Enzymatic Formation of d-Pantothemc Acid-β-Glucoside by Various β-Glucosidases

A β-gIucoside of d-pantothenic acid was formed from d-pantothenic acid and β-glucosyl donors such as cellobiose, phenyl-β-d-glucoside, salicin, and 4-methylumbelliferyl-β-d-glucoside and naphthol AS-BI-β-d-glucoside by various β-glucosidases, i.e., almond β-glucosidase, cellulase type II and III, naringinase, and hesperiginase. The compound was isolated from a reaction mixture of almond β-glucosidase by treatment with active charcoal, Amberlite CG–50, and DEAH-cellulose column chromatography, paper chromatography, and Sephadex G-IO gel filtration. Then, the compound was characterized as 4′-O-(β-d-glucopyranosyl)-d-pantothenic acid by various analytical methods including bioassay, paper chromatography, NMR and specific optical rotation. The microbiological activities of the compound were also determined.     

Hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts

Of 104 yeasts tested for the hydrolysis of rac-linalyl acetate and rac--terpinyl acetate twelve hydrolysed rac-linalyl acetate. Five of these belonged to five different species of Geotrichum. The most promising strain, Geotrichum capitatum CBS 0572.82, yielded (S)-linalool in 56% enantiomeric excess after 6h (E value 5, c = 32%). Only one unclassified yeast isolate hydrolyzed rac--terpinyl acetate with a slight preference for the S-enantiomer (E value 2). © Rapid Science Ltd. 1998     

Quantitative Study of Anomeric Forms of Glucose Produced by α-Glucosidases and Glucoamylases

A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl α-maltoside and phenyl α-glucoside were determined quantitatively. Thirteen α-glucosidases tested, including α-glucosidase from honey bee and acid α-glucosidase from pig′s liver, were found to produce α-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid α-glucosidase does not belong to the category of exo-1,4-α-glucosidase (EC 3.2.1.3).     

High-Yielding Enzymatic α-Glucosylation of Pyridoxine by Marine α-Glucosidase from Aplysia fasciata

We recently succeeded in the identification and purification of an interesting marine exo-α-glucosidase (EC 3.2.1.20) from the anaspidean mollusc Aplysia fasciata. The enzyme was characterized by good transglycosylation activity toward different acceptors using maltose as donor. High-yielding enzymatic α-glycosylation of pyridoxine using this marine enzyme is reported here; the reaction has been optimized, reaching 80% molar yield of products (pyridoxine monoglucosides 24 g/l; pyridoxine isomaltoside 35 g/l). High selectivity toward the 5′ position is observed for both monoglucoside and disaccharide formation. This is the first report describing the enzymatic production of pyridoxine isomaltoside.     

Formation of α- and β-conidia by Phaeocytostroma ambiguum

The formation of conidia in Phaeocytostroma ambiguum on different media and conditions was investigated in this study. Carnation leaf agar (CLA) and a 12 h photoperiod (24/18 °C) provided excellent conditions for the promotion of rapid formation of both alpha (α) and beta (β) conidia in a number of P. ambiguum isolates. The dimensions of α- and β-conidia amounted to 6.0–19.6 × 3.8–7.5 μm and 6.0–24.9 × 1.1–2.6 μm, respectively. They were produced on short or elongate, simple and branched conidiophores. β-conidia have not been described before in P. ambiguum. Intermediate conidia were rarely found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative Biochemical Studies of α-Glucosidases

The properties of brewer’s yeast α-glucosidase have been investigated. The enzyme was capable of hydrolyzing various α-glucosides and was active especially on aryl-α-glucosides in comparison with other α-glucosides and sugars. The rate of hydrolysis decreased in following order: phenyl-α-glucosides, sucrose, matlose and isomaltose.

The range of opt. temp, was 40~45°C and opt. pH, 6.5~7.0.

Cu⁺⁺ and Hg⁺⁺ inhibited strongly the enzyme activity and Zn⁺⁺, moderately. The enzyme was suggested to be a sulfhydryl enzyme from the inhibition experiments by SH-reagents and the effects of glutathione on the activity.

The enzyme synthesized some oligosaccharides from maltose. As the transglucosidation products, nigerose, isomaltose, kojibiose and maltotriose were detected by paperchromatography.

Pure nigerose was separated by splitting maltose with amyloglucosidase from the mixture of maltose and nigerose and by use of successive carbon column chromatography.     

Enzymatic Hydrolysis of α-Chitin

It is shown that the enzymatic preparation Celloviridin G20x can be used for hydrolyzing -chitin of various origin. The purity of the final product of hydrolysis, N-acetylglucosamine, was monitored using HPLC.     

Molecular Basis for the Recognition of Long-chain Substrates by Plant α-Glucosidases

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k_cat/K_m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)₈ barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)₈ barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.     

Anomer Selective Formation of l-Menthyl α-D-Glucopyranoside by α-Glucosidase-catalyzed Reaction

l-Menthol was glucosylated by the α-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as glucosyl donor. When 50 mg of l-menthol and 1M maltose in 10 mM citrate–phosphate buffer (pH 7.0) were incubated for 24 h at 30°C, a menthylglucoside was selectively obtained as a product. The molar conversion yield based on supplied menthol was 4.5%. The product was identified as l-menthyl α-D-glucopyranoside (α-MenG) by ¹³C-NMR analysis.     

Hydrolysis of α-chloro-substituted 2- and 4-pyridones: rate enhancement by zwitterionic structure

The hydrolysis of α-chloro-N-methyl-4-pyridone was found to be more than five times faster than that of α-chloro-N-methyl-2-pyridone. Structural studies of 2- and 4-pyridones have revealed the higher polarity and greater extent of zwitterionic content in 4-pyridone. The results are thus consistent with the hypothesis that polarization and higher zwitterionic content in the heterocyclic structures enhances the rate of hydrolysis in α-substituted pyridone and uracil derivatives.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Formation of β-Fructosyl Compounds of Pyridoxine in Growing Culture of Aspergillus niger

Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A. sydowi, when grown in a medium containing sucrose and pyridoxine. Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization. Compounds I and II were identified as 5?-O-(β-D-fructofuranosyl)-pyridoxine and 5?-O-(β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl]-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, ¹H-, and ¹³C-NMR spectra, products by hydrolysis with acid and yeast β-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Levansucrase from Microbacterium laevaniformans and yeast β-D-fructofuranosidase did not catalyze the β-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to β-D-fructofuranosyl-pyridoxine.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

Production and Properties of α-l-Arabinofuranosidase from Various Strains of Corticium rolfsii

Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

Hydrolysis of natural and synthetic substrates by α-l-fucosidase, β-d-glucuronidase and β-n-acetylhexosaminidase purified from molluscs

• 1.1. Several mollusc glycosidases have been studied for their activities towards natural substrates. α-l-Fucosidases from Chamelea gallina, Tapes rhomboideus and Mytilus edulis hydrolyze oligosaccharides (di, tri and pentasaccharides) with α1 → 2, α1 → 3 and α1 → 4 bonds, fucose-containing glycopeptides from bovine thyroglobulin and the porcine submandibular mucin (devoid of sialic acid); α-l-fucosidase from Littorina littorea hydrolyzes fucose-containing glycopeptides from bovine thyroglobulin.
• 2.2. β-d-Glucuronidase from L. littorea hydrolyzes hyaluronic acid, chondroitin 4-sulfate and heparin with a very low activity; however, it is much more active on oligosaccharides (from the above-mentioned macromolecules) containing non-reducing terminal glucuronyl residues.
• 3.3. β-N-Acetylhexosaminidase from Helicella ericetorum acts mainly with an endo-hydrolase activity on β1 → 4N-acetylhexosamine linkages of ovalbumin, ovomucoid, chitin, hyaluronic acid and chondroitin
• 4.4-sulfate; it has also a secondary exo-hydrolase activity on these substrates.

     

Partial Purification and Characterization of Acid and Neutral α-Glucosidases from Preclimacteric Banana Pulp Tissues

An acid α-glucosidase (AAG) with an optimum pH of 4.5 and two isoforms of neutral α-glucosidase (NAG I and II) with an optimum pH of 6.5 were partially purified from preclimacteric banana pulp tissues by monitoring the 4-methylumbelliferyl α-D-glucoside (4MUαG) hydrolyzing activity. The molecular weights of the AAG and the two NAG were 70,000 and 42,000, respectively, by gel filtration. By kinetic studies, the AAG was found to be a typical maltase that required substrates such as maltose, maltotriose, maltotetraose, and maltopentaose rather than soluble starch. On the other hand, the two NAGs preferred 4MUαG to maltose as substrate and their maltase activities were about 50 times lower than that of the AAG. The NAGs, as well as the AAG, did not hydrolyze isomaltose, trehalose, sucrose, or glycogen at all. Sucrose was a competitive inhibitor of the AAG but not NAGs toward 4MUαG. Glucose and maltose were also competitive inhibitors of both AAG and NAGs.