Cloning of <italic>endo-β-glucanase I</italic> gene and expression in <italic>Pichia pastoris

doi:10.1007/s11703-011-1082-6

Front Agric Chin

2011, Vol. 5

Issue (2) : 196-200 https://doi.org/10.1007/s11703-011-1082-6

RESEARCH ARTICLE

Cloning of endo-β-glucanase I gene and expression in Pichia pastoris

Yu BAI, Runfang GUO(

), Hongwei YU, Long JIAO, Shuli DING, Yingmin JIA

College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, China

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Abstract

Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70oC and 3.5, respectively.

Keywords Thermoascus aurantiacus endo-1 4-β-glucanase RACE Pichia pastoris gene expression

Corresponding Author(s): GUO Runfang,Email:runfangg@163.com

Issue Date: 05 June 2011

Cite this article:

Yu BAI,Runfang GUO,Hongwei YU, et al. Cloning of endo-β-glucanase I gene and expression in Pichia pastoris[J]. Front Agric Chin, 2011, 5(2): 196-200.

URL:

https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1082-6
https://academic.hep.com.cn/fag/EN/Y2011/V5/I2/196

Fig.1 An agarose gel electrophoresis of product from PCR. Lane M is marker-DL2000. Lanes 1-4 represent product from RT-PCR, product from 3′-RACE, product from 5′-RACE, and full-length cDNA.

Fig.2 The curves of the endo--glucanase I activities and the protein content in the culture medium of GS-EG2 after induction at different times.

Fig.3 SDS-PAGE analysis of the expression of endo--glucanase I from GS-EG2 strain. Lane M is low-molecular-mass protein standard (from top to bottom: 97.4, 66.2, 43.0, 31.0, 20.1, and 14.4 kDa). Lanes 1-9 are products after induction from 24 to 216 h. Lane 10 is negative control (GS115/pPIC9K).

Fig.4 Effects of temperature and pH on the activity of the recombinant endo--glucanase I.

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