Cloning, expression and characterization of mannanase from Armillariella tabescens EJLY2098 in Pichia pastoris

Abstract:

We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of b-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains (CBD) and glycoside hydrolase family 5 (GHF5) domains, other than the conserved b-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the b-mannanase mature peptide (re-atMAN47) into the expression vector pPICZaA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60oC and an optimal pH of 5.5. It manifested broad thermostability from 30oC~65oC, and was stable between pH 4.5~7.0. This study represents the ?rst record of a β-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.