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Title: PURIFICATION AND CHARACTERIZATION OF A BETA-GLUCOSIDASE FROM CITRUS SINENSIS VAR VALENCIA FRUIT TISSUE

Author
item Cameron, Randall - Randy
item Manthey, John
item Baker, Robert
item Grohmann, Karel

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Activity for six different glycosidases was observed in mature orange fruit peel extracts. They were a-D-glucosidase, a-D-mannosidase, a-L- arabinosidase, B-D-glucosidase, B-D-xylosidase and B-D-galactosidase. Activity for a-L-rhamnosidase was not observed. Anion exchange chromatography of rag tissue (composed of intersegmental septa, squeezed juice sacs and fruit core tissue) exhibited two peaks (one in the flow- through and one bound to the matrix) of B-D-glucosidase activity. The bound B-D-glucosidase peak was purified and characterized. B-D- glucosidase is capable of hydrolyzing a wide variety of substrates present in processed fruit juice and processing residues. Among these naturally occurring substrates are residual cell wall materials (comprising a considerable proportion of processing byproducts), glucosidic flavor and aroma precursors and glucosidic phytonutrient components. A survey with other glycosidic substrates indicates this enzyme is very specific for B-D-glucose. Of the numerous substrates tested the enzyme was most active against glucosyl-glucose disaccharides. Relatively high levels of activity were also observed for prunasin, sinigrin, salicin, hesperetin dihydrochalcone glucoside, hesperitin-7- glucoside and naringenin-7-glucoside. The wide variety of substrates from which this enzyme was capable of hydrolyzing glucose makes it problematic to assign its in-vivo role. Since the highest activity is observed against glucose disaccharides, this suggests it may have a role in cell wall metabolism. Its activity against phenolic and flavonoid glucosides indicates a potential for utilization for phytonutrient modification.

Technical Abstract: A B-glucosidase has been purified from Citrus sinensis var. Valencia fruit rag tissue (intersegmental septa, squeezed juice sacs and fruit core tissue). It was purified by ion exchange DEAE, CM-Sephadex and gel filtration (Superdex, Toyopearl HW-55S and BioGel P-100) chromatography. It has an apparent molecular mass of 64 kDa (denaturing electrophoresis) or r55 kDa (BioGel P-100). It has a pH optimum between 4.5-5.5 and a temperature optimum of 40 degrees C. It is inhibited by glucose and gluconolactone. The highest observed activity was with the disaccharides cellobiose, laminaribiose, gentiobiose and sophorose. Activity with the flavonoids hesperetin-7-glucoside and prunin was higher than with the artificial substrate p-nitrophenyl-B-D-glucopyranoside. This enzyme was also active with salicin, sinigrin, prunasin and hesperetin dihydrochalcone glucoside as substrates. Only minimal activity was observed with other p- nitrophenyl glycosides.