米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达

doi:10.3969/j.issn.2095-2341.2011.04.08

生物技术进展 ›› 2011, Vol. 1 ›› Issue (4): 276-281.DOI: 10.3969/j.issn.2095-2341.2011.04.08

米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达

王智1,宋立立2,顾金刚3,顿宝庆1*,田谷1,路明1,李桂英1

1.中国农业科学院作物科学研究所, 国家农作物基因资源与基因改良重大科学工程, 中国农业科学院生物质能源研究中心, 北京 100081;
2.沧州师范学院生命科学学院, 河北沧州 061001;
3.中国农业科学院农业资源与农业区划研究所, 中国农业微生物菌种保藏管理中心, 北京 100081

收稿日期:2011-06-17 出版日期:2011-10-25 发布日期:2011-08-30
通讯作者: 顿宝庆,副研究员,博士,主要从事生物质能源研究。E-mail:baoqingdun9@hotmail.com
作者简介:王智,博士研究生,从事生物质能源研究。E-mail:wz116280@sina.com。
基金资助:
国际先进农业科学与技术项目(农业部948项目)(No. 2011-Z09),农业部农村能源综合建设项目(No. 2130138-018),中国农业科学院作物科学研究所中央级公益性科研院所基本科研业务费专项资助项目(No. 2060302-13)共同资助。

Eukaryotic Secrete Fusion Expression of Rhizopus oryzae Glucoamylase and Paenibacillus sp. Xylanase

WANG Zhi, SONG Li-li, GU Jin-gang, DUN Bao-qing, TIAN Gu, LU Ming, LI Gui-ying

1.The National Key Facility for Crop Gene Resources and Genetic Improvement of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2.Cangzhou Normal University, Hebei Cangzhou 061001, China;
3.Agricultural Culture Collection of China, Institute of Agricultural Resources and Agricultural Divisions, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2011-06-17 Online:2011-10-25 Published:2011-08-30

摘要/Abstract

摘要： 以米根霉(Rhizopus oryzae)3.866基因组DNA为模板,克隆得到糖化酶基因(glucoamylase gene, amyA),基因全长2 049 bp,编码604个氨基酸;以类芽孢杆菌(Paenibacillus sp.)H10-3基因组DNA为模板,克隆出基因木聚糖酶基因(xylanase A gene, xynA)的成熟肽编码序列,长636 bp,编码211个氨基酸。通过重叠延伸PCR(SOE-PCR)得到拼接片段amyA-l-xynA,并将其克隆到毕赤酵母表达载体pPIC9中,得到重组质粒pPIC9-amyA-l-xynA,重组质粒线性化后经电击转化到毕赤酵母(Pichia pastoris)GS115中,得到了表达成功的工程菌AX11。在AX11发酵上清液中同时检测到糖化酶活性(5.8 U/mL)和木聚糖酶活性(32.3 U/mL)。

关键词: 糖化酶, 木聚糖酶, 融合表达, 毕赤酵母

Abstract: The glucoamylase gene (amyA) was amplified from Rhizopus oryzae 3.866 total DNA extracts. The sequence of amyA gene was consisted of 2 049 bp, and encoded 604 amino acids. The xylanase gene (xynA) was amplified from Paenibacillus sp. H10-3 total DNA extracts. The mature peptide encoding sequence of xynA was 636 bp, and encoded 211 amino acids. The mature peptide sequence of amyA gene, sequence of linker and sequence of xynA gene were spliced by overlap extension PCR (SOE-PCR), then amyA-l-xynA was obtained. The fusion gene was then inserted into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. By using electroporation, we successfully transformed the recombinant pPIC9-amyA-l-xynA into Pichia pastoris GS115, AX11 was obtained. The maximum yield of the recombinant glucoamylase and xylanase in AX11 culture medium were 5.8 U/mL and 323 U/mL, respectively.

Key words: glucoamylase, xylanase, fusion expression, Pichia pastoris

王智,宋立立,顾金刚,顿宝庆,田谷,路明,李桂英. 米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达[J]. 生物技术进展, 2011, 1(4): 276-281.

WANG Zhi1, SONG Li-li2, GU Jin-gang3, DUN Bao-qing1*, TIAN Gu1, LU Ming1, LI Gui-ying1. Eukaryotic Secrete Fusion Expression of Rhizopus oryzae Glucoamylase and Paenibacillus sp. Xylanase[J]. journal1, 2011, 1(4): 276-281.

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米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达

Eukaryotic Secrete Fusion Expression of Rhizopus oryzae Glucoamylase and Paenibacillus sp. Xylanase

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