Physicochemical characterization of pectinase activity from Bacillus spp. and their accessory role in synergism with crude xylanase and commercial cellulase in enzyme cocktail mediated saccharification of agrowaste biomass.

AIM: The aim of this study was to evaluate the physicochemical properties of the crude pectinase activity from three Bacillus isolates of ruminant dung origin and study their synergism with crude xylanases from the same Bacillus spp. and a commercial cellulase to evaluate their accessory role in improved biomass saccharification. METHODS AND RESULTS: Pectinolytic crude culture filtrate obtained from three ruminant dung isolates, Bacillus safensis M35, Bacillus altitudinis R31 and Bacillus altitudinis J208, on crude pectin containing medium possessed polygalacturonate hydrolase, pectate lyase and pectin lyase activities. Studies regarding their stability under various temperature and pH conditions revealed their mild acidic to alkaline and mesophilic nature with enzyme activity falling within the pH range 6·0-9·0 and temperature range 30-60°C. The pectinase activity was categorized as endolytic as it brought about ~50% reduction in relative viscosity of pectic polymer within initial 10 min of incubation. Synergism of pectinase activity with crude xylanase activities and/or commercial cellulase was clearly demonstrated as ~1·6 to ~1·9-fold increase in agrowaste biomass saccharification was obtained confirming the role of pectinases as accessory enzymes. CONCLUSION: Synergism of the broad-spectrum endopectinase activity obtained from three Bacillus isolates with accessory crude xylanases from the same isolates and commercial cellulase enhanced the agrowaste saccharification and confirmed the accessory role of crude pectinase as they formed an efficient enzyme cocktail functioning in a contributive manner for improvement of agrowaste biomass saccharification. SIGNIFICANCE AND IMPACT OF THE STUDY: Mesophilic crude endopectinases obtained from Bacillus spp. isolated from ruminant dung possessed activity in broad pH and temperature ranges as well as broad substrate specificity. Moreover, their synergism with crude xylanase and Primfast® 200 cellulase demonstrated the potential to form efficient enzyme cocktail for application in plant biomass saccharification process.

Xylanases of Bacillus spp. isolated from ruminant dung as potential accessory enzymes for agro-waste saccharification.

UNLABELLED: Polysaccharide hydrolase producing bacteria were isolated for biomass saccharification step in two-step bioethanol production. Xylanolytic bacteria were found to be common in ruminant dung. Seven Bacillus dung isolates exhibited high xylanolytic activity; three of which were identified as Bacillus safensis and four as Bacillus altitudinis, based on 16S rDNA and gyrB gene sequencing. Interestingly, comparison of activity profiles for B. safensis M35 and B. altitudinis R31 and J208 crude xylanases showed activity in similar temperature and pH ranges of 40-60 °C and 6·0-9·0, respectively, even though they were isolated from three different dung sources. Furthermore, 22-28% viscosity reduction of beechwood xylan substrate by all the three xylanases points towards their endo-acting nature. Endo-acting xylanases are envisaged as accessory enzymes which help expose the cellulose fibres for the subsequent action of the core enzyme cellulases. In this study, when supplemented to the commercial cellulase as a cocktail, the accessory role of the crude xylanases from the selected Bacillus strains was established as 1·3, 2·33 and 1·9 fold increase in saccharification of barley husk, sugarcane bagasse and wheat husk was achieved, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The uncontrolled use of fossil fuels and concerns about its future availability, have invoked interest over unconventional alternative energy sources like solar, hydropower, geothermal, nuclear and biomass. Plants, being largest renewable biomass on earth, have received consideration as a source of biofuels. Ruminant dung isolates M35, R31 and J208 belonging to Bacillus sp. produces majorly endo-xylanase when induced with wheat bran. Such plant cell wall degrading endo-xylanases with broad pH optima and mesophilic nature can act as accessory enzymes with cellulases to enhance the saccharification of plant biomass in biofuel industries.