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High-level expression of a novel Penicillium endo-1,3(4)-β-d-glucanase with high specific activity in Pichia pastoris

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A novel endo-1,3(4)-β-d-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-β-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized β-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l−1) with activity of 28,721 U ml−1 in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley β-glucan (12,622 U mg−1) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0–7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.

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Abbreviations

CMC-Na:

Carboxymethyl cellulose-sodium salt

TAIL–PCR:

Thermal asymmetric interlaced polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

DNS:

3,5-Dinitrosalicylic acid

RT–PCR:

Reverse-transcription polymerase chain reaction

ORF:

Open reading frame

MALDI-TOF:

Matrix-assisted laser desorption/ionization time-of-flight

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Acknowledgments

This research was supported by the National Science and Technology Support Program (2011BADB02), the China Modern Agriculture Research System (CARS-42), and the Key Program of Transgenic Plant Breeding (2009ZX08003-020B).

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Authors and Affiliations

  1. Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People’s Republic of China

    Xiaoyan Chen, Kun Meng, Pengjun Shi, Yingguo Bai, Huiying Luo, Huoqing Huang, Tiezheng Yuan, Peilong Yang & Bin Yao

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  1. Xiaoyan Chen
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  2. Kun Meng
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  3. Pengjun Shi
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Correspondence to Bin Yao.

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Chen, X., Meng, K., Shi, P. et al. High-level expression of a novel Penicillium endo-1,3(4)-β-d-glucanase with high specific activity in Pichia pastoris . J Ind Microbiol Biotechnol 39, 869–876 (2012). https://doi.org/10.1007/s10295-012-1087-z

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  • DOI: https://doi.org/10.1007/s10295-012-1087-z

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