Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an α-galactosidase, an α-mannosidase, a β-N-acetyl-glucosaminidase, a β-glucosidase, and two β-galactosidases. The pH optimum of each of these enzymes was determined. The pH optima, together with inhibition studies, suggest that each observed glycosidase activity represents a separate enzyme. Three of these enzymes, β-glucosidase, α-galactosidase, and one of the β-galactosidases, have been shown to be associated with the cell surface. The enzyme activities associated with the cell surface were shown to possess the ability to degrade to a limited extent isolated sycamore cell walls. It was found that the activities of β-glucosidase and of one of the β-galactosidases increase as the cells go through a period of growth and decrease as cell growth ceases.     

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg⁻¹. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K_m and V_max of 2.9 mmol l⁻¹ and 246.1 μmol (mg min)⁻¹, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

beta-Galactosidases in Ripening Tomatoes

Tomatoes (Lycopersicon esculentum L.) contained a high level of β-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of β-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, β-galactosidases I and III levels decreased but the β-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K_m value with p-nitrophenyl-β-galactoside, and stability with respect to pH and temperature. β-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of β-1, 4-linked galactose. The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.     

Fungal hydroxylation of (−)-santonin and its analogues

Santonin (1) was incubated with separate growing cultures of Aspergillus niger ATCC 9142, Mucor plumbeus ATCC 4740, Whetzelinia sclerotiorum ATCC 18687, Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. Three novel metabolites were isolated: 11β,13-dihydroxysantonin (3), 6,7-dehydosantonin (5) and 3,6-dihydroxy-9-keto-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (7). 11β-Hydroxysantonin (2), 14-hydroxysantonin (4) and 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (6) were also isolated. Hydroxylation at C-9 followed by a retro-aldol reaction was postulated to have produced 6 and 7. Through the synthesis and fermentation of the santonin analogues: tetrahydrosantonin (8) and α-desmotroposantonin (12), several new compounds were obtained; the most significant being 9-keto-desmotroposantonin (14), which was indicative of C-9 monohydroxylation.     

Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.     

Penicillium sp. 23 alpha-galactosidase: purification and substrate specificity

α-Galactosidase, a glycoprotein with carbohydrate and protein in ratio 1:6, has been isolated from liquid culture of micromycete Penicillium sp. 23 and purified to homogeneous state by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels. The Penicillium sp. 23 α-galactosidase specificity against a series of natural and synthetic substrates has been studied. The enzyme was found to exhibit strict specificity towards the glycon and hydrolyze exclusively α- -galactosides such as p-nitrophenyl-α- -galactopyranoside (p-NPhGal), melibiose, raffinose and stachyose. The configuration at C1 and C4 atoms of substrate as well as substitution at C2 and C6 of substrate made an important contribution to the interaction with the enzyme. The tested α-galactosidase exerted the highest affinity (K_m) with respect to the synthetic substrate p-NPhGal and maximal rate of hydrolysis (V_max), about 10 times higher, comparing with natural substrates (melibiose, raffinose and stachiose). The Penicillium sp. 23 α-galactosidase possesses wide specificity towards α-galactosidase hydrolysis link type, splitting off at varying rates the terminal galactose from disaccharides, attached by α-1,2-, α-1,3- and α-1,6-links. The enzyme is ineffective towards disaccharides with α-1,4-link. The enzyme showed potential to splitting off α-1,3-bound terminal galactose residues from antigens of the human blood group B(III) erythrocytes.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Properties of mouse α-galactosidase

α-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-α-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzyme was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a K_m value of 1.4 mM, an energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human α-galactosidase A.The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the α-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.     

Cloning and expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.     

Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth

Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wall β-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since the pH optimum for the enzyme is about 5.0. It has been suggested that enhancement of β-galactosidase and/or other glycosidase activity mediates growth promotion by auxin or low pH. This hypothesis was tested by examining the effect of inhibitors of β-galactosidase and β-glucosidase. Severe inhibition of measureable β-galactosidase or β-glucosidase activity was found to have no effect on auxin- or acid-promoted growth. It is concluded that neither β-galactosidase nor β-glucosidase plays an important role in short term growth promotion by auxin or acid. The data do not rule out the possibility that some other cell wall glycosidase is involved in auxin or acid action.     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: β-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with β-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with β-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56 000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (M_r 55 000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identify with cod cytochrome P-450b (M_r 54 000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

Synthesis, structural analysis and application of novel acarbose-fructoside using levansucrase

Acarbose-fructoside (acarbose-Fru) was newly synthesized via the acceptor reaction of a levansucrase from Leuconostoc mesenteroides B-512 FMC with acarbose and sucrose. The resultant product was separated with 10.5% purification yield via Bio-gel P-2 column chromatography and HPLC. Its structure was determined to be 1^I-β-d-fructofuranosyl α-acarbose, according to the results of ¹H, ¹³C, HSQC, and HMBC analyses. Acarbose-Fru was inhibited competitively on α-glucosidase (A. niger and baker's yeast) but mixed noncompetitively on α-amylases (A. oryzae and porcine pancreatic). Compared to acarbose, acarbose-Fru exhibited inhibition potency of 1.12 or 1.52 on A. niger α-glucosidase or A. oryzae α-amylase, respectively. Additionally, acarbose-Fru was identified as a novel substrate for dextransucrase with K_m and V_max values of 189.0 mM and 8.51 μmol/(mg min), respectively. Therefore, acarbose-Fru as a substrate might be synthesized novel acarbose derivatives by using dextransucrase.