Periplasmic Expression And Characterization Of Thermostable Lipase Of Pseudomonas Fluorescens From Raw Milk

First of all,a fast and sensitive detection method for lipase activity of dairy products was established.In the early work of our laboratory,a psychrotrophic bacteria producing heat-resistant lipase was isolated from raw milk.It was identified as Pseudomonas fluorescens and numbered BJ-10（Pseudomonas fluorescens BJ-10）.In this paper,we take the lipase produced by P.fluorescens BJ-10（lipBJ10）as research objective and employ the Escherichia coli as host bacteria to overexpress lipBJ10.In order to achieve soluble and functional lipBJ10,signal peptide fusion expression technology was applied to explore the effects of signal peptides from different secretory types on lipBJ10 secretion.The best signal peptide of lipBJ10 periplasmic expression has been screened.Based on that,we optimized the best expression conditions of periplasmic expression,and established an effective and feasible strategy for isolation and purification.Then,we studied the enzymology characteristics of lipBJ10.The main conclusions of this study are as follows:（1）A rapid detection method for lipase in dairy products was developed.The buffer,containing sodium citrate,was formulated and used to clarify milk sample.It can dissolve the insoluble casein micelles,and then the lipase activity can be directly detected by spectrophotometry after removing milk fat.In the process of detection,the method has many advantages,such as less steps,simple,easy to operate,less samples and reagents needed.In measuring milk lipase activity,the method has good correlation and high sensitivity.It can be applied to detect the lipase activities of different commercial liquid milk products,such as pasteurized milk and UHT milk.（2）The full-length nucleic acid sequence of lipBJ10 was cloned and identified by homologous comparison.It is a kind of lipase from superfamily,specifically theⅠ.3 subfamily.The sequence of nucleic acid and protein has been submitted to the NCBI database,and the number is KY939609.（3）By comparison,it is found that the effect of temperature on over-expressed soluble lipBJ10 is greater than that of IPTG,and the low IPTG induced concentration is beneficial to generate functional lipBJ10 at optimum fermentation temperature of 20℃.（4）Fusion expression to signal peptide can effectively enhance the solubility and activity of lipBJ10.When lipBJ10 were fused to DsbA,the activity of the periplasm reached the highest 265.41 U/ml.According to a comprehensive analysis,it is shown that DsbA is the most suitable signal peptide for fusion expression of lipBJ10.（5）The expression condition of lipBJ10 is optimized by response surface method.A reliable and accurate model is constructed,and the regression equation of three factors is established.Y（activity/（U/ml））=270.28+24.19A-18.82B-5.83C-55.98AB-31.42AC+2.19BC-53.58A²-60.8B²-31.84C²,A-temperature,B-IPTG induced concentration,and C-induction time.The optimal over-expression conditions deduced from the model and regression equation are as follows:the induction temperature was 22.78,the induced concentration of IPTG was 0.16 mM and the induction time was 38.1hours.The maximum lipase activity predicted by the model is 282.019 U/ml.After verification,the optimized expression condition is reliable.（6）The purification method only by two steps,affinity chromatography followed by gel filtration chromatography,has been successfully established to separate and purify high purity lipBJ10 samples,which provided a reference method and strategy for the separation and purification of recombinant protein.（7）The study of enzymatic properties showed that lipBJ10 was an alkaline lipase,and its optimum temperature and pH value are 45℃and 8.6,respectively.Ca²⁺can significantly increase the activity of lipBJ10.EDTA can reduce the enzyme activity of lipBJ10,which may be related to the chelation of Ca²⁺in the lipase structure.Heavy metal ions can significantly reduce the activity of lipBJ10.Non-ionic surfactant Triton X-100 and anionic surfactant SDS can enhance lipBJ10 enzyme activity.In particular,methanol can promote the increase of enzyme activity of lipBJ10,which makes it have the potential to be applied in the production of biodiesel.