Purification and properties of a beta-galactosidase with potential application as a digestive supplement.

Functional-based screening of crude beta-galactosidase activities from 42 yeast strains resulted in the selection of a single enzyme of potential interest as a digestive supplement. beta-Galactosidase produced by Kluyveromyces marxianus DSM5418 was purified to homogeneity by a combination of gel filtration, ion-exchange, and hydroxylapatite chromatographies. The denatured (123 kDa) and native molecular masses (251 kDa) suggest that the enzyme is a homodimer. The optimum pH and temperature of the purified enzyme were 6.8 and 37 degrees C, respectively. The unpurified beta-galactosidase in particular displayed a high level of stability when exposed to simulated intestinal conditions in vitro for 4 h. Matrix-assisted laser desorption ionization mass sectrometry analysis revealed that the enzyme's trypsin-generated peptide mass fingerprint shares several peptide fragment hits with beta-galactosidases from Kluyveromyces lactis. This confirms the enzyme's identity and indicates that significant sequence homology exists between these enzymes.

Calcium alginate entrapped preparations of Aspergillus oryzae beta galactosidase: its stability and applications in the hydrolysis of lactose.

Insoluble concanavalin A-beta galactosidase complex was obtained by using jack bean extract and this complex was crosslinked with glutaraldehyde, in order to maintain the integrity of complex in the presence of its substrate or products. Concanavalin A-beta galactosidase complex retained 92% of the initial enzyme activity whereas crosslinked complex showed 88% activity. Entrapment of concanavalin A-beta galactosidase complex into calcium alginate beads provided suitability to use this preparation in reactors. Temperature- and pH-optima of the various immobilized beta galactosidase preparations were the same as its soluble counterpart. Entrapped crosslinked concanavalin A-beta galactosidase complex retained more than 50% activity after 1h exposure with 4.0 M urea at room temperature. Moreover, entrapped crosslinked concanavalin A-beta galactosidase complex retained 81 and 62% of the original enzymatic activity in the presence of 5% calcium chloride and 5% galactose, respectively. Entrapped crosslinked concanavalin A-beta galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations. This entrapped crosslinked concanavalin A-beta galactosidase complex retained 95% activity after seventh repeated use and 93% of its original activity even after 2 months storage at 4 degrees C.

Pepper beta-galactosidase 1 (PBG1) plays a significant role in fruit ripening in bell pepper (Capsicum annuum).

During bell pepper (Capsicum annuum L.) fruit ripening, beta-galactosidase activity increased markedly as compared with other glycosidases. We purified 77.5 kDa exo-1,4-beta-D-galactanase from red bell pepper fruit classified as beta-galactosidase II. A marked decrease in galactose content appeared during fruit ripening, especially in the pectic fraction. The purified enzyme hydrolyzed a considerable amount of galactose residues in this fraction. We isolated bell pepper beta-galactosidase (PBG1) cDNA. This PBG1 protein contained the putative active site, G-G-P-[LIVM]-x-Q-x-E-N-E-[FY], belonging to glycosyl hydrolase family 35. Quantitative RT-PCR revealed that the expression of PBG1 in red fruit was significantly stronger than that from any other tissues. Moreover, expression of PBG1 occurred prior to that of pepper endo-polygalacturonase 1 (PPG1), the major fruit-ripening enzyme. Based on these results, it appears that the hydrolysis of galactose residues in pectic substances is the first event in the ripening process in bell pepper fruit.

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides.

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

beta-Galactosidase and its significance in ripening of "Saijyo" Japanese Persimmon fruit.

The fruit extracts of ripening cv. Japanese Persimmon, "Saijyo", contained a number of glycosidases and glycanases. Among them, beta-galactosidase appeared to be the most significant, and the activity increased in parallel with tissue ripening. Persimmon beta-galactosidase was presented in at least three isoforms, beta-galactosidase-I (pI = 4.88), beta-galactosidase-II (pI = 6.76), and beta-galactosidase-III (pI = 7.05). beta-Galactosidase-III had exo-type galactanase activity, while the others did not. The activity of endo-type glycanases was a maximum in immature green or yellow fruits. The firmness of the pulp tissue decreased dramatically, and the amount of water-soluble polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases, especially beta-galactosidase, appeared maximal in mature red fruits. The amount of extractable pectin remained unchanged, although the galactose content of the high-molecular-weight fraction in WSS decreased dramatically. These results suggest that the ripening of persimmon was caused by the solubilization of pectic polysaccharide by endo-type glycanases and digestion by exo-type glycosidases. beta-Galactosidase, in particular, seemed to play a major role in ripening the fruit.

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings.

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.

Preliminary characterization of a new exo-beta-(1,4)-galactanase with transferase activity.

As a prerequisite to the study of the fine chemical structure of the branched region of pectin, an exo-beta-(1,4)-galactanase was purified from a commercial preparation (Pectinex AR). Purification was carried out by precipitation with 70% saturated ammonium sulfate, preparative electrofocusing, anion-exchange chromatography and affinity chromatography on cross-linked alginate. Exogalactanase specific activity was 992 nkat mg-1 and the enzyme was devoid of beta-(1,3)- or beta-(1,6)-galactanase, arabinanase, beta-D-galactosidase and alpha-L-arabinofuranosidade activities. Residual exopolygalacturonase activity represented 2.9% of the galactanase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing showed two close bands with molecular weights of 120,000 and 90,000 and pHi of 3.8 and 4.1, respectively. The enzyme acted in an exo manner and its activity was optimum at pH 3.5 and 60 degrees C. When incubated with galacto-oligosaccharides, new oligosaccharides with a higher degree of polymerization appeared, indicating the ability of the enzyme to transfer galactose residues.

Purification of jack bean meal beta-D-galactosidase by a new affinity adsorbent.

A simple procedure has been developed for the purification of jack bean beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) by affinity chromatography employing a new affinity adsorbent. The ligand 6-N-beta-(4-aminophenyl)-ethylamino-3-O-beta-D-galactopyranosyl-6-deoxy-L-gulitol was prepared by the reaction between lactose and beta-(4-aminophenyl)-ethylamine and was coupled to cyanogen bromide activated Sepharose 4B via the amino groups of the 4-aminophenyl moiety. This affinity gel resulted in a 111-fold purification of beta-D-galactosidase with a 64% recovery of the enzyme. With p-nitrophenyl-beta-D-galactopyranoside as the substrate the apparent Km and V values were 0.59 mM and 1.87 mumol/min per mg, respectively. The method for purification of beta-D-galactosidase may be applicable to other glycosidases depending upon the choice of specific di- or oligosaccharides of known structures to be used in the preparation of ligands.