Improving The Thermostability Of Lipase Based On Rational Design

Lipase,one important industrial enzyme preparation,iswidely used in industrial processeson account of its unique catalytic properties.Howevever,lipases from nature cannot meet the high temperature conditions of biotechnological treatment,and loss rate of production as well as the manufacturing cost increases.To this end,our study intended to increase the thermostability of lipases via gene fusion technology,site-directed mutagenesis as well as protein structural analysis,including:1.Gene fusion technology was applied to improve the thermostability of lipase from two origins: Rhizopus chinensis r27 RCL with poor thermostability and TLL which from Thermomyces lanuginosus,however,cannot meet industrial requirement.In order to enhance the hydrophobicity of the protein surface,thusenhance the stability of protein,we fused the hydrophobic Self-assembling amphipathic peptides SAP(AEAEAKAKAEAEAKAK)to the Nterminus of the two lipases through a rigid linker(Linker1)and a flexible linker(Linker2).Four fusion lipases with improved thermostability(SAP-L1-r27 RCL,SAP-L2-r27 RCL,SAP-L1-TLL and SAP-L2-TLL)were obtained.Lipase r27 RCL,r27RC-SAP-L1 and SAP-L2-r27 RCL were treated at 60?,half-lives of SAP-L1-r27 RCL and SAP-L2-r27 RCL were 1.75-fold and 1.20-foldlonger than that of wild type(WT),respectively.Lipase TLL,SAP-L1-TLL,and SAP-L2-TLL were treated at 80?,half-lives of SAP-L1-TLL and SAP-L2-TLL were 1.50-fold and 1.17-foldlonger than that of WT,respectively.In addition,kinetic parameters showed that the substrate binding affinity and catalytic efficiency of four fusion lipases were significantly improved when compard with WT.In brief,thermostability of mutants increased by fusing SAP oligopeptides and Nterminus of lipase,and rigid linker is more effective than the flexible linker.2.The temperature factor(B-factor)of all amino acids in the crystal structure of lipase r27 RCL was extracted and normalized.Sequence(63-TKWDCK-6)was found extremely flexible due to their large B-factors.We downloaded all the sequences of thermostable and thermophilic lipases(92 sequences in total)from NCBI database,multiple sequence alignment was performed and conserved sequences 63-TNITCT-68 were summarised.Comparing two sequences,site-directed mutagenesis(K64N,W65 I,D66T,and K68T)was performed and two lipase mutants with improved thermostability were obtained(r27RCL-K64 N and r27RCL-K68T).r27RCL-K64 N and r27RCL-K68 T showed promising improvement in residual activity with a thermal retention of 37.88% and 48.20% compared to that of r27 RCL after incubation at 50? for 120 min.Half-life of r27RCL-K64 N and r27RCL-K68 T were increased by 2.4-fold and 3.0-fold compared with that of WT,respectively.Kinetic parameters showed that the substrate binding affinity and catalytic efficiency of the two lipase mutants were significantly higher than WT.Structural modelling was performed to explore the heat-resistant mechanism: K64 N and K68 T maintainthe hydrogen bond interaction in WT,however,the side chains of mutated sites are smaller than that of WT,which weaken the flexibility and improve stability;when W65 is mutated to I65,the hydrogen bond network disappeared in mutant,so the stability decreased in W65 I.3.One unpaired cysteine C204 was discovered in the structure of r27 RCL.Potential disulfide bonds were predicted using Disulfide by Design 2.0,it indicated that T201,3.8 ? from C204,can form disulfide bond with C204 when we mutate it to C201.Lipase mutant r27RCL-T201 C were designed by site-directed mutagenesis,and its thermostability was improved than that of WT.Half-life of r27RCL-T201 C was 1.25-time and 1.50-time longer than that of WT after treating at 50? and 60?,respectively.Besides,kinetic studies showed that the substrate binding affinity and catalytic efficiency of r27RCL-T201 C were significantly improved.Whereas,optimal temperature of r27RCL-T201 C was 5? lower than that of WT.Structural modelling discovered that the mutation site is close to Y200 which may influence the optimal temperature of r27 RCL,the newly formed disulfide bond may influence the regional stability thus impact the stability of Y200,in this way decreased the optimal temperature of r27RCL-T201 C.