Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml−1. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K m, and V max for locust bean gum substrate was 3,373 U mg−1, 1.56 mg ml−1, and 6,587.6 μmol min−1 mg−1, respectively. The enzymatic activity was not significantly affected by ions such as Ca2+, Cr3+, Co2+, Zn2+, Na+, K+, and Mg2+ and enhanced by Ni2+, Fe3+, Mn2+ and Ag+. These favorable properties make MAN5A a potential candidate for use in various industrial applications.
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This research was supported by the National High Technology Research and Development Program of China (863 program, Grant no. 2007AA100601), Chinese Program on Research for Public Good (Grant No. 2005DIB4J038), and 948 program of the Ministry of Agriculture (Grant No. 2007-Z3)
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Luo, H., Wang, Y., Wang, H. et al. A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris . Appl Microbiol Biotechnol 82, 453–461 (2009). https://doi.org/10.1007/s00253-008-1766-x
- Acidophilic fungus
- Bispora sp. MEY-1
- Pichia pastoris