A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis

The enzymatic hydrolysis of cellulose and lignocellulosic materials is marked by a rate decrease along the reaction time. Cellobiohydrolase slow dissociation from the substrate and its inhibition by the cellobiose produced are relevant factors associated to the rate decrease. In that sense, addition of β-glucosidases to the enzyme cocktails employed in cellulose enzymatic hydrolysis not only produces glucose as final product but also reduces the cellobiohydrolase inhibition by cellobiose. The digestive β-glucosidase GH1 from the fall armyworm Spodoptera frugiperda, hereafter called Sfβgly, containing the mutation L428V showed an increased k_cat for cellobiose hydrolysis. In comparison to assays conducted with the wild-type Sfβgly and cellobiohydrolase TrCel7A, the presence of the mutant L428V increased in 5 fold the initial rate of crystalline cellulose hydrolysis and reduced to one quarter the time needed to TrCel7A produce the maximum glucose yield. As our results show that mutant L428V complement the action of TrCel7A, the introduction of the equivalent replacement in β-glucosidases is a promising strategy to reduce costs in the enzymatic hydrolysis of lignocellulosic materials.     

Effect of endoxylanase and α-L-arabinofuranosidase supplementation on the enzymatic hydrolysis of steam exploded wheat straw

The cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of the bioethanol production process from lignocellulosic biomass. Hemicellulases and other accessory enzymes are becoming crucial to increase enzymatic hydrolysis (EH) yields at low cellulase dosages. The aim of this work was to evaluate the effect of two recombinant hemicellulolytic enzymes on the EH of steam pretreated wheat straw. Pretreatments at two severity conditions were performed and the whole slurry obtained after steam explosion pretreatment was employed as substrate. An endoxylanase (Xln C) from Aspergillus nidulans and an α-l-arabinofuranosidase (AF) from Aspergillus niger, have been applied in combination with cellulase enzymes. A degree of synergism of 29.5% and increases up to 10% in the EH yields were obtained, showing the potential of accessory activities to improve the EH step and make the whole process more effective.     

Effects of the addition of laminaran on β-glucosidase production by Trichoderma viride

β-Glucosidase production by Trichoderma viride WU-36B was studied in media containing laminaran. By the addition of laminaran to the medium containing glycerol as a carbon source, extracellular activities of β-glucosidase and β-1,3-glucanase increased but CMCase activity was not detected during whole culture period. Extracellular activities of β-glucosidase, CMCase, and β-1,3-gluconase were higher in the medium containing both avicel and laminaran than in the media containing avicel or laminaran as a sole carbon source.     

Ultrasonic analysis of kinetic mechanism of hydrolysis of cellobiose by β-glucosidase

High-resolution ultrasonic spectroscopy (HR–US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a β-glucosidase from Aspergillus niger (Novozyme 188) at 50 °C and pH 4.9. This technique is noninvasive, it does not require optical transparency and is suitable to continuously monitor the time dependence of the reaction progress in a broad range of experimental conditions. The time profiles of the amount of glucose released and the reaction rate were obtained from the time profile of ultrasonic velocity. The results are in good agreement with a discontinuous glucose assay (hexokinase method). The kinetic parameters of the reaction were estimated by fitting the ultrasonic time profiles of the reaction rates to several inhibition models. In addition, the equilibrium constant for the reaction of hydrolysis of cellobiose and the molar Gibbs free energy of hydrolysis were determined from the ultrasonic time profiles of concentration of glucose in the reverse reaction (glucose condensation). The results suggest the existence of more complex mechanisms regulating the activity of cellobiase than the combination of simple inhibitions. An extended kinetic model based on two sites for the competitive inhibitor (glucose) is proposed.     

Effects of cyclodextrins on the hydrolysis of ganglioside GM1 by acid β-galactosidases

The hydrolysis of ganglioside G_M1 by acid -galactosidases was greatly enhanced by the inclusion of heptakis(2,6-di-O-methyl)--cyclodextrin or -cyclodextrin in the assay mixture. The other cyclodextrins tested were not effective. The extent of stimulation by these cyclodextrins was relatively smaller than those by taurodeoxycholate and taurochenodeoxycholate. However, it is suggested that stimulation by bile salts may be partly a reflection of the detergent effects of bile salts on G_M1 and partly a reflection of the interaction between bile salts and the enzyme itself. On the other hand, the stimulation by the cyclodextrins seems to correlate to the formation of an inclusion complex between G_M1 and cyclodextrin without enzyme protein interaction.     

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1–4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1–4)Glc(α1–4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1–6)Glc(α1–4)Glc], isomaltose [Glc(α1–6)Glc], and isomaltotriose [Glc(α1–6)Glc(α1–6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).

     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

Enhancement of enzymatic hydrolysis rate of olive oil in water by dimethyl β-cyclodextrin

Summary The hydrolysis rate of olive oil byCandida cylindracea lipase in an aqueous solution without surfactants can be increased up to 6-fold by the addition of up to 60 mg/ml of dimethyl -cyclodextrin (DMCD).     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Investigation of apigenin-7-O-β-glucoside hydrolysis by β-glucosidase from almonds

Summary The enzymatic transformation of apigenin-7-O--glucoside into apigenin is effected using -glucosidase from almonds. Kinetic studies gave the following values K_m=7.63×10^–4mol/dm³, v_max=10.37 mol mg^–1 min^–1 and E₁=65.21 kJ/mol when the substrate was pure apigenin-7-O--glucoside, while in case of apigenin-7-O--glucoside from dry extract of camomile ligulate flowers E₂ = 65.57 kJ/mol.Nomenclature S_o initial substrate concentration (g/100 cm³) - P product concentration (g/100 cm³) - k reaction rate constant (min^–1) - t reaction time (min)     

Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer

Biodegradation of cellulose involves synergistic action of the endoglucanases, exoglucanases and β-glucosidases in cellulase. However, the yield of glucose is limited by the lack of β-glucosidase to hydrolyze cellobiose into glucose. In this study, β-glucosidase as a supplemental enzyme along with cellulase are co-immobilized on a pHresponsive copolymer, poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB, where MAA is α-methacrylic acid, DMAEMA is 2-dimethylaminoethyl methacrylate and BMA is butyl methacrylate). The thermal and storage stabilities of PMDB with immobilized enzymes are improved greatly, compared with those of free cellulase. Biodegradation of cellulose is carried out in a pH-responsive recyclable aqueous two-phase system composed of poly (AA-co- DMAEMA-co-BMA) (abbreviated P_{ADB 3.8}, where AA is acrylic acid) and P_MDB. Insoluble substrate and P_MDB with immobilized cellulase and β-glucosidase (Celluclast 1.5L FG and Novozyme 188, respectively) were biased to the bottom phase, while the product was partitioned to the top phase in the presence of 40 mM (NH₄)₂SO₄. When the degradation reaction of cellulose is carried out with PMDB containing immobilized cellulase and β-glucosidase, the concentration of glucose reaches 4.331 mg/mL after 108 h. The yield of glucose is 50.25% after P_MDB containing the immobilized enzymes is recycled five times.     

Immobilization of β-glucosidase in fixed bed reactor and evaluation of the enzymatic activity

Adsorption of β-glucosidase from almonds, an enzyme with big molecular size (130?kDa, 6.7?nm molecular diameter), on mesoporous SBA-15 silica in fixed bed column was studied. Previously, zeta potential analysis confirmed that the electrostatic interactions between β-glucosidase and SBA-15 were the driving force of the immobilization process. The maximum difference in the zeta potential was 25?mV at pH 3.5. Adsorption isotherm was classified as an L3 (Langmuir type 3) curve according to the Giles classification and fitted to a double Langmuir equation. The adsorbed amount in a fixed bed column was around 3.5 times higher than the amount reached in the adsorption in batch. In addition, the β-glucosidase was strongly immobilized on SBA-15 with only 7?% of leaching in the washing step with buffer solution. Immobilized β-glucosidase was catalytically active in a continuous process, reaching 100?% substrate conversion and maintaining this activity level for more than 10?h without deactivation of the enzyme. Adsorption-desorption isotherms at 77?K before and after the adsorption were carried out, concluding that the adsorption of β-glucosidase was produced blocking the pore mouth, so that a part of the enzyme penetrates inside and another part stays outside the pore.     

Effects of β-alanine supplementation on performance and body composition in collegiate wrestlers and football players

The purpose of this study was to examine the effectiveness of β-alanine as an ergogenic aid in tests of anaerobic power output after 8 weeks of high-intensity interval, repeated sprint, and resistance training in previously trained collegiate wrestlers (WR) and football (FB) players. Twenty-two college WRs (19.9 ± 1.9 years, age ± SD) and 15 college FB players (18.6 ± 1.5 years) participated in this double-blind, placebo-controlled study. Each subject ingested either 4 g·d β-alanine or placebo in powdered capsule form. Subjects were tested pre and posttreatment in timed 300-yd shuttle, 90° flexed-arm hang (FAH), body composition, and blood lactate after 300-yd shuttle. Although not statistically significant (p > 0.05) subjects taking β-alanine achieved more desirable results on all tests compared to those on placebo. Performance improvements were greatest in the FB supplement group, decreasing 300 shuttle time by 1.1 seconds (vs. 0.4-second placebo) and increasing FAH (3.0 vs. 0.39 seconds). The wrestlers, both placebo and supplement, lost weight (as was the goal, i.e., weight bracket allowance); however, the supplement group increased lean mass by 1.1 lb, whereas the placebo group lost lean mass (-0.98 lb). Both FB groups gained weight; however, the supplement group gained an average 2.1-lb lean mass compared to 1.1 lb for placebo. β-Alanine appears to have the ability to augment performance and stimulate lean mass accrual in a short amount of time (8 weeks) in previously trained athletes. Training regimen may have an effect on the degree of benefit from β-alanine supplementation.     

Effects of β-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women

Summary. This study examined the effects of 28 days of β-alanine supplementation on the physical working capacity at fatigue threshold (PWC_FT), ventilatory threshold (VT), maximal oxygen consumption ( O_2-MAX), and time-to-exhaustion (TTE) in women. Twenty-two women (age ± SD 27.4 ± 6.1 yrs) participated and were randomly assigned to either the β-alanine (CarnoSyn™) or Placebo (PL) group. Before (pre) and after (post) the supplementation period, participants performed a continuous, incremental cycle ergometry test to exhaustion to determine the PWC_FT, VT, O_2-MAX, and TTE. There was a 13.9, 12.6 and 2.5% increase (p < 0.05) in VT, PWC_FT, and TTE, respectively, for the β-alanine group, with no changes in the PL (p > 0.05). There were no changes for O_2-MAX (p > 0.05) in either group. Results of this study indicate that β-alanine supplementation delays the onset of neuromuscular fatigue (PWC_FT) and the ventilatory threshold (VT) at submaximal workloads, and increase in TTE during maximal cycle ergometry performance. However, β-alanine supplementation did not affect maximal aerobic power ( O_2-MAX). In conclusion, β-alanine supplementation appears to improve submaximal cycle ergometry performance and TTE in young women, perhaps as a result of an increased buffering capacity due to elevated muscle carnosine concentrations.     

Effects of cereal β-glucans and enzyme inclusion on the porcine gastrointestinal tract microbiota

This study was designed to evaluate the effect barley-based diets vs. oats based diets on levels of Lactobacillus, Bifidobacterium and Enterobacterium in the porcine gastrointestinal tract (GIT). In addition the effect of enzyme supplementation in both diets was explored. Twenty-eight boars were used in a 2 × 2 factorial arrangement and were assigned to 1 of 4 dietary treatments: barley-based (B) diet; barley-based diet plus an enzyme supplement (B + ES); oat-based (O) diet or oat-based diet plus an enzyme supplement (O + ES). The enzyme supplement contained endo-1,3-β-glucanase and endo-1,4-β-xylanase. Faecal samples were collected from the pigs prior to initiations of the experiment and at slaughter. At slaughter digesta samples were collected from the stomach, ileum, caecum, proximal and distal colon. Alterations in Lactobacillus species composition in the gastrointestinal tract (GIT) were analysed by genus-specific PCR – denaturing gradient gel electrophoresis (DGGE). DGGE profiles indicated that cereal source provoked shifts in Lactobacillus population. The most diverse populations of lactobacilli emerged after feeding the O diets. Enzymes inclusion altered the composition of Lactobacillus species prevalent throughout the GIT in animals fed the B diet, causing a shift in the dominant lactobacilli present in the caecum and proximal colon. No such effect was evident in animals fed the enzyme supplemented O + ES diet. Microbial plate counts revealed that the O diets gave rise to higher counts of Lactobacillus in the caecum and colon and Bifidobacterium counts in the ileum, caecum and colon than the B diets. The O diet caused a 2 log increase in Enterobacterium counts in the proximal colon, no such effects were observed in animals fed the B, the B + ES or the O + ES diets. Overall both O diets had a more positive influence on the counts of the beneficial microorganisms and richness of the Lactobacillus population in the porcine GIT.     

Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg^?1 by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l^?1 galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l^?1 glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l^?1), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l^?1) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.