Hitomi Ichinose

Effects of Cellulose Crystallinity, Hemicellulose, and Lignin on the Enzymatic Hydrolysis of Miscanthus sinensis to Monosaccharides

The effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides were investigated. A air-dried biomass was ground by ball-milling, and the powder was separated into four fractions by passage through a series of sieves with mesh sizes 250–355 μm, 150–250 μm, 63–150 μm, and <63 μm. Each fraction was hydrolyzed with commercially available cellulase and β-glucosidase. The yield of monosaccharides increased as the crystallinity of the substrate decreased. The addition of xylanase increased the yield of both pentoses and glucose. Delignification by the sodium chlorite method improved the initial rate of hydrolysis by cellulolytic enzymes significantly, resulting in a higher yield of monosaccharides as compared with that for untreated samples. When delignified M. sinensis was hydrolyzed with cellulase, β-glucosidase, and xylanase, hemicellulose was hydrolyzed completely into monosaccharides, and the conversion rate of glucan to glucose was 90.6%.

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-D-galactan backbone with β-(1→6)-D-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-L-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-L-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

A β-l-Arabinopyranosidase from Streptomyces avermitilis Is a Novel Member of Glycoside Hydrolase Family 27

Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a β-l-arabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) α-d-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-β-l-arabinopyranoside was 18 μmol of arabinose/min/mg, which was 67 times higher than that toward p- nitrophenyl-α-d-galactopyranoside. The enzyme could remove 0.1 and 45% l-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel β-domain containing Greek key motifs, another antiparallel β-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of α-d-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of β-l-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of d-galactose bound to α-galactosidase was changed in β-l-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which β-l-arabinopyranosidase is classified as a new member of the GH27 family.

Characterization of an exo-β-1,3-galactanase from Clostridium thermocellum

ABSTRACT A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including β-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β-1,3-galactobiose, β-1,4-galactosyl glucose, or β-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β-1,3-galactan and α-l-arabinofuranosidase-treated AGP.

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.

Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes.

The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.

Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes

Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15% (w/v) D-glucose, D-fructose, D-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83% observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15% (w/v) SCBC with commercial cellulase was approximately 20%. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15% SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6%. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-L-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-L-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.

Characterization of an Exo-β-1,3-D-galactanase from Streptomyces avermitilis NBRC14893 Acting on Arabinogalactan-Proteins

A gene belonging to glycoside hydrolase family 43 (GH43) was isolated from Streptomyces avermitilis NBRC14893. The gene encodes a modular protein consisting of N-terminal GH43 module and a family 13 carbohydrate-binding module at the C-terminus. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme specifically hydrolyzed only β-1,3-linkage of two D-galactosyl residues at non-reducing ends of the substrates. The analysis of the hydrolysis products indicated that the enzyme produced galactose from β-1,3-D-galactan in an exo-acting manner. When the enzyme catalyze hydrolysis of the arabinogalactan-protein, the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate β-1,6-linked D-galactosyl side chains. These properties are the same as the other previously reported exo-β-1,3-D-galactanases. Therefore, we concluded the isolated gene certainly encodes an exo-β-1,3-D-galactanase. This is the first report of exo-β-1,3-D-galactanase from actinomycetes.