Hotspot Wizard-informed engineering of a hyperthermophilic ß-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

Screening, cloning, immobilization and application prospects of a novel ß-glucosidase from the soil metagenome.

The soil environment for straw return is a rich and valuable library containing many microorganisms and proteins. In this study, we aimed to screen a high-quality ß-glucosidase (BGL) from the soil metagenomic library and to overcome the limitation of the low extraction rate of resveratrol in Polygonum cuspidatum. This includes the construction of a soil metagenomic library, screening of BGL, bioinformatics analysis, cloning, expression, immobilization, enzymatic property analysis, and application for the transformation of polydatin. The results showed that the soil metagenomic library of straw return was successfully constructed, and a novel BGL was screened. The identified 1356 bp long BGL belonged to the glycoside hydrolase 1 (GH1) family and was named Bgl1356. After successful cloning and expression of Bgl1356, it was immobilized using chitosan. The optimum temperature of immobilized Bgl1356 was 50 °C, and the pH was 5. It exhibited good tolerance for various metal ions (CO2+, Ni2+, Cu2+, Mn2+, Na2+, Ca2+, and Ag+) and organic solvents (DMSO, Triton-X-10, and ethanol). Enzymatic kinetics assays showed that Bgl1356 had good affinity for the substrate, and the specific enzyme activity was 234.03 U/mg. The conversion rate of polydatin by immobilized Bgl1356 was 95.70 ± 1.08%, facilitating the production of high amounts of resveratrol. Thus, this paper reports a novel temperature-, organic solvent-, and metal ion-tolerant BGL that has good application prospects in the pharmaceutical industry.

Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

A thermostable and inhibitor resistant ß-glucosidase from Rasamsonia emersonii for efficient hydrolysis of lignocellulosics biomass.

The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.

A comparative study on flaxseed lignan biotransformation through resting cell catalysis and microbial fermentation by ß-glucosidase production Lactiplantibacillus plantarum.

BACKGROUND: Flax lignan has attracted much attention because of its potential bioactivities. However, the bioavailability of secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, depends on the bioconversion by the colon bacteria. Lactic acid bacteria (LAB) with ß-glucosidase activity has found wide application in preparing bioactive aglycone. RESULTS: LAB strains with good ß-glucosidase activity were isolated from fermented tofu. Their bioconversion of flax lignan extract was investigated by resting cell catalysis and microbial fermentation, and the metabolism of SDG by Lactiplantibacillus plantarum C5 following fermentation was characterized by widely targeted metabolomics. Five L. plantarum strains producing ß-glucosidase with broad substrate specificity were isolated and identified, and they all can transform SDG into secoisolariciresinol (SECO). L. plantarum C5 resting cell reached a maximum SDG conversion of 49.19 ± 3.75%, and SECO generation of 21.49 ± 1.32% (0.215 ± 0.013 mm) at an SDG substrate concentration of 1 mM and 0.477 ± 0.003 mm SECO was produced at 4 mm within 24 h. Although sixteen flax lignan metabolites were identified following the fermentation of SDG extract by L. plantarum C5, among them, four were produced following the fermentation: SECO, demethyl-SECO, demethyl-dehydroxy-SECO and isolariciresinol. Moreover, seven lignans increased significantly. CONCLUSION: Fermentation significantly increased the profile and level of flax lignan metabolites, and the resting cell catalysis benefits from higher bioconversion efficiency and more straightforward product separation. Resting cell catalysis and microbial fermentation of flax lignan extract by the isolated ß-glucosidase production L. plantarum could be potentially applied in preparing flax lignan ingredients and fermented flaxseed. © 2024 Society of Chemical Industry.

Thermal Sensitivity of the Enzymatic Activity of ß-Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations.

A crucial prerequisite for industrial applications of enzymes is the maintenance of specific activity across wide thermal ranges. ß-Glucosidase (EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass to glucose. While the reaction mechanisms of ß-glucosidases from various thermal ranges (hyperthermophilic, thermophilic, and mesophilic) are similar, the factors underlying their thermal sensitivity remain obscure. The work presented here aims to unravel the molecular mechanisms underlying the thermal sensitivity of the enzymatic activity of the ß-glucosidase BglB from the bacterium Paenibacillus polymyxa. Experiments reveal a maximum enzymatic activity at 315 K, with a marked decrease in the activity below and above this temperature. Employing in silico simulations, we identified the crucial role of the active site tunnel residues in the thermal sensitivity. Specific tunnel residues were identified via energetic decomposition and protein-substrate hydrogen bond analyses. The experimentally observed trends in specific activity with temperature coincide with variations in overall binding free energy changes, showcasing a predominantly electrostatic effect that is consistent with enhanced catalytic pocket-substrate hydrogen bonding (HB) at Topt. The entropic advantage owing to the HB substate reorganization was found to facilitate better substrate binding at 315 K. This study elicits molecular-level insights into the associative mechanisms between thermally enabled fluctuations and enzymatic activity. Crucial differences emerge between molecular mechanisms involving the actual substrate (cellobiose) and a commonly employed chemical analogue. We posit that leveraging the role of fluctuations may reveal unexpected insights into enzyme behavior and offer novel paradigms for enzyme engineering.

Thermostabilizing mechanisms of canonical single amino acid substitutions at a GH1 ß-glucosidase probed by multiple MD and computational approaches.

ß-glucosidases play a pivotal role in second-generation biofuel (2G-biofuel) production. For this application, thermostable enzymes are essential due to the denaturing conditions on the bioreactors. Random amino acid substitutions have originated new thermostable ß-glucosidases, but without a clear understanding of their molecular mechanisms. Here, we probe by different molecular dynamics simulation approaches with distinct force fields and submitting the results to various computational analyses, the molecular bases of the thermostabilization of the Paenibacillus polymyxa GH1 ß-glucosidase by two-point mutations E96K (TR1) and M416I (TR2). Equilibrium molecular dynamic simulations (eMD) at different temperatures, principal component analysis (PCA), virtual docking, metadynamics (MetaDy), accelerated molecular dynamics (aMD), Poisson-Boltzmann surface analysis, grid inhomogeneous solvation theory and colony method estimation of conformational entropy allow to converge to the idea that the stabilization carried by both substitutions depend on different contributions of three classic mechanisms: (i) electrostatic surface stabilization; (ii) efficient isolation of the hydrophobic core from the solvent, with energetic advantages at the solvation cap; (iii) higher distribution of the protein dynamics at the mobile active site loops than at the protein core, with functional and entropic advantages. Mechanisms i and ii predominate for TR1, while in TR2, mechanism iii is dominant. Loop A integrity and loops A, C, D, and E dynamics play critical roles in such mechanisms. Comparison of the dynamic and topological changes observed between the thermostable mutants and the wildtype protein with amino acid co-evolutive networks and thermostabilizing hotspots from the literature allow inferring that the mechanisms here recovered can be related to the thermostability obtained by different substitutions along the whole family GH1. We hope the results and insights discussed here can be helpful for future rational approaches to the engineering of optimized ß-glucosidases for 2G-biofuel production for industry, biotechnology, and science.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the ß-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport ß-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Apoplast-Localized ß-Glucosidase Elevates Isoflavone Accumulation in the Soybean Rhizosphere.

Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing ß-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.

Comparative analysis of ß-glucosidase activity in non-conventional yeasts.

The objective of this study was to evaluate the ß-glucosidase activity in the non-conventional yeasts under cellulose, glucose and sucrose substrates. The participation of the enzyme ß-glucosidase and its contribution to the enzymatic degradation of tannins is known. Within the classification of tannins are ellagitannins, molecules of gallic acid and ellagic acid, which are considered as nutraceutical compounds due to the properties that they present and that they can be used in the design of food and new drugs, synthesis of materials with antimicrobial capacity. The extracellular ß-glucosidase activity was mainly presented in the Candida and Pichia strains, being the glucose and sucrose media the most capable for inducing the activity that showed maximum values with P. pastoris in glucose (0.1682±0.00 µmol/min mg protein), and C. utilis in cellulose (0.1129±0.1349 µmol/min mg of protein), and sucrose (0.0657±0.0214 µmol/min mg protein). Additionally, I. terricola and P. kluyvery stood out in a qualitative cellulose degradation approach measured by Congo red method (9.60±0.04 mm and 9.20±0.05 mm respectively). These indicate that P. pastoris and C. utilis have potential as ß-glucosidase producers, especially when growing under complex carbon sources for biomass conversion, new biofuels production and polyphenol degradation with more manageable bioreactor process.

Recent Advances in ß-Glucosidase Sequence and Structure Engineering: A Brief Review.

ß-glucosidases (BGLs) play a crucial role in the degradation of lignocellulosic biomass as well as in industrial applications such as pharmaceuticals, foods, and flavors. However, the application of BGLs has been largely hindered by issues such as low enzyme activity, product inhibition, low stability, etc. Many approaches have been developed to engineer BGLs to improve these enzymatic characteristics to facilitate industrial production. In this article, we review the recent advances in BGL engineering in the field, including the efforts from our laboratory. We summarize and discuss the BGL engineering studies according to the targeted functions as well as the specific strategies used for BGL engineering.

Adsorption and immobilization of ß-glucosidase from Thermoascus aurantiacus on macroporous cryogel by hydrophobic interaction.

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of ß-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF, dimensionless) were determined (1.57-5.13 and 64.19-91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, ß-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized ß-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

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 ß-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 ß-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 ß-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 ß-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, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

One-step purification and immobilization of thermostable ß-glucosidase on Na-Y zeolite based on the linker and its application in the efficient production of baohuoside I from icariin.

Baohuoside I, a minor flavonoid component of Herba Epimedii, has better bioactivities than its precursor compound icariin. In this work, we have fused the linker (4LP) to thermostable ß-glucosidase (Tpebgl3) and successfully prepared the immobilized enzyme (4LP-Tpebgl3@Na-Y) to produce baohuoside I from icariin. The activity recovery and maximum load of 4LP-Tpebgl3@Na-Y were 95.4% and 50.3 mg/g, respectively. Moreover, it exhibited four-fold improved adsorption selectivity (80.5%) with respect to native enzyme after immobilization. The maximum activity of 4LP-Tpebgl3@Na-Y was exhibited at 85 °C, pH 5.0, and it retained>80% of its initial activity after incubation at 75 °C for 2 h . It showed enhanced tolerance of organic solvent and glucose as compared to free enzymes. Kcat/Km value for 4LP-Tpebgl3@Na-Y was 1616.0 s-1•mM-1, which was 61.0% higher than that of free enzyme. Under suitable conditions (75 °C, pH 5.0, 0.1 U/mL enzyme and 120 min), 2000 mg/L icariin was transformed into baohuoside I with a molar conversion of 97.6%. 4LP-Tpebgl3@Na-Y retained 85.2% of its original activity after 10 cycles of reuse and the 768.8 mg/L/h total productivity of baohuoside I was obtained. This is the first research on one-step purification and immobilization of thermostable ß-glucosidase based on the linker and its application in the efficient production of baohuoside I from icariin.

Mechanistic Insight into the Mode of Action of Acid ß-Glucosidase Enhancer Ambroxol.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid ß-glucosidase (ß-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on ß-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits ß-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant ß-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated ß-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant ß-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional ß-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize ß-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.

Biochemical and Structural Analysis of a Glucose-Tolerant ß-Glucosidase from the Hemicellulose-Degrading Thermoanaerobacterium saccharolyticum.

ß-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p-nitrophenyl-ß-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (ß/α)8 TIM-barrel fold, and a ß8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.

Release of Soybean Isoflavones by Using a ß-Glucosidase from Alicyclobacillus herbarius.

ß-Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel ß-glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5-11) and temperatures (4-55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7-fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C.

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

Commercial applications of ß-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.

Fragment-derived modulators of an industrial ß-glucosidase.

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.

Effect of surface charge conditions of carriers on the immobilization of ß-d-glucosidase.

In this study, a series of acidic or alkaline polypeptide chains were designed and grafted onto DEG-AM resin using Fmoc solid-phase synthesis to study the relationship between enzyme conformation and carrier surface charge. ß-d-glucosidase (ßGase) was then immobilized onto these modified carriers by adsorption. Each form of immobilized ßGase showed decreasing specific activity compared to that of the free. It could be attributed to both the changes in the enzyme conformation and the decrease in mass transfer efficiency. The optimum temperature of free ßGase, DEG@B3-ßGase is 55 °C, which of DEG@A3-ßGase is 65 °C and they all have the highest activity at pH 5. The Ea values ​​of free ßGase, DEG@A3-ßGase, and DEG@B3-ßGase are 0.546 kJ/mol, 0.224 kJ/mol, and 0.446 kJ/mol, and the Km values were 1.30 mmol/L, 1.44 mmol/L and 2.63 mmol/L, respectively. It shows that free ßGase and DEG@A3-ßGase are more similar. Meanwhile, the free ßGase (1.0 g/L, pH 5.0) stored at 4 °C has a shorter half-life (t1/2), which is only 9 days. However, the half-life of DEG@B3-ßGase and DEG@A3-ßGase is 20 days and over 60 days, indicating that the negative charged surface was conducive to maintenance of the structure and catalytic property of ßGase.