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Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum , Fusarium nygamai , Trichoderma capillare , and Aspergillus calidoustus , are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of β-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare , SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 μg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus . Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low β-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized β-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The β-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the β-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, β-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

To improve the naringinase production of Aspergillus tubingensis UA13, shorten the fermentation period, and verify its industrial application value, naringinase production conditions were optimized, and 5 L scale-up study in stirred tank bioreactor was carried out. Parameters, including carbon, nitrogen sources and inducer, optimal seed age, inoculum amount, temperature and pH, were adjusted and optimized in shaking flask. Keeping pH at the optimal value 6 in bioreactor, dissolved oxygen was monitored during the fermentation and the optimal stirring rate was investigated. In 5 L scale-up study, the highest naringinase activity was 72.62 U/mL, which was 1.75 times higher than that (41.52 U/mL) in shaking flask and the fermentation period was shortened by 24 h.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A novel β-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of β-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities, and K cat /K m was 5245.316 mM -1  s -1 , 2077.353 mM -1  s -1 , 1626.454 mM -1  s -1 , and 470.432 mM -1  s -1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Trichoderma reesei has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei , leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular β-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei Four types of cellular distribution patterns were observed for β-glucosidases in T. reesei : (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi. IMPORTANCE Although β-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on β-glucosidase function. This work comprehensively studies all the β-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced β-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b , cel3e , cel3f , cel3h , cel3j , cel3c , or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of β-glucosidases in Trichoderma reesei Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of β-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study., (Copyright © 2021 Pang et al.)

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg -1 enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains., (© 2020 The Society for Applied Microbiology.)

Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates., (© 2020 Wiley Periodicals LLC.)

The hydrodynamic environment in bioreactors affects the oxygen transfer rate and the shear conditions during microbial cultivations. Therefore, assessment of the effect of the hydrodynamic environment on cellular morphology can contribute to favoring the production of metabolites of interest. The aim of this work was to use image analysis in order to quantify the fragmentation of Aspergillus niger pellets in a conventional bioreactor operated using different impeller speeds, air flow rates, and impeller configurations including Rushton turbines and Elephant Ear impellers, with evaluation of the influence of the hydrodynamic environment on the production of cellulolytic enzymes. An empirical kinetic model was proposed to describe the dynamics of pellet fragmentation and quantify the shear conditions. The results showed that the agitation speed affected the dynamics of pellet fragmentation in two ways, by accelerating the damage process and by increasing the magnitude of the fragmentation. Both endoglucanase and β-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, β-glucosidase production was favored under high shear conditions, while the highest endoglucanase production occurred under low shear conditions. These findings may be useful for defining suitable systems and operating conditions for the production of metabolites including enzymes in bioreactors, as well as defining conditions that favour a specific pre-determined enzyme cocktail., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly β-malic acid) and enzymes with pectinase, β - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.

Background: The interest for finding novel β-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years., Results: In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris, purified, and characterized. The enzyme showed good efficiency on p-nitrophenyl glucopyranoside (pNPG) (K m = 3.36 ± 0.7 mM, k cat = 898.31 s -1 ), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the β-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-D-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different pNP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG)., Conclusions: In this work, we report the characterization of a novel glucotolerant 1,2-β-glucosidase, which also has a considerable activity on 1,4-β-glucosyl bonds, that has been cloned in P. pastoris, produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications.

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer β-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a β-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

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 [BF 4 ] - 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]BF 4 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%.

The anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) is currently an attractive treatment process with energy production in the form of biogas. Hydrolysis is the rate-limiting step for the anaerobic digestion of solid wastes. Thus, in the present study fungal enzymatic pretreatment of OFMSW was applied to enhance biogas production. Two enzyme cocktails rich on β-glucosidase were produced from submerged fermentation of Aspergillus niger on basal medium using OFMSW as carbon source and urea (Urea cocktail) and Ulva rigida as nitrogen source (Ulva cocktail). Ulva cocktail displayed an important effect on OFMSW solubilization. Therefore, an increase of reducing sugar concentration about 60% was obtained which was in correlation with chemical oxygen demand (COD) increase. The performance of enzymatic pretreatment on anaerobic digestion of OFMSW was studied by conducting biochemical methane potential tests. Results showed that the enzymatic pretreatment improved methane yield of OFMSW even at high solid concentration. High methane yield about 500 ml/g total volatile solid was obtained, which corresponds up to 68% enhancement over the control., (© 2019 American Institute of Chemical Engineers.)

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum ( C. thermocellum ) by cloning in an Escherichia coli ( E. coli ) expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a K m of 19.83 mM and a V max of 0.12  μ mol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C. thermocellum a potential candidate in industrial applications., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2019 Syed Shoaib Ahmed et al.)

Naringinase has high industrial importance, and the progress in naringinase research is still quite slow. The unavailability of an effective, simple screening method, which will be applicable to different microorganisms such as bacteria, fungi, and actinomycetes, is one of the main reasons for this gap. Therefore, a simple plate assay was developed for effective screening of microorganisms for naringinase by exposing to iodine vapors. This plate assay will fill the technological void for simple screening method and will lead to screen more potent industrially important naringinase-producing microorganisms., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and β-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a β-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.

Objective: Studies concerning 2b protein from Cucumovirus showed 2b to effectively repress functioning of the plant silencing complex, current study aimed whether retransformation with 2b gene able to restore silenced transgene expression in plant cells., Results: A rolB-transgenic cell culture of Vitis amurensis Rupr. that was continuously subcultured during more than 10 years and exhibited decreased transcription of the rolB transgene was retransformed with the 2b gene of Cucumovirus-NK. Three cell lines retransformed with 2b showed a significant up-regulation of rolB expression accompanied with enhancements in their stilbenes content level in more than 2,7-fold compared to parental rolB-transgenic cell line. The mentioned increase in the level of stilbenes content was due to activation of certain stilbene synthase genes expression responsible for stilbenes biosynthesis in V. amurensis cells. Restoration of rolB expression upon 2b-retransformation led to increase in the expression levels of VaSTS2-VaSTS5 and VaSTS7 isoforms., Conclusions: 2b from CMV-NK can reactivate a silenced transgene expression, even after 10 years of subcultivation, nevertheless, optimization of the methods concerning 2b introduction in plant genomes is necessary to avoid undesirable silencing effects.

Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified β-glucosidase supplementation. In this study, recombinant bacteria expressing the β-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable β-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5 U/ml of β-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the β-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74 U/ml of β-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable β-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified β-glucosidase supplementation. Our findings indicate that the direct supplementation of β-glucosidase using bacterial cells expressing β-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an R cryst and R free of 0.18 and 0.22, respectively. NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase from Rasamsonia emersonii. NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily., (open access.)

Trichoderma reesei QM9414, Trichoderma viride 3.316, Aspergillus niger M85, and Aspergillus niger M92 were screened for hydrolyzing geniposide into genipin. T. reesei was selected according to the β -glucosidase activity of the fermentation broths using geniposide as a substrate. T. reesei was immobilized by embedding method using sodium alginate as the carrier. Geniposide was hydrolyzed by immobilized T. reesei at 28°C (200 rpm) for 34 h, and the yield of genipin was 89%. The product was purified and identified by UV, IR, EIMS, and 1 H-NMR. Since there were two sets of signals in 1 H-NMR spectra, a series of experiments were performed and verified that the existence of two conformations was the main reason. Generally, biotransformation of geniposide into genipin by immobilized T. reesei provides a promising solution to the genipin production.

This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat-precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli, but at a lower yield compared to that obtained in T. reesei. The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

In producing recombinant β-glucosidase in Escherichia coli by high-cell density cultivation (HCDC), insufficient soluble oxygen is always a problem. To address it, Vitreoscilla hemoglobin (VHb) was introduced into Escherichia coli by the bicistron and T₇ promoter expression systems, to improve soluble oxygen by bacterial cells and thereby to enhance the biomass and recombinant β-glucosidase production. In the case of bicistron expression system, cell density in shaking flask reached OD₆₀₀=(4.24±0.29), 35.03% higher than that of the control without VHb. Correspondingly, the maximum activity of β-glucosidase co-expressed with VHb was (9.78±0.55) U/mL, 25.38% higher than that of the control. In a 3-L fermentor, the maximum activity of β-glucosidase was 141.23 U/mL, 35.57% higher than that of the control. In contrast, the activity of β-glucosidase co-expressed with VHb under T₇ promoter was lower than that of the control, either in flask or in fermentor. Co-expressing β-glucosidase with VHb using the bicistron expression system may improve the tolerance of E. coli to insufficient soluble oxygen and thus promote the bacterial biomass and the enzyme yield.

Cellulose is a highly available and renewable carbon source in nature. However, it cannot be directly metabolized by most microbes including Komagataella phaffii (formerly Pichia pastoris), which is a frequently employed host for heterologous protein expression and production of high-value compounds. A K. phaffii strain was engineered that constitutively co-expresses an endoglucanase and a β-glucosidase both from Aspergillus niger and an exoglucanase from Trichoderma reesei under the control of bidirectional promoters. This engineered strain was able to grow on cellobiose and carboxymethyl cellulose (CMC) but not on Avicel. However, the detected release of cellobiose from Avicel by using the produced mixture of endoglucanase and exoglucanase as well as the released glucose from Avicel by using the produced mixture of all three cellulases at 50 °C indicated the production of exoglucanase under the liquid culture conditions. The successful expression of three cellulases in K. phaffii demonstrated the feasibility to enable K. phaffii to directly use cellulose as a carbon source for producing recombinant proteins or other high-value compounds.

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.

A Gram-negative, non-motile, aerobic, and rod-shaped bacterial strain designated as BR5-28 T was isolated from the soil of a ginseng field at Baekdu Mountain Korea, and its taxonomic position was investigated using a polyphasic approach. Strain BR5-28 T grew at 10-42°C (optimum temperature, 30°C) and pH 5.5-8.5 (optimum pH, 7.0) on R2A agar medium without additional NaCl supplementation. Strain BR5- 28T exhibited β-glucosidase activity, which was responsible for its ability to transform the ginsenosides Rb1 and Rd (the two dominant active components of ginseng) to compound-K. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Mucilaginibacter of the family Sphingobacteriaceae, and formed clusters with Mucilaginibacter frigoritolerans FT22 T (95.8%) and Mucilaginibacter gotjawali SA3-7 T (95.7%). The G+C content of the genomic DNA was 45.1%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C 16:1 ω6c and/or C 16:1 ω7c), iso-C 15:0 and anteiso-C 15:0 . The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain BR5-28 T was differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter hankyongensis sp. nov. is proposed, with the type strain BR5-28 T (=KCTC 22274 T =DSM 21151 T ).

The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A novel β-glucosidase from Streptomyces griseus was cloned and overexpressed in E. coli. The purified β-glucosidase (44 kDa) had a K m of 8.6 ± 0.5 mM and a V max of 217 ± 5.0 μmoles -1 min -1 mg at 37 °C, pH 7.2 with p-nitrophenyl-β-D glucopyranoside as substrate. The enzyme was characterised in terms of pH optimum (pH 6.9), temperature optimum (69 °C) and the influence of solvents and effectors. Purified S. griseus β-glucosidase was successfully immobilised, by simple absorption, onto zinc oxide (ZnO) nanoparticles without covalent modification. It remained tightly bound even after extensive washing and could be reused up to ten times without significant loss of activity. The immobilised enzyme had a higher optimum temperature and greater thermostability than the free enzyme. In immobilised form the enzyme readily catalysed the synthesis of alkyl glucosides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An extracellular β-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified β-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the β-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of β-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of D-glucose connected by β1-4, β1-6 and β1-2 linkage, respectively. The β-glucosidase activity was strongly enhanced by ferrous ion (Fe 2+ ) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher V MAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (K S ) for cellobiose than K S of the parental strain for glucose but also 5-times lower K S than Michaelis-Menten constant (K M ) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04 mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14 mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778 U/L at 34 hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164 U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species-P. kongii, P. olsonii, and P. viticola-have not been previously recorded in Korea.

β-Glucosidase is an essential part of cellulase enzyme system for efficient and complete hydrolysis of biomass. Psychrotolerant Pseudomonas lutea BG8 produced β-glucosidase with lower temperature optima and hence can play important role in bringing down the energy requirement for bioethanol production. To enhance β-glucosidase production, two statistical tools: Taguchi and Box-Behnken designs were applied to reveal the most influential factors and their respective concentration for maximum production of β-glucosidase under submerged fermentation. The optimal medium composition for maximum β-glucosidase production were 2.99% (w/v) bagasse, 0.33% (w/v) yeast extract, 0.38% (w/v) Triton X-100, 0.39% (w/v) NaNO 3 , and pH 8.0 at temperature 30 °C. Under optimized conditions, β-glucosidase production increased up to 9.12-fold (17.52 ± 0.24 IU/g) in shake flask. Large-scale production in 7-L stirred tank bioreactor resulted in higher β-glucosidase production (23.29 ± 0.23 IU/g) within 80 H of incubation, which was 1.34-fold higher than shake flask studies. Commercial cellulase (Celluclast ® 1.5L) when supplemented with this crude β-glucosidase resulted in improved sugar release (548.4 ± 2.76 mg/gds) from paddy straw at comparatively low temperature (40 °C) of saccharification. P. lutea BG8 therefore showed great potential for cold active β-glucosidase production and can be used as accessory enzyme along with commercial cellulase to improve saccharification efficiency., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Marine macroalgae are gaining more and more importance as a renewable feedstock for durable bioenergy production, but polysaccharides of this macroalgae are structurally complex in its chemical composition. The use of enzymatic hydrolysis may provide new pathways in the conversion of complex polysaccharides to fermentable sugars. In this study, an enzymatic cocktail with high specificity was first isolated from Aspergillus niger using the green macroalgae Ulva rigida as nitrogen source. The cocktail is rich on β-glucosidase, pectinase and carboxy-methyl-cellulase (CMCase). The highest activity was obtained with β-glucosidase (109IUmL(-1)) and pectinase (76IUmL(-1)), while CMCase present the lowest activity 4.6IUmL(-1). The U. rigida pre-treatment with this enzymatic cocktail showed high rate of reduced sugar release, and could bring promising prospects for enzymatic pre-treatment of the biogas production from U. rigida biomass which reached 1175mLgCODint(-1)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Response surface methodology was used to optimize the medium composition to improve the production of the heat-tolerant β-glucosidase from Bacillus sp. ZJ1308. Three significant factors were found to be corn cob, beef extract, and MnSO4 ·H2 O. The final medium compositions optimized were corn cob (51.8 g/L), beef extract (23.8 g/L), salicin (0.5 g/L), MnSO4 ·H2 O (0.363 g/L), MgSO4 ·7H2 O (0.4 g/L), and NaCl (5 g/L). Under the optimal conditions, the activity of β-glucosidase was up to 4.71 U/mL. β-Glucosidase was purified to homogeneity with a recovery rate of 5% and a specific activity of 110.47 U/mg. The optimal pH and temperature were 7.0 and 60 °C, respectively. β-Glucosidase was stable within a pH range of 6.0-8.0 and showed an extremely high thermostability at 80 and 90 °C, retaining 56% and 38% of its maximal activity, respectively. Ni(2+) and Ba(2+) heavily inhibited the β-glucosidase activity. The purified β-glucosidase showed a high substrate specificity. The kinetic parameters revealed that it had a high catalytic efficiency toward the substrate p-nitrophenyl-β-d-glucopyranoside (Kcat /Km = 700). It also showed a high catalytic activity toward the natural substrate salicin. This study provides a new insight into the future development and use of β-glucosidase from Bacillus sp. ZJ1308., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.

Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 β-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60 °C, and had its maximum activity against p-nitrophenyl-β-d-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-β-d-cellobioside (pNPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 μM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Quercetin is a flavonol believed to have beneficial effects on human health. Rutin, found in many plants, fruits and vegetables, is metabolized by human intestinal bacteria and converted to quercetin, where it is absorbed through the intestinal epithelium. This study aimed to isolate and characterize human intestinal bacteria capable of converting rutin to quercetin. A bacterium that can metabolize rutin was isolated from human faecal samples and identified by 16S rRNA gene sequencing. The whole-cell enzymatic activities on flavonoid glycoside and the conversion profiles of the isolate were also analysed. The bacterium was identified as Enterococcus avium EFEL009 and was shown to convert rutin to isoquercetin and then to quercetin under anaerobic conditions. Microscopic analysis revealed short chains of cocci with diameters of approx. 1 μm. β-Glucosidase was shown to be constitutively expressed in Ent. avium, while α-rhamnosidase was expressed following induction by rutin. Both enzymes were mainly localized to the cell surface. This study is the first report on the isolation of a quercetin-producing Ent. avium FEEL009, which could be a potential industrial starter bacterium., Significance and Impact of the Study: Quercetin is a member of the flavonoids family reported to have better cytoprotective abilities, stronger inhibition of lipopolysaccharide-induced nitric oxide production, and better chemoprevention than rutin. This is the first report on the isolation and characterization of Enterococcus avium EFEL009 from the human intestine which is capable of converting rutin to quercetin., (© 2015 The Society for Applied Microbiology.)

Context and Objective: Long-term prognosis of hepatocellular carcinoma (HCC) patients is challenging, and novel biomarkers are needed to predict patient risk and serve as potential therapeutic target., Results: We found β-glucosidase 1 is significantly overexpressed and activated in primary HCC tissue and multiple HCC cell lines. β-Glucosidase 1 expression is associated with predicting prognosis of HCC patients under chemotherapy. Silencing β-glucosidase 1 inhibits growth and survival of HCC cells, with preferential inhibitory effects on high β-glucosidase 1-expressing cells. Combination of chemo drug with β-glucosidase 1 inhibitor sensitized HCC cells to chemotherapy., Conclusion: Our data support β-glucosidase 1 as a HCC biomarker due to its prognosis significance.

The effect of supplementation of medium with rapeseed meal (RM) on production of biotechnologically important enzymes was investigated in submerged cultures of the white rot fungus Cerrena unicolor. The addition of RM (3.5% w/v) distinctly stimulated the activities of laccase, chitinase, and β-glucosidase. As compared to the control, the activities of chitinase, β-glucosidase, and laccase in the RM supplemented cultures were up to 4.1, 8.4, and 3.9 times higher, respectively. The results of the spectrophotometric and spectrofluorometric measurements were additionally confirmed by zymographic analysis of the samples. The level of sugars and phenolic compounds as well as the antioxidative ability of fungal preparations were also determined. The results obtained indicate that the submerged liquid fermentation of rapeseed meal can be proposed as an inexpensive and very effective method for biotechnological production of chitinase, β-glucosidase, and laccase by C. unicolor.

A mathematical model to describe the kinetics of enzyme production by the filamentous fungus Trichoderma harzianum P49P11 was developed using a low cost substrate as main carbon source (pretreated sugarcane bagasse). The model describes the cell growth, variation of substrate concentration and production of three kinds of enzymes (cellulases, beta-glucosidase and xylanase) in different sugarcane bagasse concentrations (5; 10; 20; 30; 40 gL(-1)). The 10 gL(-1) concentration was used to validate the model and the other to parameter estimation. The model for enzyme production has terms implicitly representing induction and repression. Substrate variation was represented by a simple degradation rate. The models seem to represent well the kinetics with a good fit for the majority of the assays. Validation results indicate that the models are adequate to represent the kinetics for a biotechnological process., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Coffee is among the most preferred nonalcoholic drinks, and its consumption is distributed globally. During the coffee fruiting process, however, a large amount of waste is generated in the form of pulp, mucilage, husks, and water waste. The pulp and mucilage have the chemical composition to support the growth of micro-organisms and the production of value-added product. The aim was testify pulp coffee can be considered as carbon and inductor source for β-glucosidase by Bacillus subtilis CCMA 0087. The response surface methodology (RSM) based on a central composite rotatable design (CCRD) was employed for this optimization. The methodology used in the optimization process was validated by testing the best conditions obtained and comparing them with the values predicted by the model. The highest β-glucosidase production (22·59 UI ml(-1) ) was reached in 24 h of culturing at coffee pulp concentration of 36·8 g l(-1) , temperature of 36·6°C, and pH of 3·64., Significance and Impact of the Study: Countries whose economy is based on agricultural activities generate a great deal of liquid and solid waste. Thus, it is important to develop new alternatives for using this waste rather than disposing it in the environment. The production of enzymes, and particularly cellulase, is one such alternative. In this study, we proposed to produce β-glucosidase production from pulp coffee extract using a Bacillus subtilis strain., (© 2015 The Society for Applied Microbiology.)

β-Glucosidases are important enzymes with significant prospects in the industrial biotechnology, including their use in biomass hydrolysis for bioethanol production. In this study, the use of canola meal as carbon source for β-glucosidase production by a Trichoderma viride strain in submerged fermentation was evaluated by applying central composite design and response surface methodology to optimize the production process. This statistical approach was also used to improve the passion fruit peel hydrolysis by T. viride crude extract. The model developed 3.6-fold increased β-glucosidase activity. The culture conditions that resulted in the highest β-glucosidase levels were a substrate concentration of 2.9 %, pH of medium 4.2 and cultivation time of 206 h. The β-glucosidases produced under optimal conditions showed attractive properties for industrial applications, such as activity at high temperatures and stability at 55 °C and over a wide pH range. In addition, the enzymatic hydrolysis of passion fruit peel by T. viride crude extract was very promising, resulting in glucose yields of 66.4 %. This study, therefore, presents canola meal as an inexpensive and attractive substrate for the production of microbial β-glucosidases.