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The hydrolysis of cellobiose by β-glucosidases represents the crucial step in the degradation of lignocellulosic biomass and is therefore a major target for improvement by enzyme engineering. BglA from Caldicellulosiruptor saccharolyticus (CsBglA) is among the β-glucosidases with highest known activity and thermostability, however with a temperature optimum of 70–80 °C. To improve the enzyme's activity at moderate temperatures, a semi-rational approach based on homology modeling, conservation scoring and subsequent site-saturation mutagenesis was combined with random mutagenesis, resulting in the variant CsBglA-LYTH with 150% improvement of kcat/KM at 55 °C, a specific activity of 504 U mg−1 (CsBglA: 359 U mg−1) and a KM-value of 37.1 mM (CsBglA: 61.2 mM). The temperature optimum of the enzyme was altered from 80 °C to 65 °C while retaining stability at the temperature of 55 °C as applied for cellulose hydrolysis. To our knowledge, this makes CsBglA-LYTH the bacterial β-glucosidase with highest activity and stability.

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

Here, whole genome sequencing of Microbacterium sp. CIAB417 was conducted to determine its novelty at species level and identification of genes encoding for enzymes for mannan degradation. The draft genome was predicted to have 6.53 mbp length represented by 41 contigs and 6078 genes. However, only 82.35% genes were allocated for their functions. The whole genome phylogeny, ANI score (78.84%), GGDC (genome to genome distance calculations) show probability (DDH ≥ 70%) equal to 0% and difference in advanced biochemical properties among closely predicted species. The Microbacterium sp. CIAB417 was stipulated to be novel at species level. Isolate was named as Microbacterium camelliasinensis CIAB417 (accession no JAHZUT000000000) based on its isolation from a tea garden soil. Genome was predicted for three novel mannanase coding genes man1 (MZ702740), man2 (MZ702741), and man3 (MZ702737) that belong to the GH5 and GH113 family. Besides that, mannan side chain hydrolysing enzymes alpha-galactosidase (gla1; MZ702739) and beta-glucosidase (glu1; MZ702738) were also predicted.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-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 β-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 β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-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 β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-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.

β-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-β-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (β/α)8 TIM-barrel fold, and a β8-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.

To develop a new method for enzymatic preparation of minor ginsenosides, T. stercorarium β-glucosidase (Tsbgl) was characterized and its activities of deglycosylation towards natural ginsenosides were examined. The substrates of 1 mmol l

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

Aims The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases. Methods and results The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162·20 U mg-1 ) and pNPArp (22·14 U mg-1 ). The Km and Vmax of Tpebgl1 for pNPG were 0·28 mmol l-1 and 470·2 U mg-1 and for pNPArp were 17·30 mmol l-1 and 74·28 U mg-1 . Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven by experiments in this paper then. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting β-glucosidase Tpebgl3. A quantity of 10 g l-1 of ginsenoside extract was transformed into 3·93 g l-1 of Rg3 at 90°C, pH 5·0 for 3 h, with a corresponding molar conversion of 98·19%. Conclusion The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation of ginsenoside 20(S)-Rg3 from ginsenoside extract. The three-step cooperate transformation system of ginsenoside extract was established by using Tpebgl1, Tt-Afs (a thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase) and Tpebgl3 (a thermostable ginsenoside Rb1 converting β-glucosidase). Significance and impact of the study Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.

Background Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and β-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. Results This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, β-glucosidase reduced the ceftazidime MBECs significantly (P Conclusion Considering the characteristics of the β-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

The catalytic domain (residues 128–449) of theOrpinomycessp. Y102 CelC7 enzyme (OrpCelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures ofOrpCelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site ofOrpCelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6)OrpCelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6OrpCelC7.

Gaucher disease (GD) is an inherited metabolic disorder characterised by impaired catabolism of the glycosphingolipid, glucosylceramide. The deacetylated derivative, glucosylsphingosine (GluSph, lyso-Gb1) has materialised as a biomarker for GD. Further appraisal of the clinical utility of GluSph is required in terms of its prognostic power to inform disease course and pre-symptomatic testing. In this study, we show that plasma GluSph concentrations are significantly higher in GD patients with neuronopathic disease compared with non-neuronopathic disease, even in the neonatal period. A neonate diagnosed at 1 day of age (homozygous for N370S) due to an affected older sibling, returned GluSph of 70 nmol/L compared with 1070-2620 nmol/L for four neuronopathic patients diagnosed

In Arabidopsis leaves, the stress-induced production of abscisic acid from its inactive glucose conjugate involves dynamic changes in the endoplasmic reticulum., The phytohormone abscisic acid (ABA) is produced via a multistep de novo biosynthesis pathway or via single-step hydrolysis of inactive ABA-glucose ester (ABA-GE). The hydrolysis reaction is catalyzed by β-glucosidase (BG, or BGLU) isoforms localized to various organelles, where they become activated upon stress, but the mechanisms underlying this organelle-specific activation remain unclear. We investigated the relationship between the subcellular distribution and stress-induced activation of BGLU18 (BG1), an endoplasmic reticulum enzyme critical for abiotic stress responses, in Arabidopsis thaliana leaves. High BGLU18 levels were present in leaf petioles, primarily in endoplasmic reticulum bodies. These Brassicaceae-specific endoplasmic reticulum-derived organelles responded dynamically to abiotic stress, particularly drought-induced dehydration, by changing in number and size. Under stress, BGLU18 distribution shifted toward microsomes, which was accompanied by increasing BGLU18-mediated ABA-GE hydrolytic activity and ABA levels in leaf petioles. Under non-stress conditions, impaired endoplasmic reticulum body formation caused a microsomal shift of BGLU18 and increased its enzyme activity; however, ABA levels increased only under stress, probably because ABA-GE is supplied to the endoplasmic reticulum only under these conditions. Loss of BGLU18 delayed dehydration-induced ABA accumulation, suggesting that ABA-GE hydrolysis precedes the biosynthesis. We propose that dynamics of the endoplasmic reticulum modulate ABA homeostasis and abiotic stress responses by activating BGLU18-mediated ABA-GE hydrolysis.

Background: In aquatic environments, organic polymers such as proteins, polysaccharides and organic phosphates are cleaved and up taken by microorganisms through the expression of specifi c enzymes such as Leucine Aminopeptidase (LAP), beta Glucosidase (GLU) and Alkaline Phosphatase (AP), respectively. Microbial enzymatic activities are a fundamental step in the organic matter utilization and turnover into simple monomers. The context and purpose of the study: An experiment was carried out by combining the enzymatic assay using fluorogenic substrates with microscopical counts obtained using the viability marker 5-cyano-2,3 ditolyl-tetrazolium chloride (CTC). The main objective was to verify whether the enzyme activity played by microbial cells depends of the cell viability or rather whether the hydrolytic activity is directly stimulated by the availability of organic substrates for microbial metabolism. Results: This study provides evidence that in oligotrophic waters the fluorescence signal often decreases after incubation with the substrate analogue used for the enzymatic assay. For such samples, the extension of the incubation period from 2 to 5 hours has allowed the detection of a positive fl uorescence signal. Simultaneous counts of the abundance of CTC+ cells revealed that only a fraction close to 30% of the total bacterioplankton was actively respiring, suggesting that the observed increase was related to the presence of inducible enzymes rather than of actively metabolising cells. Main findings: Since the synthesis of hydrolytic enzymes by the microbial community can be induced by the presence of the organic substrates, adapting the incubation period to the trophic condition of the examined area is required for accurate enzymatic measurements, especially for oligotrophic environments. Conclusions: This is the fi rst contribution to link the enzyme activity rates with the viability properties (in terms of actively metabolising components) of bacterioplankton inhabiting pelagic Mediterranean waters. Brief summary: Evidence of inducible enzymes in oligotrophic Mediterranean areas was provided by the increase of fl uorescence recorded after incubation up to 5 hours with specifi c fl uorogenic substrates, in concomitance with the detection of a low fraction of actively respiring cells. While microbial cells were quite active for AP synthesis, LAP and GLU were mostly inducible enzymes, activated by the addition of their specifi c polymeric organic substrate. Any potential implications: In the examined oligotrophic waters the enzymatic activities seem to be stimulated mostly by the availability of metabolisable organic substrates.

A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 β-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant β-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP β-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active β-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP β-D-mannopyranoside in comparison with 4NP β-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the β-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley β-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with β-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme-mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)-based assay exhibiting selective responses to the quantitation of β-glucosidase and the effect of its inhibitor was developed. The most common substrate, para-nitrophenyl-β-d-glucopyranoside (pNPG) was hydrolyzed by β-glucosidase to release p-nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β-glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme-triggered fluorescence turn-off/turn-on of specific CDs successfully achieved sensitive detection of β-glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β-glucosidase and monitor β-glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.

Metal–organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH2, or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes., Metal–organic frameworks (MOFs) are attractive for encapsulating enzymes for industrial purposes because they can increase selectivity, stability, and/or activity of the enzymes. Here, the authors developed an economical solid-state mechanochemical method to encapsulate enzymes during MOF synthesis.

Background β-glucosidase is an important biomass-degrading enzyme and plays a vital role in generating renewable biofuels through enzymatic saccharification. In this study, we analyzed the transcriptome of Trichoderma harzianum HTASA derived from Hainan mangrove and identified a new gene encoding β-glucosidase Bgl3HB. And the biochemically characterization of β-glucosidase activity was performed. Results Bgl3HB showed substantial catalytic activity in the pH range of 3.0–5.0 and at temperatures of 40 ℃-60 ℃. The enzyme was found quite stable at 50 ℃ with a loss of only 33.4% relative activity after 240 min of heat exposure. In addition, all tested metal ions were found to promote the enzyme activity. The β-glucosidase activity of Bgl3HB was enhanced by 2.12-fold of its original activity in the presence of 5 M NaCl. Surprisingly, Bgl3HB also showed a remarkable ability to hydrolyze laminarin compared to other measured substrates. Enzyme efficiency was examined in the sugarcane bagasse saccharification processes, in which Bgl3HB with 5 M NaCl worked better supplementing Celluclast 1.5L than the commercial Novozyme 188 ascertained it as an admirably suited biocatalyst for the utilization of agricultural waste. In this work, this is the first report of a halophilic β-glucosidase from Trichoderma harzianum, and represents the β-glucosidase with the highest known NaCl activation concentration. And adding 5 M NaCl could enhance saccharification performance even better than commercial cellulase. Conclusions These results show that Bgl3HB has great promise as a highly stable and highly efficient cellulase with important future applications in the industrial production of biofuels.

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 β-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 retained80% 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. K

Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.

Levilactobacillus (L.) brevis TMW 1.2112 is an isolate from wheat beer that produces O2-substituted (1,3)-β-D-glucan, a capsular exopolysaccharide (EPS) from activated sugar nucleotide precursors by use of a glycosyltransferase. Within the genome sequence of L. brevis TMW 1.2112 enzymes of the glycoside hydrolases families were identified. Glycoside hydrolases (GH) are carbohydrate-active enzymes, able to hydrolyse glycosidic bonds. The enzyme β-glucosidase BglB (AZI09_02170) was heterologous expressed in Escherichia coli BL21. BglB has a monomeric structure of 83.5 kDa and is a member of the glycoside hydrolase family 3 (GH 3) which strongly favoured substrates with β-glycosidic bonds. Km was 0.22 mM for pNP β-D-glucopyranoside demonstrating a high affinity of the recombinant enzyme for the substrate. Enzymes able to degrade the (1,3)-β-D-glucan of L. brevis TMW 1.2112 have not yet been described. However, BglB showed only a low hydrolytic activity towards the EPS, which was measured by means of the D-glucose releases. Besides, characterised GH 3 β-glucosidases from various lactic acid bacteria (LAB) were phylogenetically analysed to identify connections in terms of enzymatic activity and β-glucan formation. This revealed that the family of GH 3 β-glucosidases of LABs comprises most likely exo-active enzymes which are not directly associated with the ability of these LAB to produce EPS.

Until recently, four types of cellobiose-fermenting

Chemical reaction engineering is interested in elucidating the reaction kinetics through the determination of the fundamental influencing variables. The understanding of enzyme kinetics is needed to implement the potential of enzymes to satisfy determined production targets and for the design of the reactor. The quantification of the enzyme kinetics is implemented by the elucidation and building of the kinetic model (it includes one or more kinetic equations). In the context of process development, the kinetic model is not only useful to identify feasibility and for optimizing reaction conditions but also, at an early stage of development it is very useful to anticipate implementation bottlenecks, and so guide reactor setup. In this chapter we describe theoretical and practical considerations to illustrate the methodological framework of kinetic analysis. We take as study cases four archetypal kinetic cases by using as example the hydrolysis of cellobiose catalyzed by a beta-glucosidase. We show the different experimental data that can be obtained by the monitoring of enzymatic reactions in different configuration of free enzyme homogeneous ideal reactors; we show step-by-step the visualization, treatment, and analysis of data to elucidate kinetic models and the procedure for the quantification of kinetic constants. Finally, the performance of different reactors is compared in the interplay with the enzyme kinetics. This book chapter aims at being useful for a broad multidisciplinary audience and different levels of academic development.

Two variants of extracellular β-glucosidase (BGL2) were purified from the stipe and pilei of

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

Sugarcane bagasse is a natural source of phenolic compounds. However, these compounds are bound to lignocellulose components, reducing their ability to function as good antioxidants. These linkages are hydrolyzed by enzymes like

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

Steviol glycosides are fully deglycosylated to steviol in the presence of bacterial populations that were isolated from different soil samples. Heating (20 min at 80 °C) or boiling (10 min at 100 °C) of soils had little effect on the steviol formation. It is suggested that bacteria that survived with highly resistant spores are responsible for the deglycosylation of steviol glycosides. A bio-organic preparation method for steviol was developed which had a total yield of 90%. Beside deglycosylation, other reactions also occur. The steviol formed can be degraded. Under anaerobic conditions, rebaudioside A was not hydrolyzed while stevioside was degraded to steviol via rubusoside. Moreover, after an extended incubation (4 weeks) and repetitive sub-cultivation, a bacterial community was selected that converted steviol glycosides to a new and unknown ketone, given the nickname Monicanone. It appeared to be the steviol nucleus without the A-ring that underwent a Walden inversion at its original C-10. A second and related unknown compound could be isolated from an impure preparation of Monicanone by chromatographic separation and purification; this compound was a reduced form of Monicanone and named Monicanol. Steviol glycosides that were incubated with a UASB effluent of an industrial wastewater treatment system – supplemented or not – with sludge of a lab scale nitrification or denitrification unit – were completely degraded via steviol and Monicanone.

The microbial biodegradation of new PLA and PCL materials containing birch tar (1–10% v/v) was investigated. Product of dry distillation of birch bark (Betula pendula Roth) was added to polymeric materials to obtain films with antimicrobial properties. The subject of the study was the course of enzymatic degradation of a biodegradable polymer with antibacterial properties. The results show that the type of the material, tar concentration, and the environment influenced the hydrolytic activity of potential biofilm degraders. In the presence of PCL films, the enzyme activities were higher (except for α-D-glucosidase) compared to PLA films. The highest concentration of birch tar (10% v/v) decreased the activity of hydrolases produced by microorganisms to the most significant extent, however, SEM analysis showed the presence of a biofilm even on plastics with the highest tar content. Based on the results of the biological oxygen demand (BOD), the new materials can be classified as biodegradable but, the biodegradation process was less efficient when compared to plastics without the addition of birch tar.

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5–6.5 and 30–40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher β-glucosidase activity toward the β-linked glucosidic bond of stevioside than toward other β-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The β-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this β-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Corn stover is a global resource used in many industrial sectors including bioenergy, fuel, and livestock operations. However, stover removal can negatively impact soil nutrient availability, especially nitrogen (N) and phosphorus (P), biological activity, and soil health. We evaluated the effects of corn stover management combined with N and P fertilization on soil quality, using soil chemical (nitrate, ammonium and Bray-1 P) and biological parameters (β-glucosidase, alkaline phosphatase, arylsulfatase activities and fluorescein diacetate hydrolysis—FDA). The experiment was performed on a Mollisol (Typic Endoaquoll) in a continuous corn system from 2013 to 2015 in Minnesota, USA. The treatments tested included six N rates (0 to 200 kg N ha−1), five P rates (0 to 100 kg P2O5 ha−1), and two residue management strategies (residue removed or incorporated) totalling 60 treatments. Corn stover management significantly impacted soil mineral-N forms and enzyme activity. In general, plots where residue was incorporated were found to have high NH4+ and enzyme activity compared to plots where residue was removed. In contrast, fields where residue was removed showed higher NO3− than plots where residue was incorporated. Residue management had little effect on soil available P. Soil enzyme activity was affected by both nutrient and residue management. In most cases, activity of the enzymes measured in plots where residue was removed frequently showed a positive response to added N and P. In contrast, soil enzyme responses to applied N and P in plots where residue was incorporated were less evident. Soil available nutrients tended to decrease in plots where residue was removed compared with plots where residue was incorporated. In conclusion, stover removal was found to have significant potential to change soil chemical and biological properties and caution should be taken when significant amounts of stover are removed from continuous corn fields. The residue removal could decrease different enzymes related to C-cycle (β-glucosidase) and soil microbial activity (FDA) over continuous cropping seasons, impairing soil health.

In this study, a GH78 α- L -rhamnosidase AtRha from Aspergillus terreus CCF3059 was screened and expressed in Pichia pastoris KM71H. The maximum enzyme activity of AtRha was 1000 U/mL after 12 days. AtRha was most active at 65 °C and pH 6.5, displaying excellent thermal stability and pH stability. The kinetic parameters Km, Vmax, kcat and kcat/Km values for pNPR were 0.481 mM, 659 μmol/min·mg, 1065 s-1 and 2214 s-1 mM-1, respectively. AtRha could be inhibited by Fe2+, Hg2+ and Cu2+. Moreover, it displayed good tolerance to organic reagents with 52.6% activity in 15%(w/v) methanol. AtRha can hydrolyze icariin containing the α-1 rhamnoside linkage. Furthermore, AtRha and β-glucosidase TthBg3 showed excellent selectivity to cleave the rhamnose at the 3rd position and the glucosyl at the C-7 group of icariin, which established an effective and green method to produce the more pharmacological active icaritin. In addition, the optimal enzyme addition schemes and the reaction conditions were screened and optimized. After a two-stage transformation under optimized conditions, 0.5 g/L of icariin was transformed into 0.25 g/L of icaritin, with a corresponding molar conversion rate of 91.2%. Our findings provide a new, specific and cost-effective method for the production of icaritin in the industry.

Compound K (C-K) and Rh2, which are present at low levels in ginseng and ginseng extracts, have higher intestinal absorption rates than other ginsenosides. Here, we attempted to convert ginsenoside Rb1 to C-K using a β-glucosidase from Penicillium decumbens. Ten commercially available enzymes were screened to identify enzymes that can convert ginsenoside Rb1 to C-K, resulting in the selection of a P. decumbens-derived β-glucosidase. β-Glucosidase showed maximum activity at pH 4.0 and 60 °C; its substrate specificity for ginsenoside Rb1 was investigated. The main glucoside-hydrolyzing pathways were as follows: ginsenoside Rb1 or Rd → gypenoside XVII → F2 → C-K and ginsenoside Rg3 → Rh2. The P. decumbens-derived β-glucosidase was used to generate C-K and Rh2 using protopanaxadiol-type ginsenosides as substrates. Additionally, to apply this enzyme to the commercialized red ginseng extract products, the contents of C-K and Rh2 in the total ginsenosides significantly (p 0.05) increased up to 36-fold and 8.9-fold, respectively, higher than prior to subjecting to biotransformation. To the best of our knowledge, this is the first report of the dual biotransformation of C-K and Rh2 by a food-grade commercial enzyme. This study demonstrates that the use of a specific β-glucosidase may increase C-K and Rh2 contents in the ginseng extract through a simple biotransformation process and, thus, enhance its health benefits.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in

In this study, the upper critical solution temperature (UCST)-responsive polymers poly (ethylene oxide) monomethyl ether-block-poly(acrylamide-co-acrylonitrile) (PEG-b-p(AAM-co-AN) were synthesized and successfully utilized to immobilize β-glucosidase in crude enzyme solution. These UCST-responsive β-glucosidase biocatalysts (PEG-b-p(AAM-co-AN@LytA-Glu) have specific UCST with tunable transition temperature, which could be tuned the separation temperature to the desired temperature range. The P2 @ LytA-Glu with an UCST of about 42.9 ℃ was exploited by one-step covalent immobilization of β-glucosidase in crude enzyme solution. The prepared P2 @ LytA-Glu exhibited significantly improved temperature, pH, storage, and operation stabilities compared with that of free enzyme. The catalytic rate of P2 @ Glu-LytA was 14.5% higher than that of P2-Glu (immobilized pure β-glucosidase), which indicated that one-step immobilization of crude enzyme directly from crude enzyme solution was feasible, and it can greatly save the purification step and reduce the experimental cost. The engineered UCST-responsive immobilized enzymes are potentially useful for the practical green biocatalysis.