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Activity Studies of Epulopiscium Type B 1,3‐β‐glucanase
Author(s) -
Lajeunesse Karah Lynn,
Piefer Andrew John
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.835.9
Subject(s) - laminarin , glucanase , enzyme , glucan , substrate (aquarium) , biochemistry , specific activity , chemistry , enzyme assay , reagent , chromatography , biology , organic chemistry , ecology
The activity of recombinantly expressed and purified Epulopiscium 1,3‐β‐glucanase was characterized in terms of optimal temperature, pH, and β‐glucan substrate specificity. Epulopiscium is a bacterial symbiont of surgeonfish that may express and secrete a putative endo 1,3‐β‐glucanase to convert β‐glucans in seaweed to simple carbohydrates, providing an energy source for both the bacteria and host. To study enzyme function, the active site of the gene encoding 1,3‐β‐glucanase was cloned into a pET24b vector, expressed in E. coli , and purified using Ni‐NTA chromatography. Purified enzyme was incubated with buffer and carbohydrate. Enzyme reactions were stopped by the addition of DNSA reagent to quantify liberated reducing sugars. The results for average specific activity at 37 o C, 45 o C, 60 o C, and 75 o C were 90 uM/min/mg, 110 uM/min/mg, 91 uM/min/mg, and 29 uM/min/mg respectively. Interestingly, these results suggest that the enzyme has optimal activity at 45 o C, above the physiological temperature of the host. As expected, the enzyme has specificity for 1,3‐β‐glucans, as exhibited by the degradation of laminarin. Substrate specificity will be examined further by employing β‐glucan substrates with mixed linkages, and the optimal pH for activity will be found by varying the reaction pH. This research is supported through funding from a Faculty Research Grant and the Hartwick College Department of Chemistry.