Discovery and characterization of a distinctive exo-1,3/1,4-{beta}-glucanase from the marine bacterium Pseudoalteromonas sp. strain BB1.
Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-β-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-β-D-glucan with some 1,6-β branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-β-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-β-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-β linkages and 1,3-β linkages, and so ExoP is designated an exo-1,3/1,4-β-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.
Nakatani
Yoshio
Y
Biochemistry Department, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
Lamont
Iain L
IL
Cutfield
John F
JF
D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D000459:Phaeophyceae / Q000382:microbiology*; D010940:Plant Proteins / Q000235:genetics; D031161:Pseudoalteromonas / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017385:Sequence Homology; D013379:Substrate Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D010940:Plant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase