一株产角蛋白酶的铜绿假单胞菌的分离鉴定

doi:10.16431/j.cnki.1671-7236.2017.03.041

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (3): 920-927.doi: 10.16431/j.cnki.1671-7236.2017.03.041

一株产角蛋白酶的铜绿假单胞菌的分离鉴定

黄妙容¹, 钟楚红², 任广彩¹, 刘传高², 叶俊贤², 陈瑞爱^1,2

1. 农业部动物疫病防控生物技术与制品创制重点实验室, 肇庆 526238;
2. 华南农业大学兽医学院, 广州 510642

收稿日期:2016-07-21 出版日期:2017-03-20 发布日期:2017-03-21
通讯作者: 陈瑞爱 E-mail:chensa727@126.com
作者简介:黄妙容(1983-),女,广东潮州人,博士,研究方向:兽用生物制品,E-mail:miaorongh@foxmail.com;钟楚红(1992-),女,江西赣州人,硕士,研究方向:临床兽医学,E-mail:582042249@qq.com
基金资助:
大华农公司科研基金

Isolation and Identification of A Keratinase-producing Strain of Pseudomonas aeruginosa B1-2

HUANG Miao-rong¹, ZHONG Chu-hong², REN Guang-cai¹, LIU Chuan-gao², YE Jun-xian², CHEN Rui-ai^1,2

1. Key Laboratory of Biotechnology and Drug Manufacture for Animal Epidemic Prevention, Ministry of Agriculture, Zhaoqing 526238, China;
2. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China

Received:2016-07-21 Online:2017-03-20 Published:2017-03-21

摘要/Abstract

摘要：

本研究从鸡场长期堆放羽毛的土壤中筛选到一株降解羽毛能力强的菌株。该菌株在牛奶平板上培养24 h后产生透明降解圈，形状不规则，表面粗糙，边缘模糊，产生绿色荧光色素；革兰氏染色阴性，短杆状，无芽孢，有鞭毛，无荚膜。生化试验显示，该分离株能够水解牛奶、明胶，具有角蛋白酶活性，能利用柠檬酸盐，硝酸还原反应阳性。系统进化树分析显示，分离株与铜绿假单胞菌模式菌株（GenBank登录号：BAMA01000316）亲缘关系最近，综合以上结果，最终确定分离株为铜绿假单胞菌，并正式命名为Pesudomonas aeruginosa B1-2。将该分离株在以1%羽毛为唯一碳氮源的基础培养基中发酵培养，48 h后角蛋白酶活性最高达到60.3 U/mL，对羽毛降解率为85.7%。本研究中分离获得的Pesudomonas aeruginosa B1-2可用于畜禽废弃物的处理。

关键词: 分离鉴定; 生化试验; 角蛋白酶; 铜绿假单胞菌

Abstract:

A new feather-degrading bacterium was isolated from soil samples colected from pile-up feather places in this study.The transparent circle was produced after the strain was cultured on the milk medium for 24 h,the colony shape of the strain was irregular,rough surface,edge blur,and could produce green fluorescent pigment.It was gram negative,short rod,nospore and capsule and had a flagellum scanning by electron microscope. Biochemical test showed that the isolate could hydrolyze milk and gelatin,utilize citrate with keratinase activity and nitrate reduction reaction positive.Phylogenetic tree analysis showed that the isolate was most closely related to Pseudomonas aeruginosa strain (GenBank accession No.:BAMA01000316).Combined with the above results,the isolated strain was identified as Pseudomonas aeruginosa B1-2. When it was inoculated on basal medium containing 1% feather as sole carbon and nitrogen source,the highest level of keratinase activity was 60.3 U/mL after 48 h while the degradation rate of feather was 85.7%. This result indicated that the newly isolated Pseudomonas aeruginosa B1-2 could be useful in management of farm wastes.

Key words: isolation and identification; biochemical test; keratinase; Pseudomonas aeruginosa

中图分类号:

X172

黄妙容, 钟楚红, 任广彩, 刘传高, 叶俊贤, 陈瑞爱. 一株产角蛋白酶的铜绿假单胞菌的分离鉴定[J]. 《中国畜牧兽医》, 2017, 44(3): 920-927.

HUANG Miao-rong, ZHONG Chu-hong, REN Guang-cai, LIU Chuan-gao, YE Jun-xian, CHEN Rui-ai. Isolation and Identification of A Keratinase-producing Strain of Pseudomonas aeruginosa B1-2[J]. , 2017, 44(3): 920-927.

参考文献

[1] Nagal S,Jain P C. Feather degradation by strains of Bacillus isolated from decomposing feathers[J]. Brazilian Journal of Microbiology,2010,41(1):196-200.
[2] Lateef A,Adelere I A,Gueguim-kana E B.Bacillussafensis LAU 13:A new source of keratinase and its multi-functional biocatalytic applications[J].Biotechnology,Biotechnological Equipment,2015,29(1):54-63.
[3] Cao Z J,Lu D,Luo L S,et al. Composition analysis and application of degradation products of whole feathers through a large scale of fermentation[J].Environmental Science and Pollution Research Inter,2011,19(7):2690-2696.
[4] 季金殿.羽毛角蛋白质降解菌的分离、鉴定与角蛋白酶基因的克隆、表达[D].泰安:山东农业大学,2009.
[5] Hu H,Gao J,He J,et al. Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris[J].PLoS One,2013,8(3):e58393.
[6] 季金殿,岳寿松,黄亦钧,等.一株羽毛角蛋白降解菌的鉴定与系统发育分析[J].西南农业学报,2009,3:838-841.
[7] Lange L,Huang Y,Busk P K. Microbial decomposition of keratin in nature-a new hypothesis of industrial relevance[J].Applied Microbiology and Biotechnology,2016,100(5):2083-2096.
[8] 东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[9] Garrity G M,Julia A B,Timothy G L. Taxonomic Outline of the Prokaryotes Bergey's Manual of Systematic Bacteriology[M].2nd ed.New York:Springer,2004.
[10] Lin X,Lee C G,Casale E S,et al. Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformisstrain[J]. Applied and Environmental Microbiology,1992, 58(10):3271-3275.
[11] 刘柏宏.Bacillus licheniformis角蛋白酶的高效表达、热稳定性及底物特异性改造[D].无锡:江南大学,2015.
[12] Cai C G,Lou B G,Zheng X D. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis[J].J Zhejiang Univ Sci B,2008,9(1):60-67.
[13] Mazotto A M,Coelho R R,Cedrola S M, et al. Keratinase production by three Bacillus spp. using feather meal and whole feather as substrate in a submerged fermentation[J].Enzyme Research,2011, 2011:523780.
[14] Kshetri P,Ningthoujam D S. Keratinolytic activities of alkaliphilic Bacillus spp. MBRL 575 from a novel habitat,limestone deposit site in Manipur,India[J].Springerplus,2016,5:595.
[15] Macedo A J,Da silva W O,Gava R,et al. Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilities[J].Applied and Environmental Microbiology,2005,71(1):594-596.
[16] Laba W,Rodziewicz A. Biodegradation of hard keratins by two bacillus strains[J].Jundishapur Journal of Microbiology,2014,7(2):e8896.
[17] Jeevana L P,Kumari C C,Lakshmi V V.Efficient degradation of feather by keratinase producing bacillus SP[J].International Journal of Microbiology,2013,2013:608321.
[18] Anwar M S,Siddique M T,Verma A,et al.Multitrait plant growth promoting (PGP) rhizobacterial isolates from Brassica juncea rhizosphere[J]. Communicative & Integrative Biology,2014,7(1):e27683.
[19] 姚大伟.短小芽孢杆菌碱性蛋白酶酶学特性及其酶基因克隆、表达的研究[D].南京:南京农业大学,2010.
[20] 邵西群,胡博,章秀婷,等.水貂出血性肺炎病原铜绿假单胞菌的分子特征[J].中国兽医科学,2014,4:340-345.
[21] 张香斋,张艳英,李蕴玉,等.鸡源绿脓杆菌的分离鉴定及药敏试验[J].中国畜牧兽医,2015,42(6):1602-1607.
[22] 杨梅,杜崇涛,顾敬敏,等.铜绿假单胞菌噬菌体裂解酶LysYH6的表达与活性[J].吉林农业大学学报,2015,3:357-362.
[23] Yin L,Lee J,Jiang S.Isolation of a keratinase-producing bacterium and purification of its keratinase[J].J Fish Soc Taiwan,2006,33(4):377-390.
[24] 姚青,刘晓迪,顾振红,等.一株假单胞菌Pseudomonas otitidis H3及其应用201310716497.2[P].2013.

一株产角蛋白酶的铜绿假单胞菌的分离鉴定

Isolation and Identification of A Keratinase-producing Strain of Pseudomonas aeruginosa B1-2

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	卢玉葵, 刘佳琪, 钟嘉诚, 陈济铛, 朱婉君, 张溢珊, 张济培. 鹅源副鸡禽杆菌的分离鉴定及致病性研究[J]. 中国畜牧兽医, 2023, 50(6): 2562-2572.
[2]	李志杰, 赵莹, 马鑫, 王萌, 余四九, 王立斌, 潘阳阳. 动物卵泡液外泌体分离方法及主要生殖功能研究进展[J]. 中国畜牧兽医, 2023, 50(4): 1480-1488.
[3]	田常青, 张坤中, 齐玉梅, 史文静, 董志杰, 张浩浩, 何曾文, 芝吉, 赵学慧, 崇倩, 薛慧文, 苟惠天. 1株环境源单增李斯特菌的分离鉴定及生物特性分析[J]. 中国畜牧兽医, 2023, 50(4): 1543-1555.
[4]	曹颖颖, 梁亮, 曾怡蓉, 钟清华, 袁小芳, 唐海波, 冷静. 滇西树鼩源沙门氏菌的分离鉴定及生物学特性研究[J]. 中国畜牧兽医, 2023, 50(4): 1718-1728.
[5]	王冰艺, 马弘财, 邹明昊, 樊世杰, 元振杰, 拉巴次仁, 董海龙, 曾江勇. 牦牛源链球菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2023, 50(2): 745-753.
[6]	肖洋洋, 李芮芮, 马忠臣, 唐恬, 王娜, 陈创夫, 郑炜, 王勇, 王鹏雁. 新疆石河子地区牛支原体的分离鉴定及药物敏感性分析[J]. 中国畜牧兽医, 2023, 50(2): 754-763.
[7]	钟华晨, 王丽芳, 郭晨阳, 刘嘉琳, 宋洁. 内蒙古地区乳房炎奶样与环境中细菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2023, 50(2): 817-826.
[8]	孙愉, 张昊, 李畅洋, 陶燕子, 王秋菊. 牛源抗氧化芽孢杆菌的分离鉴定及生物特性研究[J]. 中国畜牧兽医, 2023, 50(1): 359-367.
[9]	魏津, 王甲, 赵浩然, 刘博, 刘元杰, 姚文生, 刘燕, 马欣. 不同地区鸡毒支原体的分离鉴定及药物敏感性研究[J]. 中国畜牧兽医, 2023, 50(1): 368-376.
[10]	杨福梅, 杨林富, 曾成, 杨志, 王康, 段纲, 代飞燕. 1株麂子源麦芽香肉杆菌的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2023, 50(1): 398-407.
[11]	张薇, 武华, 阴雅洁, 李松励, 侯绍华. 新型鸭呼肠孤病毒分离鉴定及其σC基因序列分析[J]. 中国畜牧兽医, 2022, 49(9): 3520-3529.
[12]	席静, 易继海, 王月丽, 徐朕宇, 李培东, 张江伟, 陈创夫. 牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析[J]. 中国畜牧兽医, 2022, 49(9): 3530-3538.
[13]	刘小兰, 刘昌锦, 余文洋, 李潇翔, 边彦超, 黄校花, 罗锋, 邓舜洲. 猪轮状病毒江西株AY01的分离鉴定[J]. 中国畜牧兽医, 2022, 49(8): 3151-3162.
[14]	徐民生, 柯海意, 施科达, 杨冬霞, 翟少伦, 臧莹安, 李春玲. 广东省猪传染性胸膜肺炎放线杆菌分离鉴定及耐药表型和耐药基因检测与分析[J]. 中国畜牧兽医, 2022, 49(8): 3212-3225.
[15]	杨泽敏, 李双, 金正雨, 廖义潇, 杨颖, 文明. 1株鲤鱼源植物乳杆菌的分离鉴定及其生物学特性分析[J]. 中国畜牧兽医, 2022, 49(7): 2805-2811.