[1]何小玉,刘承忠,陈明霞,等.产碱性冷适蛋白酶菌株Chryseobacterium sp. vv8的鉴定及粗酶性质[J].华侨大学学报(自然科学版),2019,40(5):646-652.[doi:10.11830/ISSN.1000-5013.201904030]
　HE Xiaoyu,LIU Chengzhong,CHEN Mingxia,et al.Identification and Preliminary Enzymatic Properties of Cold-Adapted Alkaline Protease ProducingStrain Chryseobacterium sp. vv8[J].Journal of Huaqiao University(Natural Science),2019,40(5):646-652.[doi:10.11830/ISSN.1000-5013.201904030]
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产碱性冷适蛋白酶菌株Chryseobacterium sp. vv8的鉴定及粗酶性质()
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《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第40卷
期数:
2019年第5期
页码:
646-652
栏目:
出版日期:
2019-09-20

文章信息/Info

Title:
Identification and Preliminary Enzymatic Properties of Cold-Adapted Alkaline Protease ProducingStrain Chryseobacterium sp. vv8
文章编号:
1000-5013(2019)05-0646-07
作者:
何小玉1;  刘承忠1;  陈明霞1;  李和阳2
1. 华侨大学 化工学院, 福建 厦门 361021;2. 自然资源部第三海洋研究所, 福建 厦门 361005
Author(s):
HE Xiaoyu1;  LIU Chengzhong1;  CHEN Mingxia1;  LI Heyang2
1. College of Chemical Engineering, Huaqiao University, Xiamen 361021, China; 2.Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
关键词:
冷适碱性蛋白酶;  金黄杆菌属;  生长特性;  酶特性
Keywords:
cold-adapted alkaline protease;  Chryseobacterium;  growth characteristics;  enzymatic characteristics
分类号:
Q93.9
DOI:
10.11830/ISSN.1000-5013.201904030
文献标志码:
A
摘要:
为鉴定金黄杆菌属(Chryseobacterium)菌株vv8及分析其胞外蛋白酶的酶学特性,首先,通过分子及生理生化特性对菌株vv8进行鉴定;然后,采用明胶平板筛选法和Lowry 法对菌株vv8进行产酶筛选和蛋白酶活性测定;最后,研究pH值、温度、金属离子和抑制剂对该蛋白酶活性的影响.研究结果表明:菌株vv8的16S rDNA与比目鱼黄杆菌(C. scophthalmum)具有98.24%的序列相似性,属于Chryseobacterium属;菌株vv8的最适生长条件是温度为22 ℃,NaCl质量浓度为0 mg·mL-1,pH值为6.0;菌株vv8的胞外蛋白酶在pH值为5.0~10.0,温度为0~90 ℃范围内均具有酶活性,其最适酶活温度和pH值分别为40 ℃,8.5;在最适条件下,蛋白酶具有较高的稳定性,在冻干过程中,添加葡萄糖具有明显的酶活保护作用;Fe3+,Fe2+,Cu2+等金属离子及乙二胺四乙酸(EDTA)对酶活性有明显的抑制作用,该酶对洗衣液和尿素具有较好的耐受性.
Abstract:
To identify Chryseobacterium sp. vv8 and analyze the enzymatic properties of its extracellular protease, the strain vv8 was firstly identified by molecular, physiological and biochemical characteristics. Secondly, the enzyme screening and protease activity of strain vv8 were determined by gelatin plate screening method and Lowry method. Last, the effects of pH value, temperature, metal ions and inhibitors on the protease activity were studied. The results showed that the 16S rDNA of strain vv8 had 98.24% sequence similarity with C. scophthalmum and was phylogenetically assigned as genus Chryseobacterium. The optimum culture conditions of strain were 22 ℃, the mass concentration of NaCl 0 mg·mL-1 and pH value 6.0. The extracellular protease of strain vv8 presented enzyme activity at the range of pH 5.0-10.0 and 0-90 ℃, with the highest a-ctivity at pH 8.5 and 40 ℃. Under the optimal conditions, the protease exhibited good stability, and glucose had obvious enzyme activity protection during lyophilization. Fe3+, Fe2+, Cu2+ and ethylenediaminetetraacetic acid(EDTA)showed obvious inhibitory effects on enzyme activity, but the protease exhibited good tolerance to laundry detergent and urea.

参考文献/References:

[1] 刘金龙.鱼蛋白多肽的制备及其在农业生产中的应用[D].泰安:山东农业大学,2017.
[2] 谢薇,顾龙龙,单妍.一株产耐盐碱性蛋白酶菌株的分离鉴定及最佳生产条件研究[J].包头医学院学报,2018,34(7):95-98.DOI:10.16833/j.cnki.jbmc.2018.07.041.
[3] 徐晓梅.生活污水净化耐冷菌株的分离及降解活性测定[D].武汉:武汉理工大学,2017.
[4] 姜春宇,张媱,孙燕飞,等.产低温蛋白酶酵母菌株的筛选及发酵培养基优化[J].中国酿造,2018,37(11):51-56.DOI:10.11882/j.issn.0254-5071.2018.11.010.
[5] 王继莲,李明源,任羽.布伦口湖群湿地产蛋白酶耐低温菌株的筛选及初步鉴定[J].西南农业学报,2017,30(6):1330-1334.DOI:10.16213/j.cnki.scjas.2017.6.016.
[6] FURHAN J,AWASTHI P,SHARMA S.Biochemical characterization and homology modelling of cold-active alkophilic protease from northwestern Himalayas and its application in detergent industry[J].Biocatalysis and Agricultural Biotechnology,2019,17:726-735.DOI:org/10.1016/j.bcab.2019.01.028.
[7] SINGH S K,SINGH S K,TRIPATHI V R,et al.A novel psychrotrophic, solvent tolerant Pseudomonasputida SKG-1 and solvent stability of its psychro-thermoalkalistable protease[J].Process Biochemistry,2011,46(7):1430-1435.DOI:10.1016/j.procbio.2011.03.012.
[8] 李桂英,廖祥儒,蔡宇杰.微生物转化浮萍资源生产蛋白酶的条件[J].生物资源,2018,40(1):64-69.DOI:10.14188/j.ajsh.2018.01.012.
[9] 林念炜,张锐,赵晶,等.南极产低温蛋白酶菌株Marinobacter sp. R2的发酵条件及酶学性质研究[J].厦门大学学报(自然科学版),2004,43(6):865-869.DOI:10.1161/01.ATV.0000116865.98067.31.
[10] MARGESIN R,SCHINNER F.Characterization of a metalloprotease from psychrophilic Xantho monasmaltophilia[J].Fems Microbiology Letters,1991,79(23):257-262.DOI:10.1111/j.1574-6968.1991.tb04538.x.
[11] KIM E H,CHO K H,LEE Y M,et al.Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture[J].Journal of Microbiology,2010,48(4):426-432.DOI:10.1007/s12275-010-0015-z.
[12] 叶思帆,赵媛.微生物源碱性蛋白酶的生产及其应用[J].青海科技,2018,25(2):75-78.
[13] VANDAMME P,BERNARDET J F,SEGERS P,et al.Notes: New perspectives in the classification of the Flavobacteria: Description of Chryseobacterium gen.nov. Bergeyella gen.nov. and Empedobacter nom.rev.[J].International Journal of Systematic Bacteriology,1994,44(4):827-831.DOI:10.1099/00207713-44-4-827.
[14] 刘静,闵航,章骥,等.一株产低温蛋白酶菌株的筛选鉴定及纯酶研究[J].浙江大学学报(农业与生命科学版),2006,32(3):251-256.DOI:10.3321/j.issn:1008-9209.2006.03.004.
[15] 陈明霞,李和阳,陈维维,等.68株北极产蛋白酶菌株的筛选、鉴定以及部分酶学性质[J].微生物学报,2013,53(7):702-709.DOI:10.13343/j.cnki.wsxb.2013.07.006.
[16] 陈天寿.微生物培养基的制造与应用[M].北京:中国农业出版社,1995.
[17] 晏爱芬,余丽.1株蛋白酶产生菌的分离及生理特征研究[J].安徽农业科学,2011,39(7):3793-3795.DOI:10.13989/j.cnki.0517-6611.2011.07.039.
[18] DELONG E F.Archaea in coastal marine environments [J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(12):5685-5689.DOI:10.1111/j.1753-4887.1981.tb06752.x.
[19] 陈亮,陈明霞,李和阳.一株产脂肪酶芽胞杆菌菌株SJXYZ的鉴定[J].安徽农业科学,2018,46(8):108-109.DOI:10.13989/j.cnki.0517-6611.2018.08.031.
[20] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[21] 梅源,唐佳颖,何惠容,等.霉豆瓣中优势微生物蛋白酶活性测定[J].食品与发酵工业,2017(9):134-140.DOI:10.13995/j.cnki.11-1802/ts.014512.
[22] 王耿,方柏山,张婷婷,等.一株碱性脱毛蛋白酶产生菌的筛选及系统发育分析[J].华侨大学学报(自然科学版),2009,30(3):297-301.DOI:10.11830/ISSN.1000-5013.2009.03.0297.
[23] WEISBURG W G,BARNS S M,PELLETIER D A,et al.16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703.DOI:10.1128/jb.173.2.697-703.1991.
[24] DRANCOURT M,BERGER P,RAOULT D.Systematic 16S rRNA gene sequencing of a typical clinical isolates identified 27 new bacterial species associated with humans [J].Journal of Clinical Microbiology,2004,42(5):2197-2202.DOI:10.1128/JCM.42.5.2197-2202.2004.
[25] 李晓亮,蔡明明,李阳,等.第六次北极科学考察海洋沉积物可培养细菌的多样性分析[J].微生物学通报,2016,43(5):974-983.DOI:10.13344/j.microbiol.china.150875.
[26] MUDARRIS M,AUSTIN B,SEGERS P,et al.Flavobacterium scophthalmum sp nov., a pathogen of turbot(Scophthalmus maximus L)[J].International Journal of Systematic Bacteriology,1994,44(3):447-453.
[27] 徐晓梅,薛双红,袁俊超,等.2株产低温蛋白酶耐冷菌株的筛选及酶学性质[J].江苏农业科学,2017,45(10):209-213.DOI:10.15889/j.issn.1002-1302.2017.10.058.
[28] 刘静,闵航,章骥,等.一株产低温蛋白酶菌株的筛选鉴定及纯酶研究[J].浙江大学学报(农业与生命科学版),2006,32(3):251-256.DOI:10.3321/j.issn.1008-9209.2006.03.004.
[29] 徐国英,林学政,王能飞,等.产低温蛋白酶极地菌株的筛选及Pseudoalteromonas sp.QI-1产蛋白酶粗酶性质[J].生物加工过程,2010,8(2):55-60.DOI:10.3969/j.issn.1672-3678.2010.02.011.
[30] 莫清珊,张会图,田耀,等.低温蛋白酶产生菌的筛选及其酶学性质的初步研究[J].天津科技大学学报,2015(3):19-23.DOI:10.13364/j.issn.1672-6510.20140077.
　　

备注/Memo

备注/Memo:
收稿日期: 2019-04-17
通信作者: 陈明霞(1980-),女,讲师,博士,主要从事海洋微生物的研究.E-mail:chenmx1257@163.com.
基金项目: 国家自然科学基金资助项目(41506179); 国家海洋局全球变化与海气相互作用专项资助项目(GASI-03-01-03-01); 中央级公益性科研院所基本科研业务费专项资金资助项目(海三科2014003); 福建省自然科学基金资助项目(2015J01613); 华侨大学研究
更新日期/Last Update: 2019-09-20