Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass

Dadheech, Tripti; Shah, Rohan J.; Pandit, Ramesh; Hinsu, Ankit T.; Chauhan, Prakram Singh; Jakhesara, Subhash J.; Kunjadiya, Anju; Rank, D. N.; Joshi, Chaitanya G.

doi:10.1016/j.ijbiomac.2018.02.100

Cited by 43 publications

(20 citation statements)

References 32 publications

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“…Cel5L-p50 and Cel5L-p35 showed the characteristics of acidic enzymes with maximal CMCase activity at pH 4.5 (Fig. 3A), similar to an acidic cellulase Cel-1 of buffalo rumen metagenome [40], though Cel5L-p35 was more resistant to pH changes than Cel5L-p50.…”

Section: Enzymatic Properties Of the Truncated Enzymesmentioning

confidence: 84%

Roles of Carbohydrate-Binding Module (CBM) of an Endo-��-1,4-glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

Lee¹,

Shin²,

Cho³

et al. 2018

Journal of Microbiology and Biotechnology

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An endo-β-1,4-glucanase gene, cel5L, was cloned using the shotgun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and 55°C, but had different half-lives of 4.0 and 22.8 min, respectively, at 70°C. The relative activities of Cel5L-p50 and Cel5L-p35 for barley β-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

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Section: Enzymatic Properties Of the Truncated Enzymesmentioning

confidence: 84%

Roles of Carbohydrate-Binding Module (CBM) of an Endo-��-1,4-glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

Lee¹,

Shin²,

Cho³

et al. 2018

Journal of Microbiology and Biotechnology

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“…Metagenomic approaches have been successfully used in biotechnological fields, mining new biocatalysts for industrial purposes. In particular, several efforts have been made to find cellulases with relevant characteristics for specific parameters used in ethanol industry ( Rees et al, 2003 ; Ferrer et al, 2005 ; Schröder et al, 2014 ; Garg et al, 2016 ; Lewin et al, 2017 ; Dadheech et al, 2018 ; Duque et al, 2018 ; Lee et al, 2018 ; Tiwari et al, 2018 ). Here, we report the identification and biochemical characterization of a novel ethanol- and 5-HMF-stimulated β-glucosidase, using a functional-based metagenomic screening strategy.…”

Section: Discussionmentioning

confidence: 99%

“…In this context, metagenomics allows the identification of new enzymes with specific activities without the need of previous isolation and cultivation of microorganisms, which opens the door to the huge biochemical potential of most of the microbial life existing in environments of interest ( Guazzaroni et al, 2015 ; Alves et al, 2017 ). Several examples reported in literature showed the identification of new cellulases and other enzymes from different environments through metagenomic approaches ( Mori et al, 2014 ; Montella et al, 2015 ; Yang C. et al, 2016 ; Zhou et al, 2016 ; Lewin et al, 2017 ; Tiwari et al, 2017 , 2018 ; Dadheech et al, 2018 ; Lee et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated β-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome

et al. 2018

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Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel β-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel β-glucosidase – named Lfa2 – displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing β-glucose, β-galactose, β-xylose, β-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, β-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that β-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

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“…Most of the endoglucanases belonging to GH-5 family have a conserved dyad of glutamic acid residues which take part in catalysis. Tripti et al reported that the enzyme Cel-1, which was isolated from a buffalo rumen, possessed a conserved dyad of glutamic acid residues (E314 and E179) in its active site [62]. It is observed that many families of GH do not possess catalytic proton donor/acceptor and/or nucleophile [63,64].…”

Section: Enzymes Involved In Hydrolysismentioning

confidence: 99%

Role of metagenomics in prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review

Pabbathi

Velidandi

Tavarna

et al. 2021

Biomass Conv. Bioref.

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Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass

Cited by 43 publications

References 32 publications

Roles of Carbohydrate-Binding Module (CBM) of an Endo-��-1,4-glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

Roles of Carbohydrate-Binding Module (CBM) of an Endo-��-1,4-glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated β-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome

Role of metagenomics in prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review

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