Study On The Screening、Optimization Of Its Fermentation Conditions And Enzymatic Properties Of A Cold-adapted Lipase-producing Strain

Object:Cold-adapted lipase is one of the most important enzyme for industry application withproperties of high catalytic activity at low-temperature、sensitive to heated and organic solventtolerant and so on.Generally,cold-adapted lipase is product by cold-adapted microorganisms,andthere are rich extreme environment in the nature.which contained various types of extremeenvirenmental microbial resources.Cold-adapted microorganisms had to synthesis and secretionlots of cold-adapted enzymes to adapt to low-temperature.This study is aimed at isolating andpurifying of cold-adapted lipase-producing strains from alpine frozen soils in the TianshanMountains,and choosing a high-yield strain to preliminary identificating,fermentation wasoptimized and enzymology property was researched.To establish solid foundation of acquire new cold-lipase strains from different environment,and obtain new characteristics of high activity of lipase.Methods:This study depends on the distinctive geographical environment of XinjiangProvince,isolating and purifying cold-adapted lipase producing strains from the alpine frozensoils of Glacier No.1in the Tianshan Mountains by Rhodamine-B and Tween-80medium.Combined with p-NPP colorimetric method, isolated high-yield lipase-producing strains.The growth characteristic、physiological and biochemical experiments of the a higher cold-adaptedlipase strain had been studied,and combined the16S rDNA gene sequences to determine itsgenera.The response surface method are used to rapidly optimize the lipase production conditionsand properties of enzyme was studied.Results:With the Rhodamine-B,Tween-80medium,75cold-adapted lipase producing strain wereisolated from alpine frozen soils of Glacier No.1in the Tianshan Mountains in this study.Aftersecondary screening,choosing a high-yield strain,on the basis of the morphological,physioigicaland chemotaxonomic characteristics,16S rDNA sequence homology analysis,the results is that thisstrain belong to Pseudomonas sp.,named as Pseudomonas sp.T1-39.By studying enzymatic characteristics,the optimum temperature of Pseudomonas sp.T1-39was16℃~30℃.It belongs to category of the psychrotroph.On the basis of dynamic fermentationexperiment, fermentation condition of producting lipase by Pseudomonas sp.T1-39wereoptimized.The single factor experiments were carried to determine the influence of carbon source、nitrogen source、metal ions、inducers、initial pH、fermentation time and temperature,determinedthe best enzyme production medium level of single factor:Soluble starch5.0g/L, tryptone,ammonium sulfate (1:1)10.0g/L,40ml/L olive oil emulsion, MgSO4,7H2O2.0g/L, K2HPO42.0g/L.16℃, medium initial pH9.0, the inoculum is2%, to sample weight20ml/250ml,fermentation cultivation32h reach the peak of enzyme production, speed at180rpm. Based on the results of single factor,the three important factors influencing lipase productionidentified by experiment design of Plackett-Burman were temperature, tryptone and initial pH.Inthe region as a center, Box-Benhnken design and resonse surface analysis were adapted.And threestatistically significant factors the temperature of17℃, the initial pH of8.5,4.6g/L tryptone wereselected.The lipase activity reached10.71U/mL,which was3.42folds of that before optimization.The lipase of Pseudomonas sp.T1-39was purified by ammonium sulfate fractionation,andproperties of enzyme was studied.The results show that:the optimal temperture was30℃,about70%of the original activity was maintained at20℃for1h,less than40%of the original activity wasmaintained by heated at60℃for30min.It belongs to category of the cold-lipase with aheat-sensitive.The optimum pH9.0,it showed relatively stable ability from pH7.0~9.0.the enzymeactivity was stimulated by Mg2+、Zn2+、Ni2+、Ba2+and chelating EDTA,Lip-39belongs to metalactivate lipase.The enzyme activity was stimulated by n-hexane.