BGL Activity Research Articles

Culture of Trichoderma sp. with biochar to produce high-activity cellulase in a laboratory

Biochar (BC) was used in Trichoderma sp. culture to produce high-activity cellulase on a laboratory scale. The biochar was added into the flask before being applied to the fermenter to identify the enhancement effect and to determine the best amount of addition and the most suitable incubation period. Cellulase production was performed with a working volume of 4 L, and enzymatic hydrolysis was conducted to evaluate the saccharification ability of the enzyme. During incubation, the activities of three enzymes (Endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH)) were measured for three days, and the cellulase activity was determined using a filter paper unit (FPU). In flask scale, EG, BGL, and CBH activities were increased by 1.4, 2.1, and 1.8 folds, respectively, and the incubation period was shortened by adding BC. In the fermenter scale, EG, BGL, and CBH activities were noticeably enhanced by 12.1, 5.8, and 7.2 folds, respectively, and FPU was 42.1 (9.8 folds). Additionally, the conversion rates of cellulose and steam exploded softwood and hardwood were 109.4%, 75.4%, and 87.3%, which were similar to a commercial enzyme (Cellic CTecⅡ). This study demonstrated that biochar could be used to produce high-activity cellulase in a shorter period and suggests a novel method for effective cellulase production.

NaCl treatment with low density polyethylene packaging bags can enhance the aroma of fresh-cut apple, not only inhibit its browning

NaCl is regarded as a low cost and effective anti-browning agent for fresh-cut apples, but is rarely discussed on keeping flavor. In this study, the anti-browning effect of 0.1 mol/L NaCl with low density polyethylene (LDPE) packaging bags for apple was verified by lower browning degree, the total phenolic content, polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) activity and relative genes expression. The browning score and flavor evaluation of fresh-cut apples with NaCl treatment was 1.49 and 1.47 times that of the control on the 12th d, which showed NaCl treatment increased the business value of fresh-cut apples. Butanoic acid methyl ester, butanoic acid 2-methyl-, methyl ester and hexenoic acid methyl ester were markedly higher in NaCl treatment slices than the control. Additionally, the linolenic acid content increased with NaCl treatment, which was 2.3 times that of the control on the 12th day. Enzymes activity and relative genes expression in lipoxygenase (LOX) pathway including LOX, ethanol dehydrogenase (ADH), and alcohol-acyltransferase (AAT) was activated in apple slices with NaCl treatment. Among them, AAT1 expression changed the most, eight times that of the control on day 1. However, the activity and relative expression of pyruvate decarboxylase (PDC) related to ethanol synthesis was inhibited. Moreover, NaCl enhanced β‑glucosidase (BGL) activity of fresh-cut apples by 1.9 times on day 1 and its concentration in the reaction system was proportional to the BGL activity. The results indicate NaCl treatment with LDPE packaging is an effective method to maintain appearance and regulating flavor qualities of fresh-cut apples.

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach.

β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

Inactivation and process intensification of β-glucosidase in biomass utilization

Lignocellulosic biomass has emerged as a promising environmental resource. Enzyme catalysis, as one of the most environmentally friendly and efficient tools among various treatments, is used for the conversion of biomass into chemicals and fuels. Cellulase is a complex enzyme composed of β-glucosidase (BGL), endo-β-1,4-glucanase (EG), and exo-β-1,4-glucanase (CBH), which synergistically hydrolyzes cellulose into monosaccharides. BGL, which further deconstructs cellobiose and short-chain cellooligosaccharides obtained by EG and CBH catalysis into glucose, is the most sensitive component of the synergistic enzyme system constituted by the three enzymes and is highly susceptible to inactivation by external conditions, becoming the rate-limiting component in biomass conversion. This paper firstly introduces the source and catalytic mechanism of BGL used in the process of biomass resource utilization. The focus is on the review of various factors affecting BGL activity during hydrolysis, including competitive adsorption of lignin, gas-liquid interface inactivation, thermal inactivation, and solvent effect. And the methods to improve BGL inactivation are proposed from two aspects-substrate initiation and enzyme initiation. In particular, the screening, modification, and alteration of the enzyme molecules themselves are discussed with emphasis. This review can provide novel ideas for studies of BGL inactivation mechanism, containment of inactivation, and activity enhancement. KEY POINTS: • Factors affecting β-glucosidase inactivation are described. • Process intensification is presented in terms of substrate and enzyme. • Solvent selection, protein engineering, and immobilization remain topics of interest.

Microbial cellulase production and stability investigations via graphene like carbon nanostructure derived from paddy straw

Cellulases are among the most in-demand bioprocess enzymes, and the high cost of production, combined with their low enzymatic activity, is the main constraint, particularly in the biofuels industry. As a result, low-cost enzyme production modes with high activity and stability have emerged as the primary focus of research. Here, a method for producing a graphene like carbon nanostructure (GLCNs) has been investigated utilizing paddy straw (Ps), and its physicochemical characteristics have been examined using a variety of techniques including XRD, FT-IR, SEM and TEM. Further, the pretreatment of Ps feedstock for cellulase production was done using diluted waste KOH liquid collected during the preparation of the GLCNs. To increase the production and stability of the enzyme, newly prepared GLCNs is utilized as a nanocatalyst. Using 15 mg of GLCNs, 35 IU/gds FP activity was seen after 72 h, followed by 158 IU/gds EG and 114 IU/gds BGL activity in 96 h. This nanocatalyst supported enzyme was thermally stable at 70 °C up to 15 h and exhibited stability at pH 7.0 for 10 h by holding 66 % of its half-life.

Beta-Glucosidase 1 (bgl1) Gene Analysis in Mutant and Wild-type of Penicillium sp. ID10-T065

In the previous study, Penicillium sp. ID10-T065 has been mutated using ultraviolet (UV), ethyl methyl sulfonate (EMS) and the combination of UV-EMS to increase Beta-glucosidase (bgl) activity. There were three mutants selected, UV13 (UV mutant), EM31 (EMS mutant), and UM23 (UV-EMS mutant). This study examined the mutations in the bgl gene encoding (bgl1) as well as sequence differences between mutants and wild-type of Penicillium sp. ID10-T065. The gene analysis was performed by PCR amplification and sequencing of the bgl1 gene. The results of bgl1 gene sequences (600 bp) from mutants were aligned with the wild-type, it was discovered that there were base alterations from position 2025 to 2050. Mutant UV13 showed the highest base alterations (7 bases) which occurred at position 2027 (T→C); 2036 (T→G); 2040 (T→G); 2047 (G→C); and 2048-2050 (TTG→GGA). Mutant EM31 showed alterations in five bases at positions 2034 (G→A), 2036 (T→G), 2037 (G→C), 2044 (G→C), and 2047 (G→T). Mutant UM23 showed two base alterations at position 2025 (T→A) and 2037 (G→C). UV irradiation and EMS mutation resulted in transition and transversion DNA, whereas the combination of UV-EMS mutation resulted in transversion mutations. Base alterations in UV13 and EM31 mutants, causing missense and silent mutation, while in UM23 mutant only silent mutations occur. The bgl1 gene analysis showed that mutation using UV light was more effective than using EMS or a combination of UV-EMS in Penicillium sp. ID10-T065.

Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.ResultsIn this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.ConclusionsThe synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Enhanced surface display efficiency of β-glucosidase in Saccharomyces cerevisiae by disruption of cell wall protein-encoding genes YGP1 and CWP2

Engineering of Saccharomyces cerevisiae for being capable of displaying a sufficient amount of cellulases on their cell surface needs to be accomplished for the development of an ideal microorganism for cellulosic ethanol production. This study aimed to explore the effects of two cell wall protein genes, CWP2 and YGP1, on β-glucosidase (BGL) activity on the yeast cell surface. The results showed that the disruption of YGP1 and CWP2 increased BGL activity by 63% and 24%, respectively, compared to that of the original strain. Moreover, the YGP1 disruptant produced 59% more ethanol from cellobiose than the parent strain. Further transcriptome analysis of the YGP1 disruptant strain revealed changes in the expression of genes involved in cell wall structure, biogenesis, and integrity, which might contribute to the elevated BGL display efficiency. This study provides an alternative engineering approach for enhancing the cell surface display of heterologous proteins on the yeast cell wall.

Novel findings in sea cucumber's digestive capacities: Enzymatic activities in the respiratory tree, implications for aquaculture

AbstractThere is a demand for proper feed and feeding strategies in the growing sea cucumber aquaculture. The digestive system of holothurians seems to increase nutrient assimilation through the respiratory tree (RT), but digestive functioning in this organ has not been investigated. This study evaluated the enzymatic activity of the cytosolic leucine alanine peptidase (leu‐ala) and acid phosphatase, the membrane‐linked aminopeptidase‐N (APN), the bile salt‐activated lipase, laminarinase (LAM), and β‐glycosidase (BGL), in the RT and segments of the digestive tract (DT; foregut, midgut, and hindgut) of adult Isostichopus badionotus. All enzymes, except LAM and BGL, exhibited comparable activity levels in the RT and the DT segments, but APN and BGL activities were significantly higher in the whole DT compared with those in RT, with high activity of leu‐ala in both organs. These results indicate a relevant role of the RT as an accessory organ in sea cucumber digestive physiology. Consequently, I. badionotus seems to be able to digest feeds from the water column through the RT, maximizing the use of available nutrients by luminal, intracellular, and apical membrane‐linked digestive modes. These findings expand the knowledge of sea cucumber digestive mechanisms, with implications for developing feeding strategies for all life stages in aquaculture.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus β-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

β-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

β-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Enhanced Lignocellulolytic Enzyme Activities on Hardwood and Softwood during Interspecific Interactions of White- and Brown-Rot Fungi.

Wood decomposition is a sophisticated process where various biocatalysts act simultaneously and synergistically on biopolymers to efficiently break down plant cell walls. In nature, this process depends on the activities of the wood-inhabiting fungal communities that co-exist and interact during wood decay. Wood-decaying fungal species have traditionally been classified as white-rot and brown-rot fungi, which differ in their decay mechanism and enzyme repertoire. To mimic the species interaction during wood decomposition, we have cultivated the white-rot fungus, Bjerkandera adusta, and two brown-rot fungi, Gloeophyllum sepiarium and Antrodia sinuosa, in single and co-cultivations on softwood and hardwood. We compared their extracellular hydrolytic carbohydrate-active and oxidative lignin-degrading enzyme activities and production profiles. The interaction of white-rot and brown-rot species showed enhanced (hemi)cellulase activities on birch and spruce-supplemented cultivations. Based on the enzyme activity profiles, the combination of B. adusta and G. sepiarium facilitated birch wood degradation, whereas B. adusta and A. sinuosa is a promising combination for efficient degradation of spruce wood, showing synergy in β-glucosidase (BGL) and α-galactosidase (AGL) activity. Synergistic BGL and AGL activity was also detected on birch during the interaction of brown-rot species. Our findings indicate that fungal interaction on different woody substrates have an impact on both simultaneous and sequential biocatalytic activities.

Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization

Commercial applications of β-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.

Improving β-glucosidase and xylanase production in a combination of waste substrate from domestic wastewater treatment system and agriculture residues

Cellulase and hemicellulase activities are considered to the major bottlenecks in the lignocellulosic biorefinery process, especially in an enzyme cocktail lacking β-glucosidase (BGL) and xylanase (XYL). In view of this issue, higher levels of BGL and XYL activities were obtained in the presence of wastewater and activated sludge as an induction medium mixed with 5% of rice straw by Hypocrea sp. W63. The analysis of the ionic content showed that a relatively low sludge dose could enhance the production of BGL and XYL. Most importantly, compared to a medium using freshwater, the proportion of 1:10 sludge to wastewater, which contained nutrient elements, led to 3.4-fold BGL and 3.7-fold XYL production improvements. This research describes the reuse of substrates that are largely and continuously generated from domestic wastewater treatment systems and agriculture residues, which consequently leads to the development of a simultaneous enzyme production process for sustainable biorefinery practices.

Co-evolution of β-glucosidase activity and product tolerance for increasing cellulosic ethanol yield.

β-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency ([Formula: see text]) for hydrolyzing pNPG and reduced strength of inhibition ([Formula: see text]) by glucose. To be specific, L-6C achieved a [Formula: see text] of 0.35 at a glucose concentration of 20mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68mM-1s-1, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88mM) to pNGP compared with 16BGL (1.69mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL.We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.

Influence of cellulase gene expression and cellulolytic activity on cellulose utilization in different Volvariella volvacea strains

Volvariella volvacea is delicious and nutritious while the biological efficiency is low, so the price keeps at a high level all the year round in China. V. volvacea uses crop straw enriched in cellulose as a substrate. Six cellulase genes (egI, egII, cbhI-I, cbhI-II, cbhII, bgl) encoding endoglucanase (EG) (E.C. 3.2.1.4), cellobiohydrolase (CBH) (E.C. 3.2.1.91), and β-glucosidase (BGL) (E.C. 3.2.1.21) proteins, all of which restrict the conversion of cellulose to glucose, were confirmed in genome. The investigate of enzymatic activity and biological efficiency showed better cellulose-degrading enzyme activity ensured higher biological efficiency. The cellulase genes cbhI-I, cbhI-II, and bgl clustered with those from Agaricus bisporus. Other genes, namely, egI, egII, and cbhII, clustered closely with those of wood decay fungi, which have weak cellulose-degrading activity. egI expression was significantly higher in strains 0229 and Vb1 (high biological efficiency) than in strain A8 (low biological efficiency). EG activity was positively correlated with biological efficiency in V. volvacea. EG、CBH and BGL are the three crucial endo and exo cellulytic enzymes involved in the conversion of cellulose to glucose. However, there was no relationship between CBH or BGL activity and biological efficiency. The egI gene is a key gene in the cellulase system. And there is a linear correlation between EG activity and egI expression in V. volvacea.

A new enzymatic method assessing the impact of wastewater treatment plant effluents on the assimilative capacity of small rivers

Microorganisms play an important role in maintaining a good water quality in rivers by degrading organic material, including toxic substances. In the present study, we analyzed the potential impact of municipal wastewater treatment plant (WWTP) effluents as a major stress factor on the assimilative capacity of small rivers. It was the aim to develop a new bioassay for assessing such impacts in the receiving rivers by measuring the activity of extracellular enzymes (exoenzymes) in bacteria. Therefore, we established a specific in-vitro assay to detect inhibitory effects of solid phase-enriched water samples on β-glucosidase (BGL) activity of the actinobacterium Cellulomonas uda as a proxy for the microbial decomposition of organic substances and thus for the assimilative capacity of surface waters. We found significant reductions of BGL activity in the WWTP effluents and in the receiving waters directly downstream as well as a relative quick recovery over the further course of the water bodies. The new bioassay offers a promising tool for the assessment of the assimilative capacity in surface waters and a potential impact of WWTP effluents on this key ecosystem function.AbbreviationsWWTPwastewater treatment plantBGLβ-glucosidaseEU-WFDEuropean Water Framework DirectiveFAUFormazin Attenuation UnitsPEpopulation equivalentsREFrelative enrichment factor;SPEsolid phase extractionMTBEmethyl-tert-buthyl-etherDMSOdimethyl-sulfoxideNPG4-nitrophenol-β-d-glucopyranosideDOCdissolved organic carbon

Proteolytic analysis of Trichoderma reesei in celluase-inducing condition reveals a role for trichodermapepsin (TrAsP) in cellulase production.

Filamentous fungi produce a variety of proteases with significant biotechnological potential and show diverse substrate specificities. Proteolytic analysis of the industrial enzyme producer Trichoderma reesei has been sparse. Therefore, we determined the substrate specificity of T. reesei secretome and its main protease Trichodermapepsin (TrAsP) up to P1 position using FRETS-25Xaa-libraries. The role of TrAsP was analyzed using T. reesei QM9414 and the deletant QM∆trasp in Avicel. We observed higher activities of CMCase, Avicelase, and Xylanase in QM∆trasp compared to that of QM9414. Saccharification rate of cellulosic biomass also increased when using secretome of QM∆trasp but the effect was not significant due to the absence of difference in BGL activity compared to QM9414. Higher TrAsP was produced when monosaccharides were used as a carbon source compared to cellulase inducers such as Avicel and α-sophorose. These results elucidate the relationship between TrAsP and cellulase production in T. reesei and suggest a physiological role for TrAsP.

Efficient Display of Aspergillus niger β-Glucosidase on Saccharomyces cerevisiae Cell Wall for Aroma Enhancement in Wine.

The aim of this study was to evaluate the potential application of cell-surface-displayed β-glucosidase (BGL) in wine aroma enhancement. Gene cassettes for the surface display of Aspergillus niger BGL were constructed using different promoters ( GPD and SED1) and glycosylphosphatidylinositol (GPI) anchoring regions (Sag1, Sed1, and Cwp2). The differences in surface-display cassettes, the tolerance of the displayed BGL to typical winemaking conditions, and the hydrolysis capacity for the liberation of grape aroma glycosides were analyzed. Results revealed that simultaneous utilization of GPD promoter and Sed1 anchoring domain achieved the highest BGL activity. The displayed BGL exhibited relatively high activity at pH 3.0 and at glucose concentration below 2.5% (w/v), compared to commercial enzyme (AR 2000), but exhibited no significant difference under varying ethanol concentrations. Furthermore, the surface-displayed BGL presented better ability to release free terpenols compared to AR 2000. Therefore, a surface-display system could provide a new viable solution for enhancing wine aroma.

Evaluation of Cellulolytic Enzyme Production by Indigenous Fungi in Korea

The aim of this study was to select various fungal strains indigenous to Korea that have the potential to produce cellulases, including filter paper activity (FPase), endo-β-1,4-glucanase (EG), and β-glucosidase (BGL). Among the 25 species of Ascomycetes and the 32 species of Basidiomycetes tested in this study, the Bjerkandera adusta KUC10565, Heterobasidion orientale KUC10556, Hyphoderma praetermissum KUC10609, and Trichoderma harzianum KUC1716 all exhibited remarkably high FPase activity. In addition, the T. harzianum KUC1716 showed high levels of EG and BGL activity. This strain has been selected for further study because of their enzymatic potential.