Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, Mytilus edulis, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. Both enzymes form insoluble inclusion bodies when expressed in Escherichia coli. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast Pichia pastoris with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-1985 |
| Date | January 2002 |
| Creators | Xu, Bingze |
| Publisher | Uppsala universitet, Institutionen för ytbioteknik med Centrum för ytbioteknik, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 711 |
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