BP  >> Vol. 5 No. 2 (June 2015)

    产耐高温酸性脂肪酶菌株A-16的诱变选育及其发酵条件优化
    Mutation Breeding of Thermostable Acid Lipase-Producing Strain A-16 and Optimizing of Its Lipase-Producing Condition

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作者:  

张开平,刘燕丽,马博,黄娇丽,潘月楼,欧阳秋飞:百色学院,农业与食品工程学院,广西 百色

关键词:
酸性脂肪酶诱变选育遗传稳定性发酵条件响应面分析Acid Lipase Mutation Breeding Genetic Stability Fermentation Conditions Response Surface Analysis

摘要:

从炼油厂附近土壤中筛选得到一株产耐高温酸性脂肪酶菌株A-16,通过紫外线(UV)和甲基磺酸乙酯(EMS)的复合诱变选育,获得1株产脂肪酶活力较高的突变株UE-03,产脂肪酶活力为17695.6 U/mL,比原始菌株提高了73.6%,将UE-03连续传代培养5次,产酶能力稳定。对突变株UE-03产酶条件进行了探讨,通过单因素试验筛选出最适初始pH4.0,温度为45℃,吐温-80添加量为0.15% w/v;在此基础上采用响应面分析法优化产酶条件,得到适宜产酶条件为:pH 4.28,温度46.8℃,吐温-80添加量0.15% w/v,经优化后菌株产酶活力达到19339.5 U/mL,为该菌株进一步开发利用提供优良菌种和技术参考。

A strain A-16 producing thermostable acid lipase was isolated from the soil of the refinery. By using the lipase-producing strain A-16 as starting strain, a mutant strain UE-03 with high activity of lipase were obtained and selected by the compound mutation breeding of ultraviolet (UV) and ethyl methyl sulfonate (EMS), the activity of lipase produced by that strain reached 17695.6 U/mL, and it was about 73.6% higher than that of the starting strain; the genetic stability experiment showed that the mutant UE-03 had a better genetic stability after 5 times of subculture. The fermentation conditions of mutant strain UE-03 producing lipase were investigated, the optimum results of initial pH 4.0, temperature 45℃, tween-80 0.15% (w/v) were obtained by single-factor test. On this basis, by using response surface methodology to optimize the fermentation conditions, the suitable fermentation parameters were initial pH 4.28, culture temperature of 46.8℃, tween-80 0.15% (w/v), and the acid lipase activity was up to 19339.5 U/mL after optimizing. The study provided ex-cellent strains and technology reference for the further development and utilization of the strain.

文章引用:
张开平, 刘燕丽, 马博, 黄娇丽, 潘月楼, 欧阳秋飞. 产耐高温酸性脂肪酶菌株A-16的诱变选育及其发酵条件优化[J]. 生物过程, 2015, 5(2): 15-24. http://dx.doi.org/10.12677/BP.2015.52003

参考文献

[1] 张开平, 惠明, 田青, 等 (2013) 克雷伯氏菌B-36的分离鉴定及产酸性脂肪酶特性的研究. 中国粮油学报, 10, 80-85.
[2] Tripathi, R., Singh, J., Randhir, K.B. and Thakur, I.S. (2014) Isolation, purification and characterization of lipase from Microbacterium sp. and its application in biodiesel production. Energy Procedia, 54, 518-529.
http://dx.doi.org/10.1016/j.egypro.2014.07.293
[3] Shu, Z.Y., Wu, J.G., Xue, L.Y., Lin, R.F., Jiang, Y.M., Tang, L.H., et al. (2011) Construction of Aspergillus niger lipase mutants with oil-water interface independence. Enzyme Microbial Technology, 48, 129-133.
http://dx.doi.org/10.1016/j.enzmictec.2010.10.011
[4] Hwang, H.T., Qi, F., Yuan, C.L., Zhao, X.B., Ramkrishna, D., Liuand, D. and Varma, A. (2014) Lipase-catalyzed process for biodiesel production: Protein engineering and lipase production. Biotechnology and Bioengineering, 111, 639-653.
http://dx.doi.org/10.1002/bit.25162
[5] Stergiou, P.Y., Foukis, A., Filippou, M., Koukouritaki, M., Parapouli, M., Theodorou, L.G., et al. (2013) Advances in lipase-catalyzed esterification reactions. Biotechnology Advances, 31, 1846-1859.
http://dx.doi.org/10.1016/j.biotechadv.2013.08.006
[6] 杨帆, 李福英, 何雄飞, 等 (2013) 深海产低温脂肪酶菌株Dspro004的诱变育种. 生物学杂志, 3, 20-23.
[7] 苗长林, 罗文, 吕鹏梅, 等 (2013) 脂肪酶产生菌微波-亚硝基胍复合诱变及培养条件优化. 林产化学与工业, 5, 30-34.
[8] 王美珠 (2013) 产油皮状丝孢酵母耐高温菌株的诱变筛选及发酵调控. 华东理工大学, 上海, 4.
[9] 张开平 (2013) 耐高温酸性脂肪酶产生菌的选育及其酶学特性研究.河南工业大学, 郑州, 4.
[10] 朱葛健, 王正祥 (1994) 工业微生物实验技术手册. 中国轻工业出版社, 北京.
[11] Toscano, L., Gochev, A., Montero, G. and Stoytcheva, M. (2011) Enhanced production of extracellular lipase by novel mutant strain of Aspergillus niger. Biotechnology and Biotechnological Equipment, 25, 2243-2247.
http://dx.doi.org/10.5504/BBEQ.2011.0019
[12] Fu, F., Sui, Z.-H., Zhou, W., Wang, J.-G., Chang, L.-P. and Ci, S.-F. (2014) UV-irradiation mutation of tetraspores of Gracilariopsis lemaneiformis and screening of thermotolerant strains. Journal of Applied Phycology, 26, 647-656.
http://dx.doi.org/10.1007/s10811-013-0087-3
[13] Liu, J.H., Wang, F.M., Zhao, Q. and Cheng, H. (2013) Study of mutagenesis on the strain producing cellulase. Energy Science and Technology, 5, 54-57.
[14] 宋炜, 蒋丽娟, 申爱荣, 等 (2009) 高产脂肪酶酵母菌株的分离筛选及紫外诱变. 中南林业科技大学学报, 3, 55-59.
[15] 朱琪, 王维, 兰时乐 (2013) 脂肪酶产生菌黑曲霉ZM-1复合诱变育种研究. 安徽农业科学, 18, 7796-7799.
[16] Agrawal, R., Satlewal, A. and Ashok K V. (2012) Development of a β-glucosidase hyperproducing mutant by combined chemical and UV mutagenesis. Biotechnology, 12.
[17] (2009) GB/T23535-2009, Lipase preparations[S].
[18] Ramani, K., Saranya, P., Jain, S.C. and Sekaran, G. (2013) Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies. Bioprocess and Biosystems Engineering, 36, 301-315.
http://dx.doi.org/10.1007/s00449-012-0785-2
[19] Wang, Q.F., Zhang, C.Y., Hou, Y.H., Lin, X.Z., Shen, J.H. and Guan, X.Y. (2013) Optimization of cold-Active lipase production from psychrophilic bacterium Moritella sp. 2-5-10-1 by statistical experimental methods. Bioscience Biotechnology and Biochemistry, 77, 17-21.
http://dx.doi.org/10.1271/bbb.120104