Screening Of Moderately Halophilic Bacterium For Production Function Enzyme And Optimizing Of The Fermentation Conditions

In this study, we separated and identified the moderately halophilic bacteria which produce functional enzymes from saline and alkaline soil near Daqing oil production company for the first time, understanding the types and characteristics of producing functional enzymes of moderately halophilic bacteria in this area. Combined with some physiological characters of moderately halophilic bacteria, designing screening culture medium of fuctional enzymes and optimizing the experimental method constantly in the process of screening bacteria, moderately halophilic bacteria producing fuctional enzymes were targeted screened through adding enzyme specific substrate into the medium.In this experiment, screening56moderately halophilic bacterias and of which3strains can produce amylase,2strains can produce esterase,6strains can produce lipase, After separating、purifying and identifying for the strains with high enzyme-producing activity,16SrDNA sequence analysis suggested that both production amylase strain DF-01and production lipase strain ZF-03are Nesterenkonia and thus named Nesterenkonia sp.DF-01and Nesterenkonia sp.ZF-03respectively, and production esterase strains Z-01preliminary judgment for Gracilibacillus saliphilus strain YIM91119.Though researching crude enzyme properties and producing enzyme optimization from three moderately halophilic bacterias we found that, for DF-01strains, optimum reaction of the amylase temperature was40℃, pH is8.0. The enmyze had good thermal stability and under the condition of alkaline, the enzyme was relatively stable. The optimum Nacl concentration of the enzyme reaction was5%, salt tolerance is poorer when the value is more than20%. In the condition of liquid volume100mL/250mL, the optimum conditions of producing enzymes though fermentation:1%source corn meal as the optimum carbon source,2%yeast extract as the optimum nitrogen source, medium with initial pH8.0、concentration of NaCl5%、culture temperature37℃,160rpm shaking culture for72h.For the extracellular esterase from Z-01strains, the optimum reaction temperature was45℃, pH was8.5. The enmyze had good thermal stability below50℃and good pH tolerance, and the optimum Nacl concentration of the enzyme reaction was10%, salt tolerance is better when the value is less than20%. In the condition of liquid volume100mL/250mL, the optimum conditions of producing enzymes though fermentation:5%source corn meal as the optimum carbon source,1%yeast extract as the optimum nitrogen source, medium with initial pH7.5、concentration of NaCl10%、culture temperature37℃,160rpm shaking culture for60h.For the extracellular lipase from Z-03strains, the optimum reaction temperature was50℃ pH was9.0. The enmyze had good thermal stability below70℃and good pH tolerance, and the optimum Nacl concentration of the enzyme reaction was3%, salt tolerance is better when the value is less than10%. In the condition of liquid volume100mL/250mL, the optimum conditions of producing enzymes though fermentation:1%source corn meal as the optimum carbon source,3%yeast extract as the optimum nitrogen source, medium with initial pH8.5^concentration of NaCl3%、culture temperature38℃,160rpm shaking culture for72h.