Peter Biely

Microbial xylanolytic systems

Abstract Microorganisms metabolizing xylan as a carbon source, and the enzyme systems they employ for its breakdown, may become important tools in elaborating economically and ecologically beneficial processes for using the second most abundant renewable polysaccharide. Critical factors in converting xylan into useful products with xylanolytic enzyme systems are considered.

Soluble chromogenic substrates for the assay of endo-1,4-β-xylanases and endo-1,4-β-glucanases

Abstract New soluble chromogenic substrates were prepared for specific and rapid assays of endo-1,4-β-xylanases and endo-1,4-β-glucanases. A soluble beechwood 4-O-methyl- d -glucurono- d -xylan was dyed with Remazol brilliant blue R, and hydroxyethylcellulose was coupled to Ostazin brilliant red H-3B. The assays are based on photometric measurements of the enzyme-released dyed fragments soluble in the presence of organic solvents which precipitate the original substrates and their high-molecular-weight fractions. The assays are advantageous for rapid analyses of large amount of samples and also permit evaluation of the activities of both enzymes in the presence of exo-β-glycanases and β-glycosidases, at a high level of reducing compounds and viable cells, on the cell surface and on cell membranes and organelles.

Sensitive detection of endo-1,4-β-glucanases and endo-1,4-β-xylanases in gels

Abstract A simple, highly sensitive zymogram technique for detection of endo-1,4-β-glucanases and endo-1,4-β-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl- d -glucurono- d -xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.

Specificity of cellulase and β-xylanase induction in Trichoderma reesei QM 9414

Cellulose- and xylan-degrading enzymes of Trichoderma reesei QM 9414 induced by, sophorose, xylobiose, cellulose and xylan were analyzed by isoelectric focusing. The sophorose-induced enzyme system contained two types of endo-1,4-β-glucanases (EC 3.2.1.4), one specific for cellulose and the other non-specific, hydrolyzing both cellulose and xylan, and exo-1,4-β-glucanases (cellobiohydrolases I, EC 3.2.1.91), i.e. all types of glucanases that are produced during growth on cellulose. Specific endo-1,4-β-xylanases (EC 3.2.1.8) present in the cellulose-containing medium were less abundant in the sophorose-induced enzyme system. Xylobiose and xylan induced only specific endo-1,4-β-xylanases. It is concluded that syntheses of cellulases and β-xylanases in T. reesei QM 9414 are under separate control and that the non-specific endo-1,4-β-glucanases are constituents of the cellulose-degrading enzyme system.

Cellulose- and xylan-degrading enzymes of Aspergillus terreus and Aspergillus niger

Abstract Separation of the enzyme components of Aspergillus terreus cellulolytic and xylanolytic systems by high performance gel filtration chromatography, electrophoresis, and isoelectric focusing, followed by detection of enzymes using chromogenic and fluorogenic substrates, showed the presence of (1) endo-1,4-β-glucanases, having isoelectric points around 3.3, 5.0, and 7.3, hydrolysing exclusively hydroxyethylcellulose (HEC); (2) endo-1,4-β-glucanases, with isoelectric points between 4.1 and 5.6, attacking HEC and liberating 4-methylumbelliferone from 4-methylumbelliferyl β-lactoside and 4-methylumbelliferyl β-cellobioside; (3) endo-1,4-β-xylanases, focused between pH 7.5 and 9.0, hydrolysing only xylan. All three enzyme groups were also found in a crude hemicellulase system of A. niger. Determination of the activities of endo-1,4-β-glucanase, exo-1,4-β-glucanase of the cellobiohydrolase I type (hydrolysing 4-nitrophenyl β-lactoside), β-glucosidase, endo-1,4-β-xylanase and β-xylosidase, induced in A. terreus by xylo- or glucosaccharides and by growing the fungus on polysaccharides, showed that: (1) Cellulose-degrading enzymes were induced by sophorose, laminaribiose, gentiobiose, cellobiose, and cellulose. The last two compounds, which are structurally related to xylobiose and xylan, also induced β-xylanase. (2) Xylose, xylobiose, and xylan initiated synthesis of only xylan-degrading enzymes. (3) Biosynthesis of cellulose- and xylan-degrading enzymes in A. terreus appears to be under separate regulatory control.

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile

Abstract A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5xa0kDa, with a mass of 33±1xa0kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pHxa03.1. The enzyme activity was optimal at pHxa06.0 and 55–60xa0°C. The purified esterase was stable at the pH rangexa05.0–7.0. The enzyme retained 70% of activity after 7xa0h at 50xa0°C and lost 50% of its activity after 45xa0min at 55xa0°C and after 12xa0min at 60xa0°C. Determination of kcat/Km revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1xa0h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.

Two glucuronoyl esterases of Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.

Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of β-d-xylopyranose and α-l-arabinofuranose

Measurements of esterase activity by enzyme-coupled assays on monoacetates of 4-nitrophenyl β-D-xylopyranoside and 4-nitrophenyl α-L-arabinofuranoside showed that acetylxylan esterases of families 1, 4 and 5 produced by Trichoderma reesei and Penicillium purpurogenum have a strong preference for deacetylation of position 2 in xylopyranosides. The acetylxylan esterases exhibit only weak activity on acetylated arabinofuranosides, with 2-acetate as the best substrate. Acetyl esterases of family 16 produced by the same two fungi deacetylate in xylopyranosides preferentially positions 3 and 4. Their specific activity on arabinofuranosides is also much lower than on xylopyranosides, however, substantially greater than that in the case of typical acetylxylan esterases.

Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families

Cellulose acetate (CA) was found to be a substrate of several acetyl xylan esterases (AXE). Eight AXE from different carbohydrate esterase (CE) families were tested on their activity against CA with a degree of substitution of 0.7 and 1.4. The classification of the AXEs into CE families according to their structure by hydrophobic cluster analysis followed clearly their activity against CA. Within the same CE family similar, and between the CE families different deacetylation behaviours could be observed. Furthermore, each esterase family showed a distinct regioselective mode of action. The CE 1 family enzymes regioselectively cleaved the substituents in C2- and C3-position, while CE 5 family enzymes only cleaved the acetyl groups in C2-position. CE 4 family enzymes seemed to interact only with the substituents in C3-position. Evidence was found that the deacetylation reaction of the CE 1 family enzymes proceeded faster in C2- than in C3-position of CA. The enzymes were able to cleave acetyl groups from fully substituted anhydroglucose units.

Structural Basis for Substrate Recognition by Erwinia Chrysanthemi Gh30 Glucuronoxylanase.

Xylanaseu2003A from the phytopathogenic bacterium Erwiniau2003chrysanthemi is classified as a glycoside hydrolase familyu200330 enzyme (previously in familyu20035) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β‐d‐xylopyranosyl‐(1→4)‐[4‐O‐methyl‐α‐d‐glucuronosyl‐(1→2)]‐β‐d‐xylopyranosyl‐(1→4)‐d‐xylose as a ligand. The crystal structure of the enzyme–ligand complex was solved at 1.39u2003Å resolution. The ligand xylotriose moiety occupies subsitesu2003−1, −2 and −3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β‐1,4‐xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate.