Quantitative Study of Anomeric Forms of Maltose Produced by α- and βAmylases

Anomeric forms of glucose and maltose produced from phenyl, p-nitrophenyl, p-tert-butylphenyl, p-ethylphenyl and p-chlorophenyl α-maltosides and maltopentaose by α- and β-amylases were determined quantitatively by a gas-liquid chromatographic method. All of the three kinds of α-amylases tested, B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, were found to produce only α-maltose from the maltosides. Sweet potato and barley β-amylases produced β-maltose from maltopentaose.

Saccharifying α-amylase from B. subtilis also released α-maltose from all the maltosides mentioned above, contrary to the report by Shibaoka et al. that the enzyme released β-maltose from maltosides other than phenyl α-maltoside: FEBS Lett., 16, 33 (1971); J. Biochem., 77, 1215 (1975). It appears unlikely that the α-amylase releases β-maltose, depending on the kind of substrate.     

The Formation and Hydrolysis of Pyridoxine Glucoside by Various α-Glucosidases

(—)-Epicatechin-3-gallate (ECG) and (— )-epigallocatechin-3-gallate (EGCG), major tea catechins, formed precipitates with soybean lipoxygenase (LOX) in the pH range of 4~7, although with accompanying 10 ~30% loss of the LOX activity. Yeast alcohol dehydrogenase also was precipitated by EGCG. Polyvinylpyrrolidone, Tween 20 and Triton X-100 dissociated the LOX activity from the EGCG-precipitated LOX. However, the MW of the dissociated LOX (114,000) differed from that of the native LOX (100,000). Enzyme activities of the EGCG-precipitated LOX and the dissociated LOX from the precipitate were less stable than the activity of the native LOX. These findings suggest the altered natures of proteins in the presence of tea catechins, ECG and EGCG.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

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.     

Modulation of Starch Digestion for Slow Glucose Release through "Toggling" of Activities of Mucosal α-Glucosidases

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.     

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.     

Purification and Some Properties of a Novel α-Amylase Produced by a Strain of Thermoactinomyces vulgaris

An α-amylase[α-l,4-glucan 4-glucanohydrolase, EC 3.2.1.1.], found in the culture filtrate of a strain of Thermoactinomyces vulgaris, was purified by ammonium sulfate fractionation, and DEAE-cellulose and CM-cellulose chromatographies. The purified enzyme showed a single band on disc gel electrophoresis. The optimum reaction pH and temperature were determined to be around pH 5.0 and 70°C. The isoelectric point was determined to be pH 5.2. The α-amylase was stabilized by Ca²⁺.

The α-amylase was found to hydrolyze pullulan to panose. Therefore, the hydrolytic pattern of this enzyme is different from those of pullulanase and isopullulanase.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

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.     

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.     

Red Pigment Produced by the Reaction of Dehydro-l-ascorbic Acid with α-Amino Acid

At the initial stage of the browning reaction of dehydro-l-ascorbic acid (DHA) with α-amino acid, a kind of red pigment was produced. The pigment was isolated as very hygroscopic red powder from non-aqueous reaction system, and its characterization was made. It was revealed that it had the same structure with that of the red pigment produced by the oxidation of l-scorbamic acid, an intermediate amino-reductone expected to be produced by Strecker degradation. Formation mechanism of the pigment which was considered to be an intermediate of browning reaction of DHA with α-amino acid was also discussed.     

Glucose and glycerol repression of α-amylase in Streptomyces kanamyceticus and isolation of deregulated mutants

Summary Glucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.     

Purification and Characterization of a Surface-active Macropeptide Produced from Succinylated αs1-Casein by Modification with Papain

The preceding paper described that when succinylated α_s1-casein, ca. 25,000 daltons, was modified with papain in the presence of l-leucine n-dodecyl ester (Leu-OC₁₂), an approximately 20,000-dalton macropeptide was formed as the main product. In the present work we have investigated its chemical structure and surface function. A treatment for purification at the petroleum ether/water interface gave an electrophoretically homogeneous 20,000-dalton macropeptide which functioned as a surfactant to emulsify corn oil as well as n-octane. Pulsed NMR and ESR studies demonstrated that the macropeptide, when used to emulsify n-octane in water, acted to restrict the mobility of those molecules involved in the emulsion. Various data from chemical analyses coupled with knowledge about the primary structure of α_s1-casein showed that the 20,000-dalton macropeptide was structured as succinyl-Arg¹-….-Phe¹⁴⁵-Leu-OC₁₂. A discussion is included to explain the surface function of this peptide in relation to its amphiphilic structure.     

Inactivation of Bacteriophages by ε-Poly-l-lysine Produced by Streptomyces

Degradation of a β-O-4lignin substructure model dimer by a white rot fungus, Phanerochaete chrysosporium, was investigated using a culture containing H₂¹⁸O, and the following conclusions were made. a) The direct hydrolysis at C_β of the β-O-4 dimer was not involved in formation of arylglycerol. b) About half of the oxygen at the benzyl (C_α) position of the glycerol was derived from H₂O (H₂¹⁸O) and the other half was from the oxygen at the benzyl (C_α) position of the substrate β-O-4 dimer. c) But, the oxygen at the C_α position of the substrate β-O-4 dimer did not migrate to the C_α position of the aryglycerol.     

Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

Quantitative analysis of thymosin α1 in human serum by LC-MS/MS

1 high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Tα1) concentration in human serum. Tα1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL T α1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Tα1 by the LC-MS/MS method is fast accurate, and precise.     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.The gastrointestinal tract is inhabited by a complex community of microorganisms, also referred to as the microbiota, which are believed to play an important role in human health and disease (39). This concept has been driving extensive attempts to positively influence the composition and/or activity of the intestinal microbiota through the use of so-called probiotics and/or prebiotics. A probiotic has been defined as “a preparation or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effect in this host” (48). A prebiotic has recently been (re)defined as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (42). Finally, a synbiotic is the combination of a probiotic and a prebiotic (16).One of the dominant bacteria of the intestinal microbiota of humans and animals (51) is bifidobacteria. These are gram-positive, pleomorphic, and anaerobic bacteria that have received increasing scientific attention in recent years due to their perceived probiotic activity (15, 27). The growth of gut-derived bifidobacteria has been shown to be selectively stimulated by various dietary carbohydrates that can thus be considered as prebiotics (30). In this context, it is interesting to note that more than 8% of the identified genes on bifidobacterial genomes are predicted to be involved in sugar metabolism, thus indicative of extensive carbon source-degrading abilities (55, 56).Carbohydrate degradation has been extensively studied in a variety of different Bifidobacterium species (reviewed in reference 53). For example, various α- and β-galactosidases have been characterized in Bifidobacterium breve 203 (60), Bifidobacterium adolescentis DSM20083 (20), Bifidobacterium bifidum NCIMB41171 (18), and Bifidobacterium longum MB219 (43). A number of studies have also shown that Bifidobacterium spp. produce various α- and β-glucosidase activities (reviewed in reference 53), while Bifidobacterium infantis ATCC 15697 (58), Bifidobacterium lactis DSM10140(T) (13), and B. breve UCC2003 (45) have been reported to produce β-fructofuranosidases during growth on fructooligosaccharides. Additionally, starch-, amylopectin-, and pullulan-degrading activities in bifidobacteria have been investigated (36, 44). Several β-glucosidases have been biochemically characterized from a number of strains of bifidobacteria, e.g., B. adolescentis Int-57 (8), B. breve clb (35,) and Bifidobacterium sp. strain SEN (59). To date, only two α-glucosidases (AglA and AglB) have been described from B. adolescentis DSM20083 (54). AglA was shown to preferentially hydrolyze isomaltotriose, while AglB exhibits a high preference to maltose. Both AglA and AglB were also demonstrated to have transglycosylation activity. Aside from this report, little is known about the biochemical characteristics of α-glucosidase enzymes from bifidobacteria, although it is a common activity observed among these bacteria (41).Carbohydrates other than the commercially exploited prebiotics, e.g., fructooligosaccharides (such as inulin) and trans-galactooligosaccharides (42), have received relatively little attention with regard to their possible prebiotic properties. Such potential prebiotics are, for example, honey oligosaccharides, some of which are also interesting because of their noncariogenic properties (14). One of the predominant fractions of noncariogenic sugars in honey is isomaltulose (5, 46), also called palatinose or 6-O-α-d-glucopyranosyl-d-fructose, which is a reducing disaccharide and a functional isomer of sucrose. Palatinose possesses approximately one-third of the sweetness of sucrose and is very resistant to acid and invertase hydrolysis (29, 32). The hydrolysis and adsorption of palatinose in the small intestine thus occurs at a much slower rate than does those of sucrose (17), which results in a reduction of the postprandial plasma glucose and insulin levels (3), which means that most palatinose passes through the small intestine to present a growth substrate for elements of the colonic microbiota.In this study, we describe the identification of two genes, agl1 and agl2, present in the genome of B. breve UCC2003 and responsible for the hydrolysis of α-glycosidic linkages, such as those present in palatinose.