Effect of Physical and Chemical Properties of Oil Palm Empty Fruit Bunch,Decanter Cake and Sago Pith Residue on Cellulases Production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2

The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

Protoplast Fusion of β-Glucosidase-Producing Aspergillus niger Strains

Protoplast fusion, induced by polyethylene glycol and Ca²⁺, was carried out between two auxotrophic strains ofAspergillus niger. The fusion frequency ranged from 6.2 × 10^-2-9.1 × 10^-2. After induced haploidization of a diploid, various segregants showing combinations of the parental genetic markers were isolated. Unlike diploids, haploid segregants exhibited greater variations in their morphology and β-glucosidase activities. One segregant showed a 2.5-fold increase in β-glucosidase activity over those of the parents. Thus, this method appears promising for creating new recombinant strains ofA. niger with improved β-glucosidase activities.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Crude Cellulase from Oil Palm Empty Fruit Bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for Fermentable Sugars Production

Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2 % NaOH with autoclave, which was composed of 59.7 % cellulose, 21.6 % hemicellulose, and 12.3 % lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1 % of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5 % of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33 % and 19.11 %, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.     

Genome Shuffling of Aspergillus niger for Improving Transglycosylation Activity

Isomaltooligosaccharides (IMO), the glucosylsaccharides used as food additives, are made from saccharified starch by enzymes or microbial cells with transglycosylation activity. This study aimed to generate shuffled futants of Aspergillus niger with enhanced transglycosylation activity for industrial IMO production. The starting mutant population was generated by ⁶⁰Co-γ radiation; mutants with higher transglycosylation activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by a novel high-throughput method based on detecting non-fermentable reducing sugar. After three rounds of genome shuffling, the best performing strain GS3-3 was obtained, its transglycosylation activity (14.91 U/mL) was increased by 194.1 % compared to that of original strain C-6181. In fermentor test, transglycosylation activity of GS3-3 was obtained at 16.61 U/mL. The mycelia of GS3-3 were reused ten times to produce IMO syrup from liquefied cassava starch containing about 280 g/L total sugar within 4 days. The conversion of liquefied cassava starch to IMO was at 71.3–72.1 %, which was higher than the best conversion (68 %) ever reported. GS3-3 shows a great potential for industrial IMO production.     

Response Surface Methodology Based Optimization of β-Glucosidase Production from Pichia pastoris

The thermotolerant yeast Pichia etchellsii produces multiple cell bound β-glucosidases that can be used for synthesis of important alkyl- and aryl-glucosides. Present work focuses on enhancement of β-glucosidase I (BGLI) production in Pichia pastoris. In the first step, one-factor-at-a-time experimentation was used to investigate the effect of aeration, antifoam addition, casamino acid addition, medium pH, methanol concentration, and mixed feed components on BGLI production. Among these, initial medium pH, methanol concentration, and mixed feed in the induction phase were found to affect BGLI production. A 3.3-fold improvement in β-glucosidase expression was obtained at pH 7.5 as compared to pH 6.0 on induction with 1 % methanol. Addition of sorbitol, a non-repressing substrate, led to further enhancement in β-glucosidase production by 1.4-fold at pH 7.5. These factors were optimized with response surface methodology using Box–Behnken design. Empirical model obtained was used to define the optimum “operating space” for fermentation which was a pH of 7.5, methanol concentration of 1.29 %, and sorbitol concentration of 1.28 %. Interaction of pH and sorbitol had maximum effect leading to the production of 4,400 IU/L. The conditions were validated in a 3-L bioreactor with accumulation of 88 g/L biomass and 2,560 IU/L β-glucosidase activity.     

Processing of β-Glucosidase–Silk Fibroin Nanoparticle Bioconjugates and Their Characteristics

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with excellent biocompatibility. The aim of this work was to develop silk fibroin nanoparticles (SFNs) derived from the fibrous protein, which is a novel vector for enzyme modification in food processing. Silk fibroin was dissolved in highly concentrated CaCl₂ and subjected to lengthy desalting in water. The resulting liquid silk, which contained water-soluble polypeptides with molecular mass ranging from 10 to 200 kDa, and β-glucosidase were added rapidly into acetone. The β-glucosidase molecules were embedded into silk fibroin nanoparticles, forming β-glucosidase–silk fibroin nanoparticles (βG–SFNs) with a diameter of 50–150 nm. The enzyme activity of the βG–SFN bioconjugates was determined with p-nitrophenyl-β-d-glucoside as the substrate, and the optimum conditions for the preparation of βG–SFNs were investigated. The enzyme activity recovery of βG–SFNs was 59.2 % compared to the free enzyme (specific activity was 1 U mg^-1). The kinetic parameters of the βG–SFNs and the free β-glucosidase were the same. The βG–SFNs had good operational stability and could be used repeatedly. These results confirmed that silk protein nanoparticles were good carriers as bioconjugates for the modification of enzymes with potential value for research and development. The method used in this study has potential applications in food processing and the production of flavour agents.     

Production of Cellulolytic Enzymes by Fungi Acrophialophora nainiana and Ceratocystis paradoxa Using Different Carbon Sources

Although a number of filamentous fungi, such as Trichoderma and Aspergillus, are well known as producers of cellulases, xylanases, and accessory cellulolytic enzymes, the search for new strains and new enzymes has become a priority with the increase in diversity of biomass sources. Moreover, according to the type of pretreatment applied, biomass of the same type may require different enzyme blends to be efficiently hydrolyzed. This study evaluated cellulases, xylanases, and β-glucosidases produced by two fungi, the thermotolerant Acrophialophora nainiana and Ceratocystis paradoxa. Cells were grown in submerged culture on three carbon sources: lactose, wheat bran, or steam-pretreated sugarcane bagasse, a commonly used cattle feed in Brazil. Xylanase and endo-1-4-β-glucanase (CMCase) highest production were found in A. nainiana growing on lactose and reached levels of 2,200 and 2,016 IU/L, respectively. C. paradoxa showed highest activity for xylanase when grown on wheat bran and for β-glucosidase when grown on steam-treated bagasse, at levels of 12,728 and 1,068 IU/mL, respectively.     

Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.     

Utilization of waste cellulose—III. Comparative study of the activity of the cellulases of Trichoderma viride and Aspergillus niger towards different cellulosic substrates

Filter paper, carboxymethylcellulase and β-glucosidase activities have been determined and compared for cellulases originating from Trichoderma viride (TV) and Aspergillus niger (AN). The formation of glucose and of total reducing sugar has been measured as a function of time for the hydrolysis of cellulose I by the same quantity of FP units from TV, AN or a mixture of both strains. Long term efficiency is lower for AN but an important synergistic effect has been observed for the mixture of the enzymes. This synergistic action has been assigned to a better balance of endo- and exoglucanases and essentially to the addition to TV of thermally stable endoglucanases from AN. The β-glucosidases formed in large quantity by AN have been found to be thermally unstable and susceptible to product inhibition. They do not play any role in the observed synergistic action.     

Two-Step Purification of a Novel β-Glucosidase with High Transglycosylation Activity and Another Hypothetical β-Glucosidase in Aspergillus oryzae HML366 and Enzymatic Characterization

Two novel β-glucosidases (BGHG1 and BGHG2) were purified from the enzyme extract of Aspergillus oryzae HML366 through nondenaturing gel electrophoresis and anion-exchange chromatography. The molecular weights for BGHG1 and BGHG2 were 93 and 138 kDa, respectively. The amino acid sequences were determined by matrix-assisted laser desorption/ionization tandem time of flight. The Mascot and Blast analyses indicated that BGHG1 has the same sequence as the hypothetical protein XP_001816831 from A. oryzae RIB40. Sequence comparison suggested that both enzymes belong to the glycosyl hydrolase family 3. Results from thin layer chromatography and high performance liquid chromatography showed that BGHG2 has relatively high transglycosylation activity, and after preliminary optimization, it was able to convert glucose to produce 52.48 mg/ml gentiobiose. This is the first report of production of hypothetical protein XP_001816831 and β-glucosidase with high transglycosylation activity in A. oryzae. Results provide a valuable reference for potential applications in food industry, biomass power generating industry, and many others.     

Polyploid Formation Between <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Trichoderma viride</Emphasis> For Enhanced Citric Acid Production From Cellulose

The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum beta-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.     

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0–9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg^–1). The K _m and V _max values towards p-nitrophenyl β-d-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol?min^–1?mg^–1, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl-β-d-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.     

Expression and Displaying of β-Glucosidase from Streptomyces Coelicolor A3 in Escherichia coli

Two genes encoding β-glucosidase from Streptomyces coelicolor A3(2) were cloned and expressed in Escherichia coli BL21 (DE3). Two recombinant enzymes (SC1059 and SC7558) were purified and characterized. The molecular mass of the purified SC1059 and SC7558 as determined by SDS-PAGE agrees with the calculated values (51.0 and 52.2 kDa, respectively). Optimal temperature and pH for the two enzymes were both at 35 °C and 6.0. SC7558 exhibited to be much more active than SC1059 under optimal conditions, and it was recombined with ice nucleation protein which could anchor on the surface of the cell. The optimal temperature and pH of the recombinant cells were 55 °C and 8.0, respectively. The resultant cells were to be used as material for immobilized β-glucosidase, which is convenient to catalyze substrates in various complicated conditions.     

Gene Cloning and Characterization of a Novel Salt-Tolerant and Glucose-Enhanced β-Glucosidase from a Marine Streptomycete

The gene BglNH encoding a β-glucosidase was cloned from a marine streptomycete. Sequence analysis revealed that BglNH encoded a 456-aa peptide with a calculated mass of 51 kDa. The deduced amino acid sequence of BglNH showed the highest identities of 61 % with known β-glucosidases and contained a catalytic domain which belonged to the glycoside hydrolase family 1. The gene BglNH was expressed in Escherichia coli and the recombinant enzyme (r-BglNH) was purified. The optimum pH and temperature of r-BglNH were pH?6.0 and 45 °C, respectively. The r-BglNH displayed the typical salt-tolerant and glucose-enhanced characteristics. Its activity was remarkably enhanced in the presence of 0.5 M NaCl (rose more than 1.6-fold) and 0.1 M glucose (rose more than 1.4-fold). Moreover, r-BglNH displayed good pH stability and metal tolerance. It remained stable after incubating with buffers from pH?4.0 to 10.0, and most metal ions had no significant inhibition on its activity. These properties indicate that r-BglNH is an ideal candidate for further research and industrial applications.     

Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, β-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.     

Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases

Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e., they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18, 2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.     

Characterization of Xylanase and Cellulase Produced by a Newly Isolated <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> N2 and Its Efficient Saccharification of Barley Straw

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9 U/mL) and CMCase (5.61 U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65 °C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65 °C. The FPase was optimally active at pH 5.0 and 60 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.     

Continuous cellobiose hydrolysis using self-immobilized β-glucosidase fromAspergillus phoenicis QM 329 in a fluidized-bed reactor

Aspergillus phoenicis QM 329 was grown in the shape of beads in shake flasks and in an air-lift fermentor. The production of β-glucosidase started when the carbon source, glucose, was consumed. The β-glucosidase activity was retained in the beads at a pH below 6.0. The influence of bead diameter on enzyme activity and the pH and temperature optima for cellobiose hydrolysis has been studied. The enzyme-containing beads were used in a fluidized-bed reactor for continuous cellobiose hydrolysis, and a productivity of 2.0 g/L-h at a substrate conversion of 76% was obtained. The self-immobilized β- glucosidase is a stable and reusable enzyme with a half-life of 700 h when operating at 50°C and pH 4.8.