一株氣單胞菌屬高酶活角蛋白酶生產(chǎn)菌的分離與鑒定
齊清文 張光祥* 馮婷 賴思思 黃才琴 李維
(四川師范大學(xué)生命科學(xué)學(xué)院 四川 成都 610101)
摘 要: 并非所有氣單胞菌屬(Aeromonas)細(xì)菌都是致病菌, 近年來氣單胞菌功能利用方面的研究取得了一些重要進展。用微生物降解法對廢棄羽毛加以有效利用, 既可變廢為寶又符合低碳環(huán)保的要求, 更多分泌高酶活角蛋白酶的新菌種資源的篩選與功能鑒定, 將有助于微生物降解法中降解不徹底和速度慢等難題的解決。以半腐爛羽毛為材料, 先后通過羽毛粉和酪蛋白選擇培養(yǎng)基的反復(fù)初篩和復(fù)篩,、羽毛發(fā)酵液酶活測定等篩選出相對酶活最高的菌株, 并按常規(guī)方法進行分類學(xué)鑒定和生長特性分析,。經(jīng)初篩和復(fù)篩得到的19個菌株中, FD41的羽毛發(fā)酵液相對酶活最高, 達(dá)3.864 U/OD600,。根據(jù)FD41菌株的16S rDNA序列比對,、形態(tài)特征,、革蘭氏染色和糖發(fā)酵試驗等結(jié)果, FD41鑒定為氣單胞菌屬細(xì)菌, 這是首次報道產(chǎn)高酶活角蛋白酶的氣單胞菌菌株,。研究發(fā)現(xiàn)接種量和菌株的不同都是影響培養(yǎng)液中菌體細(xì)胞增殖特性的重要因素, 并且發(fā)酵液酶活受菌體繁殖量的影響很大, 故提出在相同培養(yǎng)條件下按酶活大小篩選菌種時, 應(yīng)使用均一化的菌種接種量并以相對酶活為比較指標(biāo),。
關(guān)鍵詞: 羽毛降解菌, 角蛋白酶, 相對酶活, 氣單胞菌FD41
生物谷推薦英文摘要：
Isolation and identification of a keratinase-producing strain of Aeromonas bacterium
QI Qing-Wen ZHANG Guang-Xiang* FENG Ting LAI Si-Si HUANG Cai-Qin LI Wei
(College of Life Science, Sichuan Normal University, Chengdu, Sichuan 610101, China)
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 microor-ganism-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 ac-tivity (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.
Keywords: Feather-degrading bacterium, Keratinase, Relative enzyme activity, Aeromonas FD41
