该研究以甲醇诱导型重组毕赤酵母（Pichia pastoris） NCY-2为研究菌株，在摇瓶水平上首先考察了诱导时间、甲醇含量、诱导pH及诱导温度对蛋清溶菌酶表达的影响，然后通过正交试验设计优化出了该菌株的最佳发酵条件，并进一步研究了摇瓶发酵过程中的菌体生长和酶活力随时间的变化规律。研究结果表明，蛋清溶菌酶的最适诱导时间为96 h，甲醇含量为2%，诱导pH值为3.5，诱导温度为20 ℃；在此条件下发酵液的蛋清溶菌酶酶活力达到775 U/mL，是优化前的2.2倍。
关键词：中图分类号：Q815 文章编号：0254-5071（20１7）02-0054-04 doi：
Abstract
Using methanol inducible recombinant Pichia pastoris NCY-2 which produces egg-white lysozyme (EWL) as experimental strain, the effect of induction time, methanol concentration, induction pH and temperature on the expression of EWL were investigated by single factor experiments in shake flasks and the optimum fermentation conditions were obtained through orthogonal experiment design. The variation of cell growth and EWL activity with time in shake flask fermentation was further studied. The results showed that the optimal conditions were induction time 96 h, methanol concentration 2%, induction pH 3.5 and temperature 20 ℃. The EWL activity reached 775 U/ml under the optimized conditions, which was 2.2 times compared with that before optimization.
关键词
蛋清溶菌酶 /
毕赤酵母 /
发酵条件 /
优化
{{custom_keyword}} /
Key words
egg white lysozyme /
Pichia pastoris /
fermentation conditions /
optimization
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] 陈忠琼. 溶菌酶在畜禽生产中的应用进展[J]. 饲料博览，2014（8）：32-35.
[2] CALLEWAERT L, MICHIELS C W. Lysozymes in the animal kingdom[J]. J Bioscience, 2010, 35(1): 127-160.
[3] 傅 冰，季秀玲，俞汇颖，等. 鸡蛋清中溶菌酶的分离提取[J]. 浙江农业学报，2013，25（6）：1364-1367.
[4] POTVIN G, AHMAD A, ZHANG Z. Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review[J]. Biochem Eng J, 2012, 64(19): 91-105.
[5] 权志中，张丞斌，余 荣，等. 鸡蛋清溶菌酶基因的克隆及其在毕赤酵母中的表达研究[J]. 饲料工业，2007，28（24）：18-22.
[6] MASUDA T, UENO Y, KITABATAKE N. High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris[J]. Protein Express Purif, 2005, 39(1): 35-42.
[7] 黄继荣，傅正伟，李浙烽，等. 一种高活性鸡溶菌酶的基因克隆、表达与应用： CN 101050467A[P]. 2007-10-10.
[8] 勾连军，徐雪松，宋海泉，等. 一种鸡蛋清溶菌酶基因（RJM）的克隆及其酵母表达方法： CN104946675A[P]. 2015-09-30.
[9] CELIK E, CALIK P. Production of recombinant proteins by microbes and higher organisms[J]. Biotechnol Adv, 2009, 27(3): 297-306.
[10] ZHANG W, INAN M, MEAGHER M M. Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris[J]. Biotechnol Bioprocess Eng, 2012, 5(4): 275-287.
[11] 苟万晓，卫红伟，胡元森，等. 毕赤酵母发酵生产甲醇蛋白工艺条件的优化[J]. 中国酿造，2014，33（7）：78-82.
[12] JAHIC M, GUSTAVSSON M, JANSEN AK, et al. Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes[J]. J Biotechnol, 2003, 102(1): 45-53.
[13] ZHOU X, YING Y, TAO J, et al. Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris, employing high cell density fed-batch fermentation[J]. J Bioscience Bioeng, 2014, 118(4): 420-425.
[14] JIN H, LIU G, DAI K, et al. Improvement of porcine interferon-α production by recombinant Pichia pastoris via induction at low methanol concentration and low temperature[J]. Appl Biochem Biotechnol, 2011, 165(2): 559-71.
[15] 廖锡豪，陈明祥，谢万勇，等. 低温诱导对毕赤酵母表达重组外源蛋白的影响[J]. 中国酿造，2013，32（2）：9-12.
[16] COS O, RAM N R, MONTESINOS J L, et al. Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris, under different promoters: A review[J]. Microbial Cell Fact, 2006, 5(1): 382-391.
[17] LIM H K, CHOI S J, KIM K Y, et al. Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch[J]. Appl Microbiol Biotechnol, 2003, 62(4): 342-348.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金（81370881）；烟台大学研究生科技创新基金重点项目（YDZD1610）
{{custom_fund}}
PDF(974 KB)
