Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.
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 %.
Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh d ) and diacetyl reductase (dar d ) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.
Streptococcus thermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp. bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level.
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