Crude Β-glucosidase 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.

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

BackgroundFilamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.ResultsA novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.ConclusionsThis PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Production of Minor Ginsenosides C-K and C-Y from Naturally Occurring Major Ginsenosides Using Crude β-Glucosidase Preparation from Submerged Culture of Fomitella fraxinea.

Minor ginsenosides, such as compounds (C)-K and C-Y, possess relatively better bioactivity than those of naturally occurring major ginsenosides. Therefore, this study focused on the biotransformation of major ginsenosides into minor ginsenosides using crude β-glucosidase preparation isolated from submerged liquid culture of Fomitella fraxinea (FFEP). FFEP was prepared by ammonium sulfate (30–80%) precipitation from submerged culture of F. fraxinea. FFEP was used to prepare minor ginsenosides from protopanaxadiol (PPD)-type ginsenoside (PPDG-F) or total ginsenoside fraction (TG-F). In addition, biotransformation of major ginsenosides into minor ginsenosides as affected by reaction time and pH were investigated by TLC and HPLC analyses, and the metabolites were also identified by UPLC/negative-ESI-Q-TOF-MS analysis. FFEP biotransformed ginsenosides Rb1 and Rc into C-K via the following pathways: Rd → F2 → C-K for Rb1 and both Rd → F2→ C-K and C-Mc1 → C-Mc → C-K for Rc, respectively, while C-Y is formed from Rb2 via C-O. FFEP can be applied to produce minor ginsenosides C-K and C-Y from PPDG-F or TG-F. To the best of our knowledge, this study is the first to report the production of C-K and C-Y from major ginsenosides by basidiomycete F. fraxinea.

Optimized Biotransformation of Icariin into Icariside II by β-Glucosidase from Trichoderma viride Using Central Composite Design Method.

A crude β-glucosidase has been produced from Trichoderma viride and used to explore a simple method to prepare icariside II from icariin. The crude enzyme has been studied by zymography method and used for hydrolysis of ICA. To achieve a high conversion rate of ICA, various factors have been studied including pH, reaction time, temperature, initial concentration of enzyme, and initial concentration of ICA through central composite design experiments. In the condition of the optimum hydrolysis parameters with pH 4.0, 41°C, 1.0 mg/mL ICA, and 9.8 U/mL crude β-glucosidase, the conversion rate of ICA reached 95.03% at 1 h. Moreover, the cytotoxicity test showed that ICA II performed inhibition effects on proliferation of A549 cell, while ICA has no cytotoxicity. It indicated that the hydrolysis transformation study of ICA is valuable for exploration of active new drugs.

Two-step statistical optimization for cold active β-glucosidase production from Pseudomonas lutea BG8 and its application for improving saccharification of paddy straw.

β-Glucosidase is an essential part of cellulase enzyme system for efficient and complete hydrolysis of biomass. Psychrotolerant Pseudomonas lutea BG8 produced β-glucosidase with lower temperature optima and hence can play important role in bringing down the energy requirement for bioethanol production. To enhance β-glucosidase production, two statistical tools: Taguchi and Box-Behnken designs were applied to reveal the most influential factors and their respective concentration for maximum production of β-glucosidase under submerged fermentation. The optimal medium composition for maximum β-glucosidase production were 2.99% (w/v) bagasse, 0.33% (w/v) yeast extract, 0.38% (w/v) Triton X-100, 0.39% (w/v) NaNO3 , and pH 8.0 at temperature 30°C. Under optimized conditions, β-glucosidase production increased up to 9.12-fold (17.52±0.24IU/g) in shake flask. Large-scale production in 7-L stirred tank bioreactor resulted in higher β-glucosidase production (23.29±0.23IU/g) within 80H of incubation, which was 1.34-fold higher than shake flask studies. Commercial cellulase (Celluclast® 1.5L) when supplemented with this crude β-glucosidase resulted in improved sugar release (548.4±2.76mg/gds) from paddy straw at comparatively low temperature (40°C) of saccharification. P. lutea BG8 therefore showed great potential for cold active β-glucosidase production and can be used as accessory enzyme along with commercial cellulase to improve saccharification efficiency.

Response of β-Glucosidase and Proteases to Cadmium Addition in Andosols From a Forest and a Cultivated Field

Cadmium (Cd) is considered to be the metal with the most serious adverse effects on microbial and enzyme activities in soils polluted with heavy metals. To investigate its effects, we conducted a short-term laboratory experiment at five levels of Cd addition to the soils (0-, 5-, 50-, 100-, and 250-mg of Cd kg−1 soil) using surface soil samples from Andosols obtained from a forest and a cultivated field in an upland area of Japan's Kanto District. We determined the activities of β-glucosidase and proteases in the soils after 0, 2, 10, 20, 40, and 80 days of incubation. Availability of Cd was determined by measuring water-soluble Cd. We also measured these enzyme activities after the addition of Cd to commercial crude β-glucosidase and proteases and after the addition of these crude enzymes and Cd to autoclaved soils to evaluate the stability of the enzymes and to detect their changes in response to Cd. During the incubation experiment, Cd inhibited protease activity by up to 47% in the forest soil and 31% in the cultivated soil, where negative correlation was found between soluble Cd content and protease activity on each incubation day. In contrast, β-glucosidase activity showed little or no significant inhibition. For crude enzyme experiments, Cd did not significantly affect β-glucosidase activity, but the four proteases we tested were strongly inhibited at different levels of Cd concentrations. Thus, the responses of the β-glucosidase and protease activities of the crude enzymes to Cd addition were similar to the results for the Andosols with Cd addition. As a result, the apparent direct inhibitory effect of Cd on protease stability would result in a greater decrease in N mineralization than in cellulose decomposition by β-glucosidase.

Production and characteristics comparison of crude β-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes

The purified β-glucosidase of Aureobasidium pullulans ER-16 is one of more thermostable enzyme reported to date. Considering the unfeasibility of using purified enzyme for industrial application, it was interesting to analyze this property for the crude enzyme. Thermophilic fungus Thermoascus aurantiacus CBMAI-756 and mesophilic A. pullulans ER-16 were cultivated in different hemicellulosic materials on solid-state cultivation for β-glucosidase production. Wheat bran was most appropriate for β-glucosidase production by both microorganisms. T. aurantiacus exhibited maximum enzyme production (7.0 U/ml or 70 U/g) at 48–72 h and A. pullulans a maximum (1.3 U/ml or 13 U/g) at 120 h. Maximum activities were at 75 °C with optimum pH at 4.5 and 4.0, for T. aurantiacus and A. pullulans, respectively. A. pullulans's β-glucosidase was more pH stable (4.5–10.0 against 4.5–8.0) and more thermostable (90% after 1 h at 75 °C against 85% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t (1/2) at 80 °C were 50 and 12.5 min for A. pullulans and T. aurantiascus, respectively. These data confirm the high thermostability of crude β-glucosidase from A. pullulans. Both β-glucosidases were strongly inhibited by glucose, but ethanol significantly increased the activity of the enzyme from T. aurantiacus.

Enzymatic transformation of ginsenoside Rg 3 to Rh 2 using newly isolated Fusarium proliferatum ECU2042

A new soil isolate, Fusarium proliferatum ECU2042, was found capable of producing a ginsenoside Rg 3 hydrolase (a special β-glucosidase) which is useful for production of a very potent antitumor agent Rh 2. By investigating its fermentation progress, the optimal time for β-glucosidase biosynthesis was determined as 72 h. The crude β-glucosidase was partially purified by 49.4-fold from the cell-free extract with an activity yield of 13%. The catalytic performance of the partially purified β-glucosidase was examined, giving optima of pH and temperature at pH 5.0–6.0 and 50 °C. It was found that the F. proliferatum β-glucosidase can also hydrolyze cellobiose, but not active towards a frequently used artificial substrate p-nitrophenyl β- d-glucopyranoside (pNPG). In a lab-scale preparation, the bio-hydrolytic reaction was carried out at 50 °C for 24 h in 50 ml NaAc/HAc buffer (100 mM, pH 5.0) containing 50 mg Rg 3 and 12 mg lyophilized crude enzyme extract, giving the ginsenoside Rh 2 in 60.3% conversion.

Comparative metabolism of linamarin and amygdalin in hamsters

Rates of cyanide liberation resulting from hydrolysis of the cyanogenic glycosides linamarin, amygdalin and prunasin by a crude β-glucosidase prepared from hamster caecum were studied in vitro. In addition, hamster blood cyanide and thiocyanate concentrations were determined at 0.5, 1, 2, 3 and 4 hr after an oral dose of 0.44 mmol linamarin or amygdalin/kg body weight. Plots of cyanide liberated v. time for linamarin and prunasin yielded straight lines. A similar plot for amygdalin was curvilinear, with the rate of cyanide release increasing with time. At 10 −3 m substrate concentrations, the average rates of hydrolysis of prunasin, amygdalin and linamarin were 1.39, 0.57 and 0.13 nmol/min/mg protein, respectively. Lineweaver-Burk plots yielded apparent K m and V max values of 3.63 × 10 −5 m and 0.35 nmol/min/mg protein, respectively, for amygdalin, and 7.33 × 10 −3 m and 1.04 nmol/min/mg protein, respectively, for linamarin. Blood cyanide concentrations following amygdalin treatment reached their highest level (130 nmol/ml) 1 hr after dosing and remained elevated until 3 hr after treatment. Blood cyanide concentrations following linamarin treatment reached their highest level (116 nmol/ml) after 3 hr and then declined immediately. Area under the blood cyanide concentration-time curve was 395 nmol-hr/ml for amygdalin and 318 nmol-hr/ml for linamarin. The results suggest a faster rate of enzymatic hydrolysis and cyanide absorption for amygdalin than for linamarin.