶复筛进行筛选,测定菌株纤维素酶活性,通过形态学观察与16S rRNA序列鉴定功能菌的系统发育学地位,并对功能菌所产纤维素酶进行酶学性质研究。【结果】筛选到1株烟用纤维素酶活性高的菌株FX-1,其在37℃发酵48 h纤维素酶酶活力最高,达56.261 U·mL^(−1)。FX-1菌体呈杆状,可产芽孢,革兰氏阳性,其16S rRNA序列与Bacillus subtilis相似度最高(100%),系统发育树中与枯草芽孢杆菌位于同一分支中,初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。通过酶学性质分析,酶反应的最适温度为60℃,最适pH为5.0,在温度30~55℃或pH值3.0~7.0保温30 min后仍有较高活力,相对酶活有90%以上,具有良好的热稳定性和耐酸能力。【结论】从37份片烟样品中筛选获得1株高产纤维素酶菌株FX-1,初步鉴定为枯草芽孢杆菌。FX-1所产纤维素酶的反应条件较宽,酶学性质稳定,在烟草行业中具有良好的产业化开发利用前景。

【Objective】Bacteria with a high cellulase-producing capacity were isolated from different varieties of tobacco for the potential of upgrading the economic value of tobacco raw materials.【Methods】Candidate bacterial strains were isolated and purified from 37 flue-cured tobacco strip samples using dilution coating method.They were screened by the Congo red staining combined with shaking flask culture and cellulase activity determination.Phylogenetic property of a strain identified by morphological observation was confirmed with 16S rRNA sequencing.The cellulase activities were investigated.【Results】FX-1 was preliminarily identified as a strain that produced high cellulase activity of 56.261 U·mL^(−1) after 48h fermentation at 37℃.It was a gram-positive,rod-shape,spore-producing bacterium that showed a perfectly matching 16S rRNA sequence in a branch of Bacillus subtilis phylogenetic tree.The cellulase from FX-1 displayed a peak activity at 60℃and pH 5.0.Greater than 90%of its relative activity was retained after being held at 30-55℃or pH 3.0-7.0 for 30 min.【Conclusion】A high cellulase-producing bacterium screened from 37 flue-cured tobacco strip samples was preliminarily identified as a strain of B.subtilis.The cellulase produced by the strain FX-1 exhibited a wide-range temperature and pH stability,and thus,had a considerable potential to be developed for new commercial use in tobacco manufact.