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Second-generation biofuel production has emerged as a prominent sustainable and alternative energy. The biochemical properties of cellulolytic enzymes are imperative for cellulosic biomass conversion into fermentable sugars. In the present study, thermostable CMCase and β-glucosidase were purified and characterized from Aspergillus fumigatus JCM 10253. The enzymes were purified through 80% ammonium sulfate precipitation, followed by dialysis and DEAE-cellulose ion-exchange chromatography. The molecular masses of the purified CMCase and β-glucosidase were estimated to be 125 kDa and 90 kDa, respectively. The CMCase and β-glucosidase demonstrated optimum activities at pH 6.0 and 5.0, respectively. Their respective maximum temperatures were 50 and 60 °C. The cellulase activities were stimulated by 10 mM concentration of Ca

β-glucosidase (BGL) is a rate limiting enzyme of lignocellulose hydrolysis for second generation bioethanol production, but its inhibition by lignocellulose pretreatment products, ethanol and salt is apparent. Here, the recombinant Penicillium oxalicum 16 BGL 1 (rPO16BGL1) from Pichia pastoris GS115 kept complete activity at 0.2–1.4 mg/mL furan derivatives and phenolic compounds, 50 mg/mL sodium chloride (potassium chloride), or 100 mg/mL ethanol at 40 °C. rPO16BGL1 retained above 50 % residual activity at 30 mg/mL organic acid sodium, and 60 % residual activity at 40 °C with 300 mg/mL ethanol. Sodium chloride and potassium chloride had a complicated effect on rPO16BGL1, which resulted in activation or inhibition. The inhibition kinetics of the enzyme reaction demonstrated that organic acids and organic acid sodium were non-competitive inhibitors and that ethanol was a competitive inhibitor at < 1.5 mg/mL salicin. Moreover, substrate inhibition of the enzyme was found at > 2 mg/mL salicin, and the K(m)/K(I) and K(m)/K(SI) average values revealed that the inhibitory strength was ranked as salicin-organic acids > organic acids > salicin-organic acid sodium salt > organic acid sodium salt > salicin > salicin-KCl > salicin-NaCl > salicin-ethanol > ethanol.

β-Glucosidase (BG) hydrolyzes cellobiose into glucose, and is a vital step in converting ionic liquids (ILs)-pretreated biomass to sustainable biofuels. The inactivation mechanism of BG from Paenibacillus sp. LLZ1 induced by microcrystalline cellulose was explored in various concentrations of ILs, composed of [Emim]+ cation and [DEP]-, [OAc]-, [Br]-, [Cl]-, and [BF4]- anions. The FTIR analysis of inactivated BG indicated that the ILs altered its β-sheet content. Moreover, circular dichroism spectroscopy (CD) suggested that the α-helix content decreased, while the β-sheet content increased with the presence of ILs in general. Interestingly, the secondary structure of BG had almost no change after [Emim]DEP treatment, while ionic liquid [Emim]BF4 treatment caused the irreversible denaturation of BG. Eventually, by adding 0.4 mM of Aerosol OT surfactant, the BG activity was increased by 20.1% in the presence of 25% [Emim]DEP, and the corresponding glucose yield from hydrolysis of cellobiose was increased by 23.9%.

It is inevitably for cellobiose to be co-generated during enzymatic hydrolysis of cellulose, especially when the cellulase is lack of β-glucosidase activity. In the present study, cellobiose was found superior to glucose for cell growth by L. starkeyi, regardless of the sugar concentrations. Glucose was assimilated preferentially when cellobiose and glucose were co-fermented. Deficiency of β-glucosidase was observed to be beneficial for the simultaneous saccharification and lipid production (SSLP). High lipid titer and cellulose conversion of 9.1 g/L and 92.4%, respectively, were achieved when cellulase with low β-glucosidase activity was supplemented. The SSLP achieved higher lipid titer of 9.5 g/L when a pre-hydrolysis process was introduced. The glucosidase generated by L. starkeyi was primarily cell-bound, which contributed significantly to the cellobiose utilization and the high lipid production. These results provided a novel scheme for enhanced lipid production from lignocellulosic biomass with reduced enzyme usage, which is believed to facilitate the design of a more cost-effective lignocellulose-to-lipid route.

β-Glucosidases primarily catalyze removal of terminal glucosyl residues from a variety of glucoconjugates and also perform transglycosylation and reverse hydrolysis. These catalytic properties can be readily exploited for degradation of lignocellulosic biomass as well as for pharmaceutical, food and flavor industries. β-Glucosidases have been either isolated in the native form from the producer organism or recombinantly expressed and gaged for their biochemical properties and substrate specificities. Although almond and Aspergillus niger have been instantly recognizable sources of β-glucosidases utilized for various applications, an intricate pool of novel β-glucosidases from different sources can provide their potent replacements. Moreover, one can envisage the better efficacy of these novel candidates in biofuel and biorefinery industries facilitating efficient degradation of biomass. This article reviews properties of the novel β-glucosidases such as glucose tolerance and activation, substrate specificity, and thermostability which can be useful for their applications in lignocellulose degradation, food industry, and pharmaceutical industry in comparison with the β-glucosidases from the conventional sources. Such β-glucosidases have potential for encouraging white biotechnology.

A moderate two-step pretreatment method was investigated to improve the enzymatic saccharification of bamboo residues. SEM and FTIR were employed to characterize the structure changes. Fed-batch enzymatic saccharification was performed to obtain high concentration of fermentable sugar. Bamboo was impregnated at low severity of conditions (room temperature, 2% H2SO4 or 2% NaOH, 48 h) to initially alter the structure of bamboo, and then further pretreated by steam explosion at 1.0 MPa for 6 min. The highest delignification of 51% and the highest enzymatic hydrolysis of 47.1% were reached at 2% NaOH impregnation followed by steam explosion. The changes in the structural characteristics showed beneficial effects on the enzymatic hydrolysis. When a mixer of cellulase (30 FPU) and β-glucosidase (10 CBU) was further used, the maximum enzymatic hydrolysis of 78.9% and total glucose yield of 68.2% were obtained. The maximum sugar release from the holocellulose was 500 mg/g bamboo, approximately 83.3% conversion efficiency based on monomeric sugar recovery. With fed-batch saccharification, a final substrate loading of 30% brought 107.7 g/L glucose, 35.81 g/L xylose, and 7.82 g/L arabinose release, respectively. This study provided an effective strategy for potential utilization of bamboo residues.

A novel GH1 β-glucosidase gene (bgla) from marine bacterium was sequenced and expressed in Escherichia coli. After purification by Ni2+ affinity chromatography, the recombinant protein was characterized. The purified recombinant enzyme showed maximum activity at 40 °C, pH 7.5 and was stable between temperatures that range from 4 to 30 °C and over the pH range of 6–10. The enzyme displayed a high tolerance to glucose and maximum stimulation at the presence of 100 mM glucose. To improve glucose tolerance of the enzyme, a site-directed mutation (f171w) was introduced into β-glucosidase. The recombinant F171W showed a higher glucose tolerance than the wild type and maintained more than 40% residual activity at the presence of 4 M glucose. Additionally, the recombinant enzymes showed notable tolerance to ethanol. These properties suggest the enzymes may have potential applications for the fermentation of lignocellulosic sugars and the production of biofuels.

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme., The authors thank the support from Research Institute of Food Science (CIAL), Embrapa Instrumentation of São Carlos, Foundation for Research Support of Espírito Santo (FAPES), and Federal University of Espírito Santo (UFES).

Soybean is one of the most important commodities in the world, being applied in feed crops and food, pharmaceutical industries in different ways. Soy is rich in isoflavones that in aglycone forms have exhibited significant anti-obesity and anti-lipogenic effects. Obesity is a global problem as several diseases have been related to this worldwide epidemic. The aim of this work was to verify the effect of free and immobilized β-glucosidase, testing Lentikats, and sol–gel as carriers. Moreover, we wanted to examine if the different types of hydrolysis would generate extracts with distinct biological activity concerning lipid accumulation, PPAR-α regulation, and TNF-α, IL-6, and IL-10 concentrations using in vitro assays. Our results show that all formulations of β-glucosidase could hydrolyze soy isoflavones. Thus, after 24 h of incubation, daidzein content increased 2.6-, 10.8-, and 12.2-fold; and genistein content increased 11.7, 11.4, and 11.4 times with the use of free enzyme, Lentikats®, and sol–gel immobilized enzyme, respectively. Moreover, both methodologies for enzyme immobilization led to promising forms of biocatalysts for application in the production of soy extracts rich in isoflavones aglycones, which are expected to bring about health benefits. A mild lipogenic effect was observed for some concentrations of extracts, as well as a slight inhibition in PPAR-α expression, although no significant differences were noticeable in the cytokines TNF-α, IL-10, and IL-6 as compared with the control.

We evaluated various agricultural lignocellulosic biomass and variety of fungi to produce cellulolytic enzymes cocktail to yield high amount of reducing sugars. Solid-state fermentation was performed using water hyacinth, paddy straw, corn straw, soybean husk/tops, wheat straw, and sugarcane bagasse using fungi like Nocardiopsis sp. KNU, Trichoderma reesei, Trichoderma viride, Aspergillus flavus, and Phanerochaete chrysosporium alone and in combination to produce cellulolytic enzymes. Water hyacinth produced (U ml−1) endoglucanase (51.13) and filter paperase (0.55), and corn straw produced (U ml−1) β-glucosidase (4.65), xylanase (113.32), and glucoamylase (41.27) after 7-day incubation using Nocardiopsis sp. KNU. Production of cellulolytic enzymes was altered due to addition of various nitrogen sources, metal ions, vitamins, and amino acids. The maximum cellulolytic enzymes were produced by P. chrysosporium (endoglucanase; 166.32 U ml−1 and exoglucanase; 12.20 U ml−1), and by T. viride (filter paperase; 1.57 U ml−1). Among all, co-culture of T. reesei, T. viride, A. flavus, and P. chrysosporium showed highest β-glucosidase (17.05 U ml−1). The highest xylanase (1129 U ml−1) was observed in T. viride + P. chrysosporium co-culture. This study revealed the dependency on substrate and microorganism to produce good quality enzyme cocktail to obtain maximum reducing sugars.

A novel β-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting β-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) β-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-β-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 β-glucosidase and its applications in bioconversion and biotechnology.

Naringinase which was extracted from the fermented broth of Cryptococcus albidus was purified about 42-folds with yield 0.7% by sulfate fractionation and chromatography on Toyopearl HW-60, Fractogel DEAE-650-s, and Sepharose 6B columns. Molecular weight of protein determined by gel filtration and SDS-PAGE was 50 kDa. Naringinase of C. albidus includes high content of the dicarbonic and hydrophobic amino acids. Enzyme contains also carbohydrate component, represented by mannose, galactose, rhamnose, ribose, arabinose, xylose, and glucose. The enzyme was optimally active at pH 5.0 and 60 °C. Naringinase was found to exhibit specificity towards p-nitrophenyl-α-L-rhamnose, p-nitrophenyl-β-D-glucose, naringin, and neohesperidin. Its K m towards naringin was 0.77 mM and the V max was 36 U/mg. Naringinase was inhibited by high concentrations of reaction product—L-rhamnose. Enzyme revealed stability to 20% ethanol and 500 mM glucose in the reaction mixture that makes it possible to forecast its practical use in the food industry in the production of juices and wines.

Isoflavone aglycones have been shown to be more rapidly and efficiently absorbed into intestines than isoflavone glucosides. Helpfully, β-glucosidases can be used to convert isoflavone glucosides to aglycones. In this study, β-glucosidases from microbial (Aspergillus niger) and vegetable lima bean (Phaseolus lunatus) sources were characterized, purified, and then employed to convert isoflavone glycosides to aglycones. The microbial crude extract showed maximum activity at 60 °C and pH 5.0. It was highly stable between 40 and 60 °C and between pH 4.0 and 9.0. Optimum activity for the vegetable crude extract was achieved also at 60 °C and pH 5.5. Similarly, it presented great stability at high temperatures and a wide pH range. The microbial enzyme was purified by a factor of 14-fold to a yield of 2.2 % and a specific activity of 17 IU/mg. The vegetable enzyme was purified by a factor of fourfold to a yield of 77 % and a specific activity of 0.18 IU/mg protein. Both β-glucosidases produced satisfactory conversion rates of daidzin and genistin into daidzein and genistein; however, the microbial enzyme performed better than the vegetable enzyme. Our results suggest a potential use of these enzymes to enhance the bioavailability of isoflavones in food products.

Production costs of cellulosic biofuels can be lowered if cellulases are recovered and reused using particulate carriers that can be extracted after biomass hydrolysis. Such enzyme recovery was recently demonstrated using enzymogel nanoparticles with grafted polymer brushes loaded with cellulases. In this work, cellulase (NS50013) and β-glucosidase (Novozyme 188) were immobilized on enzymogels made of poly(acrylic acid) polymer brushes grafted to the surface of silica nanoparticles. Response surface methodology was used to model effects of pH and temperature on hydrolysis and recovery of free and attached enzymes. Hydrolysis yields using both enzymogels and free cellulase and β-glucosidase were highest at the maximum temperature tested, 50 °C. The optimal pH for cellulase enzymogels and free enzyme was 5.0 and 4.4, respectively, while both free β-glucosidase and enzymogels had an optimal pH near 4.4. Highest hydrolysis sugar concentrations with cellulase and β-glucosidase enzymogels were 69 and 53 % of those with free enzymes, respectively. Enzyme recovery using enzymogels decreased with increasing pH, but cellulase recovery remained greater than 88 % throughout the operating range of pH values less than 5.0 and was greater than 95 % at pH values below 4.3. Recovery of β-glucosidase enzymogels was not affected by temperature and had little impact on cellulase recovery.

Forty-four endophytic fungal isolates obtained from marine sponge, Hyrtios erectus, were evaluated and screened for their hydrolase activities. Most of the isolates were found to be prolific producers of hydrolytic enzymes. Only 11 isolates exhibited maximum cellular contents of lipids, rhamnolipids, and protein in the fungal isolates under the isolation numbers MERVA5, MERVA22, MERVA25, MERVA29, MERVA32, MERVA34, MERV36, MERVA39, MERVA42, MERVA43, and MERVA44. These isolate extracts exhibit the highest reducing activities against carbohydrate-metabolizing enzymes including α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase. Consequently, based on morphological and cultural criteria, as well as sequence information and phylogenetic analysis, these isolates could be identified and designated as Penicillium brevicombactum MERVA5, Arthrinium arundinis MERVA22, Diaporthe rudis MERVA25, Aspergillus versicolor MERVA29, Auxarthron alboluteum MERVA32, Dothiorella sarmentorum MERVA34, Lophiostoma sp. MERVA36, Fusarium oxysporum MERVA39, Penicillium chrysogenum MERVA42, Penicillium polonicum MERVA43, and Trichoderma harzianum MERVA44. The endophytic fungal species, D. rudis MERVA25, P. polonicum MERVA43, Lophiostoma sp. MERVA36, A. alboluteum MERVA32, T. harzianum MERVA44, F. oxysporum MERVA39, A. versicolor MERVA29, and P. chrysogenum MERVA42 extracts, showed significant hepatitis C virus (HCV) inhibition. Moreover, D. sarmentorum MERVA34, P. polonicum MERVA43, and T. harzianum MERVA44 extracts have the highest antitumor activity against human hepatocellular carcinoma cells (HepG2).

Consolidated bioprocessing of lignocellulose for ethanol production is realized by expressing cellulase enzymes on ethanologenic strain. In this study, an ethanologenic Escherichia coli ZY81 was constructed by integrating pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis into the genome of E. coli JM109 to obtain the capability of ethanol production. Then, the β-glucosidase gene bglB from Bacillus polymyxa was cloned and secretively expressed in E. coli ZY81. The recombinant strain E. coli ZY81/bglB showed an obvious activity of β-glucosidase in extracellular location with more than half in periplasmic space. EDTA was found to promote the release of the periplasmic proteins by approximately tenfold. E. coli ZY81/bglB utilized cellobiose as sole carbon source for ethanol production with 33.99 % of theoretical yield.

Exo-xylanases are a class of glycosyl hydrolases and play an important role in hydrolysis of xylan to xylose. They belong to glycosyl hydrolase (GH) family 8 with a characteristic (α/α)6 barrel fold in their molecular structures. These enzymes are generally produced by bacteria. Exceptionally, the endo-xylanases from Trichoderma reesei Rut C-30 and a few bacterial strains also show considerable exo-xylanase activities. Exo-xylanases are active on natural xylan substances, hydrolyzing long-chain xylo-oligomers from the reducing end to produce short-chain xylo-oligomers and xylose. Exo-xylanases usually show multiple enzyme functions such as β-xylosidase, exo-glucanase, β-glucosidase, and arabinofuranosidase activities, which are helpful for more efficient hydrolysis of xylan. The combined use of exo- and endo-xylanases can increase the xylose yield compared to using either of them alone. Screening new exo-xylanase-producing microbes, mining the enzyme coding sequences, genetically engineering the enzymes, and producing them in a large scale are recommended for their commercial applications in lignocellulose-based biorefinery.

Industrially, enzymatic hydrolysis of lignocellulose at high solid content is preferable over low solids due to a reduction in processing costs. Unfortunately, the economic benefits are counteracted by a linear decrease in yield with solid content, referred to as the "solid effect" in the literature. In the current study, we investigate the contribution of product inhibition to the solid effect (7-33 % solids). Product inhibition was measured directly by adding glucose to high-solid hydrolysis samples and indirectly through variation of water content and beta-glucosidase concentration. The results suggest that the solid effect is mainly controlled by product inhibition under the given experimental conditions (washed pretreated corn stover as substrate). Cellobiose was found to be approximately 15 times more inhibitory than glucose on a molar scale. However, considering that glucose concentrations are at least 100 times higher than cellobiose concentrations under industrial conditions, glucose inhibition of cellulases is suggested to be the main cause of the solid effect.

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.

In the present investigation, we have investigated the effect of zinc oxide nanoparticles (ZnONP) on the production of β-glucosidase (BGL) in Saccharomyces cerevisiae under various conditions. ZnONP was synthesized chemically and characterized using various standard techniques. The results revealed that yeast culture administered with 5 mM ZnONP enhanced the intracellular BGL activity up to 28 % compared to control with simultaneous growth of cells. However, at a higher dose of ZnONP (10 and 15 mM), both the activity of the enzyme and yeast growth was dropped. When yeast cells were grown in alcoholic medium (2, 5, and 10 % ethanol), the growth was found inhibited with substantial reduction of intracellular BGL activity. Interestingly, the administration of ZnONP further inhibited the cell growth, however, suppressed the alcoholic effect on enzyme activity. Moreover, under the same condition, ZnONP enhanced the biological activity of the enzyme in cells, indicated a higher yield of BGL production. When the mechanism of ZnONP-mediated cell growth inhibition was investigated, N-acetylcysteine (NAC)-based cell growth study proved that reactive oxygen species (ROS) was not the sole cell death mechanism induced by ZnONP, indicating a second mechanism of cell death. Our findings provide a new insight on the potential application of ZnONP as an external supplement to enhance the active production of BGL like important industrial enzyme in S. cerevisiae in both normal and alcohol stressed condition as well as to produce baker’s yeast in higher amount.

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-D-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K M = 0.24 ± 0.01 mmol L(-1)) and catalytic efficiency (k cat/K M = 1,304.92 ± 53.32 L mmol(-1) s(-1)). The activity was insensitive to Fe(+3), Cr(+2), Mn(+2), Co(+2), and Ni(2+), and 50-60 % residual activities were retained in the presence of Pb(2+), Hg(2+), and Cu(2+). Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24-48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.

As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44 ± 0.16 IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34 kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, β-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2 IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement.

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.

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0-12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO(2) and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20-1:50) were also tested. Ammonia pretreatment assisted by TiO(2)-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid=1:20, photocatalyst/biomass=1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH(3)-TiO(2)-treated solid and 82 % for glucan and 77 % for xylan with NH(3)-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2-13 % improvement over ammonia pretreatment alone.

Many different materials have been tested for β-glucosidases immobilization. Such materials, however, often show a poor activity related to a low surface area of the support or even enzyme hindrance caused by entrapment inside porous matrix. In this context, the use of nanosized zeolites as enzymes support is quite new and may be an interesting alternative. The present work evaluates the immobilization of β-glucosidases in nanosized silicalites by covalent coupling. The new biocatalyst was able to convert 100% of cellobiose into glucose in 18 h at 50 °C and pH 5, retaining 85% of its activity after five cycles of reuse. A detailed investigation of the published literature indicates that, apparently, this is the first work concerning the immobilization of β-glucosidases on nanosized zeolites ever reported.

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91–102 % increase in total phenolic contents and 53–65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

Hydrolytic enzymes were evaluated on the lipid accumulation via an oleaginous fungal species, Mortierella isabellina, cultivated on sugars released from soybean hulls. The weight loss of soybean hull, fungal growth, and lipid production were tested under different loads of hydrolytic enzymes. M. isabellina could not directly utilize cellulose and adding cellulase and β-glucosidase significantly increased the cell growth and oil accumulation of M. isabellina on soybean hulls. The highest weight loss of soybean hulls was 47.80 % and the lipid production reached 0.14 g from 1 g of soybean hull when 12 U cellulase, 27.2 U β-glucosidase, 2,278.56 U pectinase, and 15 U hemicellulase were added. Fatty acids (76.82 %) accumulated in M. isabellina were C16 and C18, which are suitable for biodiesel production. These results provide a new application for soybean hulls to be applied as the raw material for the production of biodiesel fuel, besides its traditional role as animal feed supplements.

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R(o)), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of β-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular β-glucosidase production was studied in submerged fermentation. At 30 °C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of β-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major β-glucosidase (Bgl3A) with the apparent molecular weight of ~120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 °C, and exhibited a half-life of 1 h at 60 °C. K m of Bgl3A was found to be 0.057 mM with p-nitrophenyl β-d-glucoside and V max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K i of 1.5 mM. Bgl3A readily hydrolyzed glucosides with β-linkage. Bgl3A activity was enhanced (156%) by Zn2+ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.

A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-D: -glucopyranoside (pNP-Glc) with higher efficiency (K (cat)/K (m) = 2.27 x 10(4) M(-1) s(-1)) as compared to p-nitrophenyl β-D: -galactopyranoside (pNP-Gal; K (cat)/K (m) = 1.15 x 10(4) M(-1) s(-1)). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30-65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min(-1). Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.

Previous studies have shown that oxidative lime pretreatment is an effective delignification method that improves the enzymatic digestibility of many biomass feedstocks. The purpose of this work is to determine the recommended oxidative lime pretreatment conditions (reaction temperature, time, pressure, and lime loading) for Alamo switchgrass (Panicum virgatum). Enzymatic hydrolysis of glucan and xylan was used to determine the performance of the 52 studied pretreatment conditions. The recommended condition (110°C, 6.89 bar O(2), 240 min, 0.248 g Ca(OH)(2)/g biomass) achieved glucan and xylan overall yields (grams of sugar hydrolyzed/100 g sugar in raw biomass, 15 filter paper units (FPU)/g raw glucan) of 85.9 and 52.2, respectively. In addition, some glucan oligomers (2.6 g glucan recovered/100 g glucan in raw biomass) and significant levels of xylan oligomers (26.0 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor. Combining a decrystallization technique (ball milling) with oxidative lime pretreatment further improved the overall glucan yield to 90.0 (7 FPU/g raw glucan).

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.

After screening with 0.1% esculoside and 0.03% FeCl(3), we identified from rotten wood a fungal isolate HML0366 that produces high amount of β-glucosidase. Phenotypic and rDNA internal transcribed spacer sequence analyses indicated that the isolate belongs to Aspergillus oryzae. The β-glucosidase produced by HML0366 had an activity of 128 U/g. high performance liquid chromatography analysis also demonstrated a high transglycosylation activity of the crude enzyme. The β-glucosidase was stable between pH 4-10 at 60 °C. A gentiobiose yield of 30.86 g/L was achieved within 72 h of the enzymatic reaction at pH 5 and 55 °C using 50% glucose as the substrate. For the first time, we report here the isolation of an A. oryzae strain producing β-glucosidase with high hydrolytic activities. The crude enzyme has a high transglycosylation activity, which enables the enzymatic synthesis of gentiooligosaccharides.

Diminishing fossil fuel reserve and increasing cost of fossil hydrocarbon products have rekindled worldwide effort on conversion of lignocellloloses (plant biomass) to renewable fuel. Inedible plant materials such as grass, agricultural, and logging residues are abundant renewable natural resources that can be converted to biofuel. In an effort to mimic natural cellulolytic-xylanolytic microbial community in bioprocessing of lignocelluloses, we enriched cellulolytic-xylanolytic microorganisms, purified 19 monocultures and evaluated their cellulolytic-xylanolytic potential. Five selected isolates (DB1, DB2, DB7, DB8, and DB13) were used to compose a defined consortium and characterized by 16S ribosomal RNA gene sequence analysis. Nucleotide sequence blast analysis revealed that DB1, DB2, DB7, DB8, and DB13 were respectively similar to Pseudoxanthomonas byssovorax (99%), Microbacterium oxydans (99%), Bacillus sp. (99%), Ochrobactrum anthropi (98%), and Klebsiella trevisanii (99%). The isolates produced an array of cellulolytic-xylanolytic enzymes (filter paper cellulase, β-glucosidase, xylanase, and β-xylosidase), and significant activities were recorded in 30 min. Isolates DB1 and DB2 displayed the highest filter paper cellulase: 27.83 and 31.22 U mg⁻¹, respectively. The highest β-glucosidase activity (18.07 U mg⁻¹) was detected in the culture of isolate DB1. Isolate DB2 produced the highest xylanase activity (103.05 U mg⁻¹), while the highest β-xylosidase activity (7.72 U mg⁻¹) was observed with DB13. Use of microbial consortium in bioprocessing of lignocelluloses could reduce problems such as incomplete synergistic enzymes, end-product inhibition, adsorption, and requirement for high amounts of enzymes in direct use of enzymes.

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2(3) central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60-90°C). The delignification was highly dependent on temperature and duration of pretreatment.

Conversion of lignocellulosic substrates is limited by several factors, in terms of both the enzymes and the substrates. Better understanding of the hydrolysis mechanisms and the factors determining their performance is crucial for commercial lignocelluloses-based processes. Enzymes produced on various carbon sources (Solka Floc 200, lactose and steam-pre-treated corn stover) by Trichoderma reesei Rut C30 were characterised by their enzyme profile and hydrolytic performance. The results showed that there was a clear correlation between the secreted amount of xylanase and mannanase enzymes and that their production was induced by the presence of xylan in the carbon source. Co-secretion of α-arabinosidase and α-galactosidase was also observed. Secretion of β-glucosidase was found to be clearly dependent on the composition of the carbon source, and in the case of lactose, 2-fold higher specific activity was observed compared to Solka Floc and steam-pre-treated corn stover. Hydrolysis experiments showed a clear connection between glucan and xylan conversion and highlighted the importance of β-glucosidase and xylanase activities. When hydrolysis was performed using additional purified β-glucosidase and xylanase, the addition of β-glucosidase was found to significantly improve both the xylan and glucan conversion.

Response surface methodology was used to optimize the fermentation medium for enhancing naringinase production by Staphylococcus xylosus. The first step of this process involved the individual adjustment and optimization of various medium components at shake flask level. Sources of carbon (sucrose) and nitrogen (sodium nitrate), as well as an inducer (naringin) and pH levels were all found to be the important factors significantly affecting naringinase production. In the second step, a 22 full factorial central composite design was applied to determine the optimal levels of each of the significant variables. A second-order polynomial was derived by multiple regression analysis on the experimental data. Using this methodology, the optimum values for the critical components were obtained as follows: sucrose, 10.0%; sodium nitrate, 10.0%; pH 5.6; biomass concentration, 1.58%; and naringin, 0.50% (w/v), respectively. Under optimal conditions, the experimental naringinase production was 8.45 U/mL. The determination coefficients (R 2) were 0.9908 and 0.9950 for naringinase activity and biomass production, respectively, indicating an adequate degree of reliability in the model.

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast 1.5 L, from Trichoderma reesei, supplemented with a beta-glucosidase, Novozym 188, from Aspergillus niger. Addition of glucose (0-40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48-72 h of reaction recovered from 20-40% to become approximately 70% of the rate recorded during 6-24 h of reaction. Although Michaelis-Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis-Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis-Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.

beta-Glucosidase isolated from sweet almond catalyzed syntheses of water soluble retinol glycosides were carried out in SCCO(2) media with carbohydrates-D-glucose 2, D-galactose 3, D-mannose 4, D-fructose 5, and D-sorbitol 6. Retinol glycosides yields were in the 9-34% range. Reaction with D-fructose 5 gave a highest yield of 34%. Excellent regioselectivity was observed with D-mannose 4 and D-sorbitol 6 which gave exclusively C1beta-mannoside and C1-D-sorbitolide.

The stability of the mixed enzyme preparation Naringinase from Penicillium decumbens was studied in dependence of the temperature, the pH value, and the enzyme concentration by means of response surface methodology. Deactivation kinetics by formation of an intermediate state was proposed for fitting deactivation data. Empirical models could then be constructed for prediction of deactivation rate constants, specific activity of intermediate state, and half-life values under different incubation conditions. From this study, it can be concluded that (1) Naringinase is most stable in the pH range of 4.5-5.0, being quite sensitive to lower pHs (3.5) and (2) the glyco-enzyme is a rather thermo-stable enzyme preserving its initial activity for long times when incubated at its optimal pH up to temperatures of 65 degrees C. Enriched alpha-L-rhamnosidase after column treatment and ultrafiltration presented similar deactivation kinetics pattern and half-life values as the unpurified enzyme. Thus, any influence of low molecular weight substances on its deactivation is most probably negligible. The intermediate state of the enzyme may correspond to unfolding and self-digestion of its carbohydrate portion, lowering its activity relative to the initial state. The digestion- and unfolding-grade of this intermediate state may also be controlled by the pH and temperature of incubation.

The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and beta-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691-696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197-201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538-542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267-269, 1995). In this work, two forms of beta-glucosidase (beta-glu 1 and beta-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. beta-glu 1 and beta-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of delta-gluconolactone. They were also significantly inhibited by Hg(2+) and Cu(2) at 2 mM. The addition of purified enzymes to the poor beta-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H(3)P0(4) swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, beta-glu 1 and beta-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.

The objective of this study was to evaluate the thermostability up to 70 degrees C for 1 h of selected enzymes present in fresh and composted sewage sludge (SS and SSC) or municipal solid wastes (MSW and MSWC) and their humic extract. After a thermal treatment at 70 degrees C, no beta-glucosidase activity in any humic extract was detected, whereas in SS, SSC, MSW, and MSWC, it was respectively, 35%, 68%, 17%, and 12% compared to thermally untreated samples. By contrast, o-diphenol oxidase activity was even stimulated by thermal treatment in SS samples, but in the humic extracts, this activity decreased by 75-81%. Urease activity in all humic extracts decreased by 70% or more just at 40 degrees C, whereas for organic wastes, this decrease was observed after treatment at 70 degrees C. Alkaline phosphatase (AP) activity was affected by thermal treatment only in MSW and MSWC. In humic extracts, AP activity decreased gradually to zero except for the MSW extract, where 45% activity was retained after treatment at 70 degrees C. In general, thermostability of enzymes in humic extracts was lower than the materials they were extracted from.

Research is needed to allow more efficient processing of lignocellulose from abundant plant biomass resources for production to fuel ethanol at lower costs. Potential dedicated feedstock species vary in degrees of recalcitrance to ethanol processing. The standard dilute acid hydrolysis pretreatment followed by simultaneous sacharification and fermentation (SSF) was performed on leaf and stem material from three grasses: giant reed (Arundo donax L.), napiergrass (Pennisetum purpureum Schumach.), and bermudagrass (Cynodon spp). In a separate study, napiergrass, and bermudagrass whole samples were pretreated with esterase and cellulose before fermentation. Conversion via SSF was greatest with two bermudagrass cultivars (140 and 122 mg g(-1) of biomass) followed by leaves of two napiergrass genotypes (107 and 97 mg g(-1)) and two giant reed clones (109 and 85 mg g(-1)). Variability existed among bermudagrass cultivars for conversion to ethanol after esterase and cellulase treatments, with Tifton 85 (289 mg g) and Coastcross II (284 mg g(-1)) being superior to Coastal (247 mg g(-1)) and Tifton 44 (245 mg g(-1)). Results suggest that ethanol yields vary significantly for feedstocks by species and within species and that genetic breeding for improved feedstocks should be possible.

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, beta-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH(3): 1 biomass, 100 degrees C, 30 min) or with NH(4)OH (0.5 g NH(4)OH of a 28% [v/v] per gram dry biomass; 160 degrees C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and beta-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.

To develop functional enzymes in cellulose hydrolysis at or above 70 degrees C the cellobiohydrolase (CBHI/Cel7A) of Thermoascus aurantiacus was cloned and expressed in Trichoderma reesei Rut-C30 under the strong cbh1 promoter. Cellulase production of the parental strain and the novel strain (RF6026) was examined in submerged fermentation experiments using various carbon sources, which were lactose, Solka Floc 200 cellulose powder, and steam pretreated corn stover. An industrially feasible production medium was used containing only distiller's spent grain, KH(2)PO(4), and (NH(4))(2)SO(4). Enzyme production was followed by measurements of protein concentration, total cellulase enzyme activity (filter paper activity), beta-glucosidase activity, CBHI activity, and endogenase I (EGI) activity. The Thermoascus CBHI/Cel7A activity was taken as an indication of the heterologous gene expression under the cbh1 promoter.

Barley is an abundant crop in Europe, which makes its straw residues an interesting cellulose source for ethanol production. Steam pretreatment of the straw followed by enzymatic hydrolysis converts the cellulose to fermentable sugars. Prior to pretreatment the material is impregnated with a catalyst, for example, H2SO4, to enhance enzymatic digestibility of the pretreated straw. Different impregnation techniques can be applied. In this study, soaking and spraying were investigated and compared at the same pretreatment condition in terms of overall yield of glucose and xylose. The overall yield includes the soluble sugars in the liquid from pretreatment, including soluble oligomers, and monomer sugars obtained in the enzymatic hydrolysis. The yields obtained differed for the impregnation techniques. Acid-soaked barley straw gave the highest overall yield of glucose, regardless of impregnation time (10 or 30 min) or acid concentration (0.2 or 1.0 wt%). For xylose, soaking gave the highest overall yield at 0.2 wt% H2SO4. An increase in acid concentration resulted in a decrease in xylose yield for both acid-soaked and acid-sprayed barley straw. Optimization of the pretreatment conditions for acid-sprayed barley straw was performed to obtain yields using spraying that were as high as those with soaking. For acid-sprayed barley straw the optimum pretreatment condition for glucose, 1.0 wt% H2SO4 and 220 degrees C for 5 min, gave an overall glucose yield of 92% of theoretical based on the composition of the raw material. Pretreatment with 0.2 wt% H2SO4 at 190 degrees C for 5 min resulted in the highest overall xylose yield, 67% of theoretical based on the composition of the raw material.