β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.     

α- and β-galactosidase activities in protein bodies and cell walls of lentil seed cotyledons

Cotyledons of mature Lens culinaris seeds contain two forms of both α- and β-galactosidase which can be separated by ion exchange chromatography. These forms are present in cotyledon cell walls and protein bodies except β-galactosidase II,which is undetectable in the cell walls of these organs. All the enzymatic forms were active in an acid pH range but each behaved differently with different substrates, both natural and synthetic, and in the action of different effectors on the activity. α-Galactosidase I and II were able to release free sugars from several putative substrate oligosaccharides and all the forms of α and β-galactosidase were seen to release galactose from lentil storage glycoproteins.     

Tomato α-galactosidases: conversion of human type b erythrocytes to type o

Tomato fruit contained a high level of α-galactosidase activity, and the two components found responsible for this activity, α-galactosidases I and II, were separated by chromatography on DEAE-Sephadex A-50. α-Galactosidase II, in particular, was highly purified by subsequent chromatography on a column of melibiose—agarose. The two enzymes differed markedly in MW and in inhibition by mercuric compounds but less so in other properties.α-Galactosidase II converted type B erythrocytes to type O.     

β-Galactosidase activity in ripening apples

β-Galactosidase activity has been identified in soluble and cell wall preparations from apple cortex tissue. The enzyme degrades pectin galactan and has a pH optimum of 4·0 with p-nitrophenyl-β, d-galactopyranoside as substrate. Soluble polygalacturonide increased as the applies softened with ripening and these changes were preceded by loss of galactose residues from the cell wall and an increase in β-galactosidase activity.     

Expression of β-galactosidase multiple forms during barley (Hordeum vulgare) seed germination. Separation and characterization of enzyme isoforms

β-Galactosidase (EC 3.2.1.23) activity in barley ( Hordeum vulgare ) seedlings increases moderately during the first stages of germination. The level of activity in the whole seedling is the result of increasing activity of β-galactosidase in the roots and shoots and of declining enzyme activity in the grain. β-Galactosidase was purified during different developmental stages and from various parts of the barley seedling using affinity chromatography and was resolved into multiple forms by isoelectric focusing on polyacrylamide gels. The expression of the isoforms was shown to be under temporal and tissue-specific control. Four sets of isozymes were separated by DEAE-cellulose chromatography and were shown to be functionally similar. β-Galactosidase isoforms also exhibit size microheterogeneity, the more acidic entities having higher molecular masses. The differences in molecular weight are mainly restricted to the size of the small subunit. Multiplicity can not be attributed to glycosylation, since treatment of the enzyme preparation with N- or O-glycanase did not alter the isoelectric points or the molecular weights of the isoforms.     

Multiple forms of α-galactosidase in mature leaves of Cucurbita pepo

α-D-Galactosidase has been purified from mature leaves of Cucurbita pepo using pH and ammonium sulphate fractionation, Sephadex gel filtration and DEAE Sephadex gel chromatography. Gel filtration produced one peak of α-galactosidase activity from which three distinct enzyme forms were resolved on DEAE Sephadex and designated LI, LII and LIII. Purirications obtained were ca 75, 120 and 30 fold for LI, Lll and LIII respectively. Ll was slightly contaminated with β-galactosidase and LII with β-fructosidase activity. All forms hydrolysed the α-galactosyl linkages of raffinose and stachyose. Differences between each form were found in their pH optima, reactivity toward metal ions, thermal stability and K_m values using either p-nitrophenyl-α-D-galactoside (NPG) or raffinose as substrates. All forms were inhibited by NPG at high concentrations and by α-D-galactose. It is proposed that α-galactosidases may be components of a lysosomal system in plant cells.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.     

Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Wall-bound enzymes in callus of Convolvulus arvensis

Isolated cell walls of Convolvulus callus contain α- and β-galactosidase, α- and β-glucosidase, α- and β-mannosidase, acid invertase and acid phosphatase activities. No neutral invertase or alkaline phosphatase activities could be detected. Acid invertase activity per mg cell wall increased considerably during incubation of callus fragments in nutrient solution, as opposed to the activities of the other enzymes mentioned.     

Characterization and partial purification of three glycosidases from castor bean endosperm

α-Mannosidase and β-N-acetylhexosaminidase, which could function in the cleavage of glycosidic linkages in the native Ricinus communis lectins, and β-galactosidase were purified some 100-fold from the endosperm tissue of castor bean seedlings. The procedure used ammonium sulphate precipitation followed by chromatography on CM-cellulose, hydroxyapatite and Sephacryl S-300 to separate the three activities. All three glycosidases were present, with the lectins, in the protein bodies of dry seed and increased in activity during the time that lectins are broken down in the vacuoles. The enzymes show optimal activity in the range pH 3–5.5. The α-mannosidase had a K_m of 0.77 mM for p- nitrophenyl-α-D-mannopyranoside. The β-galactosidase showed a K_m of 1.39 mM for p-nitrophenyl-β-D-galactopyranoside. The β-N-acetylhexasominidase had a K_m of 0.47 mM for p-nitrophenyl-N-acetyl-β-N-glucosamide and a K_m of 0.33 mM for p-nitrophenyl-N-acetyl-β-D-galactosamide. Effects of competitive inhibitors and cations were described.     

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

Using isoelectrofocusing (IEF), multiple forms of Petunia β-galactosidase activity could be detected. The β-galactosidase pattern showed only minor tissue-specific differences. There were, however, species-specific differences. Zea mays, for instance, showed two bands which differed from the zones obtained with Petunia preparations. Petunia and corn leaves were mixed and extracted commonly. The species-specific activity patterns remained unchanged.Petunia preparations were inactivated by 8 Murea. Following dialysis, enzymatic activity and the Petunia-specific pattern were restored. The same holds true for a mixture of Petunia and E. coli β-galactosidase preparations. On refocusing isolated Petunia zones, untreated or inactivated by 8 M urea and reactivated by dialysis, the original mobilities were shown. Therefore, it seems highly improbable that the β-galactosidase pattern was due to artefacts. Using a Petunia line which was ‘pure’, also in respect to its β-galactosidase pattern, the four main bands were preparatively separated by IEF and characterized. They showed the same pH optimum (4.3), the same temperature optimum (55°), the same inactivation kinetics by urea, the same sensitivity against Cl^?, and closely related K_m. values. In sucrose gradient centrifugation they invariably showed S values of 8–10. The multiple activities could not be separated by zone electrophoresis using various carrier systems, or by gel filtration. It seems possible that they represent forms which differ only in isoelectric points, not in MW.     

Blood Group Substance-degrading Enzymes Obtained from Streptomyces sp.

The blood group B substance-degrading activity of Streptomyces 9917S₂ is induced by galactosides as α-galactosidase activity is. Purification of the α-galactosidase was attempted by chromatography on DEAE-Sephadex and Sephadex. The purified preparation was shown to be free from α- and β-glucosidases, β-galactosidase, α- and β-glucosaminidases, and α- and β-galactosaminidases activities. The blood group B substance-degrading activity was present only in this fraction. This enzyme preparation cleaves α-(1→3)- and α-(1→6)-galactosidic linkages. The activity is inhibited by d-galactose, melibiose, and raffinose and also by l-arabinose and d-xylose.     

Production of recombinant β-galactosidase in bioreactors by fed-batch culture using DO-stat and linear control

β-Galactosidase enzymes continue to play an important role in food and pharmaceutical industries. These enzymes hydrolyze lactose in its constituent monosaccharides, glucose and galactose. The industrial use of enzymes presents an increase in process costs reflecting in higher final product value. An alternative to enhance processes’ productivity and yield would be the use of recombinant enzymes and their large-scale fed-batch production. The overexpression of recombinant β-galactosidase from Kluyveromyces sp. was carried out in 2-L bioreactors using Escherichia coli strain BL21 (DE3) as host. Effect of induction time on recombinant enzyme expression was studied by adding 1?mM isopropyl thiogalactoside (IPTG) at 12?h, 18?h and 24?h of cultivation. Glucose feeding strategies were compared employing feedback-controlled DO-stat and ascendant linear pump feeding in bioreactor fed-batch cultivations. Linear feeding strategy with IPTG addition at 18?h of cultivation resulted in approximately 20?g/L and 17,745?U/L of biomass and β-galactosidase activity, respectively. On the other hand, although the feedback-controlled DO-stat feeding strategy induced at 12?h of cultivation led to lower final biomass of 18?g/L, it presented an approximately 2.5 increase in enzymatic activity, resulting in 42,367?U/L, and most importantly it led to the most prominent specific enzymatic activity of approximately 40?U/mg_protein. Comparing to previous results, these results suggest that the DO-stat feeding is a promising strategy for recombinant β-galactosidase enzyme production.     

Patterns of growth and β-galactosidase production by Bifidobacteria

We investigated the patterns of growth and β-galactosidase production in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM and b. bifidum No.1, LVA-3, 791 on media with various carbon sources. The synthesis of β-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of β-galactosidase synthesis of 0.20 U mg^?1 h^?1 was observed in B. bifidum LVA-3 after 3–6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03–0.08 U mg^?1h^?1. On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h^?1, respectively, after 3–6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12–15 h of cultivation at 0.13 and 0.22 h^?1, respectively. The hydrolytic activity of β-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.