[1]齐小雨,陈熙,张炜.人溶菌酶重组酵母工程菌的构建和活性干粉的制备[J].江苏农业学报,2016,(05):1122-1127.[doi:10.3969/j.issn.1000-4440.2016.05.027]
　QI Xiao-yu,CHEN Xi,ZHANG Wei.Construction of recombinant yeast expressing human lysozyme and preparation of active dry powder[J].,2016,(05):1122-1127.[doi:10.3969/j.issn.1000-4440.2016.05.027]
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人溶菌酶重组酵母工程菌的构建和活性干粉的制备()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年05期
页码:
1122-1127
栏目:
畜牧兽医·水产养殖
出版日期:
2016-11-22

文章信息/Info

Title:
Construction of recombinant yeast expressing human lysozyme and preparation of active dry powder
作者:
齐小雨; 陈熙; 张炜
南京农业大学生命科学学院，江苏南京210095
Author(s):
QI Xiao-yu; CHEN Xi; ZHANG Wei
College of Life Science，Nanjing Agricultural University，Nanjing 210095，China
关键词:
人溶菌酶; 毕赤酵母; 胞内表达; 培养条件; 活性酵母干粉
Keywords:
human lysozyme; Pichia pastoris; intracellular expression; culture condition; active dry yeast powder
分类号:
S816.4
DOI:
10.3969/j.issn.1000-4440.2016.05.027
文献标志码:
A
摘要:
本试验旨在构建一种胞内表达人溶菌酶的新型酵母工程菌，并利用其高蛋白、富含溶菌酶的特点开发新型动物饲料添加剂，避免直接使用人溶菌酶制剂带来的高成本、活性不稳定等问题。将来自胎盘的人溶菌酶hLYZ基因克隆至表达载体pPICZA上，验证后的重组载体电转化至X-33毕赤酵母中，经标准培养基BMGY/BMMY培养、甲醇诱导表达后，检测蛋白表达活力和人溶菌酶活力。在此基础上，以麦芽汁-蚕蛹为培养基，通过单因素试验和正交试验来优化培养基成分和摇瓶发酵条件。结果表明，人溶菌酶基因在重组酵母工程菌中成功表达且酶活力为1 920 U。以麦芽汁和蚕蛹浸提物为碳氮源，测得蚕蛹浸提物占40%并且营养盐含量为170 μg/ml时最适合菌体生长。正交试验测得R值的大小为：生长阶段pH值＞诱导阶段甲醇含量＞接种量，最佳摇瓶发酵条件是生长阶段pH值为6，诱导阶段甲醇添加量为1%，接种量为3%。经发酵培养的新型酵母工程菌利用真空冷冻干燥技术制成干粉，活菌数为1 ml 1×109个，溶菌酶活性为1 320 U。上述研究结果为新型饲料添加剂的开发和利用奠定了基础。
Abstract:
This study aims to construct a genetically-engineered yeast intracellularly expressing lysozyme so as to develop a new animal feed additive instead of high cost and unstable lysozyme preparations. The human lysozyme gene hLYZ was cloned into the intracellular expression plasmid pPICZA which was transformed into Pichia pastoris X-33 by electronic transformation. After culture and methanol induction, the recombinant yeast was detected for enzyme activity. The culture condition was optimized by single factor experiment and orthogonal experiment. The result showed lysozyme gene was expressed and the enzyme activity was 1 920 U. Silkworm pupa accounting for 40% of the nitrogen and carbon source and nutrient content at 170 μg/ml were best for yeast thalli growth. The optimal condition for cell growth was pH of 6,methanol inducing concentration of 1% and inoculation size of 3%. By vacuum freezing and drying technology, the genetically engineered yeast was made into active powder with lysozyme activity of 1 320 U and the number of viable count of 1×109 per milliliter. The powder can be used for animal feed additive.

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　CHEN Shan-shan,DING Jian,LI Xin,et al.De novo design of α- factor signal and human lysozyme gene leads to high-level expression in Pichia pastoris[J].,2018,(05):20.[doi:doi:10.3969/j.issn.1000-4440.2018.01.003]

备注/Memo

备注/Memo:
收稿日期：2015-03-01 基金项目：江苏省科技支撑计划项目（BE2013428)；江苏省自然科学青年基金项目（BK20130672）；南京农业大学青年科技创新基金项目（KJ2013030) 作者简介：齐小雨（1990-），女，安徽滁州人，硕士，主要从事蛋白质化学研究。（Tel）17768108908；（E-mail）13865965727@163.com 通讯作者：张炜，（E-mail）wzhang@ njau.edu.cn
更新日期/Last Update: 2016-11-22