Characterization of an extremely thermo-active archaeal ß-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase.

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 ß-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant ß-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 ß-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active ß-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP ß-D-mannopyranoside in comparison with 4NP ß-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 ß-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley ß-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 ß-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.

Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

OBJECTIVES: Characterization of glucose tolerant beta glucosidase (GT-BGL) secreted by Aspergillus unguis NII 08123, determination of the gene and protein sequences of the enzyme and establishing its performance in blends for lignocellulose hydrolysis. RESULTS: Supplementation of A. unguis beta glucosidase (BGL) to cellulase released 1.6 times more sugar within 12 h during the hydrolysis of lignocellulosic biomass. The enzyme was determined to be similar to BGL-F from Emericella nidulans by MALDI-TOF analysis, and was found to be a GH3 family protein. Molecular Docking simulation studies showed that the enzyme has lesser affinity for glucose (- 5.7 kcal/mol) compared to its substrate cellobiose (- 7.5 kcal/mol). The residues present in the N-terminal domain are mostly involved in bond formation with both the substrate and the product, while the C-terminal domain contains the catalytic region. In-silico studies showed that its predicted structure is unlike that of previously reported BGLs, which might provide a clue to its exceptional catalytic activity. CONCLUSION: The GT-BGL from A. unguis NII 08123 was proven effective as a blend in for biomass hydrolyzing enzyme cocktails and the possible reasons for its glucose tolerance was determined through studies on its modeled structure.

Effects of acute hyperglycemia stress on plasma glucose, glycogen content, and expressions of glycogen synthase and phosphorylase in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂).

In the present study, the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a typical carnivorous fish, was chosen as a model to investigate the regulation of glycogen metabolism owning to its characteristic of glucose intolerance. The variation of plasma glucose concentration, glycogen content, and expressions of glycogen metabolism-related genes under acute hyperglycemia stress were measured. Following glucose administration, plasma glucose concentration increased immediately, and the glucose level remained elevated for at least 12 h. The prolonged glucose clearance and hyperglycemia revealed glucose intolerance of this fish species. Meanwhile, the glycogen content in both liver and muscle changed significantly during the clearance of plasma glucose. However, the peak value of hepatic glycogen (1 and 12 h post injection) appeared much earlier than muscle (3 and 24 h post injection). To investigate the regulation of glycogen metabolism from molecular aspect, the complete coding sequence (CDS) of glycogen synthase (GS) and glycogen phosphorylase (GP) in both liver and muscle types were obtained, encoding a polypeptide of 704, 711, 853, and 842 amino acid residues, respectively. The results of gene expression analysis revealed that the expression of liver type and muscle type GS was significantly higher than other time points at 12 and 24 h post glucose injection, respectively. Meanwhile, the highest expressions of GP in both liver and muscle types occurred at 24 h post glucose injection. The response of GS and GP to glucose load may account for the variation of glycogen content at the transcriptional level to some extent.

Structures of a glucose-tolerant ß-glucosidase provide insights into its mechanism.

Cellulose can be converted to ethanol via the fermentation of glucose, which is considered as a promising green alternative for transportation fuels. The conversion of cellulose to glucose needs three enzymes, in which ß-glucosidase (BGL) plays an essential role. However, BGL is inhibited by its own product glucose, greatly limiting its applications in industry. We previously obtained a novel BGL named Bgl6 with a high glucose tolerance. Further engineering through random mutagenesis produced a triple mutant M3 with improved thermostability. This enzyme shows promising properties for wide applications but the structural basis of the unusual properties of Bgl6 is not clear. In this study, we determined the crystal structures of Bgl6 and variants at high resolution, which provide insights into its glucose-tolerant mechanism and thermostability. Particularly, Bgl6 forms an extra channel that could be used as a secondary binding site for glucose, which may contribute to glucose tolerance. Additionally, the triple mutations could strengthen the hydrophobic interactions within the enzyme and may be responsible for the enhanced thermostability exhibited by M3, which was further confirmed by dynamic light scattering data. Lastly, structural comparison to other orthologs allows us to formulate new strategies on how to improve the catalytic efficiency of Bgl6.

Expression and characterization of a glucose-tolerant ß-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and ß-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a ß-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most ß-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2, 5.6 × 10-2, and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and ß-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.

Characterization of a new multifunctional beta-glucosidase from Musca domestica.

OBJECTIVE: To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications. RESULTS: A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l-1. The enzyme has the maximum activity with 27.6 U mg-1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg-1), cellobiose (40.1 ± 2.3 U mg-1) and cellulose (27.6 ± 3.5 U mg-1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites. CONCLUSIONS: Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803.

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ∆sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H2O2, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ∆sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ∆sigCDE than in the control strain. These results indicate that ∆sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ∆sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ∆sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ∆sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ∆sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.

Purification and characterization of three ß-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28.

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. ß-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three ß-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely ß-xylosidase (P1.1), ß-glucosidase (P1.2), and glucan 1,3-ß-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial ß-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant ß-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.

Biochemical characterization of the ß-glucosidase Glu1B from Coptotermes formosanus produced in Pichia pastoris.

ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.

Screening for cellulases and preliminary optimisation of glucose tolerant ß-glucosidase production and characterisation.

The search for a novel microbial producer of cellulases including a glucose tolerant ß-glucosidase is a challenge as most are inhibited by their product glucose. This study aims to screen for cellulolytic fungi using qualitative and quantitative screening methods. Primary screening revealed 34 of 46 fungal isolates with ß-glucosidase activity. Eleven and 13 of these also displayed endoglucanase and exoglucanase activities, respectively. During secondary screening, this number was reduced to 26 ß-glucosidase producers with 13 also having endoglucanase and exoglucanase activities. Isolate C1 displayed enhanced production of ß-glucosidases in the presence of 0.05 M glucose (69% higher activity). Optimisation of growth conditions for ß-glucosidase production by one variable at a time experiments improved production for (isolates) PS1 (64%), MB5 (84%), and C2 (69%). Isolate PS1 identified as Chaetomella sp. BBA70074 displayed the highest tolerance to glucose, retaining 10% of ß-glucosidase activity in the presence of 0.8 M glucose. Tolerance to glucose increased to 14% when produced under optimal conditions. ß-Glucosidase had a molecular weight of 170 kDa with a pH and temperature optima of 6 and 70°C, respectively. Future studies will include optimisation of the production of the glucose tolerant enzyme by Chaetomella sp. BBA70074.

Normal glucose tolerant women with low glycemia during the oral glucose tolerance test have a higher risk to deliver a low birth weight infant.

Background: Data are limited on pregnancy outcomes of normal glucose tolerant (NGT) women with a low glycemic value measured during the 75g oral glucose tolerance test (OGTT). Our aim was to evaluate maternal characteristics and pregnancy outcomes of NGT women with low glycemia measured at fasting, 1-hour or 2-hour OGTT. Methods: The Belgian Diabetes in Pregnancy-N study was a multicentric prospective cohort study with 1841 pregnant women receiving an OGTT to screen for gestational diabetes (GDM). We compared the characteristics and pregnancy outcomes in NGT women according to different groups [(<3.9mmol/L), (3.9-4.2mmol/L), (4.25-4.4mmol/L) and (>4.4mmol/L)] of lowest glycemia measured during the OGTT. Pregnancy outcomes were adjusted for confounding factors such as body mass index (BMI) and gestational weight gain. Results: Of all NGT women, 10.7% (172) had low glycemia (<3.9 mmol/L) during the OGTT. Women in the lowest glycemic group (<3.9mmol/L) during the OGTT had compared to women in highest glycemic group (>4.4mmol/L, 29.9%, n=482), a better metabolic profile with a lower BMI, less insulin resistance and better beta-cell function. However, women in the lowest glycemic group had more often inadequate gestational weight gain [51.1% (67) vs. 29.5% (123); p<0.001]. Compared to the highest glycemia group, women in the lowest group had more often a birth weight <2.5Kg [adjusted OR 3.41, 95% CI (1.17-9.92); p=0.025]. Conclusion: Women with a glycemic value <3.9 mmol/L during the OGTT have a higher risk for a neonate with birth weight < 2.5Kg, which remained significant after adjustment for BMI and gestational weight gain.

Conformational flexibility correlates with glucose tolerance for point mutations in ß-glucosidases - a computational study.

ß-glucosidases (EC 3.2.1.21) have been described as essential to second-generation biofuel production. They act in the last step of the lignocellulosic saccharification, cleaving the ß - 1,4 glycosidic bonds in cellobiose to produce two molecules of glucose. However, ß-glucosidases have been described as strongly inhibited by glucose, causing an increment of cellobiose concentration. Also, cellobiose is an inhibitor of other enzymes used in this process, such as exoglucanases and endoglucanases. Hence, the engineering of thermostable and glucose-tolerant ß-glucosidases has been targeted by many studies. In this study, we performed high sampling accelerated molecular dynamics for a wild glucose-tolerant GH1 ß-glucosidase (Bgl1A), a wild non-tolerant (Bgl1B), and a set of glucose-tolerant Bgl1B's mutants: V302F, N301Q/V302F, F172I, V227M, G246S, T299S, and H228T. Our results suggest that point mutations promissory to induce glucose tolerance trend to enhance the mobility of the flexible loops around the active site. Mutations affected B and C loops regions, and an αß-hairpin motif between them. Conformational clusters and free energy landscape profiles suggest that the mobility acquired by mutants allows a higher closure of the substrate channel. This closure is compatible with a higher impedance for glucose entrance and stimulus of its withdrawal. Based on mutants' structural analyses, we inferred that both the direct stereochemical effect on the glucose path and the changes in the mobility affect glucose tolerance. We hope these results be useful for the rational design of glucose-tolerant and industrially promising enzymes.Communicated by Ramaswamy H. Sarma.

A novel ß-glucosidase from a hot-spring metagenome shows elevated thermal stability and tolerance to glucose and ethanol.

ß-glucosidase causes hydrolysis of ß-1,4-glycosidic bond in glycosides and oligosaccharides. It is an industrially important enzyme owing to its potential in biomass processing applications. In this study, computational screening of an extreme temperature aquatic habitat metagenomic resource was done, leading to the identification of a novel gene, bglM, encoding a ß-glucosidase. The comparative protein sequence and homology structure analyses designated it as a GH1 family ß-glucosidase. The bglM gene was expressed in a heterologous host, Escherichia coli. The purified protein, BglM, was biochemically characterized for ß-glucosidase activity. BglM exhibited noteworthy hydrolytic potential towards cellobiose and lactose. BglM, showed substantial catalytic activity in the pH range of 5.0-7.0 and at the temperature 40 °C-70 °C. The enzyme was found quite stable at 50 °C with a loss of hardly 20% after 40 h of heat exposure. Furthermore, any drastically negative effect was not observed on the enzyme's activity in the presence of metal ions, non-ionic surfactants, metal chelating, and denaturing agents. A significantly high glucose tolerance, retaining 80% relative activity at 1 M, and 40% at 5 M glucose, and ethanol tolerance, exhibiting 80% relative activity in 10% ethanol, enrolled BglM as a promising enzyme for cellulose saccharification. Furthermore, its ability to catalyze the hydrolysis of daidzin and polydatin ascertained it as an admirably suited biocatalyst for enhancement of nutritional values in soya and wine industries.

The effect of obesity in pregnancy and gestational weight gain on neonatal outcome in glucose-tolerant mothers.

BACKGROUND: Most studies showing association between mothers with obesity in pregnancy or excessive gestational weight gain (GWG) and adverse neonatal outcome were cross-sectional or retrospective. Many included patients are with gestational diabetes mellitus (GDM), which is a strong risk factor for this adverse outcome. There are no prospective studies on this topic in Malaysia. This study aimed to examine prospectively the effects of obesity in pregnancy and GWG, independent of GDM, on neonatal outcome. METHODS: Pregnant mothers in the first trimester, who presented to health clinics in Kuching, were screened. Mothers with existing diabetes mellitus or GDM were excluded using 75-g oral glucose tolerance test during the first and second trimesters. Participants with the first trimester BMI ≥ 23 kg/m2 were recruited as overweight/obese group, whereas those with BMI 18.5-22.9 kg/m2 were taken as the comparison group. At every trimester visit, mothers' weights were recorded. Babies' birth weight and occurrence of adverse neonatal outcome were documented. RESULTS: There were 123 mothers recruited as overweight/obese group (mean BMI 29.0 kg/m2 ± 4.45) and 102 mothers as comparison group (mean BMI 20.4 kg/m2 ± 1.48). The number of low birth weight was similar between groups: 9.8% in overweight/obese group, 6.9% in the comparison group (p = 0.416). More than half of these babies were born to mothers with inadequate GWG (58.3% in obese group vs. 57.1% in control group, p = 0.077). There was no significant difference in the mean birth weight (3000 g ± 454.5 vs. 3038 g ± 340.8, p = 0.471), preterm delivery (8.13% vs. 3.92%, p = 0.193), and admission rate to neonatal intensive care unit (8.13% vs. 7.85%, p = 0.937) between groups. There was a positive correlation between the total GWG in overweight/obese group on baby's weight (r = 0.222, p = 0.013). Inadequate GWG was not correlated with lower birth weight (p = 0.052). CONCLUSIONS: Obesity in pregnancy was not associated with poor neonatal outcome in this small sample of women in Malaysia. Total GWG showed a weak correlation with baby's birth weight in overweight/obese group.

Unconventional ß-Glucosidases: A Promising Biocatalyst for Industrial Biotechnology.

ß-Glucosidases primarily catalyze removal of terminal glucosyl residues from a variety of glucoconjugates and also perform transglycosylation and reverse hydrolysis. These catalytic properties can be readily exploited for degradation of lignocellulosic biomass as well as for pharmaceutical, food and flavor industries. ß-Glucosidases have been either isolated in the native form from the producer organism or recombinantly expressed and gaged for their biochemical properties and substrate specificities. Although almond and Aspergillus niger have been instantly recognizable sources of ß-glucosidases utilized for various applications, an intricate pool of novel ß-glucosidases from different sources can provide their potent replacements. Moreover, one can envisage the better efficacy of these novel candidates in biofuel and biorefinery industries facilitating efficient degradation of biomass. This article reviews properties of the novel ß-glucosidases such as glucose tolerance and activation, substrate specificity, and thermostability which can be useful for their applications in lignocellulose degradation, food industry, and pharmaceutical industry in comparison with the ß-glucosidases from the conventional sources. Such ß-glucosidases have potential for encouraging white biotechnology.

Cloning, expression, biochemical characterization, and molecular docking studies of a novel glucose tolerant ß-glucosidase from Saccharomonospora sp. NB11.

Most of the presently known ß-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant ß-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding ß-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 ß-glucosidases. The recombinant enzyme was purified using a Ni-NTA column, with the molecular mass of 51 kDa, using SDS-PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co-factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 µmol/min/mg, 5042.16 s-1 and 12487.71 s-1 mM-1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. ß-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant ß-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.

Association of the extent of return to fasting state 2-hours after a glucose challenge with incident prediabetes and type 2 diabetes: The CARDIA study.

AIM: To evaluate whether the extent of return to fasting state 2-hours after a glucose challenge among normoglycemic individuals is associated with lower risk of incident prediabetes/ type 2 diabetes in the Coronary Artery Risk Development in Young Adults (CARDIA) cohort study. METHODS: We evaluated this association among 1879 normoglycemic adults who were categorized into three groups: 'Low post load' (2hPG < FPG); 'Medium post load' (2hPG ≥ FPG and < 75th percentile of the difference); and 'High post load' (2hPG > FPG and ≥ 75th percentile of the difference). We used Cox proportional hazards regression to evaluate the association of the difference in 2hPG and FPG with incident diabetes/prediabetes after adjustment for demographic and clinical covariates. RESULTS: During 20 years of follow-up, 8% developed type 2 diabetes and 35% developed prediabetes. Compared to those with 'Low post load', the risk of type 2 diabetes was higher for participants with 'High post load' [HR: 1.56, 95% CI (1.03, 2.37)] and similar for participants with 'Medium post load' [HR: 0.99, 95% CI (0.64, 1.52)]. However, HRs for incident prediabetes among participants with 'High post load' [HR = 1.2, 95 %CI = (0.98, 1.46)] was not significantly different compared to participants with 'Low post load'. CONCLUSION: Among normoglycemic individuals, a difference between 2hPG and FPG concentration > 0.9 mmol/L can be used to stratify individuals at higher risk for developing type 2 diabetes.

Molecular modeling, docking and simulation dynamics of ß-glucosidase reveals high-efficiency, thermo-stable, glucose tolerant enzyme in Paenibacillus lautus BHU3 strain.

The enzyme ß-glucosidase mediates the rate limiting step of conversion of cellobiose to glucose and thus plays a vital role in the process of cellulose degradation. The present study deals with analysis of the effective novel strain of Paenibacillus lautus BHU3 for identifying high-efficiency thermostable, glucose tolerant ß-glucosidases. Seven counterparts with elevated Tm values ranging from 64.6 to 75.8 °C with high thermo-stability, were revealed through this analysis. The blind molecular docking of the model enzymes structures with cellobiose and pNPG gave high negative interaction energies ranging from -11.33 to -13.29 and -6.43 to -9.054 (kcal mol-1), respectively. The enzyme WP_096774744.1 effectively formed 5 hydrogen bonds with the highest interaction energy (-13.29 kcal mol-1) with cellobiose at its catalytic site. Molecular dynamics simulation analysis performed for the WP_096774744.1-pNPG complex predicted Glu5, Arg7, Lue68, Gly69 and Phe325 as the major contributing residues for accomplishing hydrolysis of ß-1-4-linkage. Further, the molecular docking of WP_096774744.1 enzyme with glucose revealed a distinct glucose-binding site distant from the substrate-binding site, thus confirming the deficient competitive inhibition by glucose. Hence, WP_096774744.1 ß-glucosidase appears to be an efficient enzyme with enhanced activity to biodegrade the cellulosic materials and highly relevant for waste management and various industrial applications.

Glutantßase: a database for improving the rational design of glucose-tolerant ß-glucosidases.

Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the ß-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, ß-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of ß-glucosidase structures, called Glutantßase. Our database includes 3842 GH1 ß-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantßase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant ß-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant ß-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient ß-glucosidases. We hope that Glutantßase can help improve second-generation biofuel production. Glutantßase is available at http://bioinfo.dcc.ufmg.br/glutantbase .

Utilization of Bacillus subtilis cells displaying a glucose-tolerant ß-glucosidase for whole-cell biocatalysis.

The microbial production of industrial enzymes requires a large number of complex biochemical steps for purification which increases their production cost. Additionally, poor thermo-stability of the purified enzymes under the operational conditions along with the challenges in their recovery and subsequent reuse, limit their usage in an industrial bioprocess. Surface display of heterologous enzymes on bacterial cells appear to be a suitable alternative. Bacillus subtilis, the most well characterized Gram-positive bacterium, is being increasingly studied as a host for surface display. We displayed a glucose-tolerant ß-glucosidase (UnBgl1A) on the surface of B. subtilis cells using CWBb as the anchor protein. These cells displaying UnBgl1A (SD-01) were directly employed for biocatalysis without cell lysis and enzyme purification. The SD-01 cells elicited ∼2 times more catalytic activity compared to the cells expressing the enzyme intracellularly (IN-01). The displayed enzyme and the purified enzyme elucidated similar glucose tolerance (IC50 ∼0.9 M glucose), temperature optima (∼50 °C), and pH optima (∼6.0). The surface displayed UnBgl1A retained ∼50% activity after 4 h when stored at 50 °C whereas the purified UnBgl1A lost all its activity by the 4th hour. Additionally, the SD-01 cells could be efficiently reused for 3 sets of reactions. Further, supplementation of a cellulase cocktail with the cells of the SD-01 strain resulted in ∼2 times more glucose release from sugarcane bagasse compared to supplementation with the purified UnBgl1A. Therefore, displaying enzymes on the B. subtilis cell surface could be an attractive platform for the commercial production of industrial enzymes.