Effect Of Mutation In The Lid Domain On The Enzymatic Characterization Of Burkholderia Sp.Lipase

The activity site of lipase is covered by the lid domain of microbial lipase.Above it,which with various structural forms like no lid,single lid and double lid,etc.The lid domain makes lipase shows the characteristic of interface activation through its conformation change to control the opening and closing of the active site at the oil-water interface.Except for closely related to the interface activation,the lid domain affects many other enzymatic properties of lipase,such as the substrate specificity and the thermostability.Burkholderia sp.lipase LipA is a kind of industrial biocatalyst which widely used in enantiomeric resolution of compounds,bioenergy preparation,synthesis and degradation of polymers and other fields.Burkholderia sp.lipase LipA have two part lid domain that the lid1 stretched between residues 118-159 and the lid2 stretched between residues 214and 261.There are only sporadic reports in regard to how the two lid domains of the double lid lipase interact with each other until now,and these reports are all results from bioinformatics analysis.This research intend to investigate the interaction between two lids and the effects on enzymatic properties by introducing disulfide bonds.In addition,this research studied the effects of lid domain mutation on the thermostability of enzymes.The specific research results are as follows:1.Using bioinformatics software to predict the position of where disulfide bonds could be formed in the lid domain of Burkholderia sp lipase LipA.Using software Disulfide by Design2.0?MODIP?SSBOND and Bridge D to predict the mutation site where disulfide bond could be formed within the scope of LipA lid domain,and combining with the 3D structure of LipA,selecting the mini electronic mutant library which made up of 8 LipA mutants,including LipA-E¹¹⁸C/R²⁵⁸C?LipA-E¹¹⁸C/S²⁶⁰C?LipA-A¹²⁰C/L¹⁶⁷C?LipA-D¹³⁰C/S¹³⁵C?LipA-V¹⁴³C/A¹⁶⁰C?LipA-G¹⁴⁷C/Q¹⁵⁸C?LipA-M²⁵⁵C/S²⁶⁰C and LipA-S²⁶⁰C/S²⁶⁸C.Bioinformatics predicted that the lid domain of LipA mutants above all may introduced new disulfide bonds.2.The experimental analysis of the effects of the newly disulfide bond which formed in the LipA lid domain on its enzymatic properties.Using site-directed mutantion to obtain eight mutant genes in the electronic mutant library above;After introduced into Escherichia coli recombinantly expressed,and purification,have detected and determined that disulfide bond was formed in lid domain of six mutants by using the method of Ellman.Then have measured the kinetic constants,the thermostability and the effect of pNPB concentration on the hydrolysis activity of six mutants above,respectively.The experimental results show that:of these six mutants,only the temperature of half inactivation T₅₀¹² of LipA-G¹⁴⁷C/Q¹⁵⁸C has increased by 3.60?;The half-life extend to1.69-fold as long as wild-type ones and the value of k_cat/K_m is 1.17-fold as wild-type ones.The six mutants reached the maximum relative hydrolysis activity at pNPB concentrations of 2 mmol/L to 3 mmol/L,respectively.And the maximum relative hydrolytic activity ranged from 269.55%to 370.37%.The results of molecular dynamics simulation performed on mutant LipA-E¹¹⁸C/S²⁶⁰C show that:the root mean square deviation(RMSD)of?9-helix in the lid domain declined significantly than wild-type under the influence of the disulfide bond.3.The study on the effects of site-directed mutation of LipA lid domain on the enzymatic properties of lipase and analysis of the thermostability of lipase mutants.Our research group has constructed a series of mutants for the highly flexible lid domain previously,including LipA-E³⁵P/N¹²⁵D,LipA-E³⁵P/N¹²⁵E,LipA-E³⁵P/Q²⁶²E and LipA-F²²¹D/N¹²⁵E,intended to investigate the influences of lid domain mutation on the thermostability of lipase.Nevertheless,purification and the work on enzymatic properties research can't be finished due to the time constraint.In this research we have purified the mutants above and measured its enzymatic properties.The experimental results show that:Compared to wild type,the temperature of half inactivation T₅₀¹² of LipA-E³⁵P/N¹²⁵D?LipA-E³⁵P/N¹²⁵E?LipA-E³⁵P/Q²⁶²E and LipA-F²²¹D/N¹²⁵E have increased by 7?,9.30?,7.60?and 7.80?respectively;Under 55?heat treatment the half-life increased21.13-fold,38.14-fold,14.74-fold and 18.56-fold separately;The k_cat/K_mof LipA-E³⁵P/N¹²⁵D?LipA-E³⁵P/N¹²⁵E?LipA-E³⁵P/Q²⁶²E and LipA-F²²¹D/N¹²⁵E were:3.80×10⁵L/mmol·min,3.61×10⁵L/mmol·min,3.13×10⁵L/mmol·min and 2.69×10⁵L/mmol·min separately.