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Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

ABSTRACT: A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0-10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn²⁺ ions and was inhibited in the presence of chelating agent EDTA. The K _m and V _max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.

SUBMITTER: Shah RK

PROVIDER: S-EPMC5520813 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundLignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH3-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination.MethodsTwo endoglucanases' genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability.ResultsThe maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40 °C, while TrepCel4 was at pH 6.0 and 50 °C. They were stable over the temperature range of 30 to 60 °C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH3-N and VFA production, and MCP synthesis during in vitro rumen fermentation.ConclusionsThe TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.

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Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

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