Extracellular and cell-associated enzyme preparations were obtained from ruminal anaerobic fungi Orpinomyces sp. GMLF5 grown in culture containing microcrystalline cellulose (avicel) as sole energy source and degradation capacities of the preparations towards several polysaccharides and glycosides were studied. Fungus showed substantial increases in xylanase, carboxymethyl cellulase (CMCase), lichenase, amylase, beta-xylosidase, beta-glucosidase and alpha-L-arabinofuranosidase activities between 72 and 168 hours. High amounts of cell associated beta-xylosidase were noted in 4 and 5 days old cultures. Optimum temperature and pH of the polysaccharidases were found at 50 degrees C and 6.0-6.5, respectively. Xylanase was found to be virtually stable at 50 degrees C, CMCase and lichenase were stable at 40 degrees C for 200 min, however amylase was found more sensitive to heat treatment. The fibrolytic enzymes of the isolate GMLF5 were observed to be capable of hydrolyze the avicel.
The facultative anaerobic bacterium Lactococcus lactis has been used as a host for expression of a gene isolated from the anaerobic rumen fungus Neocallimastix sp. The coding region of the cellulase gene was obtained from the fungus with the aid of polymerase chain reaction amplification. The gene was then transformed into pCT vector system and the constructed recombinant plasmid was introduced into two L. lactis strains (IL403 and MG1363) by electroporation. The gene encoding the fungal originated cellulase was expressed in both strains successfully although the expression level was relatively lower in comparison with the original enzyme activity. Genetically modified L. lactis strains were used as silage inoculants for pre-biodegradation of the plant biomass during ensiling. That treatment resulted in a notable reduction of the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents of the plant biomass used as silage material. Inoculation with recombinant strain IL1043 resulted in 4.8 and 9.7 % decrease in NDF and ADF contents, respectively while the inoculation of silage with strain MG1363 decreased the ADF content by >5 %.
Although Lactococcus is one of the most extensively studied lactic acid bacteria, little information is available on the regulation of the catabolite receptor protein (CRP) and acid induced gene expression. The rcfB gene, encoding RcfB protein which has unknown function in Lactococcus lactis IL1403, was analysed. Promoter region contains consensus CRP binding site and acid inducible sequence at upstream of start site. A 402 bp rcfB promoter was fused to upstream of b-galactosidase gene by cloning into promoterless pAK80 plasmid. Promoter activity corresponding to b-galactosidase was measured from Lactococcus lactis IL1403 transformant grown in medium M17 containing different carbon sources. We have found that the rcfB promoter expression was highly induced by acidity and carbohydrate sources used in medium and growth phase of the culture.
-Anaerobic fungi have highly active fibrolytic enzymes and these enzymes are attractive for scientific research. We isolated a ruminal fungus of the genus Neocallimastix sp., named GMLF7, which could survive on a variety of cellulosic material such as carboxymethylcellulose (CMC), fibrous cellulose, avicel and wheat straw. Carboxymethylcellulase production was investigated with the above carbon sources and high CMCase activity was obtained with CMC (73.75 U/ml), fibrous cellulose (72.68 U/ml) and avicel (70.03 U/ml). While growth temperature of the microorganism was 39 o C, for CMCase activity optimal temperature was 50 o C and optimal pH 6.5. Enzyme reached maximum activity in 60-180 min at 50 o C. Substrate concentration also affected the enzyme activity which was increased more than 2 fold with 40 mg/ml CMC. Activity was measured in presence of various divalent ions and reagents, and Co 2+ and DTT has a positive effect on the enzyme activity.
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