海藻糖合酶在毕赤酵母表面的展示

doi:10.13523/j.cb.20160211

中国生物工程杂志

2016, Vol. 36

Issue (2): 73-80 DOI: 10.13523/j.cb.20160211

技术与方法

海藻糖合酶在毕赤酵母表面的展示

李梦悦, 王腾飞, 汪俊卿, 赵一瑾, 程成, 王瑞明

齐鲁工业大学生物工程学院济南 250353

Expression of Trehalose Synthase Gene in Pichia pastoris

LI Meng-yue, WANG Teng-fei, WANG Jun-qing, ZHAO Yi-jin, CHENG Cheng, WANG Rui-ming

Department of Biology Engineering, QILU University of Technology, Jinan 250353, China

全文: PDF(996 KB) HTML

摘要：

海藻糖合酶能够利用麦芽糖一步法转化生产海藻糖,其底物专一性较高,该酶体系生产工艺简单,不受底物麦芽糖浓度的影响,是工业生产海藻糖的首选。为获得具有生产海藻糖合酶能力的毕赤酵母表面展示载体,实验以筛选的Pseudomonas putide P06海藻糖合酶基因为模板,PCR扩增得到海藻糖合酶基因(tres,2064 bp),连接至pPICZαA质粒中,获得重组质粒pPICZαA-tres。以来自酿酒酵母的共价连接细胞壁的Pir系列蛋白的Pir1p成熟肽蛋白作为毕赤酵母表面展示的锚定蛋白,利用PCR技术扩增得到pir1p(847 bp),连接至重组质粒pPICZαA-tres中,获得重组质粒pPICZαA-tres-pir1p。将重组质粒电击转入毕赤酵母GS115中,利用α-factor信号肽将蛋白引导分泌至细胞壁展示于毕赤酵母表面。通过Zeocin抗性筛选,挑选出阳性克隆子并摇瓶发酵。发酵产物经离心、破碎并使用昆布多糖酶水解,洗脱,结果显示,SDS-聚丙烯酰胺凝胶电泳分析可见明显融合蛋白条带,表明海藻糖合酶已成功地锚定在毕赤酵母。将重组毕赤酵母使用pH 7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在30℃~60℃水浴条件下作用2 h,反应产物利用HPLC检测,能够检测到酶学活性。在优化后的条件pH 7.5,50℃,表面展示海藻糖合酶酶活达到300.65 U/g。40℃~50℃酶活较稳定,保温60 min,残留酶活相对活力达75%以上;最适反应pH值为7.5,并在碱性环境下稳定。

关键词： 表面展示; 海藻糖合酶; 海藻糖; 毕赤酵母; 重组表达

Abstract:

Trehalose synthase can be used to transform the production of trehalose in one step, and its substrate specificity is higher, the production process is simple, and can not be influenced by the concentration of substrate maltose, which is the first choice for industrial production of trehalose. To obtain the surface display vector of Pichia pastoris with the ability of producing trehalose synthase, trehalose synthase gene(tres, 2064 bp) was amplified by PCR from genome of Pseudomonas putide P06(gi=1042893, NCBI), then linked to the plasmid pPICZαA to get the recombinant plasmid pPICZαA-tres. Pir series protein Pir1p which is covalently linked cell wall of Saccharomyces cerevisiae, was used as the anchoring protein, and the pir1p(847 bp) was amplified by PCR technique, then linked to recombinant plasmid pPICZαA-tres, and the recombinant plasmid pPICZA-tres-pir1p was obtained. The recombinant plasmid was transferred into Pichia pastoris GS115 by electroporation, and the protein was directed to the cell wall by a-factor signal peptide and then was displayed on the surface of Pichia pastoris. The positive clones were selected by Zeocin resistance screening. Centrifuging, crushing and using laminarinase hydrolysis the fermentation products, SDS-polyacrylamide gel electrophoresis analysis showed obvious fusion protein bands. The trehalose synthase successfully anchored in Pichia pastoris. The Pichia pastoris strains was hang up using pH 7.5 buffer suspension and the concentration of substrate for 30% of the maltose in 30℃ to 60℃ water bath roling 2 h. Reaction products were analyzed by HPLC and the enzymatic activity can be detected. In the optimized condition, pH 7.5, 50℃, the activity of trehalose synthase reached 300.65U/g. The enzyme activity was stable in the range of 40℃ to 50℃, holding 60 min, the residual activity was more than 75%. The optimum pH was 7.5, and in alkaline environment enzyme activities remained stable.

Key words: Trehalose Surface display Recombinant expression Trehalose synthase Pichia pastoris

收稿日期: 2015-09-15 出版日期: 2015-11-19

ZTFLH:

Q819

基金资助:

国家自然科学基金(31501413)、山东省高校创新项目(J14LE02)资助项目

通讯作者: 王瑞明 E-mail: ruiming3K@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	李梦悦
	王腾飞
	汪俊卿
	赵一瑾
	程成
	王瑞明

引用本文:

李梦悦, 王腾飞, 汪俊卿, 赵一瑾, 程成, 王瑞明. 海藻糖合酶在毕赤酵母表面的展示[J]. 中国生物工程杂志, 2016, 36(2): 73-80.

LI Meng-yue, WANG Teng-fei, WANG Jun-qing, ZHAO Yi-jin, CHENG Cheng, WANG Rui-ming. Expression of Trehalose Synthase Gene in Pichia pastoris. China Biotechnology, 2016, 36(2): 73-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160211 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I2/73

[1] Elbein A D,Pan Y T,Pastuazak I,ei al. New insights on trehalose; a multifunctional molecule. Glycobiology,2003,13(4):17-27.
[2] 段作营,姚林,毛忠贵.Pseudomonas putida S1海藻糖合成酶基因在大肠杆菌中的克隆表达.工业微生物,2008,38(6):7-12. Duan Z Y,Yao L,Mao Z G. Cloning and expression of trehalose synthase gene from Pseudomonas putida S1 in Escherichia coli. Industrial Microbiology,2008,38(6):7-12.
[3] 郝昭程,王腾飞,李忠奎,等. 拟南芥硫酯酶基因在毕赤酵母中的表达. 生物工程学报,2015,31(2):1-8. Hao Z C,Wang T F,Li Z K,et al. Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris. Chin J Biotech,2015,31(2):1-8.
[4] Charoenrat T,Ketudat-Cairns M,Stendahl-Andersen H,et al. Oxygen-limitedfed-batch process:an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng,2005,27(6):399-406.
[5] 苟兴华,王卫,刘达玉,等. 麦芽寡糖基海藻糖水解酶基因在巴斯德酵母中的表达及遗传稳定性. 应用与环境生物学报,2010,16(3):408-411. Gou X H,Wang W,Liu D Y,et al. Expression of MTHase gene in Pichia pastoris and its genetic stability. Chinese Journal of Applied and Environmental Biology,2010,16(3):408-411.
[6] 张卉,袁其朋.Hepcidin的基因克隆及其在毕赤酵母中的分泌表达.生物工程学报,2007,23(3):381-385. Zhang H,Yuan Q P. Cloning and secretion expression of hepcidin in Pichia pastoris. Chinese Journal of Biotechnology,2007,23(3):381-385.
[7] 杨蕾蕾,袁其朋,李文进,等.玫瑰微球菌中treZ基因在毕赤酵母中的表达研究.生物技术通报,2009,10:173-177. Yang L L,Yuan Q P,Li W J,et al. Secretory expression of maltooligosyl trehalose trehalohydrolase Pichia pastoris from Micrococcus roseus QS412. Biotechnology Bulletin,2009,10:173-177.
[8] 韩雪清,刘湘涛,尹双辉. 毕赤酵母表达系统. 微生物学杂志,2003,23(4):35-53. Han X Q,Liu X T,Yin S H. Expression system of Pichia pastoris. Journal of Microbiology,2003,23(4):35-53.
[9] Maeauley-Patriek S,Fazenda M L,McNeil B,et al. Heterologous protein production using the Pichia pastoris expression system. Yeast,2015,22(4):249-270.
[10] 刘俊梅,聂海彦,郑薇薇,等.水生栖热菌FL-03海藻糖合酶基因的克隆及真核表达.食品科学,2010,31(23):267-270. Liu J M,Nie H Y,Zheng W W,et al. Cloning and eukaryotic expression of trehalose synthase gene from Thermus aquaticus FL-03. Food Science,2010,31(23):267-270.
[11] 陶站华,王凤芝. 酵母表面展示酶技术. 现代生物医学进展,2010,10(3):593-596. Tao Z H,Wang F Z. Yeast surface display technology. Progress in Modern Biomedicine,2010,10(3):593-596.
[12] Gasser B,Sauer M,Maurer M,et al. Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Applied and Environmental Microbiology,2007,73(20):6499-6507.
[13] Daly R,Heam M T. Expression of heterologous proteins in Pichia pastoris:a useful experimenial tool in protein engineering and production. J Mol Reeognit,2005,18(2):119-138.
[14] De Groot P W,Ram A F,Klis F M. Features and functions of covalently linked proteins in fungal cell walls. Fungal Genet Biol,2005,42(8):657-675.
[15] Fernandez M L,Murga A.Influence of the incubation temperature on the autolytic activity of Lactobacillus acidophilus.Journal of Applied Bacteriology,1995,78:426-429.
[16] Zhu T,You L,Gong F,et al. Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline β-mannanase in Pichia pastoris. Enzyme Microb Technol,2011,49(4):407-412.
[17] Jiang F,Kongsaeree P,Schilke K,et al. Effects of pH and temperature on recombinant manganese peroxidase production and stability. Appl Biochem Biotechnol,2008,146(1/3):15-27.
[18] 关波,金坚,李华钟.改良毕赤酵母分泌表达外源蛋白能力的研究进展.微生物报,2011,51(7):851-857. Guan B,Jin J,Li H Z. Genetic engineering of Pichia pastoris expression system for improved secretion of heterologous proteins. Acta Microbiologica Sinica,2011,51(7):851-857.
[19] Leonardo M Damasceno,Chung Jr Huang,Carl A Batt.Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol,2012,93(1):31-39.
[20] Inan M,Aryasomayajula D,Sinha J,et al. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnology and Bioengineering,2006,93(4):771-778.
[21] Xu P,Robinson A S. Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast. Biotechnology and Bioengineering,2009,104(1):20-29.

[1]	王惠临,周凯强,朱红雨,王力景,杨仲璠,徐明波,曹荣月. 凝血因子VII及其重组表达新进展[J]. 中国生物工程杂志, 2021, 41(2/3): 129-137.
[2]	陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.
[3]	陈素芳,夏明印,曾丽艳,安晓琴,田敏芳,彭建. 抗菌肽Cec4a的重组表达和抗菌活性研究^*[J]. 中国生物工程杂志, 2021, 41(10): 12-18.
[4]	陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[5]	石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 72-79.
[6]	郭二鹏, 张建志, 司同. 羊毛硫肽的高通量工程改造方法新进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 30-41.
[7]	岑黔鸿,高彤,任怡,雷涵. 重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析^*[J]. 中国生物工程杂志, 2020, 40(5): 15-21.
[8]	乐易林,傅毓,倪黎,孙建中. 热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 72-78.
[9]	赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. **Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^***[J]. 中国生物工程杂志, 2020, 40(3): 79-87.
[10]	薛瑞,姚林,王瑞,罗正山,徐虹,李莎. 重组贻贝足蛋白的研究进展与应用^*[J]. 中国生物工程杂志, 2020, 40(11): 82-89.
[11]	封金云,宿玲恰,吴敬. 多酶复配合成海藻糖及其分离提取的研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 65-70.
[12]	田园,李艳玲. 基于重组毕赤酵母的fusaruside生物合成 ^*[J]. 中国生物工程杂志, 2019, 39(7): 8-14.
[13]	彭强强,刘启,徐名强,张元兴,蔡孟浩. 新型重组毕赤酵母产人胰岛素前体的表达工艺研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 48-55.
[14]	严建,贾禄强,丁健,史仲平. 甲醇周期诱导控制强化毕赤酵母生产猪α干扰素 ^*[J]. 中国生物工程杂志, 2019, 39(6): 32-40.
[15]	杜立,宿玲恰,吴敬. 提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(5): 96-104.

Viewed

Full text

Abstract

Cited

Shared

Discussed