Properties And Application Of Lipase Produced By Enterobacter Ludwigii

Catering industry is an important field that can stabilize economic growth,promote all the people consumption,expand job market and facilitate people’s livelihood.The development of catering industry is of great significance to promoting market strength in our country.However,in the process of rapid development,the treatment of catering oil-bearing wastewater has become a major problem.One of the main pollution sources of Chinese water is catering oil wastewater,the removal of oil in wastewater is an urgent problem.Oily wastewater mainly comes from various restaurants,restaurants,bakery shops,food factories,etc.The main component is fatty acid ester of trihydroxyl alcohol or glycerol.When untreated wastewater is discharged directly,it will hurt aquatic organisms and pollute the aquatic environment,moreover it cause serious harm to people’s health if the gutter oil obtained from the processing and refining of oily catering wastewater go into the restaurant.In this thesis,we purified and immobilized the lipase produced by the screened lipase producing strains with high viability,and applied the immobilized lipase to oil and grease degradation experiments to provide a theoretical basis for further application of the lipase immobilization method and development of methods for degradation of oil and grease in wastewater.In this paper,a microbiology with high lipase production capacity was isolated and screened from oily wastewater of canteen.The bacterium was identified by molecular genetics as Enterobacter ludwigii.Under the optimal fermentation conditions（96 h,p H=8,35℃,4%inoculum）,the lipase yield of the strain was 13.64U/m L.The crude enzyme solution of E.ludwigii A9 was obtained by ammonium sulfate fractional precipitation,DEAE-Sepharose FF anion exchange column chromatography and Sephadex G-75 molecular sieve purification.Finally,the purified lipase ELL was obtained with a purification ratio of 9.96.The molecular weight of the purified lipase ELL is about 35 k Da and the specific activity is 90 U/mg.The optimum reaction temperature and p H of the enzyme were 30℃and 8 respectively.Ca²⁺strongly promoted the activity of lipase,but in the presence of Zn²⁺and Fe²⁺,the activity of lipase was strongly inhibited.Lipase could be inhibited by 1%（v/v）isoamyl alcohol and toluene,while Triton X-100 and polyethylene glycol can activate the enzyme.The kinetic equation of lipase showed that the Km value was 2.02 mmol/L,and the maximum reaction rate Vmax was 6.05 mmol/（L·min）.Moreover,the lipase was specific to medium and long chain substrates,and the catalytic activity of p-nitrophenylcaprylate was the highest.According to the"interfacial activation"mechanism of lipase,the hydrophobic carrier NKA macroporous resin and glutaraldehyde were used for adsorption and crosslinking immobilization of lipase ELL.The conditions of ELL adsorption and crosslinking immobilization of lipase were optimized.The optimal conditions were as follow,adsorption temperature:35℃,adsorption time:20 min,carrier volume:1.5 g,glutaraldehyde concentration:1%,When the crosslinking time was 3 h,the enzyme activity was 3.42 U.The optimal temperature of immobilized lipase NKA-ELL was30℃,while the activity of the residual lipase reached 93.5%.The thermal stability of NKA-ELL at 20-60℃was significantly higher than that of free enzyme,and the activity of the residual lipase of immobilized enzyme was all higher than 50%.The optimal p H of immobilized lipase NKA-ELL increased from 8 to 9 compared with free enzyme.The acid-base stability and p H operation range of immobilized lipase were much better than that of free enzyme,and the residual enzyme activity of immobilized lipase was higher than 80%in p H 7-10 range.The stability of organic solvents was improved to some extent,especially after the organic solvent acetone treatment,the residual enzyme activity increased by 7.16%.The Km and Vmax values were 0.86 mmol/L and 44.54 mmol/（L·min）respectively.After 8 re-cycles,the immobilized enzyme activity was still higher than 50%.After being stored at 4℃for30 days,the relative activity of immobilized enzyme was 64.91%.The optimum process conditions for lipid degradation of immobilized lipase NKA-ELL.Lipase NKA-ELL was immobilized to degrade oil under optimal conditions as degradation time 48 h;Degradation temperature was 30℃.Degradation p H was 7;When the amount of enzyme is 5%.The degradation rate of oil got 92.41%.The degradation rate is still more than 50%after recyle 5 times;It was show that immobilized lipase could degrade oil efficiently and be recovered easily.