Rameshwar Tiwari

Beta-Glucosidase: Key Enzyme in Determining Efficiency of Cellulase and Biomass Hydrolysis

Overall economics of the biomass to ethanol process is largely determined by the efficiency of biomass hydrolysis. Performance of cellulase cocktails used for saccharification of cellulose in biomass is often limited by lower amounts of β-glucosidases present, which catalyse hydrolysis of cellobiose, the product of endo and exocellulases to glucose. Inappropriate ratio of these enzymes in commercial cocktails leads to accumulation of cellobiose which inhibits the activity of cellulases. Thus, this rate limiting enzyme is of crucial importance in determining the efficiency of commercial cellulases. The saprophytic fungus Trichoderma sp., exploited for production of commercial cellulases, produces very minute quantities of β-glucosidases as compared to endo and exocellulases. However, several other organisms are known to produce β-glucosidases in higher quantities, over a broader substrate range. Strategies to get optimal ratio of exocellulases, endocellulases and β-glucosidases to enhance saccharification yields are, therefore, discussed. Appropriate levels of β-glucosidase activity in commercial cocktails have been obtained by supplementing with accessory β-glucosidases, transgenic approaches and by optimizing β-glucosidase production through manipulation of culture conditions. These approaches have resulted in achieving higher β-glucosidase activity in cellulase cocktails, facilitating higher sugar yields and thereby potentially improving enzymatic saccharification of biomass and eventually ethanol production.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

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.

Novel cold temperature active β-glucosidase from Pseudomonas lutea BG8 suitable for simultaneous saccharification and fermentation

Cold tolerant microbes play a pivotal role in the decomposition of cellulosic biomass in temperate environments. This study was aimed to evaluate β-glucosidase production from psychrotolerant microorganisms isolated from cold desert soil of the Kargil district, Himalayan region, India. A total of 12 morphotypes were isolated at 4 °C and, based on qualitative screening, seven isolates were selected for molecular identification and phenotypic microarray. Pseudomonas lutea BG8 was selected based on its β-glucosidase production potential. The characterization of β-glucosidase revealed the alkali-tolerant nature of the enzyme with a temperature optimum at 40 °C and ability to retain considerable activity over a broad pH range (5–10). The enzyme retained 95.15% activity even in the presence of 1000 mM glucose, indicating an absence of feedback inhibition and high glucose tolerance. The enzyme stability experiment revealed that the β-glucosidase enzyme retained 49.96% activity at pH 5 and 30 °C after 16 h of incubation. The Km and kcat of the β-glucosidase enzyme were found to be 0.636 ± 0.103 mM and 102.7 ± 0.004 s−1, respectively. The cold temperature active β-glucosidase from P. lutea BG8 was found to be highly efficient for bioconversion of cellobiose to ethanol along with S. cerevisiae by simultaneous saccharification and fermentation process with 91.42% (0.49 g ethanol per g cellobiose) fermentation efficiency at 4 °C. This novel β-glucosidase can lead to considerable energy savings due to its lower temperature optimum, as compared to commercial β-glucosidase.

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), β-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 β-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.

Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill

A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain (B. subtilis), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli. The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.

Evaluating the Diversity of Culturable Thermotolerant Bacteria from Four Hot Springs of India

The culturable bacterial diversity of four hot springs of India was analysed, employing different media and screened for temperature tolerance (40°C - 70°C). Sixty morphotypes from Bakreshwar, 48 from Balrampur, 46 from Chumathang and 52 from Vashist were obtained. A set of 112 isolates, tolerant to 45°C and above, were analysed employing Amplified ribosomal DNA restriction analysis (ARDRA). Sequencing of 16S rRNA gene of the representative isolates revealed that 86%, 93%, 44% and 44% of the isolates respectively from Bakreshwar, Balrampur, Chumathang and Vashist, belonged to Firmicutes. Members of Actinobacteria were present in all the four hot springs, while Proteobacteria were present only in Chumathang and Vashist and Bacteroidetes found only in Bakreshwar. This is the first report of Aurantimonas and Brevundimonas in hot springs. Biolog analyses of three isolates, growing at or above 60°C revealed unique abilities, in terms of utilization of substrates and resistance patterns.

An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of Its Binding Model toward Its Applications As Detergent Additive

A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10–70°C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50°C and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50 and 4°C with low supplementation (109 U/ml). Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

Proteomic analysis of Streptomyces sp. ssr‐198 grown on paddy straw

The filamentous bacteria Streptomyces spp. produces diverse extracellular enzymes and other secondary metabolites. Proteomic analysis of the secretome of holocellulolytic Streptomyces sp. ssr‐198 was done by tandem mass spectrometry using an Orbitrap Velos hybrid mass spectrometer. A wide range of hydrolytic enzymes, including glycoside hydrolases (17), proteases (17), polysaccharide lyases (3), esterases (2), and hypothetical proteins (14) were detected in the secretome analyzed. Overall, the secretome composition constituted of 12.50% cellulases, 17.50% hemicellulases, 21.25% proteases, 17.50% hypothetical proteins, and 31.25% other proteins. Comprehensive analysis of secretome will be useful in gaining better understanding of the unique role of hydrolytic enzymes in lignocellulose hydrolysis and helps in determining the industrial applications of these potent enzymes.

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review

Abstract Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.