Discovery and Characterization of a Distinctive Exo-1,3/1,4-β-Glucanase from the Marine Bacterium Pseudoalteromonas sp. Strain BB1

Abstract

Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-␤-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-␤-D-glucan with some 1,6-␤ 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-␤-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-␤-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-␤ linkages and 1,3-␤ linkages, and so ExoP is designated an exo-1,3/1,4-␤-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.

Structure and activity of exo‐1,3/1,4‐β‐glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C‐terminal domain

“…2B). The nearby W494 is in a position to provide a platform for a possible +2 subsite, the presence of which was previously indicated by subsite analysis of the kinetics of hydrolysis using various oligosaccharides [5]. Figure 2A,B highlights the differences in active site entrances between ExoP and ExoI, notably, sequence differences, the juxtaposition of domain 3 (ExoP) and the N‐glycosylation site at N220 (ExoI).…”

“…A second crystal form of ExoP was obtained under different crystallization conditions and following 4–5 months of incubation. It was shown that the protein had been cleaved after residue 626 to produce a fragment that included the first two domains and a short segment of the linker that connects to domain 3 [5]. The structure of this cleaved form of ExoP, only the first two domains of which were seen in the electron‐density map, was very similar to the corresponding residues observed within the intact structure.…”

“…Whereas metagenomic approaches are able to exploit the rich potential of unculturable microorganisms, particularly from the oceans [3,4], more traditional strategies will continue to lead to the discovery of new enzymes. A selection‐screening approach to try and identify exo‐1,3‐β‐glucanase activity in marine bacteria isolated from 32 different seaweeds produced just one candidate from the 90 isolates recovered [5]. Characterization of such activity had not been reported previously.…”

“…It does not appear to be an obvious carbohydrate‐binding module, of which there are many classes [11], often seen to append glycosyl hydrolases. However, because ExoP lost hydrolytic activity against laminarin following proteolytic cleavage at residue 628, the C‐terminal domain would appear to serve an essential function [5].…”

Characterization of a Broad-Specificity β-Glucanase Acting on β-(1,3)-, β-(1,4)-, and β-(1,6)-Glucans That Defines a New Glycoside Hydrolase Family

“…For determination of Michaelis-Menten constants on laminarin and pustulan, the initial velocities were measured for substrate concentrations ranging from 1.25 to 20 mg ml Ϫ1 . The degrees of polymerization of pustulan and laminarin used to calculate their molar concentrations were 94 (22) and 28 (17), respectively. The kinetic parameters were estimated using weighted nonlinear least-squares regression analysis with Grafit software (Erithacus Software, Horley, United Kingdom).…”