Purification and characterization of [beta]-galactosidase from Thai jute (Hibiscus sabdariffa L. var. altissima)

Abstract

Screening of 9 glycosidases from 10 indigenous plants from the Malvaceae family and Tiliaceae family using pNP-glycosides as substrates showed that Por Kaew Non Soong 2 (Hibiscus sabdariffa L. var. altissima) had the highest activity of β-galactosidase (1.18 U/g seed), while Krajieb Dang ( Hibiscus sabdariffa L. var. sabdariffa) had the highest activity of (1.18 U/g seed). Seeds of Por Kaew Non Soong 2 were used to study the effect of germination on β-galactosidase and activity, with the results that maximal activities of the two enzymes were detected after 2 days of imbibing. Preliminary studies on purification of Hibiscus β-galactosidase showed that the enzyme was stable at a broad pH range of 3.5 to 9.5 and at -10 C to -20C during storage. Additionally, ammonium sulfate fractionation at 35-65% saturation was useful as the first step in purification, since it removed a large amount of proteins and gave quantitative recovery of β-galactosidase and α-mannosidase. DEAE-cellulose chromatography at pH 7.0 could separate unbound β-galactosidase from bound α-mannosidase. Sephadex G-200 and Sephadex G-100 were useful for separation of β-galactosidase and α-galactosidase, and Lactosyl Sepharose 4B chromatography provided a possible additional step for purifying β-galactosidase. Complete purification of β-galactosidase required 5 steps: 35-70% ammonium sulfate fractionation, a first DEAE-cellulose chromatography, Sephadex G-100 filtration, Lactosyl Sepharose 4 B chromatography and a second DEAE-cellulose chromatography step. The final products gave a major band on SDS-PAGE and was purified with 868 fold with a yield of 13 %. With pNP-β-D-galactopyranoside as substrate, optimum activity of purified β-galactosidase was observed at pH 4.0 and 55C: Purified β-galactosidase showed 1% activity towards pNP-β-D-glucopyranoside and 6% activity towards pNP-α-L- arabinopyranoside The enzyme was strongly inhibited by FeCL(,3), HgCL(,2), p-hydroxymercuribenzoate, methyl-α-gal, methyl-β-gal, pNP-a-gal, galactono-1,4-lactone, D-galactal and D-galactose. Kinetic studies of β-galactosidase with pNP-β-D-galactopyranoside, oNP-β-D-galactopyranoside and B-lactose gave Km values of 0.80 mM, 12.8 mM and 84.7 mM respectively and V(,max) values of 63.5 nmol/min, 16.9 nmol/min and 6.0 nmol/min respectively. The molecular weight of the native form of the enzyme was 55 kD and of the denatured enzyme was 66 kD. The enzyme showed charge heterogeneity in isoelectric focusing and chromatofocusing with a pl range 6 to 9. Similar patterns and positions of β-galactosidase under cathodic gel stained with 4-methylumbelliferyl-B-D-galactopyranoside and 4-methylumbelliferyl-a-Larabinopyranoside were observed, suggesting that both activities were due to the same enzyme. Partially purified β-galactosidase and α-mannosidase were also tested for synthetic capability. Under high sugar concentrations (33% w/w) and high temperature (55C), the yields produced by β-galactosidase and α-mannosidase were 14% and 35% respectively after 7 days of incubation. Identification of synthetic products on h.p.l.c. compared to standard sugars showed that a major product produced by β-galactosidase was digalactoside and major products produced by α-mannosidase included α(1,6)-dimannoside and α(l,2)-or α(1,3)-dimannoside. Higher synthetic activity of β-galactosidase was shown at the pH range of 3.0-4.5 whereas higher synthetic activity of α-mannosidase was shown at pH 4. 0. However, β-galactosidase displayed the synthetic activity over a broader range (40-70C) than (40 60 C). For synthetic activity, β-galactosidase preferred lower concentrations of monosaccharide substrates (20-30 % w/w) than α-mannnosidase which preferred concentrations of monosaccharide over 30 % w/w. The synthetic activity of the two enzymes was increased with increasing time of incubation. Highly purified β-galactosidase was also tested for synthesis and showed lower synthetic ability than partially purified enzyme. No cross reactivity of β-galactosidase with D-glucose and D-mannose were detected.