Identification and enzyme production of a cellulolytic Bacillus-strain isolated from moose (Alces alces) rumen
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A major obstacle to industrial-scale production of fuel from lignocellulose lies in the inefficient deconstruction of plant material, due to the recalcitrant nature of the substrate toward enzymatic breakdown and the relatively low activity of currently available hydrolytic enzymes. Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Cellulases are required for cellulose degradation in nature and almost all of the biomass produced is mineralized again by enzymes which are provided by microorganisms. The crystalline material is hydrolyzed by a number of simultaneously present, interacting enzymes (endoglucanase, exoclucanase and β-glucanase), or alternatively by a multienzyme complex. Cellulosome complexes are intricate multi-enzyme machines produced by many cellulolytic microorganisms. They are characterized by having a scaffolding protein, and are typically anchored to the cell membrane through a dockerin-protein. The goal of this work involves the production, identification and initial purification of a cellulolytic and hemicellulolytic enzymes from bacterial isolates from moose (Alces alces) rumen. Five bacterial isolates (MRB 1-5) were comparatively analysed for effective producer of cellulase enzyme. Isolates were screened for cellulolytic activity using Carboxy Methyl Cellulose (CMC) agar plates and DNS reducing sugar assay, these techniques are time-efficient and reliable in identification of cellulolytic microorganisms. Screening also included growth curve characteristics under anaerobic and aerobic conditions. Among the five bacterial isolates, isolate MRB 3 was found to be the most effective cellulase producer both qualitatively and quantitatively. MRB 3 was identified by use of of DNA isolation and 16sRNA analysis as a strain of Bacillus licheniformis, tentatively named AA1. CMC- zymogram analysis of SDS-PAGE gels demonstrated two catalytically active bands at approximately 65 kDa and 45kDa. Most of the samples purified from B. licheniformis AA1 cultures showed several protein bands on SDS-PAGE with the highest band at approximately 200kDa. The presumed MEC is not attached to the cell wall but is secreted into the supernatant. The CMC-ase active, high molecular protein band and lower fragments observed in this organism, further promote the hypothesis that a MEC is present in B. licheniformis AA1. In shaking cultures supplemented with 0.5% CMC or beechwood xylan, B. licheniformis AA1 was able to regulate enzyme expression based on the substrate. A stepwise release of enzyme activity by affinity washing of cellulose-bond enzyme showed that the cellulase-activity could bind to insoluble Avicel. The protein and enzyme activity was concentrated by about two fold from culture supernatants by crossflow filtration with 95% recovery of total enzyme activity. However, significant amounts of activity passed through both 50 and 10 kDa UF membranes, indicating the presence of low-molecular cellulases. Purification of MEC from a culture supernatant was not successful. The target protein failed to bind on these otherwise standard high-yielding columns assumable not because of charge incompatibility but due to the large size of the MEC. It is concluded that a strain of B. licheniformis was isolated from the rumen of the moose (Alces alces) and was named B. licheniformis AA1. It is likely that a MEC was isolated in this organism because SDS-PAGE and zymograms were repeatedly carried out with different forms of purified MEC and results showed consistency, indicating a composition that is non-random. In addition, the inability to successfully isolate the MEC through the ion exchange chromatography was presumed to be due to size exclusion. Further experiments to verify the existence and composition of a MEC consisting of cellulases and hemicellulases in this organism are suggested.
Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2013. Master of applied and commercial biotechnology.