Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene

Using PCR, cloning and sequencing techniques, a 1.1‐kb complementary DNA fragment encoding for a β‐mannanase (mannan endo‐1,4‐β‐mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several β‐mannanases in glycoside hydrolase family 5. The β‐mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The β‐mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiaeα‐factor signal sequence. The β‐mannanase was produced in a 14‐L fermenter with an expression level of 900 mg·L−1. The expression level is strongly affected by the induction temperature. A two‐step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure β‐mannanase. However, due to post‐translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulisβ‐mannanase was close to that of the wild‐type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel β‐mannanase. This is the first time a mollusc β‐mannanase has been characterized at the DNA level.