A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

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.