Extracellular Β-glucosidase Production Research Articles

A Thermotolerant Yeast Cyberlindnera rhodanensis DK Isolated from Laphet-so Capable of Extracellular Thermostable β-Glucosidase Production.

This study aims to utilize the microbial resources found within Laphet-so, a traditional fermented tea in Myanmar. A total of 18 isolates of thermotolerant yeasts were obtained from eight samples of Laphet-so collected from southern Shan state, Myanmar. All isolates demonstrated the tannin tolerance, and six isolates were resistant to 5% (w/v) tannin concentration. All 18 isolates were capable of carboxy-methyl cellulose (CMC) degrading, but only the isolate DK showed ethanol production at 45 °C noticed by gas formation. This ethanol producing yeast was identified to be Cyberlindnera rhodanensis based on the sequence analysis of the D1/D2 domain on rRNA gene. C. rhodanensis DK produced 1.70 ± 0.01 U of thermostable extracellular β-glucosidase when cultured at 37 °C for 24 h using 0.5% (w/v) CMC as a carbon source. The best two carbon sources for extracellular β-glucosidase production were found to be either xylose or xylan, with β-glucosidase activity of 3.07-3.08 U/mL when the yeast was cultivated in the yeast malt extract (YM) broth containing either 1% (w/v) xylose or xylan as a sole carbon source at 37 °C for 48 h. The optimal medium compositions for enzyme production predicted by Plackett-Burman design and central composite design (CCD) was composed of yeast extract 5.83 g/L, peptone 10.81 g/L and xylose 20.20 g/L, resulting in a production of 7.96 U/mL, while the medium composed (g/L) of yeast extract 5.79, peptone 13.68 and xylan 20.16 gave 9.45 ± 0.03 U/mL for 48 h cultivation at 37 °C. Crude β-glucosidase exhibited a remarkable stability of 100%, 88% and 75% stable for 3 h at 35, 45 and 55 °C, respectively.

Optimization of extracellular ethanol-tolerant β-glucosidase production from a newly isolated Aspergillus sp. DHE7 via solid state fermentation using jojoba meal as substrate: purification and biochemical characterization for biofuel preparation

BackgroundThe increasing demand and the continuous depletion in fossil fuels have persuaded researchers to investigate new sources of renewable energy. Bioethanol produced from cellulose could be a cost-effective and a viable alternative to petroleum. It is worth note that β-glucosidase plays a key role in the hydrolysis of cellulose and therefore in the production of bioethanol. This study aims to investigate a simple and standardized method for maximization of extracellular β-glucosidase production from a novel fungal isolate under solid-state fermentation using agro-industrial residues as the sole source of carbon and nitrogen. Furthermore, purification and characterization of β-glucosidase were performed to determine the conditions under which the enzyme displayed the highest performance. ResultsA fungus identified genetically as a new Aspergillus sp. DHE7 was found to exhibit the highest extracellular β-glucosidase production among the sixty fungal isolates tested. Optimization of culture conditions improved the enzyme biosynthesis by 2.1-fold (174.6 ± 5.8 U/g of dry substrate) when the fungus grown for 72 h at 35 °C on jojoba meal with 60% of initial substrate moisture, pH 6.0, and an inoculum size of 2.54 × 107 spores/mL. The enzyme was purified to homogeneity through a multi-step purification process. The purified β-glucosidase is monomeric with a molecular mass of 135 kDa as revealed by the SDS-PAGE analysis. Optimum activity was observed at 60 °C and pH of 6.0, with a remarkable pH and thermal stability. The enzyme retained about 79% and 53% of its activity, after 1 h at 70 °C and 80 °C, respectively. The purified β-glucosidase hydrolysed a wide range of substrates but displaying its greater activity on p-nitrophenyl-β-D-glucopyranoside and cellobiose. The values of Km and Vmax on p-nitrophenyl β-D-glucopyranoside were 0.4 mM and 232.6 U/mL, respectively. Purified β-glucosidase displayed high catalytic activity (improved by 25%) in solutions contained ethanol up to 15%. Conclusionβ-glucosidase characteristics associated with its ability to hydrolyse cellobiose, underscore its utilization in improving the quality of food and beverages. In addition, taking into consideration that the final concentration of ethanol produced by the conventional methods is about 10%, suggests its use in ethanol-containing industrial processes and in the saccharification processes for bioethanol production. Graphical abstract▪

Characterization of Thermophilic β-Glucosidase of Rhizospheric Bacterial Strain (LSKB15) Isolated from Cholistan Desert, Pakistan

Fifty thermophilic bacterial strains isolated from rhizospheric soil of Cholistan desert, Pakistan, and designated as LSKB01-LSKB50 were screened for β-glucosidase gene (bgl) belonging to glycoside hydrolase family 1 (GH 1) using PCR technique. Subsequently, the same strains were screened for extracellular β-glucosidase production using esculin as substrate. All fifty strains were shown to be amplified for conserved region of bgl gene and to secrete extracellular β-glucosidase. One strain (LSKB15) secreted relative high amount of this enzyme as indicating by size of ferric-esculetin precipitate. This strain was further cultivated on cellulose containing media and β-glucosidase was purified by ammonium sulfate, dialysis and gel filtration chromatography. The purified enzyme showed an optimal temperature of 60°C and an optimal pH of 7. It also showed excellent temperature and pH stability retaining > 90% activity after incubation for 2 h at pH 5-8 and 40-60°C. Finally, the purified enzyme was run on Native-PAGE and subsequently incubated in phosphate buffer containing 5 mM of 4-methylumbelliferyl-β-D-glucoside (4-MUG) for 15 min at 50°C and visualized by UV light as white band. We concluded that thermophilic LSKB15 β- glucosidase may work with other cellulase to degrade available cellulose synthesized by plant and the properties exhibited by it such as high temperature and pH stability pointed out its potential industrial importance.

Over-Expression of the Thermobifida fusca β-Glucosidase in a Yarrowia lipolytica Transformant to Degrade Soybean Isoflavones

A gene (bgl) encoding a β-glucosidase in thermophilic actinomycete Thermobifida fusca NTU 22 was cloned into a Yarrowia lipolytica expression system. Heterologous expression resulted in extracellular β-glucosidase production with activity as high as 630 U/mL in a Hinton flask culture filtrate. This recombinant β-glucosidase was purified 9.2-fold from crude culture filtrate by DEAE-Sepharose FF column chromatography as measured by its increase in specific activity. The overall yield of the purified enzyme was 47.5%. The molecular weight of the purified β-glucosidase estimated by SDS-PAGE was 45 kDa, which agreed with the predicted molecular weight based on the nucleotide sequence. About 15% enzyme activity loss was observed after the enzyme was heat-treated at 50 °C for 180 min. It was also found that the activity of the enzyme was inhibited by Hg2+, Cu2+, Ba2+, Ag+, p-chloromercuribenzene, and iodoacetate. The β-glucosidase from T. fusca had the most activity for daidzein-7-glucoside and genistein-7-glucoside among the tested flavonoid glycosides, but there was moderate or little activity for luteolin-7-glucoside, cyanidine-3-glucoside, and quercetin-3-glucoside. These properties are important for the soybean isoflavone applications of this β-glucosidase.

Optimization of cultivation conditions in banana wastes for production of extracellular β-glucosidase by Trichoderma harzianum Rifai efficient for in vitro inhibition of Macrophomina phaseolina

ABSTRACTThe present study was carried out to optimize the physical and nutritional parameters for cultivation of Trichoderma harzianum Rifai for production of extracellular β-glucosidase, an alternative to chemcial fungicides to control Macrophomina phaseolina, which causes charcoal rot disease in crops. Response surface methodology by the Box–Behnken design (BBD) based on four factors (temperature, carbon sources, inoculum size and pH) was used for optimization of the cultivation conditions to produce β-glucosidase using agro-industrial banana wastes. The highest β-glucosidase activity (1483.27 U/mL) was attained under optimized cultivation conditions: ∼36 ºC and pH 7.3, using carbon source and inoculum size of 10% (w/v) and 5% (w/v), respectively. It is noteworthy that the low deviation values (0.09−0.44%) in the verification experiments (R1, R2 and R3) inferred the generated model was accurate to predict optimal cultivation conditions of T. harzianum Rifai. Likewise, the obtained diameters of inhibition zones ranging from 58.00−38.66 mm following treatment with β-glucosidase was found comparable to other in vitro tests using pure T. harzianum isolates and chemical fungicides. Hence, the findings indicated that it was feasible to use β-glucosidase as a greener alternative to chemical fungicides for control of M. phaseolina infection and consequently, for protection of crops against charcoal rot disease.

Comparative study of the production of extracellular β-glucosidase by four different strains of Aspergillus using submerged fermentation

ABSTRACTFour strains of Aspergillus (Aspergillus niger CDBB-H-176, A. niger CDBB-H-175, A. niger ATCC 9642, and Aspergillus terreus CDBB-H-194) were used to produce extracellular β-glucosidase. Using an orthogonal experimental design (L9), we optimized the parameters of culture medium to maximize the activity of β-glucosidase. The optimal conditions (same for the four strains) were as follows: temperature, 30°C; pH, 6.0; orbital agitation, 200 rpm; concentration of sucrose, 0.5% (w/v). The most productive strain was A. niger CDBB-H-175, with a yield of 701.2 U/mL. In a second stage, we optimized (L18) the concentration of nutrients in the culture medium to determine whether this modification would increase the production of β-glucosidase. The optimal conditions for A. niger CDBB-H-175 were as follows (%, w/v): NaNO3, 0.3; KCl, 0.3; KH2PO4, 0.15; NH4NO3, 0.1; NH4H2PO4, 0.1; MgSO4 · 7H2O, 0.05; yeast extract, 0.1. The production of β-glucosidase under these conditions was 1207.9 U/mL. Enzymatic assays were used to characterize the enzyme; the optimum temperature and pH of β-glucosidase produced by the four selected micro-organisms were found to be 65°C and 5.0, respectively. We determined the Michaelis–Menten constants (Km) only for A. niger CDBB-H-175 and CDBB-H-176; the values were 2.7 and 2.2 mM, respectively.

Penicillium miczynskii β-glucosidase: A Glucose-Tolerant Enzyme Produced Using Pineapple Peel as Substrate

Abstract β-Glucosidases have great potential to be used in various biotechnological processes, such as those used by the food and feed industries, biomass hydrolysis for bioethanol production, and enhancing the flavor of wine, tea, and fruit juice. This work describes β-glucosidase production by Penicillium miczynskii. The effects of pH, time, and condition of cultivation, temperature, different carbon sources, and concentration of the selected agro-industrial waste on extracellular β-glucosidase production were studied in submerged fermentation. Additionally, the enzyme produced was biochemically characterized. The best conditions for P. miczynskii β-glucosidase production were 3% pineapple peel as carbon source, under stationary conditions, for 9 days, at pH 5.5 and temperature of 20°C, which yielded 2.82 U/mL. The enzyme showed best activity at pH 4.5–5.0, and 65°C. It was stable at 55°C and 60°C, with a half-life of 50 min and 40 min, respectively. The β-glucosidase was slightly activated in the prese...

Production, Purification, and Characterization of a β-Glucosidase of Penicillium funiculosum NCL1

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of β-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular β-glucosidase production was studied in submerged fermentation. At 30 °C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of β-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major β-glucosidase (Bgl3A) with the apparent molecular weight of ~120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 °C, and exhibited a half-life of 1 h at 60 °C. K(m) of Bgl3A was found to be 0.057 mM with p-nitrophenyl β-D-glucoside and V(max) was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K(i) of 1.5 mM. Bgl3A readily hydrolyzed glucosides with β-linkage. Bgl3A activity was enhanced (156%) by Zn²⁺ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.

Extracellular β-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Extracellular β-glucosidase production by a marine Aspergillus sydowii BTMFS 55 under solid state fermentation using statistical experimental design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as Aspergillus sydowii BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of Aspergillus sydowii in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ∼95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards pNPG and enzyme has a Km and Vmax of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a Ki of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of Saccharomyces cerevisiae in presence of cellulase and the purified β-glucosidase of Aspergilus sydowii BTMFS 55.

The characterisation of a novelPichia anomala β-glucosidase with potentially aroma-enhancing capabilities in wine

The production and characterisation of β-glucosidase from an isolated yeast strain classified as aPichia anomala MDD24 were studied. The result shows that cellobiose is a good inducer for extracellular β-glucosidase production and optimum concentration is 1.5 percent cellobiose in yeast peptone dextrose medium. The purified β-glucosidase fromPichia anomala MDD24 exhibited a specific activity of 614±14 U mg−1 of protein and a molecular mass of 42 kDa. This enzyme was slightly inhibited by fructose and sucrose in the range of 4 to 20% (w/v). An ethanol concentration between 4 and 20% (v/v) activated β-glucosidase activity, at presence 16% (v/v) ethanol, β-glucosidases obtained maximum relative activity around 150%. The optimum pH and optimum temperature for β-glucosidase activity were 4.5 and 40 °C, respectively. Although the activity under the pH and temperature of wine production (pH 3.5–4.0 and 15–20 °C) was quite low, the enzyme was stable and the relative activities were higher than commercial enzyme under those conditions. The extracellular β-glucosidase fromPichia anomala MDD24 makes it possible to release glucosidically-bound monoterpenes, which are the major contributors to floral and fruity aromas in wines fromMuscat-type varieties, at final stage of alcoholic fermentation.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low beta-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express beta-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing beta-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher beta-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.

Production of extracellular β-glucosidase by Monascus purpureus on different growth substrates

Various agro-industrial residues in combination with peptone, NH 4Cl and/or soy bran were screened as substrates for extracellular β-glucosidase (BGL) production by Monascus purpureus NRRL1992 on submerged fermentations (SmF). Higher BGL production was achieved when the agro-industrial residues were combined with peptone, and the utilization of NH 4Cl (inorganic nitrogen source) had not supported high enzyme production. The combination between grape waste and peptone was the best for enzyme production, and was selected as the growth substrate for further investigations. The evaluation of the effects of the medium components on enzyme production showed that the influence of peptone was more important than grape waste. The production of extracellular BGL by M. purpureus was inducible and controlled by carbon (glucose) catabolite repression.

Induction and carbon source control of extracellular β-glucosidase production in Acremonium persicinum

The effect of carbon source on the production of a single extracellular β-glucosidase by Acremonium persicinum was investigated using fast protein liquid chromatography. The enzyme was detected in the medium during growth of the fungus on a wide range of β-glucans, particularly those containing a high proportion of (1 ± 6)-β-linkages. While growth of A. persicinum on gentibiose, cellobiose and laminari-oligosaccharides moderately increased β-glucosidase production, growth on sophorose provoked 10-fold greater levels of this enzyme in the culture medium. Levels of the β-glucosidase were minimal in the presence of even low concentrations of glucose, sucrose, fructose or galactose. The results are consistent with the extracellular A. persicinum β-glucosidase being an inducible enzyme, subject to carbon source control by readily metabolizable sugars, with sophorose possibly directly inducing its synthesis.

Research Note: Sauerkraut Brine: a Potential Substrate for Production of Yeast β-Glucosidase

Sauerkraut brine could serve as a substrate for production of extracellular β-glucosidase by Candidayeasts. Of the four species examined, Candida wickerhamiiand Candida molischianaproduced significantly more β-glucosidase activity at 95% confidence level than Candida lipolyticaand Candida utilis. pH had a profound effect on yeast β-glucosidase production. Candida wickerhammiiyielded the highest level of β-glucosidase activity (768±4.03 units/L) after 48 h of growth at pH 3.8, and the enzyme had a specific activity of 24 units/mg protein.