Bioprospecting of novel thermostable ß-glucosidase from Bacillus subtilis RA10 and its application in biomass hydrolysis.

BACKGROUND: Saccharification is the most crucial and cost-intensive process in second generation biofuel production. The deficiency of ß-glucosidase in commercial enzyme leads to incomplete biomass hydrolysis. The decomposition of biomass at high temperature environments leads us to isolate thermotolerant microbes with ß-glucosidase production potential. RESULTS: A total of 11 isolates were obtained from compost and cow dung samples that were able to grow at 50 °C. On the basis of qualitative and quantitative estimation of ß-glucosidase enzyme production, Bacillus subtilis RA10 was selected for further studies. The medium components and growth conditions were optimized and ß-glucosidase enzyme production was enhanced up to 19.8-fold. The ß-glucosidase from B. subtilis RA10 retained 78% of activity at 80 °C temperature and 68.32% of enzyme activity was stable even at 50 °C after 48 h of incubation. The supplementation of ß-glucosidase from B. subtilis RA10 into commercial cellulase enzyme resulted in 1.34-fold higher glucose release. Furthermore, ß-glucosidase was also functionally elucidated by cloning and overexpression of full length GH1 family ß-glucosidase gene from B. subtilis RA10. The purified protein was characterized as thermostable ß-glucosidase enzyme. CONCLUSIONS: The thermostable ß-glucosidase enzyme from B. subtilis RA10 would facilitate efficient saccharification of cellulosic biomass into fermentable sugar. Consequently, after saccharification, thermostable ß-glucosidase enzyme would be recovered and reused to reduce the cost of overall bioethanol production process.

Glycoside hydrolase production by Aspergillus terreus CM20 using mixture design approach for enhanced enzymatic saccharification of alkali pretreated paddy straw.

A successful lignocellulosic ethanol production process needs to address the technological impediments such as cost-competitiveness and sustainability of the process. Effective biomass utilization requires a repertoire of enzymes including various accessory enzymes. Developing an enzyme preparation with defined hydrolytic activities can circumvent the need for supplementing cellulases with accessory enzymes for enhanced hydrolysis. With this objective, mixture design approach was used in the present study to enhance glycoside hydrolase production of a fungal isolate, Aspergillus terreus CM20, by determining the proportion of different lignocellulosic components as enzyme inducers in the culture medium. A mixture of paddy straw and wheat straw (1.42:1.58) resulted in improved cellulolytic activities. The precipitated crude enzyme showed higher CMCase (365.03 18 IU g-1), FPase (161.48 IU g-1), avicelase (15.46 IU g-1), ß-glucosidase (920.92 IU g-1) and xylanase (9627.79 IU g-1) activities. The potential of the crude enzyme for saccharification of alkali pretreated paddy straw was also tested. Under optimum conditions, saccharification released 25.0 g L-1 of fermentable sugars. This indicates the superiority of the crude enzyme produced with respect to its hydrolytic enzyme components.

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

ß-Glucosidase is an essential part of cellulase enzyme system for efficient and complete hydrolysis of biomass. Psychrotolerant Pseudomonas lutea BG8 produced ß-glucosidase with lower temperature optima and hence can play important role in bringing down the energy requirement for bioethanol production. To enhance ß-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 ß-glucosidase under submerged fermentation. The optimal medium composition for maximum ß-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, ß-glucosidase production increased up to 9.12-fold (17.52 ± 0.24 IU/g) in shake flask. Large-scale production in 7-L stirred tank bioreactor resulted in higher ß-glucosidase production (23.29 ± 0.23 IU/g) within 80 H of incubation, which was 1.34-fold higher than shake flask studies. Commercial cellulase (Celluclast® 1.5L) when supplemented with this crude ß-glucosidase resulted in improved sugar release (548.4 ± 2.76 mg/gds) from paddy straw at comparatively low temperature (40 °C) of saccharification. P. lutea BG8 therefore showed great potential for cold active ß-glucosidase production and can be used as accessory enzyme along with commercial cellulase to improve saccharification efficiency.

Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology.

Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production.

Harnessing the hydrolytic potential of phytopathogenic fungus Phoma exigua ITCC 2049 for saccharification of lignocellulosic biomass.

Phytopathogenic fungi develop unique systems for fast invasion by producing hydrolases, which may be explored as a source of hydrolytic enzymes for biofuel research. The present work deals with evaluation of a potato pathogen Phoma exigua ITCC 2049 for its potential to produce cellulase and xylanase enzyme. Taguchi methodology was applied to reveal the influence and contribution of five important factors (carbon source, organic and inorganic nitrogen source, surfactant, and pH) on hydrolytic enzyme production by Phoma. Cultivation of fungus under optimized condition produced endoglucanase (37.00 IU/ml), FPase (1.13 IU/ml), ß-glucosidase (2.67 IU/ml) and xylanase (24.92 IU/ml) within 8 days of submerged fermentation. Saccharification of biopretreated Parthenium and paddy straw with cocktail of Phoma secretome supplemented with commercial ß-glucosidase resulted in the significantly higher reducing sugar yield (651.04-698.11 mg/gds). This study proves the potential of Phoma as an alternative source of enzymes for biomass saccharification.