Molecular cloning, expression and characterization of novel cellulases from metagenomic DNA of buffalo rumen | |
| Author | Akbar, Tanzeem |
| Call Number | AIT Diss no.FB-12-05 |
| Subject(s) | Cellulase DNA |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Engineering and Bioprocess Technology, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. FB-12-05 |
| Abstract | Microbial communities residing in the rumen of herbivores are capable of hydrolyzing lignocellulosic residues to absorbable sugars. Unfortunately, large sections of such microbial communities are not yet culturable using conventional microbiological laboratory techniques and available nutrient media. However, a culture-independent approach, called metagenomics, can now mine, without in vitro culturing, the wealth of unraveled genetic information contained in such complex microbial communities comprising of both culturable and unculturable microorganisms. In the present study, metagenomic DNA was isolated from a pooled sample of swamp buffalo (Bubalus bubalis) rumen contents following the direct lysis method without prior separation of microbial cells using culture-independent techniques of metagenomics. Three sets of PCR primers, based on consensus regions of glycolsyl hydrolase 5 (GH5) family of cellulases, were used for PCR amplification. Seven partial and two full-length cellulase genes, Umcel5B25 and Umcel5B29, were amplified from the metagenomic DNA. The Umcel5B29 contained a complete open reading frame (ORF) and was defined as functional gene, whereas Umcel5B25, lacking a proper ORF, was defined as a pseudogene. The full-length Umcel5B29 had a complete ORF of 1611 bp. Similarity research indicated that Umcel5B29 is closely related to the cellulases of ruminal unculturable microorganisms (73% to 98% similarity) of GH5 family of cellulases. The sequence homology to the consensus pattern within the catalytic domain of GH5 family corroborated phylogenetic affiliation of Umcel5B29 to GH5 family of cellulases. Furthermore, two highly-conserved Glu residues were also identified within the active site of Umcel5B29. The amino acid alignment indicated that Umcel5B29 does not comprise a carbohydrate-binding module (CBM). Subsequently, Umcel5B29 was overexpressed in E. coli using pBAD202/D-TOPO expression vector and the expressed enzyme was characterized. The recombinant enzyme exhibited moderate level of activity when the enzyme was incubated at pH values 3-4, indicating that the enzyme can tolerate acidic to neutral pH better than that of the basic pH as negligible or no activity was detected above pH 8. The recombinant enzyme exhibited moderate thermal stability, because more than 50% of catalytic activity remained after incubation at 30 to 60°C for 30 minutes. However, at temperature above 60°C, the enzyme was inactivated/denatured quickly, showing less than 10% of residual activity and complete inactivation after incubation of 1 hour. The relative hydrolysis rates of various substrates indicated that Umcel5B29 most likely functions as an endoglucanase. The Km and Vmax values of Umcel5B29, using carboxymethyl cellulose (CMC) as substrate, were determined as 1.21±0.33 mM and 236±0.42U/mg, respectively. Lacking a CBM and having optimal stability and functionality along a wide range of temperature and pH makes Umcel5B29 cellulase a potential candidate for applications in denim and other textile industries. The study also provided the conclusive evidence of the diversity and functions of cellulases produced by microbial population in the buffalo rumen sampled. It was possible to relate the enzyme to other metagenomic cellulases reported earlier from other rumens as well, indicating the similarity in the rumen microbial communities of such animals. |
| Year | 2012 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. FB-12-05 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Rakshit, Sudip Kumar; |
| Examination Committee(s) | Athapol Noomhorm;Anal, Anil Kumar;Yakupitiyage, Amararatne ; |
| Scholarship Donor(s) | Higher Education Commission of Pakistan; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2012 |