Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.     

Characterization and biotechnological application of an acid alpha-galactosidase from Tachigali multijuga Benth. seeds

Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.     

Cloning and characterization of a novel alpha-galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal-alpha-1,4 linkage.

A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.     

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.     

Production, Purification, and Characterization of alpha-Galactosidase from Monascus pilosus

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.     

Cloning of the gene encoding a novel thermostable alpha-galactosidase from Thermus brockianus ITI360.

An alpha-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53, 810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the alpha-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93 degrees C with para-nitrophenyl-alpha-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80 degrees C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The K(m)s for melibiose and raffinose at 80 degrees C were determined as 4.1 and 11.0 mM, respectively. The alpha-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no alpha-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.     

A galactomannan-hydrolysing α-galactosidase from jack fruit (Artocarpus integrifolia) seed: Affinity chromatographic purification and properties

An acid α-galactosidase from the seeds of the jack fruit seed (Artocarpus integrifolia) has been purified to homogeneity by affinity chromatography on a matrix formed by cross-linking the soluble α-galactose-bearing guar seed galactomannan. The 35kDa enzyme was a homotetramer of 9.5kDa subunits. Its carbohydrate part (5.5%) was composed of galactose and arabinose. TheK _m withp-nitrophenyl α-D-galactoside as substrate was 0.35 mM. TheK _i values indicated inhibition by galactose, 1-O-methyl α-galactose and melibiose in the decreasing order. Among α-galactosides, the enzyme liberated galactose from melibiose, but not from raffinose or stachyose at its pH optimum (5.2). The guar seed galactomannan was however efficiently degalactosidated; limited enzyme treatment abolished the precipitability of the polysaccharide by the α-galactose-specific jack fruit seed lectin, and complete hydrolysis yielded insoluble polysaccharide. Though similar in sugar specificity and subunit assembly, α-galactosidase and the lectin coexisting in the jack fruit seed gave no indication of immunological identity.     

Production of recombinant alpha-galactosidases in Thermus thermophilus

A Thermus thermophilus selector strain for production of thermostable and thermoactive alpha-galactosidase was constructed. For this purpose, the native alpha-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (alpha-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal(+) phenotype was assumed to be essential for growth on melibiose. In a Gal(-) background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant alpha-galactosidase. Moreover, the AgaT(-) strain had to be Km(s) for establishment of the plasmids containing alpha-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT(-) Gal(+) Km(s)) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous alpha-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Purification and characterization of the raffinose oligosaccharide chain elongation enzyme,galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.     

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Characterization of extremely thermostable enzymatic breakers (alpha-1,6-galactosidase and beta-1,4-mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum

An alpha-galactosidase and a beta-mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified alpha-galactosidase was found to have a temperature optimum of 100-105 degrees C with a half-life of 130 minutes at 90 degrees C and 3 min at 100 degrees C, while the purified beta-mannanase was found to have a temperature optimum of 91 degrees C and a half-life of 13h at this temperature and 35 min at 100 degrees C.These represent the most thermostable versions of these enzymes yet reported. At 25 degrees C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5-h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100-fold after 1.5 h and 375-fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85 degrees C, the TN5068 enzymes reduced viscosity by 30-fold after 1.5 h and 100-fold after 5 h compared to a 2.5-fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85 degrees C (1.6-fold reduction after 1.5 h and a 4.2-fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified beta-mannanase alone can substantially reduce viscosity of guar solutions, while the alpha-galactosidase alone had limited viscosity reduction activity. However, the alpha-galactosidase appeared to minimize residual particulate matter when used in conjunction with the beta-mannanase. This could be the result of extensive hydrolysis of the alpha-1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the beta-mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery. (c) 1996 John Wiley & Sons, Inc.     

Production and characterization of raffinose-hydrolysing and invertase activities ofAspergillus fumigatus

Raffinose-type galactose oligosaccharides constitute a substantial part (40%) of the soluble sugars present in soybean seeds and are responsible for flatulence following ingestion of soybean and other legumes. Enzymic hydrolysis of these oligosaccharides would improve the nutritional value of soybean milk.Aspergillus fumigatus produces substantial raffinose-hydrolysing and invertase activities when grown on wheat straw. Three proteins displaying maximal activity at pH 4.5–5.5 and 55–60°C and having molar mass of 66.8, 50.3 and 30.2 kDa were purified. Raffinose and sucrose were hydrolyzed with equivalent affinities by each protein. Nevertheless, theK _m andV _lim values determined for hydrolysis of sucrose by the 66.8 kDa enzyme differed from those determined with the 50.3 kDa protein. Glucose was produced when sucrose was the substrate. The three proteins hydrolyzed also stachyose but not melibiose, maltose, inulin or 4-nitrophenyl α-d-galactopyranoside.A. fumigatus enzymes may be candidates for processing of soybean milk to reduce its flatulence potential.     

Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus

The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation. This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.     

Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

General Properties of Beta-Galactosidase of Xanthomonas campestris

Partially purified beta-galactosidase of Xanthomonas campestris required 32 to 37 degrees C and pH 5.5 to 5.8 for optimum activity. The enzyme had low affinity for lactose hydrolysis (K(m) = 22 mM) and was inhibited by thiol group reagents, ethylenediaminetetraacetic acid, galactose, and d-galactal.