Improving The Stability Of Candida Rugosa Lipase1 By Active Site Stabilization Strategy

The mechanism of enzyme stabilization is an important scientific problem.Protein engineering is an efficient approach to explore the complex mechanism between enzyme stability and catalytic efficiency/substrate selectivity,which not only reveals the natural evolution mechanism of enzymes,but also lays a foundation for the industrial application of biocatalysts.Structural biology research shows that there is smearing of atomic electron densities around their equilibrium position,namely B-factor.Recently,Scientists are highly concerned about the study for stability by substituting amino acids with high B-factor.But there is still a lack of general guidelines.Particularly,stabilizing the large mass protein with complex structure is challenge.Our lab improved the stability of lipase B from Candida antarctica by mutating residues with high B-factor within active site and revealed the base knowledge of the structure for ACS.In order to study on the mechanism of ACS more in-depth and comprehensively,we selected LIP1 with more complex structure as the research object and studied systematically the effects of the residues with high B-factor within active site for enzyme stability,activity,substrate selectivity.Compared to the related enzymes,we explored the factors of the flexibility,spatial distribution of the residues which affected the enzyme stability seriously.Firstly,LIP1 gene was successfully cloned in shuttle vector pGAPZ?A and expressed in the form of secretion in Pichia pastoris.Characterization of enzymatic properties shows that LIP1 has high hydrolytic activity to the substrates with medium and long chain pNP-esters and triglycerides;The optimum temperature and pH of LIP1 are 45?and8.0,respectively.Its half-life is 6 min at 60?and its stability is poor.Molecular mutation technology will improve its properties.In order to construct the mutant library,we developed a method based on two-step PCR for constructing the library in eukaryotic cell,and removed the use of E.coli and restriction enzyme.At the same time,the efficiency of constructing library was improved from one week to one day.The transformation rate reached 6.9×10³/?g.We analyzed the B-factor of residues within active site and selected 18 residues within 10?of catalytic residue Ser209 with the highest B-factor for saturation mutagenesis.The following are two aspects to explore the mechanism of the change of enzyme function.1)The relativity between stability and substrate selectivityBased on the sequence alignment between LIP1 and its isoenzyme,Gly414 in LIP1 is different from any one of other enzymes?Ala/Thr?.The residue has a high B-factor and sites the bend connecting two helixes,which is flexible region.By screening the saturation library in position 414,four mutants?G414A,G414M,G414H and G414W?with improved stability from 200 clones were obtained.The optimum temperature was increased to 50?,55?,58?and 60?compared to 45?of WT,respectively.The mutant G414W,its optimum substrate was changed into pNP-C4 from pNP-C8.Modelling and docking showed that the multiple interactions formed by hydrophobic clusters and hydrogen bond in the acyl-binding tunnel might lead to the observed stability improvement.The tunnel is interrupted by the bulky?-indolyl side chain of tryptophan,which might inhibit the entrance of long chain substrate and be the reason of substrate selectivity change.2)Study systematic on relativity between enzyme stability and activity18 residues within 10?of Ser209 were mutated for studying relativity between enzyme stability and activity.By three-tier screening which included culture dish screening and two-step 96 well-plate,under two screening pressures of high stability and activity,five mutants with improved stability from 2.0×10⁴ clones were obtained.Following ordered recombination mutagenesis,which the best single-site mutant combined the next best mutant in turn,was introduced and the best mutant Var3?F344I/F434Y/F133Y/F121Y?was obtained rapidly.Its half-life at 60°C was increased by 40 folds without compromising its activity.In the process of combination,the double mutant Var1?F344I/F434Y?exhibited the highest catalytic efficiency(k_cat/K_m)2.2 times of WT and showed a half-life 10-fold longer than WT.The modelling structure of the mutant demonstrated that the formation of hydrogen-bond network and compact packing are the main reasons to improve stability.For more in-depth understanding the phenomenon from the ACS,we compared the results of the mutations on the stability of the different lipases with various structural complexities,and studied the effect of the relative B-factor,the distance between the mutant site and catalytic residue on the enzyme stability.The result showed that residues with high B-factor?relative B-factor:60¹00?in the region of 6¹0?within catalytic residue might be the hot region for increasing the success rate of the enzyme mutation.Our study indicated that the ACS is an efficient strategy for improving the enzyme stability.Systematic analysis on the relationship between the structure and function of the mutant will lay the foundation for further research of the enzyme regional stabilization rule and development of the enzyme stabilization technology.