Abstract
Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol.
Highlights
Hemicellulose is the second most highly abundant polysaccharide in lignocellulosic biomass
Uncultured archaeal sequences belonging to methanogens of “uncultured marine bacteria” groups II and III were identified in the Caatinga goat rumen [12]
The free ranging Caatinga goats from which samples were taken had access to high-salt water ponds, which may be the reason for the presence of these groups in the Substrate pNPG pNPX pNPGal pNPC pNPαG pNPM pNPR Cellobiose Maltose Lactose Salicin Xylobiose
Summary
Hemicellulose is the second most highly abundant polysaccharide in lignocellulosic biomass. Unlike cellulose, it is not homogeneous, being composed of pentoses (e.g., xylose, arabinose), hexoses (e.g., mannose, glucose, galactose), and sugar acids [1,2,3]. One type of hemicellulose is xylan, which is β-1,4-linked xylopyranosyl units often modified by acetyl, arabinofuranosyl and glucuronyl groups [1, 4]. Endo-1,4-β-xylanases (EC 3.2.1.8), which cleave the xylan backbone, and β-D-xylosidases (EC 3.2.1.37), which act on xylobiose to release two xylose monomers, are two fundamental enzymes for the degradation of hemicellulose. For its complete degradation, several accessory enzymes are needed, such as exo-oligoxylanases, α-arabinofuranosidases, and α-glucuronidases, as well as esterases, acetyl xylanesterases and feruloyl esterases [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
