Characterization of a ß-N-acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34.

In applications of chitin, one of the most abundant resources on earth, human milk oligosaccharides with many health functions were synthesized by transglycosylation of ß-N-acetylhexosaminidase. Synthesis of new transfer products can be expected by other ß-N-acetylhexosaminidases in nature. A total of 38 microorganisms that secrete ß-N-acetylhexosaminidases with transglycosylation activity were isolated from a soil screen. Using N,N'-diacetylchitobiose as the substrate, the transfer ratio increased with a decrease in substrate degradation when it was less than 60%. Metarhizium sp. A34 ß-N-acetylhexosaminidase had high transglycosylation activity and showed a maximum production of the oligosaccharides against the substrate degradation where (GlcNAc)5 and (GlcNAc)4 were produced in addition to (GlcNAc)3 . The maximum curve was attributed to a sequential reaction of transglycosylation followed by hydrolysis where oligosaccharides are an intermediate product and are hydrolyzed in a second step. The purified ß-N-acetylhexosaminidase from Metarhizium sp. A34 had an optimal pH of 5 and was stable from pH 7 to 8. At pH 5, it had an optimal temperature of 40°C and was stable up to 30°C for 30 min. This enzyme had high thermostability up to 55°C when bound to the cell wall. The acceptor specificity for the transglycosylation reaction was enhanced for lower molecular weight sugar alcohols in the order of glycerin (C3), erythritol (C4), and xylitol (C5). The transfer product with glycerin was identified as 1-O-ß-d-N-acetylglucosaminyl glycerin, which may prove useful as a starting material for new glycolipids in food applications. PRACTICAL APPLICATION: Metarhizium sp. A34 ß-N-acetylhexosaminidase produced 1-O-ß-d-N-acetylglucosaminyl glycerin through the transglycosylation. Chitin oligosaccharides of the donor are obtained by hydrolysis of chitin. 1-O-ß-d-N-Acetylglucosaminyl glycerin may be useful to start material for the synthesis of new glycolipids. High thermostability of this enzyme is useful to prevention of contamination in the transglycosylation reaction.

Relationship between pellet formation by Aspergillus oryzae strain KB and the production of ß-fructofuranosidase with high transfructosylation activity.

Aspergillus oryzae KB produces two ß-fructofuranosidases (F1 and F2). F1 has high transfructosylation activity (Ut) to produce fructooligosaccharides. F2 has high hydrolysis activity (Uh), releasing glucose and fructose. It is desirable to selectively produce F1, which can be used for production of fructooligosaccharides. Here, the relationship between filamentous pellet size and selective production of F1 in liquid culture was investigated. Our finding revealed that: (i) The mean particle size of pellets (5.88 ± 1.36 mm) was larger, and the ratio of Ut to Uh was improved (Ut/Uh = 5.0) in 10% sucrose medium compared with 1% sucrose medium (pellet size = 2.60 ± 0.37 mm; Ut/Uh = 0.96). (ii) The final culture pH of the 1% sucrose medium was 8.7; on controlling the pH of 1% sucrose medium at 5.0, increased pellet size (9.69 ± 2.01 mm) and Ut/Uh (7.8) were observed. (iii) When 3% glycerin was used as carbon source, the pellet size decreased to 1.09 ± 0.33 mm and Ut/Uh was 0.57. (iv) In medium containing 1% sucrose, the pellet size was dependent on the number of spores used in the culture inoculum, but, in these experiments, Ut/Uh was almost constant (1.05 ± 0.08). Collectively, the data show that the value of Ut/Uh is proportional to the pellet size when liquid culture of A. oryzae strain KB is performed in some conditions (such as in the presence of high sucrose concentration, low pH, or added Tween surfactant), but in other conditions Ut/Uh is independent of pellet size.

Characteristics of an ß-N-Acetylhexosaminidase from Bacillus sp. CH11, Including its Transglycosylation Activity.

ß-N-Acetylhexosaminidase was identified from Bacillus sp. CH11 and found to have relatively high transferring activity. In this study, its enzymatic properties and transglycosylation activity including its acceptor specificity were investigated. Its molecular weight was estimated to be 90 kDa by SDS-PAGE and its optimal pH was approximately 7 with good stability from pH 6 to 8. Its optimal temperature was 40 °C, and its activity was stable at temperatures of up to 40 °C. To analyze its acceptor specificity for transglycosylation, N, N'-diacetylchitobiose was used as a donor substrate and alcohols, sugar alcohols, sugars and polyphenols were used as acceptors. Dialcohols, which have 2 hydroxyl groups on the outside of the carbon chains, were good acceptors. The molecular size of the acceptor did not influence the transglycosylation up to at least 1,5-pentanediol (carbon number: C5). Glycerin (C3), erythritol (C4), and xylitol (C5), all small molecular weight sugar alcohols, had high acceptor specificity. Transglycosylation to mono- and disaccharides and polyphenols was not observed except for L-fucose. For the ß-N-acetylhexosaminidase-catalyzed transglycosylation of chitin oligosaccharides and xylitol, the transfer product was identified as 1-O-ß-D-N-acetylglucosaminyl xylitol. The optimal ratio of xylitol was 24% to 2% N, N'-diacetylchitobiose and 226 mg per 1 g N, N'-diacetylchitobiose was produced. CH11 ß-N-acetylhexosaminidase efficiently produced 1-O-ß-D-N-acetylglucosaminyl xylitol via transglycosylation. PRACTICAL APPLICATION: The new transfer products including 1-O-ß-D-N-acetylglucosaminyl xylitol are attractive compounds for their potential physiological functions. 1-O-ß-D-N-Acetylglucosaminyl xylitol was produced effectively from chitin-oligosaccharides and xylitol by ß-N-acetylhexosaminidase from Bacillus sp. CH11. This enzyme may be useful for the development of food materials for health-related applications such as oligosaccharides with intestinal functions and noncariogenic sugars.

Enzymatic Properties of Alginate Lyase from Paenibacillus sp. S29.

Paenibacillus sp. S29 was isolated from soil and produces an alginate lyase. The molecular weight of this enzyme was 32 kDa and the N-terminal amino acid sequence was ASVTKST. The optimal pH was approximately 8.7 and the enzyme was stable over a pH range of 5.6 to 8.8 at 40 °C for 60 min. The optimal temperature was approximately 50 °C, and the residual activity was not decreased at temperatures of up to 40 °C at pH 8 for 30 min. Paenibacillus sp. S29 alginate lyase had also a little activity toward hyaluronic acid. Poly G and poly M separated from alginate were degraded efficiently, and poly M was the more susceptible substrate. The maximum amount of reducing sugar released by the enzyme was 261 mg per gram of sodium alginate. The main sugar released was monosaccharide (unsaturated uronate) and small amounts of oligosaccharides of degree of polymerization 2-6 were also released.

Effects of nonionic surfactants on pellet formation and the production of ß-fructofuranosidases from Aspergillus oryzae KB.

Aspergillus oryzae KB produces two ß-fructofuranosidases (F1 and F2). F1 has high transferring activity and produces fructooligosaccharides from sucrose. Mycelial growth pellets were altered by the addition of Tween 20, 40 and 80 (HLB=16.7, 15.6 and 15.0, respectively) in liquid medium cultures to form small spherical pellets. The particle size of the pellets decreased with the HLB value, which corresponds to an increase in surfactant hydrophobicity. Selective F1 production and pellet size were maximized using Tween 20. Adding polyoxyethylene oleyl ethers (POEs) with various degrees of polymerization (2, 7, 10, 20 and 50: HLB=7.7, 10.7, 14.7, 17.2 and 18.2, respectively) was investigated. A minimum mean particle size was obtained using a POE with DP=10, HLB=14.7. The POE surfactants had little effect on the selective production of F1. The formation of filamentous pellets depended on the surfactant HLB value, and F1 enzymes were produced most efficiently using Tween 20.

Synthesis of galactosyl glycerol from guar gum by transglycosylation of α-galactosidase from Aspergillus sp. MK14.

A guar gum-hydrolyzing strain, Aspergillus sp. MK14, secreted α-galactosidase selectively in liquid culture. Its α-galactosidase activity (0.820 U/ml) was much higher than its ß-mannosidase and ß-mannanase activities (0.027 and 0.050 U/ml, respectively). The molecular weight was estimated to be 59,000 Da by SDS-PAGE. The optimal pH was 5 and it was active from pH 2.2 to 6.2. The optimal temperature was 60 °C and the activity was stable below 50 °C. Enzyme activity toward melibiose was much lower than that with pNP-α-D-galactopyranoside. The activities toward 6(1)-α-D-galactosyl-mannobiose and 6(3),6(4)-α-D-galactosyl-mannopentaose were relatively high (86.2% and 48.4% relative to pNP-α-D-galactopyranoside, respectively). MK14 crude enzyme released only the monosaccharides, galactose and mannose (Gal/Man: 0.64) from guar gum. When glycerol was added to the reaction mixture, the transglycosylation proceeded efficiently, and the synthesis of galactosyl glycerol was 76.6 mg/g of guar gum. MK14 α-galactosidase could use guar gum as a good substrate (donor) in the transglycosylation.

Characteristics of α-L-arabinofuranosidase from Streptomyces sp I10-1 for production of L-arabinose from corn hull arabinoxylan.

Streptomyces sp I10-1 α-L-arabinofuranosidase efficiently produced L-arabinose from high arabinose-content corn hull arabinoxylan (ratio of arabinose to xylose, 0.6). The optimum pH at 40 °C was around 6, and the enzyme was stable from pH 5 to 11. The optimum temperature was 50 °C at pH 5, and the activity was stable at 40 °C. The enzymatic activity against corn hull arabinoxylan was 2.3 times higher than towards p-nitrophenyl-α-L-arabinofuranoside. Approximately 45% L-arabinose recovery was achieved from corn hull arabinoxylan. It was considered that L-arabinose residues not removed by the enzyme were attributable to those linked with ferulic acid. The open reading frame of the enzyme gene consisted of 1,224 bp, and the predicted peptide was 408 amino acids, which corresponded to a molecular size of 45, 248 Da. It was presumed that the smaller molecular size (31,000 Da) estimated on SDS-PAGE resulted from proteolysis by proteases. I10-1 α-L-arabinofuranosidase belongs to the Alpha-L-AF C superfamily, which is associated with glycoside hydrolase family 51, but the properties were unique.

Enzymatic properties of ß-1,3-glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo-ß-1,3-glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N-terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only ß-1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of ß-1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of ß-1,6-linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo-ß-1,3-glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the ß-1,3 linkage between the 3rd and 4th glucosyl residue.

Synthesis of sugar fatty acid esters using a ß-xylosidase from Aspergillus awamori for transxylosylation.

Aspergillus awamori K4 ß-xylosidase has broad acceptor specificity. It has been used to synthesize a sugar fatty acid ester via its transxylosylation activity. One xylosyl residue was initially transferred to hexamethylene glycol as a linker with a yield of 0.36 g/g xylobiose. Linoleic acid was subsequently linked to one terminal hydroxyl side of the transfer product hydroxyhexyl xyloside through an esterification reaction catalyzed by a lipase. The synthesis of hexyl linoleoyl xyloside was confirmed by TOF-MS analysis. The binding with a linker improved the esterification reaction because of the hydrophobic hexamethylene chain and also prevented steric hindrance by the xylosyl residue. This sugar fatty acid ester synthesis method using transglycosylation should facilitate the production of emulsifiers or surfactants with various functions.

Production of L-arabinose from corn hull arabinoxylan by Arthrobacter aurescens MK5 α-L-arabinofuranosidase.

Arabinoxylans, which are comprised of a xylan backbone to which are attached glycosyl units that are primarily L-arabinofuranosyl units, are ubiquitous among plant species where it is a constituent of the cell wall. Arabinoxylan has attracted much attention as a potential biomass resource and L-arabinose has recently been reported to possess functional properties that are effective in the treatment of diabetes. Here, we report an α-L-arabinofuranohydrolase, isolated from the soil microbe Arthrobacter aurescens strain MK5, effective in releasing L-arabinose from corn hull arabinoxylan. When A. aurescens strain MK5 was grown in a liquid medium, corn hull arabinoxylan, which has a higher arabinose content (Ara/Xyl = 0.6) than oat spelts xylan (Ara/Xyl = 0.12), induced more efficient arabinoxylan hydrolase production. Analysis of enzyme activity in the culture broth revealed that arabinoxylan hydrolase activity was high, and α-L-arabinofuranosidase and ß-xylosidase activities were low. The optimum pH of the MK5 arabinoxylan hydrolase at 40 °C was around 7 and enzyme activity was relatively stable at an alkaline pH up to 9.5. The optimum temperature at pH 7 was around 50 °C and enzyme activity was stable under 50 °C. During the hydrolysis of corn hull arabinoxylan, only L-arabinose was released and 45.1% maximum sugar recovery was achieved. The A. aurescens MK5 enzyme was a typical arabinoxylan α-L-arabinofuranohydrolase and was most effective at releasing L-arabinose from corn hull arabinoxylan, which has a high arabinose content. This enzyme may have important industrial applications.

Production of fructooligosaccharides by beta-fructofuranosidases from Aspergillus oryzae KB.

Aspergillus oryzae KB produces two types of beta-fructofuranosidases: F1 and F2. F1 produces the fructooligosaccharides (FOSs) 1-kestose, nystose, and fructosyl nystose from sucrose through a transfructosylation action, whereas F2 mainly hydrolyzes sucrose to glucose and fructose. F1 and F2 enzymes were more selectively produced from the KB strain in liquid media with a sucrose concentration>2% and <2%, respectively. Immobilization using an anion-exchange resin (WA-30; polystyrene with tertiary amine) and cross-linking with glutaraldehyde depressed the hydrolysis reaction of F2 (high hydrolyzing enzyme) alone and enhanced the thermal stability of F1 (high transferring enzyme). F1 enzyme produced in the high sucrose medium was immobilized, cross-linked, and packed in a tubular reactor for continuous production of FOSs (24.6% 1-kestose, 21.6% nystose, 5.7% and fructosyl nystose). In a long-term operation in which 60% sucrose was imputed at 55 degrees C, the composition of FOSs produced was 51.9% (transfer ratio: 92%), and production by the immobilized enzyme was maintained for 984 h.

Two types of beta-fructofuranosidases from Aspergillus oryzae KB.

Aspergillus oryzae KB produces two types of beta-fructofuranosidases, F1 and F2. F1 produces 1-kestose, nystose, and fructosyl nystose from sucrose through its transfructosylation action. F2 hydrolyzes sucrose to glucose and fructose. N-Terminal amino acid sequences of the purified enzymes were DYNAAPPNLST for F1 and YSGDLRPQ for F2. Each enzyme encoding gene was identified in the genome of Aspergillus oryzae. Although the KB strain showed a higher production of F2 than F1 in a low sucrose liquid medium, F2 production gradually decreased, whereas F1 production increased with increasing sucrose concentration in the medium. Synthesis of F1 and F2 mRNAs analyzed on reverse-transcription polymerase chain reaction corresponded to individual enzymatic production. During liquid culture of the KB strain, F1 synthesizes fructooligosaccharides from sucrose through transfructosylation, and F2 gradually hydrolyzes it. In a highly concentrated sucrose medium, intake of sucrose into the KB strain was depressed by F1 through synthesis of transfer products, fructooligosaccharides.

Biological pretreatment with two bacterial strains for enzymatic hydrolysis of office paper.

The cellulose-hydrolyzing strains, Sphingomonas paucimobilis MK1 and Bacillus circulans MK2, were separated from soil and were grown together in a single culture plate. Growth B. circulans MK2 in liquid culture required symbiosis with S. paucimobilis MK1. Biological pretreatment with the combined strain suspension after the liquid culture improved enzymatic hydrolysis of office paper from municipal wastes. Sugar recovery by S. paucimobilis MK1 (51%) was 1.4 times higher than that of the untreated sample (30%) and in the strain combination with B. circulans MK2, recovery was further improved by 2.5 times (75%). The sugar recovery in maximum condition was enhanced up to 94% for office paper. Furthermore, biological pretreatment effects were confirmed for more than 1 day less time. In X-ray diffraction patterns for the crystallinity of cellulose in office paper changed after biological pretreatment, the crystallinity was increased in comparison to that in untreated paper. The mechanism of biological pretreatment effect was explained by the fact that the strain acted as an endoglucanase, which hydrolyzes amorphous areas randomly.

Production of galacto-manno-oligosaccharides from guar gum by beta-mannanase from Penicillium oxalicum SO.

Beta-mannanase from Penicillium oxalicum SO efficiently hydrolyzed guar galactomannan to galacto-manno-oligosaccharides. Gel filtration estimated the molecular weight of the beta-mannanase as 35 000 and SDS-PAGE as 29 000. The optimum pH was around 5 while a stable pH was reached in the range of 3-6. Optimum temperature was around 60 degrees C at pH 5, while under 60 degrees C activity was stable. HPLC analysis detected oligosaccharides with degrees of polymerization (DP) of 2 to 7 and 2 to 6 released on hydrolysis of guar and locust bean gums, respectively; about 92% of the released sugars were oligosaccharides. In analysis of the sugar distribution on MALDI-TOF-MS, major products of DP 6 and 7 and DP 5 and 6 were confirmed in hydrolysates of guar gum and locust bean gum, respectively. One of the main oligosaccharides released from guar gum, with DP 7, had a high galactose content (Gal/Man = 0.76) and corresponded to a blockwise galactose-substituted mannan type in galactomannan.

Characteristics of transxylosylation by beta-xylosidase from Aspergillus awamori K4.

The Aspergillus awamori K4 beta-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger beta-xylosidase gene with only five amino acid differences. K4 beta-xylosidase had the same catalytic mechanism as family 3 beta-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 beta-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-beta-D-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-beta-D-xylopyranoside, 6S-O-beta-D-xylosyl maltitol (S: sorbitol residue) and 6G-O-beta-D-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl-enzyme intermediate.

Synthesis of new glycosides by transglycosylation of N-acetylhexosaminidase from Serratia marcescens YS-1.

Serratia marcescens YS-1, a chitin-degrading microorganism, produced mainly N-acetylhexosaminidase. The purified enzyme had an optimal pH of approximately 8-9 and remained stable at 40 degrees C for 60 min at pH 6-8. The optimum temperature was around 50 degrees C, and enzyme activity was relatively stable below 50 degrees C. YS-1 N-acetylhexosaminidase hydrolyzed p-nitrophenyl beta-N-acetylgalactosamide by 28.1% relative to p-nitrophenyl beta-N-acetylglucosamide. The N-acetylchitooligosaccharides were hydrolyzed more rapidly, but the cellobiose and chitobiose of disaccharides that had the same beta-1,4 glycosidic bond as di-N-acetylchitobiose were not hydrolyzed. YS-1 N-acetylhexosaminidase efficiently transferred the N-acetylglucosamine residue from di-N-acetylchitobiose (substrate) to alcohols (acceptor). The ratio of transfer to methanol increased to 86% in a reaction with 32% methanol. N-Acetylglucosamine was transferred to the hydroxyl group at C1 of monoalcohols. A dialcohol was used as an acceptor when the carbon number was more than 4 and a hydroxyl group existed on each of the two outside carbons. Sugar alcohols with hydroxyl groups in all carbon positions were not proper acceptors.