Screening And Identification Of Lipase Producing Bacteria And Optimization Of Enzyme Producing Conditions

The animal husbandry level in China has exceeded the world average,but the impact of large-scale animal husbandry on the ecological environment is increasingly serious.Animal husbandry has become a major source of pollution.It not only puts pressure on the environment but also affects human health.The disposal of sick and dead animals is particularly serious.According to the statistics,98.2% of farmers cause air pollution,of which 36.4% is serious pollution.However,there is a lack of scientific and systematic disposal measures for the carcasses of farm animals that have not died after slaughter in China.At present,animal carcasses are mainly treated by traditional burial and open burning methods.Landfills and incineration not only occupy land resources but also cause secondary pollution.Compared with the traditional physical and chemical methods,the biodegradation method for the treatment of animal carcasses and wastewater has the advantages of mild reaction,high product recycling rate and environmental friendliness,and can bring huge ecological and economic benefits.As one of the main components of animal body,the effective degradation of fat will bring great improvement to ecological benefits and economic benefits.In this study,the strains producing lipase were first screened by using the screening medium with olive oil as the only carbon source and the fermentation medium with animal body fat as the only carbon sourc.A high lipase strain would be selected from the selected strain.We optimized the fermentation process,and tried to improve the enzyme activity by using uv mutagenesis and chemical mutagenesis.The application of lipase in biodegradation of animal fat was discussed in this study.In this study,samples werecollected from farms,kitchen wastewater,poultry market and other environments,and 13 lipase producing strains were screened out by Victorian blue emulsion plate.There were 5strains of bacteria: B-YYT-8,B-TTY-11,B-YYT-19,B-YYT-32 and B-YYT-34.8 strains of fungi: F-YYT-1,F-YYT-2,F-YYT-3,F-YYT-4,F-YYT-5,F-YYT-6,F-YYT-7,F-YYT-10.Through morphological observation,biochemical identification test,16 S r DNA sequence determination and phylogenetic analysis,they were finally determined that: B-YYT-8 was acinetobacter baumannii,B-YYT-11 was bacillus cereus,B-YYT-19 and B-YYT-32 were pail chicken enterococcus,and B-YYT-34 was escherichia coli.After morphological observation and ITS identification,they were finally determined that: F-YYT-1,F-YYT-2,F-YYT-3,F-YYT-4,F-YYT-5 were Candida parasitosis,and F-YYT-6,F-YYT-7 and F-YYT-10 were aspergillus fumigatus.The enzyme activities were measured: B-YYT-8:33.5 U/m L,B-YYT-11: 50.0 U/m L,B-YYT-19: 29.3 U/m L,B-YYT-32: 26.4 U/m L,B-YYT-34: 42.2 U/m L,F-YYT-1: 53.4 U/m L,F-YYT-2: 95.1 U/m L,F-YYT-3: 52.2 U/m L,F-YYT-4: 54.1 U/m L,F-YYT-5:42.5 U/m L,F-YYT-6: 54.5 U/m L,F-YYT-7: 44.3 U/m L,F-YYT-10: 78.3 U/m L.Based on the above experiments,it was concluded that F-YYT-2 is a high-yield lipase strain.According to the mutagenesis principle,uv and DES mutagenesis were performed to obtain a mutant strain F-YYT-2C.The average enzyme activity was 104.69 U/m L.This experiment conducted preliminary purification of lipase produced by the fermentation of two mutagenic strains F-YYT-2U,F-YYT-2C and the original strain F-YYT-2,and tracked the protein concentration and enzyme activity of strains with different degrees of purification,and finally verified the reliability of breeding high-yield lipase strains with complex mutagenesis The application value in biodegradation was preliminarily verified through the method of national standard CJ/ t51-2004.In the experiment,the oil content before fermentation was 0.2808mg/m L,and after 48 h fermentation,the oil content decreased to 0.1031mg/m L.Detection of F-YYT-2C products after fermentation in olive oil fermentation.One high lipase strain F-YYT-2 was selected from the strains.The fermentation condition was optimized and the optimum fermentation period was determined to be 48 h.The optimum inoculation amount of F-YYT-2 was 2%.The optimal initial PH value ofF-YYT-2 was 8.0.F-YYT-2 best fermentation temperature 28?;The optimum shaking speed of F-YYT-2 was 220 r/min.In this study,13 lipase producing strains were isolated and identified,among which 8were fungi and 5 were bacteria.High lipase strain F-YYT-2 was obtained from fungi,and high lipase strain B-YYT-11 from bacteria.What's more,this study lays a foundation for the construction of genetically engineered bacteria,the construction of efficient production strains and the improvement of the environment of livestock and poultry.