Study On Screening Of A High-yielding Lactate Dehydrogenase Strain And Characterizing Of The Produced Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is an important oxidoreductase in vivo glycolysis.It catalytic the pyruvate and lactate in the reversible conversion. Studies on lactate dehydrogenase has been quite extensive, but few studies of Bacillus subtilis lactate dehydrogenase were reported. Bacillus subtilis is not pathogenic and has only a single cell membrane which could benefit the direct secretion of many proteins into the medium. Gram-positive bacteria have been used as a typical representative of the physiological, biochemical, genetic and molecular biology for over 40 years. The aims of present study was to breed a high-yielding lactic dehydrogenase producer and to purify and characterize the lactic dehydrogenase for providing the basis of developing the food-original Bacillus subtilis lactic dehydrogenase.Through extensive screening of food samples, the environment hays and soil samples, a high-yielding lactate dehydrogenase strain was obtained and identified as Bacillus subtilis. And purification parameters of LDH and its enzymatic properties were studied. The obtained conlusions were listed as following:1. The strain BJ07, which can produce high activity of LDH, was isolated from these samples and identified as Bacillus subtilis.2. The separation and purification methods of LDH from Bacillus subtilis BJ07 were investigated and optimized.The optimized purification process included removing cells by the centrifugation,40% saturation ammonium sulfate precipitation, dialysis, gel filtration chromatography with Superdex-75 and ion exchange chromatography with DE52. Sedium dodecyl-sulfate-polyacrylamide gel electrophovesis(SDS-PAGE) was used to examine the purification effect, and the results indicated that the final product obitained was homogeneous and had a molocular weight around 28.4KD. The purification rate and activity recovery of LDH was 25.7 and 10.7%, respectively.3. The enzyme was sensitive to the temperature and pH, and the maximum enzyme activity existed at 45C, and the optimized pH was 7.5. The stability temperature of LDH enzyme was blew 35℃.4. The cultivation of strains to produce enzymes optimal time for 60h, culture temperature 31℃,1.0% fermentation of urea as a nitrogen source, 0.5% in powder chitosan and 1.0% glucose as carbon sources, the LDH enzyme activity were highest.