海洋源蛋白酶产生菌筛选及酶学特性的初步研究
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摘要
从海洋源鱿鱼中筛选高产蛋白酶菌株。通过检测蛋白酶产生水解圈结合蛋白酶活性测定的方法筛选高产蛋白酶菌株,采用PCR技术对筛选菌株进行16S rRNA鉴定,并构建目的菌株的系统发育树,同时研究粗蛋白酶的酶学特性。结果表明:筛选得到的10株产蛋白酶活力较高的菌株,经鉴定分别属于芽孢杆菌属(Bacillus sp.)、普罗威登斯菌属(Providencia sp.)和假单胞菌属(Pseudomonas sp.)。其中SW5菌株产酶活性最高达257.67±2.44 U/mL,为甲基营养型芽孢杆菌(Bacillus methylotrophicus)。该菌株所产粗蛋白酶的酶学特性研究发现,其最适pH为8.0,最适温度为40℃,终离子浓度为1 mmol/L时Mn2+、Ba2+和Ca2+对该蛋白酶活性有较高的激活作用,而Fe~(2+)和Zn~(2+)能明显抑制该蛋白酶活性。
The purpose of this study is to screen high protease production strain from the squid,with generation protease hydrolysis circle and protease activity determination method. The typical strains are identified by 16 S rRNA sequencing,and subject to phylogenetic analysis. Meanwhile,the properties of crude protease enzyme are researched. The results show that after screening 10 strains had a high yield protease activity,identified belong to Bacillus sp.,Providencia sp.,Pseudomonas sp.,Among them SW5 strains produced highest enzyme activity up to 257.67±2.44 U/mL,identified as Bacillus methylotrophicus. Investigation on the crude enzymology characteristics further indicate that the enzyme exhibites the highest activity at pH 8.0 and the optimum temperature is 40 ℃. The protease activity is greatly inhibited by Mn~(2+)、Ba~(2+)and Ca~(2+),but is not inhibited by Fe~(2+)and Zn~(2+)at the final ion concentration 1 mmol/L.
The purpose of this study is to screen high protease production strain from the squid,with generation protease hydrolysis circle and protease activity determination method. The typical strains are identified by 16 S rRNA sequencing,and subject to phylogenetic analysis. Meanwhile,the properties of crude protease enzyme are researched. The results show that after screening 10 strains had a high yield protease activity,identified belong to Bacillus sp.,Providencia sp.,Pseudomonas sp.,Among them SW5 strains produced highest enzyme activity up to 257.67±2.44 U/mL,identified as Bacillus methylotrophicus. Investigation on the crude enzymology characteristics further indicate that the enzyme exhibites the highest activity at pH 8.0 and the optimum temperature is 40 ℃. The protease activity is greatly inhibited by Mn~(2+)、Ba~(2+)and Ca~(2+),but is not inhibited by Fe~(2+)and Zn~(2+)at the final ion concentration 1 mmol/L.
引文
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[25]ZHANG Yan,WU Yanyan,LI Laihao,et al. Identification and antibacterial components of bacterial strain F35 isolated from pinctada fucata mucus[J]. Food Science,2012,33(21):217-220.(in Chinese)
[26]YANG Jia,ZU Baidai,YANG Qing,et al. Isolation and aroma-producing study of bacteria from xiaoerbulake high-temperature daqu[J]. Liquor-Making Science&Technology,2014(10):47-50.(in Chinese)
[27]HUANG Ziyan,LIU Chunhua,LUO Tingting,et al. Screening of proteinase-producing Lactic acid bacteria from fish sauce and the application[J]. Food and Fermentation Industries,2010(11):88-92.(in Chinese)
[28]MONTRIWONG A,RODTONG S,YONGSAWATDIGUL J. Detergent-stable salt-activated proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation[J]. Applied Biochemistry and Biotechnology,2015,176(2):505-517.
[29]PARK J N,FUKUMOTO Y,FUJITA E,et al. Chemical composition of fish sauces produced in Southeast and East Asian countries[J]. Journal of Food Composition and Analysis,2001,14(2):113-125.
[30]ZHANG Hao,ZHANG Chaohua,CAO Wenhong. Optimization of koji-making technology for preparing fish sauce and its fermentation of blue scad[J]. Science and Technology of Food Industry,2014,35(14):215-220.(in Chinese)
[2]RHEE S J,LEE J E,LEE C H. Importance of lactic acid bacteria in Asian fermented foods[J]. Microbial Cell Factories,2011,10(1):1.
[3]WANG Dong,ZHANG Jie,ZHENG Zhiyong,et al. Characteristic comparison of two neutral proteases used for soy sauce fermentttion[J]. Journal of Food and Biological Technology,2012,31(5):479-485.(in Chinese)
[4]FENG Jie,ZHAN Xiaobei,ZHOU Chaohui,et al. Comparative analysis of flavor compounds in draft soy sauce origin from two different membranes[J]. Journal of Food and Biological Technology,2010,29(1):33-39.(in Chinese)
[5]AKOLKAR A V,DURAI D,DESAI A J. Halobacterium sp. SP1(1)as a starter culture for accelerating fish sauce fermentation[J]. Journal of Applied Microbiology,2010,109(1):44-53.
[6]TAO Xingwu,GAO Bing. Study on deodorization of fish sauce during fermentation by external koji addition[J]. China Brewing,2007(12):46-47.(in Chinese)
[7]WICHAPHON J,THONGTHAI C,ASSAVANIG A,et al. Volatile aroma components of Thai fish sauce in relation to product categorization[J]. Flavour and Fragrance Journal,2012,27(2):149-156.
[8]KLOMKLAO S,BENJAKUL S,VISESSANGUAN W,et al. Effects of the addition of spleen of skipjack tuna(Katsuwonus pelamis)on the liquefaction and characteristics of fish sauce made from sardine(Sardinella gibbosa)[J]. Food Chemistry,2006,98(3):440-452.
[9]CHANCHAROONPONG C,HSIEH P C,SHEU S C. Enzyme production and growth of Aspergillus oryzae S. on soybean koji fermentation[J]. Apcbee Procedia,2012,2:57-61.
[10]GUAN L,CHO K H,LEE J H. Analysis of the cultivable bacterial community in jeotgal,a Korean salted and fermented seafood,and identification of its dominant bacteria[J]. Food Microbiology,2011,28(1):101-113.
[11]UDOMSIL N,RODTONG S,CHOI Y J,et al. Use of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation[J]. Journal of Agricultural and Food Chemistry,2011,59(15):8401-8408.
[12]SINSUWAN S,RODTONG S,YONGSAWATDIGUL J. A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce[J]. Food Chemistry,2010,119(2):573-579.
[13]GAO Ruichang,LU Wenting,LIU Xiangdong et al. Comparison on characteristics of four kinds of bombay duck fish sauce fermented by Halophilic archaea[J]. Food and Fermentation Industries,2014,40(11):52-58.(in Chinese)
[14]TIAN Y,UMEZAWA E,DUAN R,et al. Three types of proteinases in Japanese common squid Todarodes pacificus hepatopancreas as studied by using carp myofibrils as substrate[J]. Fisheries Science,2010,76(2):365-373.
[15]TAMURA K,PETERSON D,PETERSON N,et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Molecular Biology and Evolution,2011,28(10):2731-2739.
[16]KUMAR S,NEI M,DUDLEY J,et al. MEGA:a biologist-centric software for evolutionary analysis of DNA and protein sequences[J]. Briefings in Bioinformatics,2008,9(4):299-306.
[17]TOMAC A,MASCHERONI R H,YEANNES M I. Modeling total volatile basic nitrogen production as a dose function in gamma irradiated refrigerated squid rings[J]. LWT-Food Science and Technology,2014,56(2):533-536.
[18]HUAN Huijie,ZHONG Hongbo,LEI Fenfen,et al. Study on isolation and identification of protease-producing marine bacteria and optimization of fermentation medium[J]. Science and Technology of Food Industry,2013,34(24):181-185.(in Chinese)
[19]DENG A,WU J,ZHANG Y,et al. Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp.B001[J]. Bioresource Technology,2010,101(18):7100-7106.
[20]JAOUADI B,ABDELMALEK B,FODIL D,et al. Purification and characterization of a thermostable keratinolytic serine alkaline proteinase from Streptomyces sp. strain AB1 with high stability in organic solvents[J]. Bioresource Technology,2010,101(21):8361-8369.
[21]KAZAN D,BAL H,DENIZCI A A,et al. Studies on alkaline serine protease produced by Bacillus clausii GMBE 22[J].Preparative Biochemistry&Biotechnology,2009,39(3):289-307.
[22]SUNGSRI-IN R,BENJAKUL S,KIJROONGROJANA K. Pink discoloration and quality changes of squid(Loligo formosana)during iced storage[J]. LWT-Food Science and Technology,2011,44(1):206-213.
[23]FRITSCH M J,KREHENBRINK M,TARRY M J,et al. Processing by rhomboid protease is required for Providencia stuartii TatA to interact with TatC and to form functional homo-oligomeric complexes[J]. Molecular Microbiology,2012,84(6):1108-1123.
[24]ZHANG T,LI R,QIAN H,et al. Biosynthesis of levan by levansucrase from Bacillus methylotrophicus SK 21.002[J].Carbohydrate Polymers,2014,101:975-981.
[25]ZHANG Yan,WU Yanyan,LI Laihao,et al. Identification and antibacterial components of bacterial strain F35 isolated from pinctada fucata mucus[J]. Food Science,2012,33(21):217-220.(in Chinese)
[26]YANG Jia,ZU Baidai,YANG Qing,et al. Isolation and aroma-producing study of bacteria from xiaoerbulake high-temperature daqu[J]. Liquor-Making Science&Technology,2014(10):47-50.(in Chinese)
[27]HUANG Ziyan,LIU Chunhua,LUO Tingting,et al. Screening of proteinase-producing Lactic acid bacteria from fish sauce and the application[J]. Food and Fermentation Industries,2010(11):88-92.(in Chinese)
[28]MONTRIWONG A,RODTONG S,YONGSAWATDIGUL J. Detergent-stable salt-activated proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation[J]. Applied Biochemistry and Biotechnology,2015,176(2):505-517.
[29]PARK J N,FUKUMOTO Y,FUJITA E,et al. Chemical composition of fish sauces produced in Southeast and East Asian countries[J]. Journal of Food Composition and Analysis,2001,14(2):113-125.
[30]ZHANG Hao,ZHANG Chaohua,CAO Wenhong. Optimization of koji-making technology for preparing fish sauce and its fermentation of blue scad[J]. Science and Technology of Food Industry,2014,35(14):215-220.(in Chinese)