Characterization of a Novel Mesophilic Bacterial Amylase Secreted by ZW2531-1, a Strain Newly Isolated from Soil

A novel mesophilic bacterial amylase, named oligosaccharide-producing multifunctional amylase(OPMA), was discovered and characterized. OPMA is an extracellular enzyme secreted by ZW2531-1, a strain newly isolated from Chinese soil. It could be purified to homogeneity from the culture supernatant of ZW2531-1 by 30%―60% saturated ammonium sulfate precipitation, followed by twice Sephadex gel filtration chromatography. OPMA is a 66 kDa protein based on SDS-PAGE and has an isoelectric point(pI) at pH=5.3 by Iso...     

Enzymatic synthesis of prebiotic oligosaccharides

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.     

Effect of structure of the amino acids and amines on the rate and mechanism of their condensations with pyridoxal

Kinetics and mechanism of condensation of amino acids and amines of different structure and their derivatives with pyridoxal were studied. It was established that the amino acid with secondary amino group, proline, adds to pyridoxal with the formation of amino alcohol. α-Amino acids in the course of condensation with pyridoxal form amino alcohols which transform to Schiff bases. The latter compounds by elimination of the α-hydrogen atom or CO₂ from the amino acid fragment and the subsequent hydrolysis of the quinoid structure form the final products. β- And ɛ-amino acids react with pyridoxal to form Schiff bases which are stable to chemical transformations. The possibility was shown of their conversion to the quinoid structure. It was established that the guanidine structure of the molecule of L-arginine unlike its α-NH₂ group did not take part in the condensation with pyridoxal. The quantitative evaluation of the condensation rates of triptamine, Ltriptofane, and its methyl ester in the stage of dehydration of their amino alcohols was carried out.     

Novel fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAc) for the resolution of oligosaccharides in glycopeptides, based on enzyme transglycosylation reaction

Several fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAcs), i.e., DBD-Asn-GlcNAc, NBD-Asn-GlcNAc, NDA-Asn-GlcNAc, PS-Asn-GlcNAc, FITC-Asn-GlcNAc, DMEQ-Asn-GlcNAc and DBD-PZ-Boc-Asn-GlcNAc, were synthesized as the acceptors for the resolution of oligosaccharides in glycopeptides. The resolution of oligosaccharides is based on the transglycosylation reaction with end-β-N-acetylglucosaminidase (Endo-M), isolated from Mucor hiemalis. The synthesis of each fluorescent acceptor was carried out in a one-pot reaction of Asn-GlcNAc and the corresponding fluorescent tagging reagent. The transglycosylation reaction using Endo-M proceeds at RT in neutral phosphate buffer (pH 6.0) and reached maxima at around 30 min. When Fmoc-Asn-GlcNAc (acceptor) was enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M, the ratio of Disialo-Asn-Fmoc (transglycosylation product) was almost comparable with the decreasing ratio of Fmoc-Asn-GlcNAc. Therefore, the transglycosylation activity of Endo-M from Disialo-Asn (donor) and fluorescent-Asn-GlcNAc (acceptor) was calculated from the decreasing ratio of fluorescent-Asn-GlcNAc. The order was NDA-Asn-GlcNAc > DBD-Asn-GlcNAc Fmoc-Asn-GlcNAc > NBD-Asn-GlcNAc DMEQ-Asn-GlcNAc > DNS-Asn-GlcNAc > PS-Asn-GlcNAc > FITC-Asn-GlcNAc. On the other hand, the activity with a fluorescent acceptor (DBD-PZ-Boc-Asn-GlcNAc), labeled to a carboxylic acid group in the Asn residue, was the strongest among the synthesized acceptors.     

Glycopeptide synthesis through endo-glycosidase-catalyzed oligosaccharide transfer of sugar oxazolines: probing substrate structural requirement

An array of sugar oxazolines was synthesized and tested as donor substrates for the Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A)-catalyzed glycopeptide synthesis. The experiments revealed that the minimum structure of the donor substrate required for Endo-A catalyzed transglycosylation is a Man beta1-->4-GlcNAc oxazoline moiety. Replacement of the beta-D-Man moiety with beta-D-Glc, beta-D-Gal, and beta-D-GlcNAc monosaccharides resulted in the loss of substrate activity for the disaccharide oxazoline. Despite this, the enzyme could tolerate modifications such as attachment of additional sugar residues or a functional group at the 3- and/or 6-positions of the beta-D-Man moiety, thus allowing a successful transfer of selectively modified oligosaccharides to the peptide acceptor. On the other hand, the enzyme has a great flexibility for the acceptor portion and could take both small and large GlcNAc-peptides as the acceptor. The studies implicate a great potential of the endoglycosidase-catalyzed transglycosylation for constructing both natural and selectively modified glycopeptides.     

A HPLC method for specific determination ofα-amylase and glucoamylase in complex enzymatic preparations

Summary In the hydrolysis of soluble starch by mixtures of α-amylase and glucoamylase, the ratios maltose/glucose and maltoriose/glucose linearly depend, over a wide range, on the relation between both enzymes and are independent on the activity level of the enzymatic preparation. HPLC determination of hydrolysis products (glucose, maltose and maltotriose) of soluble starch by mixtures of these enzymes, after incubation under controlled conditions, is a rapid method for the evaluation of the relative levels of each enzyme in the mixtures. The method, first developed using pure commercial amylases, is applied, with consistent results, to cell free media ofAspergillus niger cultures on a glycogen-rich effluent.     

N-(2-aminobenzoyl)-N-methylhydrazones of aldehydes and aldoses and their cyclization to benzo-1,3,4-triazepine derivatives

The products of the condensation of aliphatic aldehydes with N-(2-aminobenzoyl)-N-methylhydrazine exist in DMSO-d₆ solution as tautomeric mixtures of linear aldohydrazone and cyclic benzo-1,3,4-triazepine forms. The linear tautomer predominates for 2-aminobenzoyl-N-methylhydrazones of aromatic aldehydes. A tautomeric equilibrium is observed in DMSO-d₆ for the products of the condensation of the hydrazide of 2-aminobenzoic acid with a series of aldoses. This equilibrium exists between α,β-isomeric pyranose forms and the open aldosohydrazone form. Isomeric conversion to the seven-membered benzo-1,3,4-triazepine form is observed for the products of the condensation of aldoses with N-(2-aminobenzoyl)-N-methylhydrazine.     

Docking and molecular dynamics studies on the stereoselectivity in the enzymatic synthesis of carbohydrates

Glycosidases constitute a vast family of enzymes that catalyze the breaking and formation of glycosidic bonds. The synthesized oligosaccharides, being crucial to life, are involved in many biochemical processes, particularly in the pharmaceutical and food industries. The proposed catalytic mechanism of retaining glycoside hydrolases (glycosidases) occurs via a double displacement mechanism involving a covalent glycosyl enzyme intermediate. During the transglycosylation reactions, the control of the stereoselectivity for the formation of the new bond remains a complicated problem in the chemical synthesis of oligosaccharides. In this paper, docking and molecular dynamics methods were used to study the second step of the mechanism of transglycosylation in retaining glycosidases from six microorganisms with known stereoselectivity. Using the natural substrates as donor and acceptor molecules, we were able to corroborate and provide structural information about the active site, the trapped monosaccharide acceptor and the bound intermediates during the step that precedes transglycosylation, as well as identify and understand the commonly displayed stereoselectivity by these glycosidases in nature. The information obtained with this procedure helps to recognize, explain and predict the stereoselectivity of the sugars studied. These kind of procedures can be used to improve the efficiency of large-scale industrial synthesis of a specific sugar.     

Purification and Characterization of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides

α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4–7. Maximal activities were determined at 60, 55, and 50°C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50°C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg²⁺, Ag⁺, and Cu²⁺. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes     

Resolution of oligosaccharides in glycopeptides using immobilized Endo-M and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

The resolution of asparagine-linked oligosaccharides in glycopeptides was carried out by combination of the transglycosylation reaction and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The resolution of the oligosaccharides is based on the enzymic transglycosylation reaction with Endo-beta-N-acetylglucosaminidase (Endo-M) isolated from Mucor hiemalis. The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. In the present research, the enzyme was also immobilized in the well of a microassay plate by the sol-gel technique. The transglycosylation reaction was easily managed due to the immobilization. Furthermore, multiple use was possible by the encapsulated Endo-M. The resulting fluorescent oligosaccharides were separated by UPLC and efficiently detected by ESI-TOF-MS. Several oligosaccharides in ovalbumin were successfully identified by the proposed procedure.     

Transferase activity of endo-β-1,6-glucanases from the crystalline stalks of bivalve marine molluscs

It has been shown that endo-β-1,6-glucanases from marine molluscs perform a transglycosylation reaction. When o-nitrophenyl β-D-glucopyranoside (Np glucoside) was used as acceptor, among the newly formed products Np gentiobioside, -trioside, and -tetraoside with a total yield of up to 20% on the initial Np glucoside were detected.     

Chemical synthesis of cyclodextrins by using intramolecular glycosylation

An efficient synthesis of cyclodextrins (CDs) by using the intramolecular glycosylation is demonstrated. alpha-CD, an alpha(1-->4)linked hexaglucoside, was prepared via a block condensation of three maltose units. A modified key maltose intermediate as a precursor to both glycosyl donor and acceptor components was prepared in 6 steps starting from maltose. All the glycosylation for chain elongation and cyclization of saccharides was carried out after tethering the donor to the acceptor by the phthaloyl bridge to give the desired saccharides in good yields with complete alpha-selectivity. delta-CD composed of 9 glucose units was synthesized by the same manner from three maltotriose units.     

Hyperthermostable,Ca<Superscript>2+</Superscript>-Independent,and High Maltose-Forming α-Amylase Production by an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>: Whole Cell Immobilization

The synthesis of extracellular α-amylase in Geobacillus thermoleovorans was constitutive. The enzyme was secreted in metabolizable carbon sources as well as non-metabolizable synthetic analogues of glucose, but the titers were higher in the former than that in the latter. G. thermoleovorans is a fast-growing facultatively anaerobic bacterium that grows under both aerobic and anaerobic conditions and produces an extracellular amylolytic enzyme α-amylase with the by-product of lactic acid. G. thermoleovorans is a rich source of various novel thermostable biocatalysts for different industrial applications. α-Amylase synthesis was subject to catabolite repression in the presence of high concentrations of glucose. The addition of cAMP to the medium containing glucose did not result in the repression of α-amylase synthesis. The addition of maltose (1%) to the starch arginine medium resulted in a twofold enhancement in enzyme titers. Polyurethane foam (PUF)-immobilized cells secreted α-amylase, which was higher than that with the free cells. PUF appeared to be a better matrix for immobilization of the thermophilic bacterium than the other commonly used matrices. The repeated use of PUF-immobilized cells was possible over 15 cycles with a sustained α-amylase secretion. The use of this enzyme in starch saccharification eliminates the addition of Ca²⁺ in starch liquefaction and its subsequent removal by ion exchangers from the product streams.     

Ion chromatographic methods for the detection of starch hydrolysis products in ruminal digesta.

Dionex high-performance ion chromatographic methods were evaluated for separation and quantitation of plant sugars and starch digestion products in the ruminal digesta of cattle. Mono- and disaccharides were eluted from a Dionex CarboPac PA1 column with sodium hydroxide used isocratically or as a pH gradient. Maltooligosaccharides which had a degree of polymerization (DP) less than 30 glucose residues were eluted in 60 min by a sodium hydroxide eluent containing a sodium acetate gradient. Carbohydrates were detected amperometrically. Responses were linear (r2 greater than 0.99) for glucose, disaccharides and maltooligosaccharides (DP less than 8). Precipitation and solid-phase extraction methods were evaluated for clean-up of samples of feedstuffs, ruminal contents, and bacterial culture fluids. Perchloric acid precipitation hydrolyzed sucrose but did not affect recoveries of cellobiose, isomaltose or maltose. Ethanol in concentrations of 79 and 86% precipitated maltooligosaccharides having chain lengths larger than 14 and 9 glucose residues, respectively. Maltooligosaccharide recoveries from solid-phase extraction columns varied with maltooligosaccharide size and column packing. Recoveries were greater than 94% for short chains (DP less than 6) eluted from phenyl-substituted columns and variable for all oligosaccharides eluted from C18 columns. Applications of these methods are presented and include: (1) detection of sugars in ruminant feed, (2) monitoring changes in ruminal sugars after feeding and (3) monitoring changes in extracellular sugars and oligosaccharides in the culture fluids of the ruminal bacterium, Bacteroides ruminicola.     

Synthesis of 3-methyl-2-cyclohexenones catalyzed by mercury(II) salts and their microwave assisted BiCl3 catalyzed aldol condensations

HgSO₄ catalyzed hydrative cyclization of 1,6-heptadiynes is present. This reaction proceeded smoothly under the mild condition for differently 4-sustituted 1,6-diynic substrates giving corresponding 3-methyl-2-cyclohexenones with high to excellent yield. The microwave assisted aldol condensation of cyclohexenones under the catalysis of BiCl₃ afforded 3-styryl-cyclohexenones with high regio- and stereo-selectivity.     

Biosynthetic activity of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II

The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii β-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described. With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively. In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively. The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides. In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained. The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10%. Detailed kinetic analysis indicated a significant (about twofold) increase in V _max/K _M of synthetic reactions in the presence of DMSO. A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose.     

Evaluation of Enzymatic Reactors for Large-Scale Panose Production

Panose is a trisaccharide constituted by a maltose molecule bonded to a glucose molecule by an α-1,6-glycosidic bond. This trisaccharide has potential to be used in the food industry as a noncariogenic sweetener, as the oral flora does not ferment it. Panose can also be considered prebiotic for stimulating the growth of benefic microorganisms, such as lactobacillus and bidifidobacteria, and for inhibiting the growth of undesired microorganisms such as E. coli and Samonella. In this paper, the production of panose by enzymatic synthesis in a batch and a fed-batch reactor was optimized using a mathematical model developed to simulate the process. Results show that optimum production is obtained in a fed-batch process with an optimum production of 11.23 g/l h of panose, which is 51.5% higher than production with batch reactor.     

Chemoselective coupling of sugar oximes and α-ketoacids to glycosyl amides and N-glycopeptides

The reaction of unprotected sugar hydroxylamines and oximes with α-ketoacids leads to the chemoselective formation of glycosyl amides following the decarboxylative condensation pathway of Bode’s ketoacid hydroxylamine (KAHA) ligation. Sugar oximes with gluco configuration stereoselectively form β-linked products. This method can be used for the convergent synthesis of N-acylated sugars and oligosaccharides bearing small labels, spacers, or peptides in the acyl part.     

Characterisation of two novel cyclodextrinases using on-line microdialysis sampling with high-performance anion exchange chromatography

In this work, a real-time sampling/analytical method for on-line measurements of two newly discovered cyclomaltodextrinases (CDases) has been developed and evaluated. This novel methodology not only allows the final products to be investigated, but it also reveals enzyme-specific differences in the degradation pathways during the hydrolysis of different substrates, which is a great advantage in the important tasks of investigating the mechanisms of and classifying new hydrolases, and is an advantage that conventional techniques cannot offer. Two different enzymes, one CDase from Laceyella sacchari (LsCda13) and one from Anoxybacillus flavithermus (AfCda13), were investigated during the hydrolysis of α-, β- and γ-cyclodextrin, and the hydrolysis products were sampled via a microdialysis probe and injected on-line every 30 min into a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector (HPAEC–PAD), where they were identified. The enzymes yielded the same end-products, maltose and glucose, in an approximate molar ratio of 2:1, but they exhibited distinctly different patterns of intermediate product formation before reaching the end-point. LsCda13 had a more random distribution of the intermediate products, whereas AfCda13 showed the distinct intermediate production of maltotriose, which in some cases accumulated.     

Resolution of N-linked oligosaccharides in glycoproteins based upon transglycosylation reaction by CE-TOF-MS

The resolution of asparagine-type oligosaccharides in glycoproteins was carried out by combination of the transglycosylation reaction and CE-TOF-MS. The oligosaccharides enzymatically transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M. The resulting fluorescent-oligosaccharides were separated by CE and detected by TOF-MS. Disialo-Asn was successfully identified by the proposed procedure. Application to oligosaccharides in ovalbumin was also described in this communication.