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

Abstract

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 beta-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-beta-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 degrees 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 micromol min(-1) mg(-1), respectively. The enzymatic activity was not significantly affected by ions such as Ca(2+), Cr(3+), Co(2+), Zn(2+), Na(+), K(+), and Mg(2+) and enhanced by Ni(2+), Fe(3+), Mn(2+) and Ag(+). These favorable properties make MAN5A a potential candidate for use in various industrial applications.

A new acidophilic fungus Teratosphaeria acidotherma (Capnodiales, Ascomycota) from a hot spring

“…are anamorphic fungi of Teratosphaeriaceae and that T. acidotherma forms a sister clade of these anamorphic fungi. Bispora and Acidomyces strains are similar to the present fungus in having acidophilic properties (Sigler and Carmichael 1974;Baker et al 2004;Luo et al 2008;Selbmann et al 2008). We found that the Bispora sp.…”

“…Bispora sp. MEY-1 was isolated from acidic waste water of the ''721'' uranium mine in China, and its optimum pH for growth was reported as pH 2.5-3 (Luo et al 2008). Bispora sp.…”

“…From the description about Bispora sp. MEY-1 strain (Luo et al 2008) and our phylogenetic analysis, we consider the strain Bispora sp. MEY-1 is possibly identical with T. acidotherma.…”

“…and Bispora sp., which are all nested in the Teratosphaeria clade, including We found the strain Bispora sp. MEY-1 has the identical sequence of 547 base pairs in the ITS region to those of our four isolates, though no description about the teleomorph was given for MEY-1 by Luo et al (2008). Because there is a possibility MEY-1 is in the same taxon as our strains, we tried to obtain the MEY-1 (CGMCC 2500) from the Chinese Academy of Sciences.…”

Biodiversity, evolution and adaptation of fungi in extreme environments

“…In general, extremozymes have attracted a great deal of interest due to their potential for practical applications. However, studies on fungal acidic enzymes still remain limited with a few exceptions including mannanase, xylanase (Luo et al 2009a(Luo et al , 2009b, and polygalacturonase (Yang et al 2011) from the acidophilic Bispora sp. strain MEY-1, or laccase from H. acidophila (Tetsch et al 2005).…”