枯草芽孢杆菌产中性蛋白酶发酵条件的优化
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摘要
选用玉米粉、豆饼粉、KH_2PO_4及Na_2HPO_4为基础配方,通过单因素实验对枯草芽孢杆菌发酵产中性蛋白酶的发酵培养基、培养条件、发酵条件进行优化。确定了优化的发酵培养基为:20g·L~(-1)甘油,30g·L~(-1)豆饼粉,2mmol·L~(-1)氯化钙,0.3g·L~(-1 )KH_2PO_4,4g·L~(-1 )Na_2HPO_4;优化的培养条件为:培养温度30℃,接种量5%。调控发酵培养基pH值7.0控制发酵,发酵24h酶活达到7 344U·mL~(-1)。进行流加甘油实验,培养温度30℃,发酵过程控制pH值7.0,调整转速和通风量控制溶氧30%～35%,接种量5%,经过30h发酵,酶活达到10 654U·mL~(-1),较未补料提高了45%。
Using corn flour,soybean cake powder,KH_2PO_4,and Na_2HPO_4as the basic ingredients,we optimized the fermentation medium,culture conditions,and fermentation conditions of Bacillussubtilisfor producing neutral protease by single-factor experiments.The optimum fermentation medium can be determined as follows:the glycerin of 20g·L~(-1),the soybean cake powder of 30g·L~(-1),the CaCl_2of 2mmol·L~(-1),the KH_2PO_4of 0.3g·L~(-1),and the Na_2HPO_4 of 4g·L~(-1).Furthermore,the optimum culture conditions can be determined as follows:the culture temperature of 30℃,the inoculum of 5%.Under above conditions,the neutral protease activity reaches 7 344U·mL~(-1 )when the pH value of fermentation medium is 7.0,and the fermentation time is24h.While the glycerol fed-batch experiment indicates that the neutral protease activity can reach 10 654U·mL~(-1),which has increased by 45%compared to that of the control group without fed-batch,when the pH value of fermentation medium is 7.0,the inoculum is 5%,the fermentation time is 30h,and the dissolved oxygen concentration is 30%～35%through adjusting the speed and air flow.
Using corn flour,soybean cake powder,KH_2PO_4,and Na_2HPO_4as the basic ingredients,we optimized the fermentation medium,culture conditions,and fermentation conditions of Bacillussubtilisfor producing neutral protease by single-factor experiments.The optimum fermentation medium can be determined as follows:the glycerin of 20g·L~(-1),the soybean cake powder of 30g·L~(-1),the CaCl_2of 2mmol·L~(-1),the KH_2PO_4of 0.3g·L~(-1),and the Na_2HPO_4 of 4g·L~(-1).Furthermore,the optimum culture conditions can be determined as follows:the culture temperature of 30℃,the inoculum of 5%.Under above conditions,the neutral protease activity reaches 7 344U·mL~(-1 )when the pH value of fermentation medium is 7.0,and the fermentation time is24h.While the glycerol fed-batch experiment indicates that the neutral protease activity can reach 10 654U·mL~(-1),which has increased by 45%compared to that of the control group without fed-batch,when the pH value of fermentation medium is 7.0,the inoculum is 5%,the fermentation time is 30h,and the dissolved oxygen concentration is 30%～35%through adjusting the speed and air flow.
引文
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[5]赵丛,张敏,王建玲,等.N+离子注入诱变选育中性蛋白酶高产菌株及发酵条件的研究[J].工业微生物,2008,38(3):12-16.ZHAO C,ZHANG M,WANG J L,et al.Breeding of neutral protease producing strain and fermentation conditions by N+ion implantation mutagenesis[J].Industrial Microbiology,2008,38(3):12-16.
[6]许波,黄遵锡,陈金全,等.枯草芽孢杆菌AS1.398中性蛋白酶基因的克隆及其在毕赤酵母中的高效表达[J].云南师范大学学报(自然科学版),2005,25(3):51-56.XU B,HUANG Z X,CHEN J Q,et al.Cloning of Bacillus subtilis AS1.398neutral proteinase gene and its high expression in Pichia pastoris[J].Journal of Yunnan Normal University(Natural Sciences Edition),2005,25(3):51-56.
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[8]张敏,赵丛,路福平,等.中性蛋白酶基因在大肠杆菌中诱导表达条件的研究[J].食品与发酵工业,2007,33(10):31-34.ZHANG M,ZHAO C,LU F P,et al.Research on expression condition of neutral protease in the E.coli[J].Food and Fermentation Industries,2007,33(10):31-34.
[9] KATZ M E,MASOUMI A,BURROWS S R,et al.The Aspergillus nidulans xprFgene encodes a hexokinase-like protein involved in the regulation of extracellular proteases[J].Genetics,2000,156(4):1559-1571.
[10] MANSFELD J,PETERMANNL E,DURRSCHMIDT P,et al.The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus[J].Protein Expression and Purification,2005,39:219-228.
[11] YATSUDA A P,BAKKER N,KRIJGSVELD J,et al.Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors[J].Infection and Immunity,2006,74(3):1989-1993.
[12] NILEGAONKAR S S,ZAMBARE V P,KANEKAR P P,et al.Production and partial characterization of dehairing protease fromBacillus cereus MCMB-326[J].Bioresource Technology,2007,98:1238-1245.
[13]程伟,吴丽华,徐亚磊,等.浓香型白酒酿造微生物研究进展[J].中国酿造,2014(3):1-4.CHENG W,WU L H,XU Y L,et al.Research progress on brewing microbes in the production process of Luzhou-flavor liquori[J].China Brewing,2014(3):1-4.
[14] RAO M B,TANKSALE A M,GHATGE M S,et al.Molecular and biotechnological aspects of microbial proteases[J].Microbiology and Molecular Biology Reviews,1998,62(3):597-635.
[15]秦鹏,计成,郭宏.不同能量蛋白水平对褐壳蛋鸡生产性能的影响[J].饲料工业,2001,22(10):22-24.QIN P,JI C,GUO H.Effects of different energy protein levels on the performance of brown-shell hens[J].Feed Industry,2001,22(10):22-24.
[16]钱萍,沈海丽,张璐,等,提高AS1.398中性蛋白酶发酵水平的研究[J].江苏食品与发酵,2005(2):10-15.QIAN P,SHEN H L,ZHANG L,et al.Study on improving the fermentation level of AS1.398neutral protease[J].Jiangsu Food and Fermentation,2005(2):10-15.
[17] SHAFEE N,ARIS S N,RAHMAN R,et al.Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacteriumBacillus cereus strain146[J].Journal of Applied Sciences Research,2005,1(1):1-8.
[18] HUANG G R,DAI D H,HU W L,et al.Optimization of medium composition for thermostable protease production by Bacillus sp.HS08with a statistical method[J].African Journal of Biotechnology,2008,7(8):1115-1122.
[19]陈鸿图,傅金磊,杨月明,等.枯草芽孢杆菌产中性蛋白酶的特性及补料研究[J].食品与发酵工业,2012,38(3):43-48.CHEN H T,FU J L,YANG Y M,et al.Characteristics and feeding research of neutral protease produced by Bacillus subtilis[J].Food and Fermentation Industries,2012,38(3):43-48.
[20] GB/T 23527—2009,蛋白酶制剂[S].
[2]姜锡瑞,段刚,周红伟.酶制剂应用技术问答[M].北京:中国轻工业出版社,2008:45-70.
[3]由德林.酶工程原理[M].北京:科学出版社,2011:40-47.
[4]刘东旺.芽孢杆菌N19中性蛋白酶发酵条件及其酶学性质研究[D].保定:河北农业大学,2008.LIU D W.Fermentation conditions and characteristics of neutral protease fromBacillus N19[D].Baoding:Hebei Agricultural University,2008.
[5]赵丛,张敏,王建玲,等.N+离子注入诱变选育中性蛋白酶高产菌株及发酵条件的研究[J].工业微生物,2008,38(3):12-16.ZHAO C,ZHANG M,WANG J L,et al.Breeding of neutral protease producing strain and fermentation conditions by N+ion implantation mutagenesis[J].Industrial Microbiology,2008,38(3):12-16.
[6]许波,黄遵锡,陈金全,等.枯草芽孢杆菌AS1.398中性蛋白酶基因的克隆及其在毕赤酵母中的高效表达[J].云南师范大学学报(自然科学版),2005,25(3):51-56.XU B,HUANG Z X,CHEN J Q,et al.Cloning of Bacillus subtilis AS1.398neutral proteinase gene and its high expression in Pichia pastoris[J].Journal of Yunnan Normal University(Natural Sciences Edition),2005,25(3):51-56.
[7] WANG L F,RODNEYJ D.Expression of Bacillus subtilis neutral protease gene(nprE)in Saccharomyces cerevisiae[J].Journal of General Microbiology,1993(139):343-347.
[8]张敏,赵丛,路福平,等.中性蛋白酶基因在大肠杆菌中诱导表达条件的研究[J].食品与发酵工业,2007,33(10):31-34.ZHANG M,ZHAO C,LU F P,et al.Research on expression condition of neutral protease in the E.coli[J].Food and Fermentation Industries,2007,33(10):31-34.
[9] KATZ M E,MASOUMI A,BURROWS S R,et al.The Aspergillus nidulans xprFgene encodes a hexokinase-like protein involved in the regulation of extracellular proteases[J].Genetics,2000,156(4):1559-1571.
[10] MANSFELD J,PETERMANNL E,DURRSCHMIDT P,et al.The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus[J].Protein Expression and Purification,2005,39:219-228.
[11] YATSUDA A P,BAKKER N,KRIJGSVELD J,et al.Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors[J].Infection and Immunity,2006,74(3):1989-1993.
[12] NILEGAONKAR S S,ZAMBARE V P,KANEKAR P P,et al.Production and partial characterization of dehairing protease fromBacillus cereus MCMB-326[J].Bioresource Technology,2007,98:1238-1245.
[13]程伟,吴丽华,徐亚磊,等.浓香型白酒酿造微生物研究进展[J].中国酿造,2014(3):1-4.CHENG W,WU L H,XU Y L,et al.Research progress on brewing microbes in the production process of Luzhou-flavor liquori[J].China Brewing,2014(3):1-4.
[14] RAO M B,TANKSALE A M,GHATGE M S,et al.Molecular and biotechnological aspects of microbial proteases[J].Microbiology and Molecular Biology Reviews,1998,62(3):597-635.
[15]秦鹏,计成,郭宏.不同能量蛋白水平对褐壳蛋鸡生产性能的影响[J].饲料工业,2001,22(10):22-24.QIN P,JI C,GUO H.Effects of different energy protein levels on the performance of brown-shell hens[J].Feed Industry,2001,22(10):22-24.
[16]钱萍,沈海丽,张璐,等,提高AS1.398中性蛋白酶发酵水平的研究[J].江苏食品与发酵,2005(2):10-15.QIAN P,SHEN H L,ZHANG L,et al.Study on improving the fermentation level of AS1.398neutral protease[J].Jiangsu Food and Fermentation,2005(2):10-15.
[17] SHAFEE N,ARIS S N,RAHMAN R,et al.Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacteriumBacillus cereus strain146[J].Journal of Applied Sciences Research,2005,1(1):1-8.
[18] HUANG G R,DAI D H,HU W L,et al.Optimization of medium composition for thermostable protease production by Bacillus sp.HS08with a statistical method[J].African Journal of Biotechnology,2008,7(8):1115-1122.
[19]陈鸿图,傅金磊,杨月明,等.枯草芽孢杆菌产中性蛋白酶的特性及补料研究[J].食品与发酵工业,2012,38(3):43-48.CHEN H T,FU J L,YANG Y M,et al.Characteristics and feeding research of neutral protease produced by Bacillus subtilis[J].Food and Fermentation Industries,2012,38(3):43-48.
[20] GB/T 23527—2009,蛋白酶制剂[S].