Screening Of Low Temperature Lipase Producing Strain,Optimization Of Fermentation Conditions And Study On Enzymatic Properties

Extreme microorganisms have formed a series of special mechanisms to adapt to extreme temperature,pH,salinity,pressure and other environmental conditions,which are attracting more and more attention.Daxing'anling area is a typical low-temperature environment,there are a large number of low-temperature microorganisms in such high-cold areas,which is a valuable resource worthy of research and development.Cryogenic enzymes have become the focus of enzyme research because of their unique cold adaptability and catalytic activity.This experiment takes the soil under the Xing'an Larix gmelinii forest in Mohe County,Heilongjiang Province as the research object.The microorganisms in the soil were isolated and purified by traditional plate culture method,and the highly active strains producing low temperature lipase were screened.The fermentation conditions of the strain were optimized by single factor test combined with response surface method.Lipase was purified by ammonium sulfate precipitation method,and its enzymatic properties were studied.The test results are as follows:1.Using different plate culture medium,30 different bacterial strains were isolated from the soil under the Xing'an larch forest in Mohe County,Heilongjiang Province.Five lipase-producing strains were obtained by plate screening medium culture,and a high-yield strain X16 was obtained by re-screening.By observing the morphology of the strain,the corresponding physiological and biochemical experiments and 16s rDNA sequencing results.the strain was determined to be Serratia plymuthica.2.Through the test of the growth characteristics of the strain,it was determined that the optimum growth temperature of strain X16 was about 20? and grew well at 15-30?,which belonged to the category of low temperature bacteria.Through the single factor experiment,the different factors of enzyme production conditions(temperature,pH,liquid volume,inoculation amount)and enzyme production medium(carbon source,nitrogen source,metal ion,inducer)were optimized.The optimum enzyme production conditions of strain X16 were as follows:maltose 20g/L,peptone 15g/L,Mg2+ 0.5g/L,olive oil 40mL/L,culture temperature 20?,pH 7.5,liquid volume 20ml/50ml,inoculum 0.5%.The experiment designed by Plackett-Burman was further verified,and the results showed that the eight factors had no significant effect on enzyme production.3.The lipase produced by strain X16 was preliminarily purified by ammonium sulfate precipitation method,and the purification result was the best in ammonium sulfate precipitation with 70%saturation.To explore its enzymatic properties,the optimum reaction temperature of strain X16 was 30? and the enzyme activity was relatively stable.The enzyme activity was sensitive to heat,and the enzyme activity decreased significantly after 40?treatment of 40min and 50? treatment of 30min.Although the enzyme activity changed little at 60 ?,the enzyme activity was not high.It still has certain enzyme activity at 0?,which belongs to the category of low temperature lipase.The optimum pH value is 7.0,and the enzyme activity is relatively stable under neutral or weak alkaline conditions.Among the metal ions,Mg2+,Zn2+ and Mn2+ can obviously promote the enzyme.N-hexane in organic solvents significantly promoted the enzyme,while methanol and ethanol had obvious inhibitory effect on the enzyme.