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A Novel endo-1,4-β-Mannanase from Bispora antennata with Good Adaptation and Stability over a Broad pH Range

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Abstract

An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg−1. MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0–8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0–11.0). The K m and V max values were 1.33 mg ml−1 and 444 μmol min−1 mg−1 and 1.17 mg ml−1 and 196 μmol min−1 mg−1 for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co2+, Ni2+, and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.

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Acknowledgements

This work was supported by the Earmarked Fund for the Key Program of Transgenic Plant Breeding (2009ZX08003-020B) and the Agricultural Science and Technology Conversion Funds (2009 GB23260444).

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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

    Qiong Liu, Huiying Luo, Pengjun Shi, Huoqing Huang, Kun Meng & Bin Yao

  2. Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China

    Qiong Liu & Peilong Yang

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  1. Qiong Liu
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Correspondence to Peilong Yang or Bin Yao.

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Fig. S1

The cDNA nucleotide and deduced amino acid sequences of man5. The putative signal peptide is underlined. The stop codon is indicated with an asterisk (JPEG 98 kb)

Fig. S2

The tertiary structure of MAN5 predicted with Accelrys Discovery Studio software using the GH 5 β-mannanase (PDB: 1QNO) from T. reesei (41% identity) as the template. The putative catalytic residues are indicated in black (JPEG 64 kb)

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Liu, Q., Yang, P., Luo, H. et al. A Novel endo-1,4-β-Mannanase from Bispora antennata with Good Adaptation and Stability over a Broad pH Range. Appl Biochem Biotechnol 166, 1442–1453 (2012). https://doi.org/10.1007/s12010-011-9537-z

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