Shojiro Iwahara

Microbial degradation of dehydrodiconiferyl alcohol, a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran-γ′-aldehydic (II) and γ′-carboxylic compounds, 2) phenylcoumaran-α′-aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran-γ′-carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the α′-aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the γ′-ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.

TRANSFER OF MAN9GLCNAC TO L-FUCOSE BY ENDO-BETA -N-ACETYLGLUCOSAMINIDASE FROM ARTHROBACTER PROTOPHORMIAE

We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man9GlcNAc, tol-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man9GlcNAc-l-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man9GlcNAc-l-Fuc; and ii) preparation of Man9GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man9GlcNAcβ(1,2)-l-Fuc. Methyl β-l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl α-l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.

Degradation of d,l-syringaresinol, a ?-?? linked lignin model compound, by Fusarium solani M-13-1

A β-β′ linked lignin model compound, d,l-syringaresinol monobenzyl ether (Ib) was incubated with Fusarium solani M-13-1 in a shaking culture. From the culture filtrates, three compounds II, IIIb and IV were isolated and identified. Substrate Ib was oxidized at the α-position of the side chain to give a hemiketal, an α-hydroxylated compound IIA, which was then transformed to the ketoalcohol, 3-hydroxymethyl-2-(4-benzyloxy-3,5-dimethoxyphenyl)-4-(4-hydroxy-3,5-dimethoxybenzoyl)-tetrahydrofuran (IIB). These products were converted to a γ-lactone derivative, 6-oxo-2-(4-benzyloxy-3,5-dimethoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane (IIIb), via alkyl-aryl cleavage. The syringyl moiety released from II by the cleavage reaction was identified as 2,6-dimethoxy-p-benzoquinone (IV). Incubation of 2,6-dimethoxyphenol (V) in fungal culures did not give the p-quinone IV. d,l-Syringaresinol dimethyl ether was not degraded and the etherated moiety of Ib was not attacked by the fungus, indicating that the degradation of d,l-syringaresinol was catalyzed by phenol oxidizing enzymes. The oxidation products of Ib with peroxidase/H2O2 was investigated and discussed in relation to the degradation products of the fungus.

Purification and characterization of exo-α-d-mannosidase from a Cellulomonas sp.

Abstract A Gram-positive bacterium isolated from a soil sample was found to produce a high level of α-mannosidase in culture media. The organism was identified as Cellulomonas sp. from various bacteriological characteristics. The production of the enzyme was strongly induced by yeast extract. To purify the enzyme, the bacterium was first grown in a glucose medium, and then the cells were transferred to an inducing medium containing only yeast extract. The enzyme had two different molecular weight forms. The high-molecular-weight form was purified from the inducing medium by column chromatography. The enzyme was purified to homogeneity by ultracentrifugal analysis. The enzyme was strongly inactivated by Hg2+, Cu2+, Zn2+, EDTA andp-chloromercuribenzoic acid. The molecular weight of the enzyme was estimated to be about 450 000 by gel filtration. The purified enzyme could liberate only mannose from native yeast mannan and the optimum pH was 6.5–8.0. The enzyme rapidly cleaved α-1,2- and α-1,6-linked mannose chains, but the hydrolysis rate for α-1,3 linkage was very low. In addition, the purified enzyme showed only slight activity towardsp-nitrophenyl-α- d -mannoside and did not hydrolyzeO-α- d -mannopyranosyl(1 → 2)- d -mannitol.

Purification and properties of chondroitinase produced by a bacterium isolated from soil

Abstract A Gram-positive bacterium isolated from a soil sample capable of growing on chondroitin sulfate as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as either an Aureobacterium or a Curtobacterium. When grown on chondroitin sulfate, the bacterium secreted a chondroitinase (EC 4.2.2.4 or EC 4.2.2.5) into the culture medium. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 81,000. It was inhibited by Hg2+, Zn2+, Fe2+ and Cu2+ ions. The enzyme hydrolyzed chondroitin sulfate-A, -C, chondroitin and hyaluronic acid, although the hydrolysis rate for hyaluronic acid was low. On the other hand, the enzyme did not act on iduronic acid-containing mucopolysaccharides such as dermatan sulfate, heparin and heparan sulfate. A decrease in viscosity and gel filtration analyses showed that the purified enzyme degrades the substrate endolytically at the initial reaction.

Activation of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae by various sugars

Abstract Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was activated by the addition of glucose, mannose, N-acetylglucosamine, and β-allose. While the enzyme did not appear to be significantly affected by the addition of galactose or N-acetylgalactosamine. These results indicate that the C-4 and C-6 positions of the monosaccharide are the most important for enzyme activation. Moreover, the enzyme was activated by the addition of disaccharides such as cellobiose, gentiobiose, and di-N-acetylchitobiose, but not by polysaccharides such as starch and yeast mannan. In the presence of N-acetylglucosamine, the enzyme activation occurred well over pH 4.0 and the Km value of the enzyme for (Man)6(GlcNAc)2-Asn-dansyl changes from 1.2 mM to 3.2 mM.

The Active Oxygen Response of Raspberry Protoplasts to O-glycans of Fusarium sp. M7-1

Summary O -glycans isolated from glucuronic acid-containing glycoproteins of Fusarium sp. M7-1 had been used as a signaling molecule. The role of the oxidative burst, fast and transient production of activated oxygen species, was investigated in Rubus protoplasts. Two assays, based on Cyt c reduction and on pyranine quenching, were used to scrutinize the generation of O 2 .− and of H 2 O 2 , respectively, and agents known to scavenge active oxygen species or to alter the lipoxygenase pathway. The results suggested that the burst did not involve peroxidase but rather a trans-plasma membrane superoxide synthase formed O 2 .− dismurating in H 2 O 2 .

Production and characterization of extracellular uronic acid-containing glycoproteins from Fusarium oxysporum

Fusarium and Giberella species were found to secrete acidic polysaccharides when grown in Czapex-Dox medium. Fusarium oxysporum showed the highest rate of secretion of acidic sugars into the growth medium. The acidic sugar-containing glycoprotein from F. oxysporum was purified from the culture filtrate to apparent homogeneity on ultracentrifugation analysis, by ammonium sulfate precipitation, Toyopearl HW-55 gel filtration and charcoal chromatography. The purified acidic sugar-containing glycoprotein has a 10% protein content and a content of 40% acidic sugars as glucuronic acid, and the neutral sugars present in this glycoprotein are mainly mannose, galactose, and glucose. The protein moiety of the glycoprotein contains a high proportion of serine and threonine residues. Treatment of the glycoprotein with alkaline solution resulted in an increase in absorbance at 241 nm, and alkaline borohydride treatment resulted in a marked decrease in the number of serine and threonine residues. We conclude that the glycoprotein has O-linked sugar chains which are mainly attached to serine and threonine residues of the protein moiety. The primary structure of the acidic polysaccharide from F. oxysporum was analyzed mainly by NMR spectrometry. The main parts of this polysaccharide have structures similar to those of uronic acid-containing polysaccharide from Fusarium sp. M7-1.

Preparation of β(1→6)-linked galactofuranoside oligomers from the acidic polysaccharide of Fusarium sp. M7-1

Abstract This paper describes a method of preparating β(1→6)-linked galactofuranoside oligomers. The main structure of the acidic polysaccharide of Fusarium sp. M7-1 has a linear chain composed of β(1→6)-linked galactofuranose residues. For preparation of the β(1→6) galactofuranoside oligomers, glucuronic acid residues were converted to unsaturated sugars using an acidic polysaccharide lyase from Cellulomonas sp. Mild acid hydrolysis was found to be very effective in preparing the β(1→6) galactofuranoside oligomers from the unsaturated polysaccharide of Fusarium sp. M7-1. After treatment with 1 M acetic acid at 100°C for 5 h, the unsaturated polysaccharide was separated into oligomers, and the primary structures of the β(1→6) galactofuranoside oligomers were resolved, mainly by 400-MHz 1 H-NMR spectrometry. These oligomers were resistant to commercial β- d -galactosidases.

Degradation of unsaturated polysaccharide derived from acidic polysaccharide of Fusarium sp. M7-1 by a bacterium isolated from soil

Abstract A soil bacterium, strain no. 19, capable of using unsaturated polysaccharide derived from acidic polysaccharide of Fusarium sp. M7-1 as a sole source of carbon was isolated. The bacterium degraded about 70% of the total sugar content. Results from analysis of the degraded polysaccharide showed that the bacterium degraded the β(1→6) galactofuranoside linkage as well as the unsaturated glucuronic acid residues linked to the galactofuranoside residues via the α(1→2) linkage.