Transgalactosylation by thermostable β-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus (original) (raw)
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Eur J Biochem, 2001
The hyperthermostable b-glycosidases from the Archaea Sulfolobus solfataricus (SsbGly) and Pyrococcus furiosus (CelB) hydrolyse b-glycosides of d-glucose or d-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbGly show a marked preference for making new b(133) and b(136) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbGly and yields b-d-Galp-(136)-d-Glc and b-d-Galp-(133)-d-Glc in a molar ratio of < 1 : 2. The partitioning of galactosylated SsbGly between reaction with sugars [k Nu (m 21´s21 )] and reaction with water [k water (s 21 )] is about twice that of CelB. It gives a mixture of linear b-d-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are b(136) and b(133) for the reactions catalysed by SsbGly and CelB, respectively. The accumulation of b-d-Galp-(136)-d-Glc at the end of lactose hydrolysis reflects a 3±10-fold specificity of both enzymes for the hydrolysis of b(133) over b(136) linked glucosides. Galactosyl transfer from SsbGly or CelB to d-glucose occurs with partitioning ratios, k Nu /k water , which are seven and . 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new b-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by < 2.5 log 10 units from structure±reactivity correlations based on the pK a of the leaving group. q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5057 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5063 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5065
Carbohydrate Research, 2015
Broad regioselectivity of a-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 a-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2-and 1,3-linkage (DDG à 0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (DDG à 0 = 1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (DDG à F À DDG à H ), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower.
Applied Microbiology and Biotechnology, 2014
Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galactooligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70°C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from Ho-BGLA transgalactosylation are β-D-Galp-(1→6)-D-Lac (6GALA) and β-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type Ho-BGLA and shown to be favorably accommodated.
Incorporation of galactose into galactosyltransferase
Biochimica et Biophysica Acta (BBA) - General Subjects, 1977
Bovine skim milk galactosyltransferase (EC 2.4.1.22) retained its catalytic activity after partial enzymatic removal of sialic acid and galactose. Desialylated and degalactosylated galacto~yltransferase was a galactosyl acceptor in the galactosyltransferase reaction. [14C]Galactose from UDP-[~4C]galactose was incorporated into the carbohydrate-depleted galactosyltransferase by native galactosyltransferase. The results suggest that galactosyltransferase participates in the biosynthesis of its glycopeptides of the sialic acid-galactose-N-acetylglucosamine type.
Applied Biochemistry and Biotechnology, 2006
β-Galactosidase from the fungus Talaromyces thermophilus CBS 236.58 was immobilized by covalent attachment onto the insoluble carrier Eupergit C with a high binding efficiency of 95%. Immobilization increased both activity and stability at higher pH values and temperature when compared with the free enzyme. Especially the effect of immobilization on thermostability is notable. This is expressed by the half-lifetime of the activity at 50°C, which was determined to be 8 and 27 h for the free and immobilized enzymes, respectively. Although immobilization did not significantly change kinetic parameters for the substrate lactose, a considerable decrease in the maximum reaction velocity V max was observed for the artificial substrate o-nitrophenylβ-D-galactopyranoside (oNPG). The hydrolysis of both oNPG and lactose is competitively inhibited by the end products glucose and galactose. However, this inhibition is only very moderate as judged from kinetic analysis with glucose exerting a more pronounced inhibitory effect. It was evident from bioconversion experiments with 20% lactose as substrate, that the immobilized enzyme showed a strong transgalactosylation reaction, resulting in the formation of galactooligosaccharides (GalOS). The maximum yield of GalOS of 34% was obtained when the degree of lactose conversion was roughly 80%. Hence, this immobilized enzyme can be useful both for the cleavage of lactose at elevated temperatures, and the formation of GalOS, prebiotic sugars that have a number of interesting properties for food applications.
The catalytic potential of b-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of b-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl b-D-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of b-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.
Process Biochemistry, 2010
Bacillus stearothermophilus secretes ,-mannanase and oa-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of aL-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified P-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The P-D-mannanase activity exhibited thermostabiity, retaining nearly full activity after incubation for 24 h at 70°C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for V.. and Km of 455 60 U/mg and 1.5 0.3 mg/ml, respectively, at 70°C and pH 6.5.
Applied Microbiology and Biotechnology, 2005
The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-D-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-D-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct inter-molecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.