Enzymatic Synthesis of 1,5-Anhydro-4-O-β-D-glucopyranosyl-D-fructose Using Cellobiose Phosphorylase and Its Spontaneous Decomposition via β-Elimination

Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-β-D-glucopyranosyl-D-fructose [βGlc(1→4)AF] from 1,5-anhydro-D-fructose and α-D-glucose 1-phosphate. βGlc(1→4)AF decomposed into D-glucose and ascopyrone T via β-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of βGlc(1→4)AF was 17 h at 30 °C and pH 7.0. Because βGlc(1→4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.     

Enzymatic Synthesis and Structural Confirmation of Novel Oligosaccharide,D-Fructofuranose-linked Chitin Oligosaccharide

Utilizing transglycosylation reaction catalyzed by β- N -acetylhexosaminidase of Stenotrophomonas maltophilia , β-D-fructofuranosyl-(2↔1)-α- N , N ´diacetylchitobioside (GlcNAc ₂ -Fru) was synthesized from N -acetylsucrosamine and N , N ´-diacetylchitobiose (GlcNAc ₂ ), and β-D-fructofuranosyl-(2↔1)-α- N , N ´, N ´´-triacetylchitotrioside (GlcNAc ₃ -Fru) was synthesized from GlcNAc ₂ -Fru and GlcNAc ₂ . Through purification by charcoal column chromatography, pure GlcNAc ₂ -Fru and GlcNAc ₃ -Fru were obtained in molar yields of 33.0 % and 11.7 % from GlcNAc ₂ , respectively. The structures of these oligosaccharides were confirmed by comparing instrumental analysis data of fragments obtained by enzymatic hydrolysis and acid hydrolysis of them with known data of these fragments.     

Synthesis of 3-Keto-levoglucosan Using Pyranose Oxidase and Its Spontaneous Decomposition via β-Elimination

3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction. This study investigated the ability of pyranose oxidase to convert LG into 3ketoLG by oxidizing the C3 hydroxyl group. During the oxidation of LG, 3ketoLG was spontaneously crystallized in the reaction mixture. Starting with 500 mM LG, the isolation yield of 3ketoLG was 80 %. Nuclear magnetic resonance analyses revealed that a part of 3ketoLG dimerized in aqueous solution, explaining its poor solubility. Even under normal conditions, 3ketoLG was unstable in aqueous solution, with a half-life of 16 h at pH 7.0 and 30 °C. The decomposition proceeded through β-elimination of the C–O bonds at both C1 and C5, as evidenced by decomposition products. This instability explains the difficulty in obtaining 3ketoLG via the LGDH reaction.     

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity

α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and Agl1 was inhibited in the presence of 1 % glucose, while Foagl1 was more tolerant to glucose than the other two enzymes were. The three fungal isomaltases did not show transglycosylation when isomaltose was used as the substrate and a similar result was observed for AgdC and Agl1 when p-nitrophenyl-α-glucoside was used as the substrate.     

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.     

Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser–His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X₂ and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser–His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser–His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.     

Epimerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2-O-α-D-glucopyranosyl-D-mannose and 2-O-β-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds. Following these experiments, we propose a kinetic model for the epimerization and decomposition of kojibiose and sophorose by heat treatment under neutral pH and alkaline conditions. The proposed model shows a good fit with the experimental data collected in this study. The rate constants of a reversible epimerization of kojibiose at pH 7.5 and 90 °C were (1.6 ± 0.1) × 10⁻⁵ s⁻¹ and (3.2 ± 0.2) × 10⁻⁵ s⁻¹ for the forward and reverse reactions, respectively, and were almost identical to those [(1.5 ± 0.1) × 10⁻⁵ s⁻¹ and (3.5 ± 0.4) × 10⁻⁵ s⁻¹] of sophorose. The rate constant of the decomposition reaction for kojibiose was (4.7 ± 1.1) × 10⁻⁷ s⁻¹ whereas that for sophorose [(3.7 ± 0.2) × 10⁻⁶ s⁻¹] was about ten times higher. The epimerization reaction was not significantly affected by the variation in the buffer except for a borate buffer, and depended instead upon the pH value (concentration of hydroxide ions), indicating that epimerization occurred as a function of the hydroxide ion. These instabilities are an extension of the neutral pH conditions for keto-enol tautomerization that are often observed under strong alkaline conditions.     

Innovative Preparation of Biopharmaceuticals Using Transglycosylation Activity of Microbial Endoglycosidases

Most functional biopharmaceuticals such as antibodies are glycoproteins carrying N-linked oligosaccharides (N-glycans). In animal cells, these glycans are generally expressed as heterogeneous glycoforms that are difficult to separate into a pure form. The structure of these glycans directly affects several biological aspects of the glycoproteins, especially binding affinity. Therefore, the preparation of glycoproteins with well-defined and homogeneous glycoforms is necessary for functional studies and improved efficacy, particularly for biopharmaceuticals. This review describes the recent remarkable progress in the development and production of biopharmaceutical glycan-modified antibodies, through the use of glycan remodeling using microbial endoglycosidases and sophisticated glycoengineering techniques utilizing microbial enzymatic reaction mechanisms.     

Preparation of a Molecular Library of Branched β-Glucan Oligosaccharides Derived from Laminarin

To study the structure of β-glucans, we developed a separation method and molecular library of β-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a β-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the β-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer β-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the β-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of β-glucans.     

Evaluation of Ammonia Pretreatment for Enzymatic Hydrolysis of Sugarcane Bagasse to Recover Xylooligosaccharides

Sugarcane bagasse is a useful biomass resource. In the present study, we examined the efficacy of ammonia pretreatment for selective release of hemicellulose from bagasse. Pretreatment of bagasse with aqueous ammonia resulted in significant loss of xylan. In contrast, pretreatment of bagasse with anhydrous ammonia resulted in almost no xylan loss. Aqueous ammonia or anhydrous ammonia-pretreated bagasse was then subjected to enzymatic digestion with a xylanase from the glycoside hydrolase (GH) family 10 or a xylanase from the GH family 11. The hydrolysis rate of xylan in bagasse pretreated with aqueous ammonia was approximately 50 %. In contrast, in the anhydrous ammonia-treated bagasse, xylan hydrolysis was > 80 %. These results suggested that anhydrous ammonia pretreatment would be an effective method for preparation of sugarcane bagasse for enzymatic hydrolysis to recover xylooligosaccharides.     

Purification,Characterization, and cDNA Cloning of a Prominent β-Glucosidase from the Gut of the Xylophagous Cockroach Panesthia angustipennis spadica

Abstract: In this study, a β-glucosidase (PaBG1b) with high specific activity was purified from gut extracts of the wood-feeding cockroach Panesthia angustipennis spadica using Superdex 75 gel filtration chromatography and High-Trap phenyl hydrophobic chromatography. The protein was purified 14-fold to a single band identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular mass of 56.7 kDa. The specific activity of the purified enzyme was 708 μmol/min/mg protein using cellobiose as substrate. To the best of our knowledge, this is the highest specific activity reported among β-glucosidases to date. The purified PaBG1b showed optimal activity at pH 5.0 and retained more than 65 % of the activity between pH 4.0 and 6.5. The activity was stable up to 50 °C for 30 min. Kinetic studies on cellobiose revealed that the K_m was 5.3 mM, and the V_max was 1,020 μmol/min/mg. The internal amino acid sequence of PaBG1b was analyzed, and two continuous sequences (a total of 39 amino acids) of the C-terminal region were elucidated. Based on these amino acid sequences, a full-length cDNA (1,552 bp) encoding 502 amino acids was isolated. The encoded protein showed high similarity to β-glucosidases from glycoside hydrolase family 1. Thus, the current study demonstrated the potential of PaBG1b for application in enzymatic biomass-conversion as a donor gene for heterologous recombination of cellulase-producing agents (fungi or bacteria) or an additive enzyme for cellulase products based on the high-performance of PaBG1b as a digestive enzyme in cockroaches.     

Control of pH by CO 2 Pressurization for Enzymatic Saccharification of Ca(OH) 2 -Pretreated Rice Straw in the Presence of CaCO 3

The aim of this study was to investigate the effect of pH control by CO ₂ pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) ₂ -pretreated/CO ₂ -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO ₂ partial pressure ( p CO ₂ ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger β-glucosidase, indicated that pH 5.5–6.0 is optimal for solubilizing sugars from Ca(OH) ₂ -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO ₂ conditions revealed that the highest soluble sugar yields were obtained at p CO ₂ 0.4 MPa and over, which is consistent with the expected pH at the p CO ₂ without enzymes and demonstrates the effectiveness of pH control by CO ₂ pressurization.     

Characterization of a GH36 β-L-Arabinopyranosidase in Bifidobacterium adolescentis

β-L-Arabinopyranosidases are classified into the glycoside hydrolase family 27 (GH27) and GH97, but not into GH36. In this study, we first characterized the GH36 β-L-arabinopyranosidase BAD_1528 from Bifidobacterium adolescentis JCM1275. The recombinant BAD_1528 expressed in Escherichia coli had a hydrolytic activity toward p-nitrophenyl (pNP)-β-L-arabinopyranoside (Arap) and a weak activity toward pNP-α-D-galactopyranoside (Gal). The enzyme liberated L-arabinose efficiently not from any oligosaccharides or polysaccharides containing Arap-β1,3-linkages, but from the disaccharide Arap-β1,3-L-arabinose. However, we were unable to confirm the in vitro fermentability of Arap-β1,3-Ara in B. adolescentis strains. The enzyme also had a transglycosylation activity toward 1-alkanols and saccharides as acceptors.     

In Vivo Digestibility of Carbohydrate Rich in Isomaltomegalosaccharide Produced from Starch by Dextrin Dextranase

Slowly digestible carbohydrates are needed for nutritional support in diabetic patients with malnutrition. They are a good source of energy and have the advantage that their consumption produces a low postprandial peak in blood glucose levels because they are slowly and completely digested in the small intestine. A high-amount isomaltomegalosaccharide containing carbohydrate (H-IMS), made from starch by dextrin dextranase, is a mixture of glucose polymers which has a continuous linear structure of α-1,6-glucosidic bonds and a small number of α-1,4-glucosidic bonds at the reducing ends. It has a broad degree of polymerization (DP) distribution with glucans of DP 10－30 as the major component. In our previous study, H-IMS has been shown to exhibit slow digestibility in vitro and not to raise postprandial blood glucose to such levels as that raised by dextrin in vivo. This marks it out as a potentially useful slowly digestible carbohydrate, and this study aimed to evaluate its in vivo digestibility. The amount of breath hydrogen emitted following oral administration of H-IMS was measured to determine whether any indigestible fraction passed through to and was fermented in the large intestine. Total carbohydrate in the feces was also measured. H-IMS, like glucose and dextrin, did not result in breath hydrogen excretion. Carbohydrate excretion with dietary H-IMS was no different from that of glucose or water. These results show that the H-IMS is completely digested and absorbed in the small intestine, indicating its potential as a slowly digestible carbohydrate in the diet of diabetic patients.     

4-O-Methyl Modifications of Glucuronic Acids in Xylans Are Indispensable for Substrate Discrimination by GH67 α-Glucuronidase from Bacillus halodurans C-125

A GH67 α-glucuronidase gene derived from Bacillus halodurans C-125 was expressed in E. coli to obtain a recombinant enzyme (BhGlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. BhGlcA67 showed maximum activity at pH 5.4 and 45 °C. When BhGlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4-O-methyl-glucuronic acid from these substrates. BhGlcA67 acted only on 4-O-methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcA³Xyl₃), which has a glucuronic acid side chain with a 4-O-methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcA³Xyl₄) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcA³Xyl₃). The environment for recognizing the 4-O-methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4-O-methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4-O-methyl group is critical for the activities of the GH67 family.     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

Characterization of Cell Wall α-1,3-Glucan–Deficient Mutants in Aspergillus oryzae Isolated by a Screening Method Based on Their Sensitivities to Congo Red or Lysing Enzymes

We previously reported that sensitivity to Congo Red (CR) or Lysing Enzymes (LE) is affected by the loss of cell-wall α-1,3-glucan (AG) in Aspergillus nidulans. We found that the amount of CR adsorbed to AG was significantly less than the amount adsorbed to β-1,3-glucan (BG) or chitin, suggesting that loss of cell-wall AG would increase exposure of BG on the cell surface, and thereby increase the sensitivity to CR. Generally, fungal BGs are known as biological response modifiers because of their recognition by Dectin-1 receptors in human immune systems. Therefore, isolation of AG-deficient mutants in Aspergillus oryzae has been used in the Japanese fermentation industry to create strains with increased ability to promote immune responses. Here, we aimed to isolate AG-deficient strains by mutagenizing A. oryzae conidia with chemical mutagens. Based on the increased sensitivity to CR in AG-deficient strains of A. nidulans and A. oryzae, we established a screening method for isolation of AG-deficient strains. Several candidate AG-deficient mutants of A. oryzae were isolated using the screening method; these strains showed increased sensitivity to CR and/or LE. Cytokine production was increased in the dendritic cells co-incubated with germinated conidia of the AG-deficient mutants. Furthermore, according to a Dectin-1 NFAT (nuclear factor of activator T cells)-GFP (green fluorescent protein) reporter assay, Dectin-1 response levels in the AG-deficient mutants were higher than those in wild-type A. oryzae. These results suggest that we successfully isolated AG-deficient mutants of A. oryzae with immunostimulatory effects.     

Functional Characterization of the GH10 and GH11 Xylanases from Streptomyces olivaceoviridis E-86 Provide Insights into the Advantage of GH11 Xylanase in Catalyzing Biomass Degradation

We functionally characterized the GH10 xylanase (SoXyn10A) and the GH11 xylanase (SoXyn11B) derived from the actinomycete Streptomyces olivaceoviridis E-86. Each enzyme exhibited differences in the produced reducing power upon degradation of xylan substrates. SoXyn10A produced higher reducing power than SoXyn11B. Gel filtration of the hydrolysates generated by both enzymes revealed that the original substrate was completely decomposed. Enzyme mixtures of SoXyn10A and SoXyn11B produced the same level of reducing power as SoXyn10A alone. These observations were in good agreement with the composition of the hydrolysis products. The hydrolysis products derived from the incubation of soluble birchwood xylan with a mixture of SoXyn10A and SoXyn11B produced the same products as SoXyn10A alone with similar compositions. Furthermore, the addition of SoXyn10A following SoXyn11B-mediated digestion of xylan produced the same products as SoXyn10A alone with similar compositions. Thus, it was hypothesized that SoXyn10A could degrade xylans to a smaller size than SoXyn11B. In contrast to the soluble xylans as the substrate, the produced reducing power generated by both enzymes was not significantly different when pretreated milled bagasses were used as substrates. Quantification of the pentose content in the milled bagasse residues after the enzyme digestions revealed that SoXyn11B hydrolyzed xylans in pretreated milled bagasses much more efficiently than SoXyn10A. These data suggested that the GH10 xylanases can degrade soluble xylans smaller than the GH11 xylanases. However, the GH11 xylanases may be more efficient at catalyzing xylan degradation in natural environments (e.g. biomass) where xylans interact with celluloses and lignins.     

Cross-linking of β-casein by Trichoderma reesei tyrosinase and Streptoverticillium mobaraense transglutaminase followed by SEC–MALLS

Enzymatic modification of proteins, in order to produce functional materials such as hydrogels, adhesives and films via cross-linked networks or scaffolds of proteins, is a constantly evolving technology to create tailored micro- and nanostructured materials for food, cosmetic, and medical applications. For the successful utilization of oxidoreductases or transferases such as tyrosinases and transglutaminases, respectively, it is crucial to understand the action of these enzymes on protein substrates. In this study, cross-linking of the milk protein β-casein by Trichoderma reesei tyrosinase (TrTyr) was studied using size-exclusion chromatography (SEC) equipped with multi-angle light scattering (MALLS) and ultraviolet/visible (UV/Vis) detectors in order to determine the molecular mass (MM), radius of gyration (R_G) and degree of polymerization (DP) of the reaction products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect early polymerization states. The widely used Streptoverticillium mobaraense transglutaminase (Tgase) was used for comparison to tyrosinase from T. reesei. The results showed that cross-linking of β-casein by these two different types of enzymes resulted in the formation of polymerized reaction products with MM ranging from 500 to 1700 kg mol⁻¹ depending on the enzyme dosage and incubation time. The DP varied from 21 to 71, respectively. In the case of TrTyr the polymerized reaction products were slightly colored, and formation of the covalent cross-linking of β-casein could be monitored by UV/Vis as a function of incubation time.