一株气单胞菌属高酶活角蛋白酶生产菌的分离与鉴定

Isolation and identification of a keratinase-producing strain of Aeromonas bacterium

摘要:

并非所有气单胞菌属(Aeromonas)细菌都是致病菌, 近年来气单胞菌功能利用方面的研究取得了一些重要进展。用微生物降解法对废弃羽毛加以有效利用, 既可变废为宝又符合低碳环保的要求, 更多分泌高酶活角蛋白酶的新菌种资源的筛选与功能鉴定, 将有助于微生物降解法中降解不彻底和速度慢等难题的解决。以半腐烂羽毛为材料, 先后通过羽毛粉和酪蛋白选择培养基的反复初筛和复筛、羽毛发酵液酶活测定等筛选出相对酶活最高的菌株, 并按常规方法进行分类学鉴定和生长特性分析。经初筛和复筛得到的19 个菌株中, FD41 的羽毛发酵液相对酶活最高, 达3.864 U/OD₆₀₀。根据FD41 菌株的16S rDNA 序列比对、形态特征、革兰氏染色和糖发酵试验等结果, FD41 鉴定为气单胞菌属细菌, 这是首次报道产高酶活角蛋白酶的气单胞菌菌株。研究发现接种量和菌株的不同都是影响培养液中菌体细胞增殖特性的重要因素, 并且发酵液酶活受菌体繁殖量的影响很大, 故提出在相同培养条件下按酶活大小筛选菌种时, 应使用均一化的菌种接种量并以相对酶活为比较指标。

Abstract:

Not all of Aeromonas species were pathogenic, and some bacteria of this genus were found to have important utilization value in recent years. Effective utilization of discarded feather by microorganism-decomposing method could meet the requirements of environment and Low-carbon economy. However, screening and evaluation of more microbe resources should be favourable for solving the thorny problem on poor efficiency and low speed of biodegradation. In the study, keratinase-producing strains were isolated from decaying feather using feather selective medium, casein plate and enzyme activity assay of keratinase in fermentation liquid of feather. The morphological observation of cell and colony, sugar fermentation experiments, Gram stain, PCR and sequence analysis of 16S rDNA were performed for taxonomic classification. The strain named FD41 with the highest relative keratinase activity (RKA, viz. the ratio of keratinase activity unit to A₆₀₀ value of feather fermentation liquid) of 3.864 U/OD₆₀₀ was isolated. The BLAST using the 16S rDNA sequence (GenBank accession No. HM587254) of FD41 showed that the first 100 sequences, all from Aeromonas, shared high homology to HM587254 with 99% identity. According to the results of taxonomic classification, the strain FD41 was identified as an Aeromonas bacterium. This is the first report on keratinase-producing strain of Aeromonas bacterium. The measurement values of keratinase activity of fermentation liquid were found to be influenced signally by cell proliferation. The different strains and inoculation amounts resulted in very distinct proliferation curves of bacterium in liquid medium. These results suggested that the RKA was appropriate index and equalizing inoculation amount was prerequisite for screening and identifying of strains with high enzyme activity at same condition of liquid culture.