Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

Enzymatic synthesis of D-glucosaminic acid from D-glucosamine

D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides and a chiral synthon, is easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) catalysed by glucose oxidase.     

Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity

1,6-Anhydro-3,4-O-isopropylidene-1-thio-D-mannitol was converted into its sulfoxide which after hydrolysis, acetylation and subsequent Pummerer rearrangement gave the penta-O-acetyl-1-thio-D-mannoseptanose anomers in excellent yield. This anomeric mixture was used as donor for the glycosylation of 4-nitro- and 4-cyanobenzenethiol in the presence of boron trifluoride etherate and trimethylsilyl triflate, respectively, to yield the corresponding thioseptanosides in high yield. The same strategy was applied for the synthesis of the corresponding L-idothioseptanosides using 1,6-anhydro-3,4-O-isopropylidene-1-thio-L-iditol as starting material. The penta-O-acetyl-D-glucothioseptanose donors could not be synthesised the same way, as the Pummerer reaction of the corresponding tetra-O-acetyl-1,6-thioanhydro-1-thio-D-glucitol sulfoxides led to an inseparable mixture of the corresponding L-gulo- and D-glucothioseptanose anomers. Therefore, D-glucose diethyl dithioacetal was converted via its 2,3,4,5-tetra-O-acetyl-6-S-acetyl derivative into an anomeric mixture of its 6-thio-septanose and -furanose peracetates which could be separated by column chromatography. Condensation of the 6-thio-glucoseptanose peracetates with 4-cyano- and 4-nitrobenezenethiol in the presence of boron trifluoride etherate afforded anomeric mixtures of the corresponding thioseptanosides. The D-manno-, L-ido- and D-glucothioseptanosides obtained after Zemplén deacetylation of these mixtures were tested for their oral antithrombotic activity.     

Enzymatic method for determination of the degree of deacetylation of chitosan.

A new method was developed for the accurate determination of the degree of deacetylation of chitosan. The method involves the complete hydrolysis of chitosan to glucosamine and N-acetylglucosamine by a cooperative action of chitosanolytic enzymes exo-beta-D-glucosaminidase, beta-N-acetylhexosaminidase, and chitosanase, and subsequent determination of the monosaccharides by specific colorimetric assays or HPLC. The conditions required for the complete hydrolysis of chitosan were examined and the degree of deacetylation of several chitosan samples was determined.     

Effective production of chitinase and chitosanase byStreptomyces griseus HUT 6037 using colloidal chitin and various degrees of deacetylation of chitosan

The advantages of the organismStreptomyces griseus HUT 6037 is that the chitinase and chitosanase using chitinaceouse substrate are capable of hydrolyzing both amorphous and crystalline chitin and chitosan. We attempted to investigate the optimization of induction protocol for high-level production and secretion of chitosanase and the influence of chitin and partially deacetylated chitosan sources (75–99% deactylation). The maximum specific activity of chitinase has been found at 5 days cultivation with the 48 hours induction time using colloidal chitin as a carbon source. To investigate characteristic of chitosan activity according to substrate, we used chitosan with various degree of deacetylation as a carbon source and found that this strain accumulates chitosanase in the culture medium using chitosanaceous substrates rather than chitinaceous substrates. The highest chitosanase activity was also presented on 4 days with 99% deacetylated chitosan. The partially 53% deacetylated chitosan can secrete both chitinase and chitosanase which was defined as a soluble chitosan. The specific activities of chitinase and chitosanase were 0.89 at 3 days and 1.33 U/mg protein at 5 days, respectively. It indicate that chitosanase obtained fromS. griseus HUT 6037 can hydrolyze GlcNAc-GlcN and GlcN-GlcN linkages by exo-splitting manner. This activity increased with increasing degree of deacetylation of chitosan. It is the first attempt to investigate the effects of chitosanase on various degrees of deacetylations of chitosan byS. griseus HUT 6037. The highest specific activity of chitosanase was obtained with 99% deacetylated chitosan.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy-D-glucose from beta-chitin

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine, GlcNAc) from chitin. Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin and beta-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc and N,N'-diacetylchitobiose ([GlcNAc](2)). The product ratio, GlcNAc:[GlcNAc](2), increased while the total yield decreased as the pH was raised from 3. All of the [GlcNAc](2) produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A. cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.     

Alternansucrase acceptor reactions with D-tagatose and L-glucose

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are D-tagatose and L-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide alpha-d-glucopyranosyl-(1-->1)-beta-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from L-glucose, but the trisaccharide alpha-D-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-(1-->4)-l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, alpha-D-glucopyranosyl-(1-->4)-L-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of alpha-D-glucopyranosyl-(1-->4)-L-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Depolymerization of beta-chitin to mono- and disaccharides by the serum fraction from the para rubber tree, Hevea brasiliensis

The serum fraction of latex from Hevea brasiliensis, the para rubber tree, is known to contain an endo-chitinolytic enzyme, hevamine. Herein the activity of the rubber serum towards beta-chitin is investigated. The serum contained 6 mg/mL of protein and a chitinolytic activity of 18 mU permg of protein. The optimum ratio of enzyme to chitin was 0.22 mU/mg, and the optimum substrate concentration was 60 mg/mL. The optimum pH range was pH2-4, and the optimum temperature was 45 degrees C. At these conditions both (GlcNAc)2 and GlcNAc were produced in a molar ratio of approximately 2:1. The hydrolysis of 300 mg of chitin with 64 mU of the rubber serum for 8 days under the optimum conditions gave 39 mg of GlcNAc and 108 mg of (GlcNAc)2 as determined by HPLC. Mixing the rubber serum preparation with an Aspergillus niger pectinase preparation containing beta-N-acetylhexosaminidase can be used to produce almost exclusively the GlcNAc monomer in about 50% yield.     

Synthesis of the 6-deoxytalose-containing tri- and hexasaccharides of the O-antigen polysaccharide from Mesorhizobium huakuii IFO15243T

The synthesis of a trisaccharide and a hexasaccharide, the monomer and dimer of the repeating unit of O-antigen polysaccharide from Mesorhizobium huakuii IFO15243, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation reactions starting from commercially available l-rhamnose. The target oligosaccharides in the form of their p-methoxyphenyl glycosides are suitable for further glycoconjugate formation via selective cleavage of this group.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.     

Aminolytic reaction catalyzed by D-stereospecific amidohydrolases from Streptomyces spp

From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe-d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe-d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30-60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity.