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In the present work, the immobilized footprint of β-glucosidase (BGL) on silica-coated iron oxide was explored to produce reusable catalysts with flexible active sites for high activity and heightened storage stability. Synthesized iron oxide particles were coated with silica and functionalized with various densities of (3-aminopropyl)triethoxysilane (APTES) to obtain particles with amine densities ranging from 0 to 3 × 10

A combined intake of cooked sweet potato and fried onion in humans was found to suppress the increase of plasma quercetin metabolite concentration. Experiments using rat β-glucosidase indicated that excess carbohydrate digestion products, especially glucose-containing saccharides, interfere with the deglycosylation of quercetin glucosides during intestinal epithelial uptake. Combined meals of sweet potato and onion may lower the bioavailability of onion quercetin glucosides.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78 kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-β-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50 °C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5 mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1 M of glucose, displaying its outstanding glucose tolerance. The K

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) β-glycosidase that has β-glucosidase, β-fucosidase, and β-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The β-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: • Metagenomic glycosidase, MeBglD2, recognizes various saccharides • Structures of metagenomic MeBglD2 complexed with various saccharides are determined • MeBglD2 has a unique substrate recognition mechanism at the positive subsites.

Abstract An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases. Key points • Strain A9 can secrete a thermostable β-glucosidase that has high glucose tolerance • A coculture of strain A9 and C. thermocellum showed high cellulose degradation • Strain A9 achieves biological saccharification without addition of β-glucosidase

β-Glucosidase is a key enzyme that hydrolyzes nonvolatile glycosylated precursors of aroma compounds and enhances the organoleptic quality of wines. In this study, a novel β-glucosidase from Hanseniaspora uvarum Yun268 was localized, purified, and characterized. Results indicated that β-glucosidase activity was mainly distributed within the cells. After purification via ammonium sulfate precipitation combined with chromatography, β-glucosidase specific activity increased 8.36 times, and the activity recovery was 56.90%. The enzyme had a molecular mass of 74.22 kDa. It has a Michaelis constant (K

In this work, a convenient and dual-signal readout optical sensing platform for the sensitively and selectively determination of beta-glucosidase (β-Glu) activity was reported using protein-inorganic hybrid nanoflowers [BSA-Cu

Enzyme immobilization provides ideal operating conditions for enzymes stabilization and sustainable recycling. In this work, as a kind of clay material, montmorillonite (MTL) was chosen for immobilizing the β-glucosidase extracted from Agrocybe aegirit. The immobilized β-glucosidase via partly cross-linking enzyme aggregates (pCLEAs) formed by self-catalysis provided biocatalysts with satisfactory thermal and pH stability. Compared to the glutaraldehyde cross-linked, the immobilized β-glucosidase (β-G-pCLEAs@MTL) exhibited significantly higher immobilization efficiency (IE) and immobilization yield (IY), which were 80.6% and 76.9%, respectively. The β-G-pCLEAs@MTL also showed better stability and preferable reusability. And the activity of the β-G-pCLEAs@MTL remained 85.0% after 5 cycles and 74.7% after 10 cycles. Therefore, the method based on the pre- crosslinking to form pCLEAs and after-immobilization can effectively improve IY and IE. In addition, MTL seems to be a good alternative carrier to immobilize other enzymes for industrial application.

A liposome based nanosensor Lipo-1 for the detection of multianalytes such as copper, cyanide and ATP has been described. The binding performance of Bzimpy changed and tuned in the liposome Lipo-1 system, compared to homogeneous solution.

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen or herbivore attack. Via co-expression analysis with public transcriptomes, we determined that the model legume Medicago truncatula has evolved a two-component system composed of a β-glucosidase, denominated G1, and triterpene saponins, which are physically separated from each other in intact cells. G1 expression is root-specific, stress-inducible, and coregulated with that of the genes encoding the triterpene saponin biosynthetic enzymes. However, the G1 protein is stored in the nucleolus and is released and united with its typically vacuolar-stored substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates. We dub this two-component system the saponin bomb, in analogy with the mustard oil and cyanide bombs, commonly used to describe the renowned β-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named

Background Beta-Glucosidases (3.2.1.21) play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. However, the full potential and different applications of recombinant high-yield microbial β-glucosidase-producing systems remain to be tackled. Results A β-glucosidase gene designated as Mg132 was isolated from a coral microorganism by high-throughput sequencing and functional screening. The deduced amino acid sequences of Mg132 showed a highest identity of 97% with β-glucosidase predicted in the GenBank database. This gene was cloned and overexpressed in Escherichia coli BL21 (DE3) for the first time. The optimal pH and temperature of purified recombinant Mg132 were 8.0 and 50 °C, respectively. It exhibited a high level of stability at high concentration of glucose and ethanol, and glucose concentrations below 300 mM distinctly stimulated p-nitrophenyl-β-D-glucopyranoside hydrolysis, reaching 200% at 15% ethanol. The Km and Vmax values were 0.293 mM and 320 μmol min-1 mg-1 , respectively, while using p-nitrophenyl-β-D-glucopyranoside as a substrate. Wine treated with Mg132 had an obvious positive catalytic specificity for glycosides, which give a pleasant flavor of temperate fruity and floral aromas. The total concentration of fermentative volatiles was 201.42 ± 10.22 μg·L-1 following Mg132 treatment and 99.21 ± 7.72 μg·L-1 in control samples. Conclusion Good tolerance of winemaking and aroma fermentative properties suggest that Mg132 has potential application in aroma enhancement in wine for the further study. This article is protected by copyright. All rights reserved.

Plants possess many glycoside hydrolase family 1 (GH1) β-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high kcat/Km not only on scopolin, but also on various β-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 Å resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.

During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT. In summary, we show that within the root meristem, xylem cells act as a local organizer of vascular development by non-autonomously regulating cytokinin levels in neighbouring procambium cells via sequential induction and repression modules.

Ginsenosides are the main active components of ginseng, including many types and different contents. Among them, minor ginsenosides have better biological functions and pharmacological activities than those of the major ginsenosides. However, minor ginsenosides cannot be obtained in large quantities, but by means of enzymatic transformation technology, some major ginsenosides can be de-glycosylated at a specific position to generate minor ginsenosides. In this study, we report two glycosidase genes associated with the conversion of ginsenoside Rd to ginsenosides F

To illustrate the effects of long-term straw returning on the fungal community, soil enzyme activity, and crop yield in a fluvo-aquic soil area typical of the Huang-Huai-Hai Plain, a 10-year field experiment (established in 2010) located in Dezhou City, Shandong province, was performed, including three fertilization regimes (NF, no fertilization control; NPK, fertilization with chemical N, P, and K fertilizers; NPKS, straw returning combined with chemical N, P, and K fertilizers). This study aimed to explore the regulation mechanisms of fungal communities on soil fertility, enzyme activities, and crop yield by employing co-occurrence network and structural equation model analyses. Our results showed that long-term straw returning significantly improved soil nutrients, enzyme activity, and wheat yield. Compared with the NPK and NF treatments, soil organic matter (SOM) increased by 9.20% and 34.75%, alkali-hydrolyzed nitrogen (AN) increased by 12.03% and 39.17%, dehydrogenase (DHA) increased by 37.21% and 50.91%

The utilization of sugarcane bagasse (SB) in fermentation requires pretreatment processes to render fermentable components available to microorganisms. Pretreatment by using ionic liquids (ILs) is considered promising but the high cost is an impediment in its adoption, therefore, a mixture of IL pretreated and untreated SB was utilized to obtain bacterial multienzyme under solid-state fermentation (SSF). Bacillus aestuarii UE25, a thermophilic strain was utilized for that purpose. Fermentation conditions were optimized by adopting a central composite design. The model showed a good correlation between the predicted and the experimental values for amylase, xylanase, endoglucanase, and β-glucosidase. Volumetric and specific productivity of xylanase (4580 IU ml-1 h-1 , 244.25 IU mg-1 substrate, and 50 IU mg-1 protein) were higher than the other enzymes. Changes in lignin content and reduced cellulose crystallinity due to IL pretreatment, followed by fermentation, were visualized by scanning electron microscopy, Fourier transform infrared spectroscopy, and Nuclear magnetic resonance. The strategy adopted by utilizing a mixture of IL pretreated and untreated SB under SSF proved promising to obtain high titers of different enzymes simultaneously. Since the bacterial strain used is thermophilic, therefore, the multienzyme can find its application in commercial processes which are carried out at high temperatures.

A novel β-glucosidase was purified from pumpkin (Cucurbita moschata) seed by anion exchange chromatography and gel permeation chromatography, and its molecular mass was determined to be 42.8 kDa by gel permeation chromatography. The heterodimeric structure consisting of two subunits, free from disulfide bonds, was determined by native-PAGE analysis followed by zymography. The enzyme was maximally active at pH 4.0 and 70°C, and Vmax, Km, and kcat values were 0.078 units mg−1 protein, 2.22 mM, and 13.29 min−1, respectively, employing p-nitrophenyl-β- d -glucopyranoside as the substrate. The high content of glycine determined by amino acid analysis implies that the enzyme possesses flexible conformations and interacts with cell membranes and walls in nature. Circular dichroism studies revealed that the high stability of the enzyme within the pH range of 2.0–10.0 is due to its reversible pH-responsive characteristics for α-helix–antiparallel β-sheet interconversion.

A visible and sensitive assay for the quantitative detection of β-glucosidase (β-glu) activity based on Au@CeO

Enzyme-like catalysts by design have been a long sought-after goal of chemists but difficult to realize due to the challenges in the construction of multifunctionalized active sites with accurately positioned catalytic groups for complex substrates. Hydrolysis of cellulose is a key step in biomass utilization and requires multiple enzymes to work in concert to overcome the difficulty associated with hydrolyzing the recalcitrant substrate. We here report methods to construct synthetic versions of these enzymes through covalent molecular imprinting and strategic postmodification of the imprinted sites. The synthetic catalysts cleave a cellulose chain endolytically at multiple positions or exolytically from the nonreducing end by one or three glucose units at a time, all using the dicarboxylic acid motif found in natural cellulases. By mimicking the endocellulase, exocellulase, and β-glucosidase, the synthetic catalysts hydrolyze cellulose in a synergistic manner, with an activity at 90 °C in pH 6.5 buffer more than doubled that of

Poor management of crop residues leads to environmental pollution and composting is a sustainable practice for addressing the challenge. However, knowledge about composting with pure crop straw is still limited, which is a novel and feasible composting strategy. In this study, pure corn straw was in-situ composted for better management. Community structure of β-glucosidase-producing microorganisms during composting was deciphered using high-throughput sequencing. Results showed that the compost was mature with organic matter content of 37.83% and pH value of 7.36 and pure corn straw could be composted successfully. Cooling phase was major period for cellulose degradation with the highest β-glucosidase activity (476.25 μmol·p-Nitr/kg·dw·min) and microbial diversity (Shannon index, 3.63; Chao1 index, 500.81). Significant compositional succession was observed in the functional communities during composting with Streptomyces (14.32%), Trichoderma (13.85%) and Agromyces (11.68%) as dominant genera. β-Glucosidase-producing bacteria and fungi worked synergistically as a network to degrade cellulose with Streptomyces (0.3045**) as the key community revealed by multi-interaction analysis. Organic matter (-0.415***) and temperature (-0.327***) were key environmental parameters regulating cellulose degradation via influencing β-glucosidase-producing communities, and β-glucosidase played a key role in mediating this process. The above results indicated that responses of β-glucosidase-producing microorganisms to cellulose degradation were reflected at both network and individual levels and multi-interaction analysis could better explain the relationship between variables concerning composting cellulose degradation. The work is of significance for understanding cellulose degradation microbial communities and process during composting of pure corn straw.

Icaritin is a rare and high-value isopentane flavonoid compound with remarkable activities. Increasing yields while reducing cost has been a great challenge in icaritin production. Herein, we first reported a high titer icaritin biosynthesis strategy from epimedin C through co-immobilizing α-l-rhamnosidase (Rha1) and β-glucosidase (Glu4) using cross-linked enzyme aggregates (CLEAs). The created CLEAs exhibited excellent performances in terms of catalytic activity, thermal stability, pH stability, and reusability. Notably, Rha1-CLEAs (

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by β-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable β-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of β-glucosidase, the whole genome of

Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl beta-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.

Bermudagrass (Cynodon spp.) is one of the most widely distributed warm-season grasses globally. The growth habits and plant type of bermudagrass are strongly associated with the applied purpose of the landscape, livestock, and eco-remediation. Therefore, persistent efforts are made to investigate the genetic basis of plant type and growth habits of bermudagrass. Here, we dissect the genetic diversity of 91 wild bermudagrass resources by genome-wide association studies (GWAS) combined with weighted gene co-expression analysis (WGCNA). This work is based on the RNA-seq data and the genome of African bermudagrass (Cynodon transvaalensis Burtt Davy). Sixteen reliable single-nucleotide polymorphisms (SNPs) in transcribed regions were identified to be associated with the plant height and IAA content in diverse bermudagrass by GWAS. The integration of the results from WGCNA indicates that beta-glucosidase 31 (CdBGLU31) is a candidate gene underlying a G/A SNP signal. Furthermore, both qRT-PCR and correlation coefficient analyses indicate that CdBGLU31 might play a comprehensive role in plant height and IAA biosynthesis and signal. In addition, we observe lower plant height in Arabidopsis bglu11 mutants (homologs of CdBGLU31). It uncovers the breeding selection history of different plant types from diverse bermudagrass and provides new insights into the molecular function of CdBGLU31 both in plant types and in IAA biosynthetic pathways.

Due to the poor enzyme thermal stability, the efficient conversion of high crystallinity cellulose into glucose in aqueous phase over 50 °C is challenging. Herein, an enzyme-induced MOFs encapsulation of β-glucosidase (β-G) strategy was proposed for the first time. By using various methods, including SEM, XRD, XPS, NMR, FTIR and BET, the successful preparation of a porous channel-type flower-like enzyme complex (β-G@MOFs) was confirmed. The prepared enzyme complex (β-G@MOFs) materials showed improved thermal stability (from 50 °C to 100 °C in the aqueous phase) and excellent resistance to ionic liquids (the reaction temperature was as high as 110 °C) compared to the free enzyme (β-G). Not only the catalytic hydrolysis of cellulose by single enzyme (β-G) in ionic liquid was realized, but also the high-temperature continuous reaction performance of the enzyme was significantly improved. Benefiting from the significantly improved heat resistance, the β-G@MOFs exhibited 32.1 times and 34.2 times higher enzymatic hydrolysis rate compared to β-G for cellobiose and cellulose substrates, respectively. Besides, the catalytic activity of β-G@MOFs was retained up to 86 % after five cycles at 110 °C. This was remarkable because the fixation of the enzyme by the MOFs ensured that the folded structure of the enzyme would not expand at high temperatures, allowing the native conformation of the encapsulated protein well-maintained. Furthermore, we believe that this structural stability was caused by the confinement of flower-like porous MOFs.

Background Cellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens, TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro. Results TL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including β-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and β-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and β-glucan of wheat and highland barley . Conclusions The genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and β-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and β-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.

The enzymatic pathway of xanthan depolymerization has been predicted previously; however, the β-glucosidase and unsaturated glucuronyl hydrolase in this system have not been cloned and characterized. This lack of knowledge hinders rational modification of xanthan and exploration of new applications. In this work, we report on the properties of Mibgl3, a xanthan-degrading enzyme isolated from

Glycoside hydrolase family 12 endocellulase (GH family12) plays a key role in the degradation of β-glucan and cellulose. Hyperthermostable GH family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolysis of β(1 → 4) glucosidic linkages in cellulose and β-glucan containing β(1 → 3),(1 → 4) mixed-linkages. Therefore, in the combination with the hyperthermophilic β-glucosidase from P. furiosus (BGLPf), non-crystalline cellulose and β-glucan can be degraded to glucose completely by EGPf at high temperature. X-ray crystallography and protein engineering were used to reveal how the β(1 → 4) and β(1 → 3) linkages in β-glucan substrates are recognized by the enzyme. Structural and functional analyses clarified that the active site of EGPf consists of six subsites: the reducing end subsites (+1 and + 2) recognize both β(1 → 4) and β(1 → 3) linkages of various substrates in a productive binding mode, and recognition is controlled by Trp121 and Gln208 located at subsite +2. It was also revealed that the deep cleft in subsite -4 can accommodate the torsion angles of substrates consisting of β(1 → 3),(1 → 4) mixed-linkages due to the changing tilt of the Trp62 side chain. From the structural similarity, it is proposed that the substrate specificity of family 12 endocellulases towards β(1 → 3),(1 → 4) mixed-linkage substrates are controlled by the subsites (+1, +2, and -4). Furthermore, the function of family 12 endocellulase could be improved by protein engineering method using the information of the analysis.

β-glucosidases (BGLs) belong to the group of enzymes of cellulases and acts 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 β-glucosidase from Moniliophthora perniciosa in the hydrolysis of pre-treated 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. This article is protected by copyright. All rights reserved.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. β-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa β-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of the G6P-β-glucosidase interactions have not described so far. We investigated the molecular details of G6P binding into B. polymyxa β-glucosidase A through in silico docking using the site-identification by ligand competitive saturation (SILCS) technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift towards the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of β-glucosidase A.

Background Some patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) or wheat allergy showed negative ω-5 gliadin-specific IgE test and high level of grass pollen-specific IgE. It was presumed that these patients developed allergic reaction upon cross-reaction of their IgE antibodies raised against grass pollen allergens to wheat allergens. This study aimed to clarify clinical characteristics and wheat allergens of this phenotype of WDEIA/wheat allergy, which were tentatively diagnosed as grass pollen-related wheat allergy (GPWA). Methods A total of six patients with GPWA were enrolled, and controls were 17 patients with grass pollen allergy but no episode of wheat allergy, and 29 patients with other wheat allergies: 18 with conventional WDEIA and 11 with hydrolyzed wheat protein allergy. Sensitization to wheat proteins was determined by basophil activation test (BAT). IgE-binding proteins in wheat flour were identified by immunoblotting followed by mass spectrometry. Wheat allergen-specific IgE tests were established by CAP-FEIA system. Results All the six patients with GPWA were sensitized to water-soluble wheat proteins in BAT and IgE-immunoblotting, and peroxidase-1 (35 kDa) and beta-glucosidase (60 kDa) were identified as specific IgE-binding wheat proteins. The binding of patient IgE to these proteins was inhibited by pre-incubation of patient sera with grass pollen. The peroxidase-1- and beta-glucosidase-specific IgE tests identified three and four of six patients with GPWA, respectively, but only two of 29 controls, indicating high specificity of these tests. Conclusions Peroxidase-1 and beta-glucosidase are specific wheat allergens for GPWA among grass pollen allergy and other types of wheat-induced food allergies.

6 Figuras, BACKGROUND Hydrolysis of the fruit phenolic glucosides occurring during the oil extraction process is the main biochemical reaction affecting the biosynthesis and accumulation of secoiridoid compounds in virgin olive oil. An integrated approach at the molecular, biochemical, and metabolic level was used to study the olive β‐glucosidase gene family in seven olive cultivars selected by their different phenolic profiles. RESULTS Eight β‐glucosidase genes have been identified by in silico analysis of an olive transcriptome. Their expression levels were analyzed by reverse transcription quantitative polymerase chain reaction in olive fruits at different ripening stages: I, green fruits, 16–19 weeks after flowering (WAF); II, yellow–green fruits, 22–25 WAF; III, turning fruits, 28–31 WAF; and IV, fully ripe fruits, 35–40 WAF. Gene expression was compared with the level of β‐glucosidase activity in the fruit and with the phenolic composition of fruits and oils from different olive cultivars. Phylogenetic analysis of the encoded proteins and differences found among the β‐glucosidase genes based on Gene Ontology enrichment analysis data suggests maximum involvement of two genes, OeBGLU1A and OeBGLU1B, in the phenolic composition of virgin olive oil. Positive correlation coefficients were found within each olive cultivar between OeBGLU1A and OeBGLU1B gene expression data and the phenolic content of the oil. CONCLUSION The results obtained suggest that the expression pattern of specific β‐glucosidase genes may be an accurate predictor for the phenolic content of virgin olive oil that could be used in olive breeding programs. © 2021 Society of Chemical Industry., This research was funded by the research project AGL2015‐67652‐R from the ‘Programa Estatal de I+D+i Orientada a los Retos de la Sociedad’ funded by the Government of Spain. We are grateful to Mar Pascual for her technical assistance.

Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous β-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a β-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.

The aim of this work was to determine the effect of different drying treatments: freeze-drying, room-drying, and oven-drying on the grape phenolic composition of the 'Regent' and 'Cabernet Sauvignon' varieties. After drying, the samples were grounded, submerged to ultrasound-assisted extraction, and analyzed with high-performance liquid chromatography (HPLC). This study demonstrated that the use of different drying methods significantly affects the content of polyphenols in grape skins extracts. The greatest content of anthocyanins-diglucoside was preserved using freeze-drying, i.e. 15706.86 mg/kg for 'Regent', while the content of anthocyanins-monoglucoside was best preserved by room-drying, i.e. 216778.68 and 9220.30 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively. The highest content of flavonol glycosides (2583.04 and 1429.64 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively), hydroxycinnamic acids (1303.31 and 544.88 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively), and stilbene (2321.52 and 79.36 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively) was observed in the oven-dried samples. By applying freeze-drying the most optimal content of flavan-3-ol was preserved. Contents of polyphenolic compounds in oven-dried samples after 6 months of storage were almost identical to those in the samples analyzed immediately after drying. The greatest rate of degradation was observed in the room-dried samples while it was moderate in the freezedried ones. The results of this experiment demonstrate that it is necessary to dry samples in different ways to obtain the highest content of a certain polyphenolic group of compounds. The application of a drying method is determined by the goal of the final dried product in terms of content and composition of different polyphenolic compounds; thus, the obtained results could have an application in scientific research and for commercial purposes as well., Cilj ovog rada bio je utvrditi učinak različitih postupaka sušenja: liofilizacije, sušenja u sobi i sušenja u pećnici na fenolni sastav grožđa sorata 'Regent' i 'Cabernet Sauvignon'. Nakon sušenja uzorci su usitnjeni, podvrgnuti ultrazvukom potpomognutoj ekstrakciji te potom analizirani primjenom tekućinske kromatografije visoke djelotvornosti. Ovo je istraživanje jasno pokazalo da uporaba različitih metoda sušenja značajno utječe na sadržaj polifenola u ekstraktima kožice grožđa. Najveći sadržaj antocijan-diglukozida sačuvan je liofilizacijom, 15706.86 mg/kg u slučaju 'Regenta', dok je sadržaj antocijan-monoglukozida najbolje očuvan sušenjem pri sobnoj temperaturi, 216778.68 i 9220.30 mg/kg u slučaju 'Regenta' odnosno 'Cabernet Sauvignona'. Najveći sadržaj flavonol-glikozida (2583.04 i 1429.64 mg/kg u slučaju 'Regenta' odnosno 'Cabernet Sauvignona'), hidroksicimetnih kiselina (1303.31 i 544.88 mg/kg u slučaju 'Regenta' odnosno 'Cabernet Sauvignona') i stilbena (2321.52 i 79.36 mg/kg u slučaju 'Regenta' odnosno 'Cabernet Sauvignona') zabilježen je u uzorcima sušenim u pećnici. Primjenom liofilizacije očuvan je najoptimalniji sadržaj flavan-3-ola. Sadržaj polifenolskih spojeva u uzorcima sušenim u pećnici, nakon razdoblja od 6 mjeseci skladištenja, bio je gotovo identičan sadržaju u uzorcima analiziranim neposredno nakon sušenja. Najveća razgradnja zabilježena je u uzorcima sušenim u sobi, dok je u liofiliziranim uzorcima bila umjerena. Rezultati ovog eksperimenta pokazuju da je uzorke potrebno sušiti na različite načine kako bi se dobio najveći sadržaj određenih skupina fenolnih spojeva. Primjena metode sušenja određena je konačnim ciljem osušenog proizvoda s obzirom na sadržaj i sastav različitih polifenolskih spojeva; stoga bi dobiveni rezultati mogli imati primjenu u znanstvenim istraživanjima, ali i u komercijalne svrhe.

Introduction Gaucher disease (GD) is caused by a deficiency of β-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD. Objectives This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples. Methods Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49). Results The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%–8.2% and 3.8%–10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing. Conclusions The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

In the present work, enzymes pectinase and naringinase were simultaneously co-immobilized on an eco-friendly chitosan coated magnetic nanoparticles (chitosanMNPs) by cross-linking using chitosan as a macro-molecular cross-linker. The maximum activity recovery of both enzymes in the co-immobilized form was obtained at chitosanMNPs to enzymes ratio of 1:3, 3% cross-linker concentration and 150 min cross-linking time. The synthesized MNPs before and after co-immobilization were characterized using different techniques. The prepared biocatalyst was found spherical with an average size below 200 nm and showed supermagnetic property with saturation magnetization of 38.28 emu/g. The optimum pH and temperature of both enzymes in co-immobilized form was found at 5.5 and 65 °C. The prepared biocatalyst exhibited an improved thermal stability with 1.8-fold increase in the half-life. The secondary structural analysis revealed that, prepared co-immobilized biocatalyst undergone changes in the conformational and structural rigidity due to macro-molecular cross-linker. The co-immobilized biocatalysts were evaluated for one pot clarification and debittering of grapefruit juice and found ~52% reduction in turbidity and ~85% reduction in the naringin content. The co-immobilized enzymes were recycled up to 7th cycle and can be easily stored at room temperature for 30 days retaining up to 64% and 86% residual activities respectively.

β-Glycosidases, which catalyse the hydrolysis of glycoside bonds, have a wide spectrum of industrial applications. However, the reaction product glucose inhibits the activities of many β-glucosidases. Consequently, the reduced catalytic activities of the enzyme limit the industrial applications of the enzymes. For that reason, the studies dealing with maintaining the activities of the relevant enzymes at high glucose concentrations are a great interest among the researchers. In this context, herein, protein-inorganic hybrid nanoflowers were synthesized using β-glucosidase and copper ion by fast sonication method for 10 min. After characterization of synthesized nanoflowers, pH/temperature studies, glucose tolerance, anti-protease activity, recyclability and total antioxidant and total oxidative capacity levels were estimated. Accordingly, the optimum pHs of free β-glucosidase and hybrid nanoflower (β-GNF) were found to be 6 and 5, respectively, and the optimum temperature values for both hybrid nanoflowers and free enzyme were 40 °C. β-GNF exhibited better activity than free enzyme in low acidic and alkaline environment and at high temperature. The nanoflower retained nearly all (99 %) of its initial activity at all glucose concentrations (0.01, 0.05 and 0.1 mg/mL), especially at pH 5 and 6. Also, β-GNF maintained more than 90 % of initial activity at 0.01 and 0.05 mg/mL glucose at pH 4 and 7. It also displayed about 96 % high residual activity after proteinase K treatment for 3 h at 37 °C, while that of the free β-glucosidase was about 87 %. The reusability studies showed that β-GNF only lost ∼28 % of its initial activities at the end of five cycles. The hybrid nanoflowers at 5 mg/mL concentration exhibited the high total antioxidant capacity. In addition, low total oxidant capacity and oxidative stress index levels were recorded at the same concentration of the hybrid nanoflower. The findings of the present study revealed that β-GNFs may be evaluated as a candidate for various industrial applications due to its high glucose tolerance, anti-protease activity, reusability and resistance to low acidic/alkaline environment and high temperature.

To explore the influence mechanisms of polystyrene-microplastics (PS-MPs) on sewage sludge composting and put forward relevant composting adjustment strategies, a 30-day sewage sludge (SS) composting experiment was conducted by adding 0%, 0.5%, and 1% (w/w) PS-MPs. The addition of PS-MPs reduced compost temperature, microbial biomass carbon (MBC), and the degradation of volatile solids (2.6%-4.8%), and inhibited the activities of key enzymes (β-glucosidase and alkaline phosphatase) but increased urease activity in the thermophilic phase. Moreover, PS-MPs altered the relative abundance of dominant bacteria and changed the relevance of main enzymes and bacterial communities. Moreover, high levels of PS-MPs inhibited the contribution of dominant bacterial to alkaline phosphatase and β-glucosidase. Redundancy analysis revealed that PS-MPs affected the composting process mainly through reduced MBC at the mesophilic phase and temperature at the thermophilic phase. Thus, regulating MBC and temperature in specific phases could help overcome the adverse effects of PS-MPs on composting.

Bacteriophages and phage polysaccharide-degrading enzymes (depolymerases) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of bacteriophage KpV74 and phage depolymerase Dep_kpv74 specific to the hypervirulent Klebsiella pneumoniae of the K2 capsular type. The depolymerase Dep_kpv74 was identified as a specific glucosidase that cleaved the K2 type capsular polysaccharides of the K. pneumoniae by a hydrolytic mechanism. This depolymerase was effective against thigh soft tissue K. pneumoniae infection in mice without inducing adverse behavioral effects or toxicity. The depolymerase efficiency was similar to or greater than the bacteriophage efficiency. The phage KpV74 had a therapeutic effect only for treating the infection caused by the phage-propagating K. pneumoniae strain and was completely inactive against the infection caused by the K. pneumoniae strain that did not support phage multiplication. The depolymerase was effective in both cases. A mutant resistant to phage and depolymerase was isolated during the treatment of mice with bacteriophage. A confirmed one-base deletion in the flippase-coding wzx gene of this mutant is assumed to affect the polysaccharide capsule, abolishing the KpV74 phage adsorption and reducing the K. pneumoniae virulence.

To investigate the fermentative characteristics of Bacillus and lactic acid bacteria, the key microbes known to be involved in doenjang-meju (a Korean traditional fermented soybean brick) fermentation, we prepared and analyzed two sets of doenjang-meju inoculated with either Aspergillus oryzae and Bacillus velezensis (BDM) or A. oryzae and Leuconostoc mesenteroides (LDM). A large decrease in pH was observed during the early fermentation period in LDM, whereas the pH remained relatively constant in BDM. Although observed in higher levels in BDM during the early fermentation period, free sugar and amino acid contents and Aspergillus abundance were higher in LDM thereafter, which aligned with α-amylase and protease activity profiles in LDM and BDM, suggesting their association with Aspergillus. Higher levels of isoflavone aglycones and glycerol along with greater β-glucosidase and lipase activities in LDM and BDM, respectively, were suggestive of the characteristics of Leuconostoc and Bacillus, respectively. More diverse and higher amounts of volatile compounds were observed in BDM than in LDM. The α-amylase, lipase, protease, β-glucosidase, and antimicrobial activities of A. oryzae, B. velezensis, and L. mesenteroides were examined through genomic analyses and in vitro assays, which well supported the results of their fermentative characteristics in LDM and BDM.

As one of the most important conjugated mycotoxins, zearalenone-14-glucoside (Z14G) has received widespread attention from researchers. Although the metabolism of Z14G in animals has been extensively studied, the intracellular toxicity and metabolic process of Z14G are not fully elucidated. In this study, the cytotoxicity of Z14G to human ovarian granulosa cells (KGN) and the metabolism of Z14G in KGN cells were determined. Furthermore, the experiments of co-administration of β-glucosidase and pre-administered β-glucosidase inhibitor (Conduritol B epoxide, CBE) were used to clarify the mechanism of Z14G toxicity release. Finally, the human colon adenocarcinoma cell (Caco-2) metabolism model was used to verify the toxicity release mechanism of Z14G. The results showed that the IC50 of Z14G for KGN cells was 420 μM, and the relative hydrolysis rate of Z14G on ZEN was 35% (25% extracellular and 10% intracellular in KGN cells). The results indicated that Z14G cannot enter cells, and Z14G is only hydrolyzed extracellularly to its prototype zearalenone (ZEN) by β-glucosidase which can exert toxic effects in cells. In conclusion, this study demonstrated the cytotoxicity of Z14G and clarified the toxicity release mechanism of Z14G. Different from previous findings, our results showed that Z14G cannot enter cells but exerts cytotoxicity through deglycosylation. This study promotes the formulation of a risk assessment and legislation limit for ZEN and its metabolites.

OBJECTIVES: To estimate the existence of phyto-chemicals and then to determine the antidiabetic activity against α-amylase and β-glucosidase inhibition in vitro. METHODS: The study was carried out by following standard procedures. RESULTS: Phytochemicals analysis indicated the presence of different phytochemicals. The total phenolic content was 6.055 mg GAE/g and the total flavonoid content was 5.706 mg RU/g in the plant extract. The total saponins, alkaloids, and tannins contents were (0.044%), (2.88%) and (2.862 nm) respectively. α-amylase inhibition activity of Calligonum polygonoides (CP) extract was 70% with IC50 of 610 μg/mL and that of β-gluco-sidase inhibition activity was 65% with IC50 of 640 µg/mL. CONCLUSION: The findings reported for the first time the antidiabetes-promoting effects of an extract of CP in vitro, thus validating their promising anti-diabetes potential.

We investigated the effects of deleting major extracellular protease-encoding genes on cellulolytic and xylanolytic enzyme production in Aspergillus aculeatus. We first investigated the effect of prtT deletion, a positive transcription factor for extracellular protease-encoding genes in Aspergillus, on extracellular protease production in A. aculeatus. Genetic analysis indicated that among the major extracellular proteases, pepIIa and pepIIb are controlled by PrtT, but pepI is not. Thus, we generated a mutant with deletion of the two genes prtT and pepI (ΔprtTΔpepI) and one with deletion of the three genes pepI, pepIIa, and pepIIb (ΔpepIΔIIaΔIIb). Extracellular protease activities decreased in both ΔprtTΔpepI and ΔpepIΔIIaΔIIb to 3% of that in the control strain (MR12). Comparative time-course analyses indicated that endoglucanase activity in ΔprtTΔpepI increased to double that in MR12. Xylanase activities increased in both ΔprtTΔpepI and ΔpepIΔIIaΔIIb to fourfold higher than that in MR12 at maximum. β-Glucosidase activities were increased in ΔprtTΔpepI and ΔpepIΔIIaΔIIb 1.3- and 1.4-fold higher than that in MR12 at maximum, respectively. Residual activities of endoglucanase, xylanase, and β-glucosidase after 7 days of incubation at 37°C in the culture supernatant were 63%, 36%, and 48% of the original in MR12. Residual endoglucanase activities were more than 80% of the original in ΔprtT, ΔprtTΔpepI, and ΔpepIΔIIaΔIIb. Residual xylanase activities were not improved in all test strains. β-Glucosidase remained almost 97% of the original in ΔprtTΔpepI. These findings indicated that the reduction of extracellular proteases effectively improved cellulolytic and xylanolytic enzyme production and stability in A. aculeatus.

β-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant β-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant β-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower KM value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.

Background A total of 11 β-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each β-glucosidase remained unknown. Results We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. Conclusions Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the β-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

Fungal cellulases generally contain a reduced amount of β-glucosidase (BG), which does not allow for efficient cellulose hydrolysis. To address this issue, we implemented an easy co-immobilization procedure of β-glucosidase and cellulase by adsorption on wrinkled mesoporous silica nanoparticles with radial and hierarchical open pore structures, exhibiting smaller (WSN) and larger (WSN-p) inter-wrinkle distances. The immobilization was carried out separately on different vectors (WSN for BG and WSN-p for cellulase), simultaneously on the same vector (WSN-p), and sequentially on the same vector (WSN-p) in order to optimize the synergy between cellulase and BG. The obtained results pointed out that the best biocatalyst is that prepared through simultaneous immobilization of BG and cellulase on the same vector (WSN-p). In this case, the adsorption resulted in 20% yield of immobilization, corresponding to an enzyme loading of 100 mg/g of support. 82% yield of reaction and 72 μmol/min·g activity were obtained, evaluated for the hydrolysis of cellulose extracted from

A novel membraneless β-glucan/O