Heterologous Expression And Esterase Properties Of Potato Patatin

Patatin is a potato glycoprotein.It has broad application potential because of its superior protein characteristics such as solubility,foaming,emulsifying,antioxidant and anti-cancer,as well as the lipolytic acyl hydrolase activity different from general storage proteins.With the rapid development of potato staple food in China,patatin has attracted much attention in the field of food.However,due to its low content in natural potato,complex extraction,purification methods and high preparation cost,its enzymatic properties had not been fully studied,which also restricted its wide application in food,medicine and other industries.Therefore,its preparation methods and enzymatic properties need to be further studied.At present,the technology of foreign protein expression through eukaryotic and prokaryotic expression system has been mature,and a large number of esterases with excellent properties and high utilization have been prepared by these methods.In this study,patatin gene was cloned from potato tubers and expressed in E.coli system and P.pastoris system,the enzyme expression system was established and expression condition were studied,enzymatic properties were explored,and then excavated its effect on natural substrate and its decomposition products were preliminarily explored,in order to open up a new way for its utilization in food,medicine and other fields.The main results are as follows:1.Bioinformatics analysis of patatin gene.Using bioinformatics analysis tools,homologous sequence alignment,evolutionary tree analysis and rare codon analysis of patatin gene were carried out on patatin gene,and then its basic physical and chemical properties were predicted:the protein was composed of 386 amino acids with a relative molecular weight of 42.49 k Da.It was a hydrophilic protein with signal peptides and transmembrane domains.2.Construction and expression of Escherichia coli expression system for patatin.The gene patatin was cloned from potato tuber,connected with vector p ET-28a（+）to construct recombinant plasmid,transferred into Escherichia coli BL21（DE3）host for induction and expression,and the optimal induction and culture conditions of patatin-E were determined:the induced culture temperature was 24°C,the induced culture time was 26 h,the initial culture p H was 7,and the concentration of inducer IPTG was 20μmol·L^-1,inoculation amount of 10%,liquid filling amount of 100 m L and rotation number of 180 r·min^-1.At the same time,the best medium was SB medium.the optimum nutrient components were determined:10 mmol·L^-1glucose,5mmol·L^-1tyrosine and 10 mmol·L^-1Mg Cl₂.Under this component,the total enzyme activity of patatin-E increased by 6.2 times.After purification,the specific enzyme activity of patatin-E reached 18.68 U·mg^-1,enzyme activity was increased 85-fold and the recovery of the purified protein was about 70.2%.The yield was 29.35 U pure enzyme in 100 m L medium.3.Construction and expression optimization of Pichia pastoris expression system for patatin.The target gene was linked with vector p GAPZαA to construct recombinant plasmid and transferred into Pichia pastoris X-33 host for secretory expression,and the optimal culture conditions of patatin-P were determined:the culture temperature was 28°C,the culture time was 144 h,the initial culture p H was8,the inoculation amount was 2.0%,and the liquid volume was 100 m L.At the same time,the optimum nutrient components were determined:50 mmol·L^-1sucrose,50mmol·L^-1tyrosine and 10 mmol·L^-1Mg Cl₂.Under this component,the enzyme activity of patatin-P increased by 2.2 times.After purification,the specific enzyme activity of patatin-P reached 1.63 U·mg^-1,enzyme activity was increased 8-fold and the recovery of the purified protein was about 85.7%.The yield was 219.91 U pure enzyme in 100 m L medium.4.The enzymatic properties of patatin and preliminary study on its decomposition in natural oil.The properties of patatin from two expression systems were explored in this chapter.Firstly,the optimum reaction conditions.The optimum reaction temperature of patatin-E and patatin-P was 37°C,the optimum p H was 9,the reaction time of patatin-E was 25 min,and the reaction time of patatin-P was 30 min.Secondly,the stability of the protein was relatively stable in the range of30-90°C and p H of 6.0-9.0,and the residual relative enzyme activity was still more than 60%.Patatin was placed at room temperature for 28 days,the enzyme activity decreased by only about 30%,so the storage stability was superior.The enzyme activity was significantly promoted by Al³⁺,Mg²⁺and Fe²⁺and the enzyme activity was obviousiy inhibited by Ni²⁺and Fe³⁺.Except ethyl acetate and chloroform,most organic solvents had no significant effect on enzyme activity and patatin had good tolerance to organic solvents.The enzyme activity of patatin was obviousiy inhibited by EDTA,SDS,β-ME and DTT,especially patatin-E.Patatin had strong hydrolysis ability to short chain substrates（p-NPC₂and p-NPC₄）and still had certain hydrolysis activity to long chain substrates(p-NPC₁₂and p-NPC₁₆).The two proteins had also the highest affinity for p-NPC₂,V_maxwere 49.75 mmol·L^-1·min^-1and 50.00mmol·L^-1·min^-1,respectively.K_mwere 1.01mmol·L^-1and 1.14 mmol·L^-1,respectively.Patatin-E had better storage stability,and patatin-P had more stronger tolerance to chemical reagents.The enzyme had higher hydrolysis activity for natural oils rich in saturated fatty acids.It was found that the two had also better stability,patatin-E had better storage stability,and patatin-P had stronger tolerance to chemical reagents.To sum up,patatin gene was expressed in Escherichia coli system and induced for 26 h,and secretory expression in Pichia pastoris system,and it taked 144 h to culture,patatin-E had higher specific enzyme activity and purification multiple than patatin-P,but the purification recovery of the latter was higher and the yield is 7.5times that of patatin-E.The results would help to reduce the fermentation cost and obtain a large number of patatin with high biological activity and stability,which will be conducive to its industrial production.At the same time,the enzymatic properties of patatin were fully studied,and it was found that patatin had the bifunctional activity of esterase and lipase,it could be widely used in food,medicine and other fields.