对一株高温蛋白酶高产菌株枯草芽孢杆菌BY25的发酵培养基组成与产酶条件进行了优化。正交试验结果显示培养基中各因子对产酶影响从高到低为:豆饼粉、葡萄糖、硫酸镁、麸皮、磷酸氢二钠、氯化钙。在此基础上进行培养基组成优化,将豆饼粉、麸皮混合氮源改为以豆饼粉为单一氮源进行蛋白酶发酵。单因素试验发现,在单一氮源培养条件下,培养基中各因子对产酶影响从高到低依次为:豆饼粉、葡萄糖、氯化钙、磷酸氢二钠。除微量氯化钙外,金属盐,尤其是金属硫酸盐的添加对产酶有显著抑制作用。此外,培养初始pH和培养时间对产酶有显著影响,接种量也有一定影响。通过绘制120 h产酶曲线发现,BY25产酶曲线为双峰,产酶曲线顶峰出现在发酵后72 h,优化后BY25发酵培养基各组分添加量为豆饼粉60 g/L、葡萄糖60 g/L、氯化钙 0.5 g/L、磷酸氢二钠 4 g/L,接种量为4.5%～5%,初始培养pH为8.0。优化产酶培养基和产酶条件后,发酵液酶活力可达到101.1 μmol/(min·mL)。
Abstract
The fermentation media components and culture conditions of the thermostable protease from a thermophilic strain Bacillus subtilis BY25 were further studied. A six factor and three level orthogonal experiment was conducted to observe the effects of fermentation medium components on thermostable protease yield. Wheat bran was replaced by soybean cake powder for its low contribution rate. Single factor experiment was carried out to optimize the protease production. The optimized culture condition was at 30 ℃, with initiating pH of 8.0, 4.5%-5% inoculum size for 72 h; and the optimized fermentation medium components were described as follows:glucose 60 g/L, soybean cake powder 60 g/L, Na2HPO4 4 g/L and CaCl2 0.5 g/L. After fermentation, the casein hydrolysis activity of the enzyme was estimated up to 101.1 μmol/(min·mL), much higher than other domestic reports.
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Sana B, Ghosh D, Saha M,et al. Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-proteobacterium isolated from the marine environment of the sundarbans [J]. Process Biochem, 2006, 41(1):208-215.
[2] Yagasaki M,Hashimoto S. Syntesis and application of dipeptides; current status and perspectives[J]. Appl Microbiol Biot, 2008, 81(1):13-22.
[3] Madingan M T, Martinko J M, Parke J. 微生物生物学 [M]. 杨文博,译.北京:科学出版社,2001.
[4] 彭素萍, 林先贵, 王一明. 一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究[J]. 土壤, 2010, 42(3):410-414. Peng S P, Lin X G, Wang Y M. Fermentation condition and some properities of a thermostable protease from moderate thermophilic strain BY25[J]. Soils, 2010, 42(3):410-414.
[5] 戴玄, 唐兵, 陈向东, 等. 产高温蛋白酶微生物菌种资源的研究[J]. 微生物学杂志, 1997, 17(3):25-29. Dai X, Tang B, Chen X D,et al. Study on microbial strain resources for thermophilic protease production[J]. Journal of Microbiology, 1997, 17(3):25-29.
[6] Heussen C,Dowdle E B. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates [J]. Anal Biochem, 1980, 102(1):196-202.
[7] Wilson E D. Studies in bacterial proteases I. The relation of protease production to the culture medium[J]. J Bacteriol, 1930, 20(1):41-59.
[8] Semets E V, Glenn A R, May B K, et al. Accumulation of messenger ribonucleic acid specific for extracellular protease in Bacillus subtilis 168 [J]. J Bacteriol, 1973, 116(2):531-534.
[9] Fisher S H. Regulation of nitrogen metabolism in Bacillus subtilis:vive la difference [J]. Mol Microbiol, 1999, 32(2):223-232.
[10] 活泼. 高温中性蛋白酶及其产生菌的初步研究[J]. 工业微生物, 2003, 33(2):30-34. Huo P. Preliminary studies on the thermostable neutral protease[J]. Industrial Microbiology, 33(2):30-34.
[11] 舒丹,李宏,严建华, 等. 高温芽孢杆菌碱性蛋白酶发酵条件及酶性质研究[J]. 四川大学学报, 2004, 41(4):856-860. Shu D, Li H, Yan J H, et al. Study on fermentation condition and properity of alkaline protease from thermoliphic Bacillus sp. SD-142[J]. Journal of Sichuan University, 2004, 41(4):856-860.
[12] 朱泓, 王一明, 林先贵, 等. 一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究[J]. 土壤, 2013, 45(5):899-904. Zhu H, Wang Y M, Lin X G, et al. Differences in enzyme production induced by fermentation media of a moderate thermophilic strain BY25[J]. Soils, 2013, 45(5):899-904.
[13] 朱泓, 王一明, 林先贵. 一株产高温蛋白酶高产菌株菌体增殖和产酶关系的响应面模型研究[J]. 微生物学通报, 2013-10-11,http://www.cnki.net/kems/detail/11.1996.Q.20131011.2259.039.html. Zhu H, Wang Y M, Lin X G. RSM modeling research of the relationship between cell growth and protease yield of a a moderate thermophilic strain Bacillus subtilis BY25 [J]. Microbiology, 2013-10-11,http://www.cnki.net/kems/detail/11.1996.Q.20131011.2259.039.html.
[14] Tokunaga T, Rashid M H, Kuroda A, et al. Effect of degS-degU mutations on the expression of sigD, encoding an alternative sigma factor and autolysin operon of Bacillus subtilis [J]. J Bacteriol, 1994, 176(16):5177-5180.
[15] Dod B, Balassa G, Raulet E, et al. Spore control(Sco)mutations in Bacillus subtilis[J]. Molecular and General Genetics, 1978, 163(1):45-56.
[16] Ogura M,Tanaka T. Transcription of Bacillus subtilis degR is σ D-dependent and suppressed by multicopy proB through σD [J]. J Bacteriol, 1996, 178:216-222.
[17] Perego M,Hoch J A. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis [J]. J Bacteriol, 1988, 170:2560-2567.
基金
收稿日期:2013-03-29 修回日期:2013-11-07
基金项目:中国科学院知识创新工程项目(KSCX2-EW-N-08); 国家林业局“948”项目(2011-G25)
第一作者:朱泓,博士生。*通信作者:林先贵,研究员。E-mail: xglin@issas.ac.cn。
引文格式:朱泓,王一明,林先贵. 一株高温蛋白酶高产菌株产酶条件的优化[J]. 南京林业大学学报:自然科学版,2014,38(1):31-35.
PDF(1603073 KB)
