Screening And Identification Of Lipase Production Strain And Optimization Of Fermentation Conditions And Its Enzymatic Properties

Lipases known as triacylglycerol acylhydrolases(E.C.3.1.1.3)are ubiquitous carboxylic ester hydrolases that can catalyze hydrolysis of the long chain triglycerides to fatty acids,diacylglycerol,monoacylglycerol,and glycerol in the aqueous phase.In non-aqueous systems,lipase has good properties such as lipidation,transesterification,alcoholysis and amineysis,and this feature allows lipase to be widely used in pharmaceutical synthesis,papermaking,textiles,food,cosmetics and other industries.In this paper,olive oil was used as the sole carbon source to select high yield lipase strain.Then,the optimize the fermentation conditions,purification and characterization of the lipase,and application for bioremediation of restaurant wastewater had also been reaserched in the paper.The main researching contents are as follows:A strain of HFE733 producing lipase was screened from the waste residue by means of lipid assimilation plate and tributyrate glyceride tablet,and the determination of enzyme activity was 1.93 U/mL.The strain was identified as Pseudomonas aeruginosa by morphological,physiological,biochemical and phylogenetic methods.The optimal mutant strain D13 was obtained by the methods of UV and diethyl sulfate mutagenesis through the original strain HFE733.The enzyme activity of lipase was 5.03 U/mL by shake flask fermentation,which was 160.62% higher than that of strain HFE733.The genetic stability test showed that D13 strain had good genetic stability,and then named as HFED13.The lipase fermentation conditions of HFED13 were optimized by single factor experiments.The optimum fermentation conditions were as follows: fermentation temperature 30 ?,fermentation period 60 h,initial pH 7.0,inoculation amount 5%,loading volume 50 mL/250 mL,shaking speed 220 r/min.Then,the fermentation medium components were optimized by Plackett-Burman experiments,the steepest ascent experiments and response surface methodology.The optimum formulation of the fermentation medium was as follows: yeast extract 34 g/L,olive oil 5.92 m L/L,sucrose12.5 g/L,copper sulfate 0.4 g/L,manganese sulfate 0.2 g/L.The activity of lipase was9.27 U/mL,and the results were in good agreement with the predicted values,which was 84.29% higher than that before optimization.The enzyme was isolated and purified from the fermentation broth of HFED13 strain by purification methods of ammonium sulfate precipitation,Sephandex G-25 gel filtration desalting and DEAE-cellulose A-52 ion exchange chromatography.SDS-PAGE analysis showed that the purified lipase was a single protein with molecular weight of 51.02 kDa,a purification factor of 9.97,a recovery of 31.54%,and a specific activity of 79.25 U/mL.The optimum pH of the purified lipase was 7.0~8.5,and the optimum reaction temperature was 40 ?,and the enzyme had a wide range of pH stability.Different metal ions and chemical agents had different effects on lipase.In addition,the purifiedenzyme was highly activated by several metal ions and chemical reagents such as Fe3+,Al3+,?-mercaptoethanol,cysteine and dithiothreitol.Whereas,the enzyme was strongly inhibited by Co2+?Cu2+?Tween 80 and Triton X-100.10 mM sodium dodecyl sulfate completely inhibited the activity of the enzyme.The strains and enzymes had good degradation effect on the restaurant wastewater.The oil content and COD(chemical oxygen demand)in the wastewater decreased from 4296.00 mg/L and11449.42 mg/L to 195.79 mg/L and 1243.97 mg/L respectively after six days of mixing with waste water and fermentation broth.Down 95.44% and 89.14%,basically reached the emission standards.