Co-production of schizophyllan and cellulolytic enzymes from bagasse by Schizophyllum commune.

Schizophyllum commune is a mushroom-forming fungus well-known for its ability to degrade lignocellulosic materials and production of schizophyllan, a high added-value product for cosmeceutical, pharmaceutical, and biomaterial industries. Conventionally, schizophyllan is produced by submerged fermentation using glucose as a carbon source. In this work, we demonstrate that alkaline pretreated bagasse can be used by Schizophyllum commune as an alternative carbon source for the production of schizophyllan. The influence of different factors was investigated including cultivation time, biomass loading, and culturing media component and a co-product correlation model was proposed. In this lab-scale study, a yield of 4.4 g/L of schizophyllan containing 89% glucose was achieved. In addition to schizophyllan, the cellulolytic enzymes co-produced during this process were isolated and characterized and could find applications in a range of industrial processes. This demonstrates the potential of using agricultural waste as a cheaper alternative feedstock for this biorefinery process.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and ß-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external ß-xylosidase.

Biochemical characterization and synergism of cellulolytic enzyme system from Chaetomium globosum on rice straw saccharification.

BACKGROUND: Efficient hydrolysis of lignocellulosic materials to sugars for conversion to biofuels and chemicals is a key step in biorefinery. Designing an active saccharifying enzyme system with synergy among their components is considered a promising approach. RESULTS: In this study, a lignocellulose-degrading enzyme system of Chaetomium globosum BCC5776 (CG-Cel) was characterized for its activity and proteomic profiles, and synergism with accessory enzymes. The highest cellulase productivity of 0.40 FPU/mL was found for CG-Cel under the optimized submerged fermentation conditions on 1% (w/v) EPFB (empty palm fruit bunch), 2% microcrystalline cellulose (Avicel®) and 1% soybean meal (SBM) at 30 °C, pH 5.8 for 6 d. CG-Cel worked optimally at 50-60 °C in an acidic pH range. Proteomics analysis by LC/MS/MS revealed a complex enzyme system composed of core cellulases and accessory hydrolytic/non-hydrolytic enzymes attacking plant biopolymers. A synergistic enzyme system comprising the CG-Cel, a ß-glucosidase (Novozyme® 188) and a hemicellulase Accellerase® XY was optimized on saccharification of alkaline-pretreated rice straw by a mixture design approach. Applying a full cubic model, the optimal ratio of ternary enzyme mixture containing CG-Cel: Novozyme® 188: Accellerase® XY of 44.4:20.6:35.0 showed synergistic enhancement on reducing sugar yield with a glucose releasing efficiency of 256.4 mg/FPU, equivalent to a 2.9 times compared with that from CG-Cel alone. CONCLUSIONS: The work showed an approach for developing an active synergistic enzyme system based on the newly characterized C. globosum for lignocellulose saccharification and modification in bio-industries.

Production of high activity Aspergillus niger BCC4525 ß-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis.

A cDNA encoding ß-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high kcat/Km (461 mg-1 s-1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-ß-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant ß-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.

ß-Mannanase production by Aspergillus niger BCC4525 and its efficacy on broiler performance.

BACKGROUND: Mannan is a hemicellulose constituent commonly found in plant-derived feed ingredients. The gum-like property of mannan can obstruct digestive enzymes and bile acids, resulting in impaired nutrient utilisation. In this study, ß-mannanase production by Aspergillus niger strain BCC4525 was investigated using several agricultural residues under solid state condition. The biochemical properties of the target enzyme and the effects of enzyme supplementation on broiler performance and energy utilisation were assessed. RESULTS: Among five carbon sources tested, copra meal was found to be the best carbon source for ß-mannanase production with the maximum yield of 1837.5 U g(-1) . The crude ß-mannanase exhibited maximum activity at 80 °C within a broad range of pH from 2 to 6. In vitro digestibility assay, which simulates the gastrointestinal tract system of broilers, showed that ß-mannanase could liberate reducing sugars from corn/soybean diet. Surprisingly, ß-mannanase supplementation had no significant effect on broiler feed intake, feed conversion rate or energy utilisation. CONCLUSION: A high level of ß-mannanase was produced by A. niger BCC4525 under solid state condition using copra meal as carbon source. Although the enzyme has the desired properties of an enzyme additive for improving broiler performance, it does not appear to be beneficial.

Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris.

Plant-based animal feed contains antinutritive agents, necessitating the addition of digestive enzymes in commercial feeds. Enzyme additives are costly because they are currently produced separately from recombinant sources. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. A coexpression system for the extracellular production of phytase and xylanase was established in Pichia pastoris yeast. The genes for each enzyme were fused in-frame with the α-factor secretion signal and linked by the 2A-peptide-encoding sequence. Each enzyme was expressed extracellularly as individual functional proteins. The specific activities of 2A-expressed phytase (PhyA-2A) and 2A-expressed xylanase (XylB-2A) were 9.3 and 97.3 U mg(-1) , respectively. Optimal PhyA-2A activity was observed at 55 degreesC and pH 5.0. PhyA-2A also exhibited broad pH stability from 2.5 to 7.0 and retained approximately 70% activity after heating at 90 degreesC for 5 min. Meanwhile, XylB-2A exhibited optimal activity at 50 degreesC and pH 5.5 and showed pH stability from 5.0 to 8.0. It retained >50% activity after incubation at 50 degreesC for 10 min. These enzyme properties are similar to those of individually expressed recombinant enzymes. In vitro digestibility test showed that PhyA-2A and XylB-2A are as efficient as individually expressed enzymes for hydrolyzing phytate and crude fiber in feedstuff, respectively.

Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement.

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris.

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris. The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL(-1), respectively. Both recombinant phytases exhibited high affinity for phytate but not for p-nitrophenyl phosphate. Optimal phytase activity was observed at 50 degrees C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 degrees C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.

A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars.

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.