Cloning Cellulase Genes from Victoria Falls Rainforest Decaying Logs Metagenome

Abstract

The Victoria Falls rainforest is a protected site whose forest floors harbor a host of cellulolytic microorganisms involved in biomass degradation. This study collected decaying logs and soil from the rainforest for bioprospecting cellulases from their metagenomes. Metagenomic DNA was isolated from the compound sample. Degenerate cellulase primers were used to amplify cellulase genes in the metagenome. The resulting amplicons cloned into Z-competent Escherichia coli DH5α were analyzed by functional screening for the production of cellulase extracellularly. Functional screening of the clones resulted in one clone (Clone-i) testing positive for extracellular cellulase production. Submerged fermentation of Clone-i was carried out for cellulase production. The cellulases were characterized to determine their activity's optimum pH and temperature. The diversity of the cellulases produced by Clone-i was determined. Clone-i's optimum enzyme activity was observed after 72 hours of incubation at 50°C and pH 5. Clone-i produced 80% more exoglucanases as compared to endoglucanases. The cellulolytic Clone-i' isolate shows Victoria Falls rainforest's potential as an enzyme bioprospecting site, reflecting that metagenomics is a valuable tool in microbial ecology.

Keywords: Victoria Falls rain forest; bioprospecting; cellulases; decaying logs; metagenomics.

Fig. 1.

Gel electrophoresis of the metagenomic DNA isolated from the Victoria Falls decaying compound logs sample. M – the 1 Kb gene ladder (Thermo Fisher Scientific, USA), lane 1 – the Fosmid Control DNA, lane 2 – the metagenomic DNA isolated from the Victoria Falls compound logs sample

Fig. 2.

Gel electrophoresis of cellulase Primer 1 and Primer 2 PCR products. M – the 100 bp molecular weight marker (Thermo Fischer Scientific), lanes 1, 2, and 3 – the reactions with MgCl₂ concentrations of 2 mM, 3 mM, and 4 mM, respectively. Amplicons from Lane 1 of fragment size 400 bp to 1,200 bp were subsequently used for cloning.

Fig. 3.

Functional screening of the Clones-i, -c, and -g on Mandel’s media after 18 hours of incubation followed by flooding with Gram’s iodine. Clone-i had a zone of clearance indicating the production of extracellular cellulases.

Fig. 4.

Production of exoglucanase () endoglucanase () by Clone-i over time. Enzyme production was carried out over 120 hours at 37°C.

Fig. 5.

Effect of pH on exoglucanase (), endoglucanase (), and total cellulase activity () produced by Clone-i grown at 37°C for 72 hours at pH 4 to 9.

Fig. 6.

Effect of temperature on exoglucanase (), endoglucanase (), and total cellulase activity () of enzyme produced by Clone-i grown at 37°C for 72 hours.

Fig. 7.

Activities of the diverse cellulases produced by Clone-i.