Jürgen Puls

Two major xylanases of Trichoderma reesei

Abstract The two main xylanases produced by Trichoderma reesei were purified to electrophoretic homogeneity by ion-exchange and gel chromatography. The enzymes had isoelectric points of 9.0 and 5.5 and their molecular masses were 20 and 19 kDa, respectively. The purified xylanases were most probably distinct gene products, as they were also serologically dissimilar and had different proteolytic cleavage patterns. The pI 9.0 xylanase tolerated higher pH values and temperatures than the pI 5.5 xylanase. Both enzymes clearly preferred polymeric substrates to xylo-oligosaccharides. The substrate preference for polymeric xylans decreased with decreasing substitution (decreasing solubility). The apparent K m values for different xylans varied from 3.0 to 6.8 mg ml -1 for the pI 9.0 xylanase and from 14.8 to 22.3 mg ml -1 for the pI 5.5 xylanase. The pI 9.0 xylanase also showed transxylosidase activity with xylotetraose and xyloheptaose as substrates.

Characteristics of Trichoderma reesei ?-xylosidase and its use in the hydrolysis of solubilized xylans

SummaryThe β-xylosidase (EC 3.2.1.37) of Trichoderma reesei was purified and its characteristics and use in the hydrolysis of steamed birch xylan were studied. The enzyme was a glycoprotein with a molecular weight of 100000 as determined by SDS-gel electrophoresis and its isoelectric point was 4.7. The pH optimum was 4.0 and temperature optimum 60°C. β-Xylosidase was competitively inhibited by xylose and the inhibition constant was 2.3 mM. The purified enzyme also showed α-arabinofuranosidase activity.

Biotechnical utilization of wood carbohydrates after steaming pretreatment

SummaryBirch wood was used as raw material to study the effect of steaming pretreatment on the characteristics, enzymatic hydrolysis and fermentation of cellulose and hemicellulose. The cellulose remained undissolved in the fibres after steaming, but the degree of polymerization decreased and the surface area increased with increasing steaming temperature. The yield in enzymatic hydrolysis with T. reesei and A. niger cellulases increased from 40 to 75% of theoretical when the pretreatment temperature was increased from 170 to 210°C at a residence time of 10 minutes. The glucose released was fermented to ethanol by yeast without interference of toxic compounds. After steaming, the hemicellulose was mainly in the form of xylo-oligomers. The average chain length decreased with increasing temperature. Only the monomeric sugars were fermented to ethanol by Fusarium oxysporum. After steaming at 210°C toxic decomposition products inhibited the fermentation completely. In aerobic conditions also the xylo-oligomers were metabolized.

Evaluation of different microbial xylanolytic systems

The xylanolytic enzymes produced by Trichoderma reesei QM 9414, Aspergillus awamori VTT-D-75028, Fusarium oxysporum VTT-D-80134, Bacillus subtilis ATCC 12711 and Streptomyces olivochromogenes ATCC 21713 differed with respect to β-xylosidase activity and side-group cleaving activities. The highest xylanase activity was produced by T. reesei. All the fungi produced β-xylosidase, whereas in the bacterial culture filtrates β-xylosidase activity was negligible. T. reesei culture filtrate contained all the side-group cleaving activities assayed (acetyl esterase, α-glucuronidase and α-arabinosidase) and those of F. oxysporum and S. olivochromogenes contained esterase. All the side-group cleaving activities were low in the culture filtrates of A. awamori and B. subtilis. The differences between the xylanolytic systems were reflected in the hydrolysis of steamed birchwood hemicellulose. The xylose yields obtained ranged from 0 (with B. subtilis) to 90% (with T. reesei) of the theoretical maximum. The best enzyme for complete hemicellulose hydrolysis was therefore that of T. reesei. However, in some applications in which complete hydrolysis is not needed or in which hydrolysis of cellulose is to be avoided, one of the other xylanases may be more suitable than that of T. reesei.

Production, purification and characterization of an esterase liberating phenolic acids from lignocellulosics

Abstract Production of extracellular esterase cleaving phenolic sidegroups from xylan was investigated using strains of Aspergillus. The activity levels produced were unrelated to the ferulic acid content of the lignocellulosic raw material in the cultivation medium. No correlation between the production of feruloyl esterase, acetyl xylan esterase and acetyl esterase activities was observed either. An esterase produced by Aspergillus oryzae was purified to electrophoretic homogeneity. The enzyme was an acidic monomeric protein having an isoelectric point of 3.6 and a molecular mass of 30 kDa. It was most active in the pH-range from 4.5 to 6.0 and was stable at temperatures up to 45°C. The esterase had a wide substrate specificity, liberating ferulic, p-coumaric and acetic acids from steam-extracted wheat straw fragments and acetic acid from acetylated xylo-oligomers and α-naphthyl acetate. The enzyme acted in synergism with other xylanolytic enzymes, which was reflected in increased production of phenolic acids from wheat straw xylo-oligosaccharides in the presence of xylanases of Trichoderma reesei.

Deacetylation of xylans by acetyl esterases of Trichoderma reesei

SummaryTwo previously purified esterases of Trichoderma reesei were used to study the deacetylation of polymeric, oligomeric and dimeric acetylated xylan fragments. For the first time nearly complete enzymatic deacetylation of polymeric xylan with purified acetyl xylan esterase was demonstrated, resulting in precipitation of the remaining polymer structure. The esterases had very different substrate specifities, one having a preference for high molecular weight substrates and the other showing high activity only towards acetyl xylobiose. The latter enzyme was also regioselective, cleaving off the acetyl substituent only from the C-3 position of the xylopyranose ring. The highest xylose yield from acetylated xylan was obtained by the synergistic action of xylanase, \-xylosidase and acetyl xylan esterase.

An α-glucuronidase from Trichoderma reesei RUT C-30

Abstract The major α-glucuronidase of T. reesei Rut C-30 was purified by chromatographic methods. The molecular and hydrolytic properties of the purified enzyme were studied. The enzyme had a molecular weight of 91,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a pI of 5.0–6.2 as determined by chromatofocusing. The pH optimum was pH 4.5–6.0 and the enzyme was stable for 24 h at 40°C at pH 4.8–5.5. The purified α-glucuronidase preferred low-molecular-weight xylooligomers as substrate. The enzyme seemed to act almost exclusively on the bond between the terminal xylose at the nonreducing end of a xylose chain and the methyl glucuronic acid attached to it. Minor activity against long-chain glucuronoxylan was also detected. A significant enhancing effect of α-glucuronidase on the hydrolysis of glucuronoxylan by pure xylanases was observed.

Topochemical characterisation of phenolic extractives in discoloured beechwood (Fagus sylvatica L.)

Summary The topochemical distribution of phenolic extractives in steamed and kiln-dried beechwood with discolourations was investigated on a cellular level by using scanning UV microspectrophotometry (UMSP). For the chemical characterisation of accessory compounds, acetone and methanol extracts of the discoloured beechwood were separated by accelerated solvent extraction (ASE) and analysed with high performance liquid chromatography (HPLC). The UV microscopic investigations reveal that the accessory compounds responsible for the discolouration of beechwood are mainly restricted to the longitudinal and ray parenchyma cells and the lumen of vessels. The detected extractives are characterised by high UV absorbance values and an absorbance maximum in a wavelength range between 280 and 290 nm. The separation of the acetone and methanol extracts of discoloured beechwood shows the presence of different low molecular phenols such as catechin and 2,6-dimethoxybenzochinon, which are transformed into high condensation compounds during steaming and kiln-drying.

Enzyme-aided characterisation of carboxymethylcellulose

Two conventionally prepared carboxymethylcelluloses (CMCs) with differing degrees of substitution (DS: 0.6 and 1.2) were fragmented by endoglucanase treatment until no further degradation was possible. This treatment brought about a decisive improvement in the water solubility of the polymers. The degraded samples were separated into 18 fractions by size exclusion chromatography (SEC). The quality of the preparative SEC was monitored by analytical SEC equipped with a multiple detector system. After acidic hydrolysis of each individual fraction, the carboxymethyl derivatives of the anhydroglucose units were determined by anion exchange chromatography and pulsed amperometric detection. The accessibility of endoglucanase action was clearly a function of the degree of substitution (DS). As the DS increased the efficiency of the enzyme became more limited. It could also be demonstrated that the polysaccharide chain of both CMC samples included some regions that were highly substituted and some other regions of low substitution.

Evaluation of new organosolv dissolving pulps.Part I: Preparation, analytical characterization and viscose processability

New acidic organosolv pulping processes, such as Acetosolv, Formacell and Milox, promise to have superior potential in terms of purification selectivity and specific investment costs. Consequently, a thorough investigation of these new acidic pulping processes in comparison to state-of-the-art acidic magnesium sulfite technology was conducted. The impact of pulping and bleaching parameters on the physical and chemical characteristics was studied to compare process efficiency and selectivity for each type of pulp made from Eucalypt wood. In addition to a detailed analysis of the chemical composition and physical properties on a molecular and supramolecular level, the TCF-bleached dissolving pulps were tested for their applicability in viscose fiber production. The influence of pulp properties as determined by standard and advanced analytical methods on process performance and selected fiber properties is emphasized.