Salt Bridge Designed By Computer Aid Contributed To Thermostability Of Lipase LipA From Burkholderia Sp.

The poor temperature tolerance of Burkholderia bacteria(Burkholderia sp.)ZYB002 lipase A limit it's use effect and application scope severely.There is a vital significance to enhance the stability of the enzyme protein for extending the shelves half-life of enzyme protein and improving the enzyme reaction rate.In this paper,with the aid analysis of FoldX,Rosetta and YASARA,we build a thermostable mutant electronic library for lipase LipA.Then,combining with the 3 D structure information,we screen out the mutant with salt bridge effect from the thermostable mutant electronic library of lipase lipA.Next,we use site-directed mutagenesis technique to construct the mutant with salt bridge effect and determine its' mutation effect for screening out thermostablle mutant.Finally,in order to screen out higher thermostable mutant of lipase LipA,we biuld superposition mutants(the part of thermostable mutant screened out previously in our grounp)furtherly.Detailed experimental results are as follows:Firstly,YASARA software is used to determine the active center of lipase A and protected.FoldX and Rosetta software are used to make virtual saturated mutations for amino acid sites other than the central area.According the gibbs free energy analysis of every mutant by FoldX,Rosetta software and the structure rational visual inspection of every mutant by YASARA software,we built a mutant electronic library which contains 341 potential thermostable mutants.Secondary,through the " show interaction" function of YASARA software,we screened out 27 mutants which may have salt bridge effect from the 341 mutants5 electronic library.Due to some sites is mutated into the same properties of amino acids or after introducing mutations cause insertional mutagenesis,we only built 20 mutants successfully.After inducing expression and thermostablity analysis,we got 3 thermostable mutants from 20 mutant expression proteins.They are LipA-N125D,LipA-N125E and LipA-Q262E respectly.Compared to wide type,their temperature of half inactivation 1501O increased 4.0 ?,5.5 ? and 5.5 ? respectly;under 55 ? heat treatment,their Half-life increased 1.2-fold,2.8-fold and 1.6-fold respectively.Thirdly,we selected the most thermostable mutant(LipA-E35P and LipA-L218E,LipA-V220E,LipA-F221D and LipA-V223D)from the five saturated mutant libraries which are built in the prophase experiment work respectively.Then,we overlaped these mutants with the above three mutant(LipA-N125D,LipA-N125E and LipA-Q262E)respectively and constructed 15 positive double mutant.By the determination of thermal stability,15 double mutants' tempreture of half inactivation T5012 increased from 5.5 ?(LipA-V223D/N125D)to 11.1 ?(LipA-F221D/N125E)than wide type;under 55 ? heat treatment,their half-life t1/2 increased from 2.1-fold(LipA-V223D/N125D)to 49.57-fold(LipA-F221D/N125E)than wide type.