| Popis: |
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. |
