Research On The Enzymatic Characterization And Structure-function Relationship Of Lipase AFLB From Aspergillus Fumigatus

Lipase?E.C.3.1.1.3?is a kind of serine hydrolases that catalyze a variety of reactions?such as hydrolysis,esterification,transesterification,etc.?which are widely involved in important industrial processes.With the rapid development of industry,the demand for new lipases is growing day by day.The lipase CALB from Candida antarctica is the most important enzyme used in the industry,revealing an efficient catalyst for preparation of single isomer chiral drugs,synthesis of biodegradable polyesters and so on.It's found that the lipase AFLB from Aspergillus fumigatus GIM 3.19 is homologous to CALB,revealing a great industrial application prospects.But its enzymatic properties and molecular structure are still unknown.In this study,lipase AFLB was used as the research object.The preparation,enzymatic properties and three-dimensional structure of the recombinant enzyme were studied.The structure and function relationship of the particular lid structure and N-terminal subdomain of lipase AFLB were studied by means of site-directed mutagenesis and molecular dynamics simulation.All of these will help to better understanding of the lipases of CALB family and lay a solid foundation for its future protein engineering.The main research contents are as follows:?1?Cloning,expression and characterization of lipase AFLBThe gene aflb coding lipase AFLB from Aspergillus fumigatus was successfully cloned by reverse transcription PCR,and the expression vector pET28a-aflb and its E.coli expression strain were constructed successfully.Recombinant lipase AFLB was successfully expressed in E.coli cells at the condition of 20°C,0.05 mmol/L IPTG,induced at 200 rpm for 24 h.The recombinant protein obtained by combining metal chelate affinity chromatography and anion exchange chromatography was in high purity?>90%?.And the yield is about 1 mg/L fermentation liquor.The enzymatic properties of lipase AFLB showed that lipase AFLB is a mesophilic lipase,with a optimum reaction temperature of 40°C,good stability below 45°C,and optimum pH of 7.5.Lipase AFLB is highly resistant to high concentration of NaCl?6 mol/L?and exhibits strong tolerance to non-polar organic solvents?n-hexane,diethyl ether and toluene?.But its tolerances for polar organic solvents?DMSO,methanol,ethanol,acetone and acetonitrile?and surfactants?Triton X-100,Tween-80/60/20,SLS and SDS?were weak.The most suitable substrate for lipase AFLB is tributyrin which contains short-chain fatty acids,followed by triglycerides containing medium-long chain long fatty acids?C8-18?,showing strict sn-1,3-specificity toward tricaprylin and triolein in same time.?2?Preparation of lipase AFLB crystal and its structure determinationThe optimal protein concentration for lipase AFLB crystallization determined by protein precrystallization experiments was 12 mg/mL.After further screening of crystallization conditions,the growth conditions of lipase AFLB crystals were determined as follows:ambient temperature 16°C,10 mmol/L Tris?pH 8.0?,100 mmol/L NaCl and 1.8 mol/L ammonium sulfate.A high diffraction quality crystal can be obtained by a hanging drop method.The crystal structure of lipase AFLB was resolved by molecular replacement method with a final resolution of 2.0?.The PDB ID of lipase AFLB structure is 6IDY.The structure of lipase AFLB is a traditional?/?hydrolase fold structure,which is formed by16?helices and 7?sheets.The catalytic triad of lipase AFLB is composed of three residues-S233,D318 and H361.The"oxygen ion hole"is composed by the NH on the backbone of residues T169 and Q234.In the the?/?hydrolase core region,lipase AFLB shows high similarity to the homologous lipase CALB,and their RMSD of the 235 backbone?carbon atoms is 1.407?.The"lid"structure of lipase AFLB is a random coil with a disulfide bond in it.The large hydrophobic side chain of residue W275 in the middle of the"lid"penetrates deeply into its hydrophobic catalytic pocket,thereby it separates the hydrophobic catalytic pocket from the polar environment to protect the enzyme from viability under non-operating conditions.The N-terminal subdomain of lipase AFLB is one of its important features,and it is also the unique N-terminal subdomain of this lipase subfamily.For the position and composition of the secondary structure,the N-terminal subdomain of lipase AFLB has a certain similarity with the"cap"subdomain of thermophilic esterase and CALA-like lipase.It indicates that the N-terminal subdomain of lipase AFLB may play an important role in the recognition of ester substrates.?3?Regulation of enzyme activity by lipase AFLB lid structureCompared with the wild type lipase AFLB,the K_m value AFLB lid mutants toward triacetin was decreased by 30%to 63%,and the k_cat value increased of 1.6 to 5.6 times.However,the half-life value(t_1/2)and melting temperature?T_m?of lid mutants showed a different degree of decline,and the decline of the mutants AFLB-CALBlid and C273A/C281A is the most obvious.These results indicate that the lid of the lipase AFLB plays an important role in protecting enzyme stability.Through the mutation of a single key amino acid residue on the lid?AFLB-W275A?or disulfide bond cleavage?AFLB-C273A/C281A?,the affinity and catalytic activity of lipase AFLB for glyceride substrate can be significantly improved,but it will damage its structural stability,in turn,negatively affects thermal stability.?4?Regulation of enzyme activity by the N-terminal subdomain of lipase AFLBAFLB mutants lacking helix?1?AFLB-D1-25?and?1-?2?AFLB-D1-59?can still be successfully expressed in E.coli hosts.Deletion of the helix?1-?3 and longer fragments comprising residues 60-128 resulted in failure of AFLB soluble activity expression.The specific activity of the mutant AFLB-D1-59 to tributyrin was 2.14 times that of AFLB wt,but its optimal reaction conditions?pH and temperature?,salt tolerance,thermostability and substrate specificity were almost as same as that of wild type.The maximum insertion pressure?MIP?values of lipase AFLB wt and AFLB-D1-59 to phospholipid monolayer were 14.59 and 58.66 mN/m,respectively.It indicates that AFLB-D1-59 inserted more deeply into the phospholipid monolayer than AFLB wt in the same phospholipid monolayers with same surface density.At the same time,it was found that the synergistic factor of AFLB-D1-59?0.6824?was higher than that of AFLB wt?0.0298?,indicating that AFLB-D1-59 is more inclined to adsorb onto DOPC monolayer membrane.Therefore,the reason for the improvement of the hydrolysis activity of the mutant AFLB-D1-59 may be that the improved adsorption specificity of the mutant to the oil-water interface promoted the whole hydrolysis reaction.The RMSD and RMSF of AFLB wt and AFLB-D1-59 are close,meaning that the N-terminal subdomain can be stably attached to the core domain.The conformation of the enzyme molecule remains stable even in the absence of the N-terminal 1-59 amino acid residues.However,amino acid residues 48-62?IGKTEFSRSTKDAKS?form a large,positively charged and irregular loop structure on the surface of the enzyme molecule that can be cleaved by endopeptidases.Thus,in the natural environment,the helices?1 and?2 of the N-terminal subdomain of the lipase AFLB may mediate the regulation of enzyme activity by being retained or excised.