An acidic β-mannanase from Penicillium sp. C6: gene cloning and over-expression in Pichia pastoris

Abstract

Using degenerate polymerase chain reaction (PCR), thermal asymmetric interlaced (TAIL)-PCR, and reverse transcription (RT)-PCR techniques, a new β-mannanase gene, denoted as man5C6, was obtained from Penicillium sp. C6. The gene has an open reading frame of 1,155 bp, and codes for a polypeptide (Man5C6) of 384 amino acids including a putative 26-residue signal peptide. The deduced amino acid sequence showed highest identity (59.2%) with an experimentally verified β-mannanase from Podospora anserine belonging to glycoside hydrolase family 5. Man5C6 was successfully expressed in Pichia pastoris, and secreted up to 2.5 g in 1 l medium. Recombinant Man5C6 was easily purified to electrophoretic homogeneity by a sing-step chromatography. The purified recombinant enzyme exhibited optimal activity at pH 4.5, and remained >55% of its maximum activity at pH 3.0–7.0. The temperature optimum was found to be 70°C. The specific activity, and K _m and V _max values were 226.5 U mg⁻¹, 12.3 mg ml⁻¹ and 2,400.2 μmol min⁻¹ mg⁻¹, respectively, for locust bean gum, and 78.7 U mg⁻¹, 0.2 mg ml⁻¹ and 894.6 μmol min⁻¹ mg⁻¹, respectively, for konjac flour. These properties make Man5C6 a potential candidate for high-level production of β-mannanase with low cost and simple processing technology.