Birch pulp xylan works as a food hydrocolloid in acid milk gels and is fermented slowly in vitro.

The objective was to evaluate the potential of birch xylan as a food hydrocolloid and dietary fibre. High-molecular weight xylan was isolated from birch kraft pulp by alkaline extraction, and enzymatically hydrolysed. Fermentability of xylans was evaluated using an in vitro colon model and performance as a hydrocolloid was studied in low-fat acid milk gels (1.5% and 3% w/w). Texture of the gels and water holding capacity of xylans were compared with inulin, fructooligosaccharide and xylooligosaccharide. Xylans showed slower fermentation rate by faecal microbiota than the references. Xylan-enriched acid milk gels (3% w/w) had improved water holding capacity (over 2-fold) and showed lower spontaneous syneresis, firmness and elasticity when compared to control (no hydrocolloids) or to references. In conclusion, birch xylan improved texture of low-fat acid milk gel applications, and the slow in vitro fermentation rate predicts lower incidence of intestinal discomfort in comparison to the commercial references.

Impact of total solid content and extraction pH on enzyme-aided recovery of protein from defatted rapeseed (Brassica rapa L.) press cake and physicochemical properties of the protein fractions.

Pectinase treatment was used to facilitate protein recovery from defatted rapeseed (Brassica rapa) cold-pressing residue in water-lean conditions and without pH adjustment. Effect of extraction pH on protein yield and physiochemical properties of the protein concentrates was assessed. Enzymatic hydrolysis of carbohydrates was feasible at high (40%) solid content and improved protein recovery at pH 6. Comparable protein yields (40-41% of total protein) from enzyme-aided water extraction (pH 6) and nonenzymatic alkaline extraction (pH10) at 10% solid content suggested that after enzymatic treatment, rapeseed protein could be extracted without exposure to alkali. However, water extraction required dilute conditions, whereas alkaline extraction was feasible also at 20% solid content. The water extracts possessed better protein solubility, higher ζ-potential, and smaller particle size than isoelectric precipitates from alkaline extraction, indicating higher dispersion stability. This is suggested to be mediated by electrostatic interactions between proteins and pectic carbohydrates in the water extracts.

Effect of enzyme-aided cell wall disintegration on protein extractability from intact and dehulled rapeseed (Brassica rapa L. and Brassica napus L.) press cakes.

Cell-wall- and pectin-degrading enzyme preparations were used to enhance extractability of proteins from rapeseed press cake. Rapeseed press cakes from cold pressing of intact Brassica rapa and partially dehulled Brassica napus seeds, containing 36-40% protein and 35% carbohydrates, were treated with pectinolytic (Pectinex Ultra SP-L), xylanolytic (Depol 740L), and cellulolytic (Celluclast 1.5L) enzyme preparations. Pectinex caused effective disintegration of embryonic cell walls through hydrolysis of pectic polysaccharides and glucans and increased protein extraction by up to 1.7-fold in comparison to treatment without enzyme addition. Accordingly, 56% and 74% of the total protein in the intact and dehulled press cakes was extracted. Light microscopy of the press cakes suggested the presence of pectins colocalized with proteins inside the embryo cells. Hydrolysis of these intracellular pectins and deconstruction of embryonic cell walls during Pectinex treatment were concluded to relate with enhanced protein release.

Enzyme-aided alkaline extraction of oligosaccharides and polymeric xylan from hardwood kraft pulp.

In this paper we describe the effect of enzyme treatments on the production of polymeric xylan, oligosaccharides and hemicellulose lean pulp by alkaline extraction of bleached hardwood kraft pulp. Enzyme treatments were carried out before one or in between two subsequent alkaline extractions by purified Trichoderma reesei xylanase and endoglucanase II (Cel 5a) as well as by a commercial monocomponent endoglucanase (FibreCareR). Without enzyme pre-treatment 61% and 7% of the pulp xylan was extracted in high purity in the first and second alkaline stage, respectively. Higher molecular mass xylan was obtained in the second than in the first alkaline extraction. Xylanase treatment before alkaline extraction hydrolyzed up to 12% of xylan to xylooligosaccharides. According to our results, preparation of polymeric xylan, and/or oligosaccharides as well as hemicellulose lean pulp with cellulose content of 93-94%, is possible by enzyme-aided alkaline extraction process.

Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides.

In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86-95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.

Thermotolerant and thermostable laccases.

Laccases are phenol-oxidizing, usually four-copper containing metalloenzymes. For industrial and biotechnological purposes, laccases were among the first fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibres. In order to facilitate novel and more efficient bio-catalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability and thermotolerance. This review gives a current overview on the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.

Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus.

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60 degrees C. Lac-4.8 was thermally activated at 50 degrees C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0-3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus.

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn(2+), veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn(2+) and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.

Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood.

Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.

Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata.

Lignin peroxidase (LiP) is the first enzyme connected to oxidative breakdown of the aromatic plant heteropolymer lignin and related xenobiotics. However, this extracellular enzyme has been described in only a few species of wood-decaying basidiomycetous fungi. The white rot basidiomycete Phlebia radiata 79 readily produces a versatile set of lignin-oxidizing enzymes including lignin and manganese peroxidases (LiPs and MnPs) and laccases. Here we describe genomic and primary structure of two new LiP-encoding genes, Pr-lip1 and Pr-lip4, and genomic characterization for isozyme LiP3/LIII of P. radiata, encoded by the gene depicted Pr-lip3. Pr-lip1 and Pr-lip4 code for 370- and 361-amino-acid long proteins beginning with 26- and 24-amino-acid secretion pre-propeptides, respectively. Translated LiP1 and LiP4 share the highest protein sequence identity (74 and 86%) with P. radiata LiP3, and 70% identity with the one deduced LiP from Bjerkandera adusta. The three P. radiata LiP sequences form a coherent phylogenetic cluster, which is further supported by similarities within gene organization interrupted by 11-introns. To find out the significance of LiP upon fungal growth on natural lignocellulose, such as wood, we studied ligninolytic gene expression on hardwood (milled alder) and softwood (spruce chips). All the LiP-encoding genes were expressed on wood with predominance of Pr-lip3 transcript abundance, in particular on spruce wood chips, where also time-dependent expression of the multiple lip genes was observed.