Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene.
Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-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 beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-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 beta-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. edulis beta-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 beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.
Xu
Bingze
B
Center for Surface Biotechnology, Uppsala Biomedical Center, Uppsala University, Sweden.
Sellos
Daniel
D
Janson
Jan-Christer
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000201:enzymology / Q000235:genetics*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044902:beta-Mannosidase
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008361:Mannosidases; D044902:beta-Mannosidase