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Cloning Cellulase Genes from Victoria Falls Rainforest Decaying Logs Metagenome

Makhosazana Nyathi

Makhosazana Nyathi

Department of Applied Biology and Biochemistry, National University of Science and TechnologyBulawayo, Zimbabwe
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Nyathi, Makhosazana
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Zephaniah Dhlamini

Zephaniah Dhlamini

Department of Applied Biology and Biochemistry, National University of Science and TechnologyBulawayo, Zimbabwe
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Dhlamini, Zephaniah
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Thembekile Ncube

Thembekile Ncube

Department of Applied Biology and Biochemistry, National University of Science and TechnologyBulawayo, Zimbabwe
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Ncube, Thembekile
  
Jul 29, 2024
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Article Category: ORIGINAL PAPER
Published Online: Jul 29, 2024
Page range: 343 - 348
Received: Sep 20, 2022
Accepted: Dec 28, 2022
DOI: https://doi.org/10.33073/pjm-2024-029
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© 2024 Makhosazana Nyathi et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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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
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.
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.
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.
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.
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.
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.
Activities of the diverse cellulases produced by Clone-i.
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