Research On The Preparation And Characterization Of Natural And Recombinant Bacillus Licheniformis Lipases

Lipase（EC.3.1.1.3）can catalyze the hydrolysis and synthesis of ester compounds,so it has good application prospects in many industries,such as food processing,waste treatment,biofuels,detergents,and pharmaceuticals.At present,many lipases have been isolated from sources such as animals,plants,and microorganisms,among which microbial lipase is the enzyme with the most diverse enzymatic properties and great potential for industrial applications.However,most natural microbial lipases have low activity and are difficult to purify,and the free form cannot meet harsh reaction conditions,so only a small amount has been commercially developed and utilized.To cope with the growing industrial demand and overcome the defects of enzymes,the preparation and characterization of natural and recombinant microbial lipases to replace traditional chemical and synthetic catalysts and test their application potential have become current research hotspots.In this study,a lipase-producing Bacillus licheniformis strain was isolated,and its extracellular lipase was partially purified and characterized to explore its potential for the biodegradation of food pesticide residue 2,4-D butyl ester.The Bacillus licheniformis lipase was selected as the research object which has relatively high coverage in the peptide mass spectrometry analysis of partially purified lipase.For the first time,recombinant lipase Lip was prepared by prokaryotic expression technology,purified and enzymatic property characterization was performed,and the lipase Lip was applied to enhance the release of fatty acid flavor in low-fat cheese.The functionalized magnetic mesoporous biochar was prepared with Cinnamomum camphora seed shell as the substrate and the lipase lip was modified by enzyme immobilization technology.The biochemical and structural properties of immobilized lipase were characterized and further applied to catalyze the transesterification of Cinnamomum camphora seed kernel oil and perilla seed oil to produce structural lipids.The molecular docking was used to study the pathway of the interaction between the lipase Lip and the substrates p-nitrophenyl palmitate（p-NPP）and tributyrin,and the catalytic mechanism of the lipase was investigated.Attempting to modify lipase Lip through protein engineering technology based on molecular recognition and the key residues of the conserved region of enzyme.Then explored the influence of effective site-directed mutagenesis on its enzymatic and structural properties and analyzed the potential reasons for the enhancement of mutant activity and performance combined with molecular dynamics simulation.The main findings are as follows:（1）In this study,Bacillus licheniformis NCU CS-5 was isolated from the spoiled Cinnamomum camphora seed kernel,and its extracellular lipase was isolated and partially purified with an activity of 192.98 U/mg and a molecular weight of approximately 27 k Da.Lipase is active over a wide range of temperatures（5～55℃）and p H values（6.0～9.0）,with optimum temperatures of 40℃and a p H of 8.0,respectively.The enzyme remains active in the presence of various organic solvents,metal ions,enzyme inhibitors,and surfactants.In the presence of commercial detergents,both crude lipase and partially purified lipase remained more than 80%active after 5 hours.The isolated lipases showed high activity against medium-and long-chain fatty acid(C₈-C₁₈)substrates.Peptide mass spectrometry analysis of partially purified lipases showed 34.80%coverage of Lip from the Bacillus licheniformis lipase family.In addition,it degraded more than 90%of the food pesticide residue 2,4-D butyl ester to its hydrolysate 2,4-D within 24 hours,indicating that the lipase can be used for the biodegradation of organic ester pesticide residues.（2）The partial purification of lipase from wild bacteria results in poor activity and low purity,and this difficulty is caused by the limited amount of available experimental equipment;therefore,the lipase Lip derived from Bacillus licheniformis,which has relatively high coverage in peptide mass spectrometry analysis,was selected as the research template,and then prokaryotic expression was performed in different Escherichia coli strains.Under the optimal expression conditions,the recombinant bacteria obtained highly active recombinant lipase after 12 hours of large-scale expression in 3-L shake flasks（with an enzyme activity of 599.03 U/mg,which was3.10 times that of partially purified lipase）.The activity of recombinant lipase was optimal at 40℃and p H 10.0,with a relative activity of more than 80%at p H 9.0～10.0and a relative activity of more than 50%at medium temperature（30～45℃）.Lipase exhibits some tolerance to various organic solvents and metal ions and high specificity for short-and medium-carbon chain fatty acid triglycerides.Among the different substrates tested,lipase had the strongest binding affinity for p-nitrophenyl palmitate（p-NPP,p-nitrophenyl palmitate）(K_m=0.674 m M,V_max=950.196μM/min).Subsequently,lipase Lip was applied to a test to enhance the flavor release of fatty acids in low-fat cheeses,and it was found that it could hydrolyze low-fat cheeses more efficiently than the conventional commercial lipase Novozyme 435 to increase the levels of butyric,caproic,caprylic and capric acids.（3）To improve the enzymatic properties of the recombinant lipase Lip and broaden the scope of enzyme use caused by the free form,polydopamine-functionalized magnetic mesoporous biochar（MPCB-DA）was prepared as a carrier for the preparation of the immobilized Bacillus licheniformis lipase Lip by covalent fixation.Under the optimized immobilization conditions,the maximum immobilization rate and efficiency were 45%and 54%,respectively.The immobilized lipase MPCB-DA-Lipase exhibited good thermal stability and alkali resistance.MPCB-DA-Lipase retained 56%of its initial activity after 10 reuse cycles and more than 85%of its relative activity after 70 days of storage at 4 or 25℃.MPCB-DA-Lipase was applied to catalyze the transesterification reaction between Cinnamomum camphora seed kernel oil and Perilla seed oil,with a maximum transesterification efficiency of 46%,and the structural lipids produced were medium-chain-monounsaturated long-chain fatty acid glycerides（MUM）and mono medium-chain-unsaturated medium-chain fatty acid glycerides（UMU）,which are new types of medium-and long-chain triglycerides.（4）To explore the reaction mechanism of the binding and hydrolysis between the lipase Lip and the substrate and to pave the way for molecular modification of the lipase Lip,a molecular docking method was used to explore the pathway of the interaction between the lipase Lip and the ligand and to determine the catalytic mechanism of the lipase Lip.Two representative ester substrates,p-NPP and tributyrin,can form stable catalytic conformations with the catalytic active center of the lipase lip-catalytic triplet（composed of Ser77,Asp129,and His152）.According to the results of molecular docking,the reaction mechanism of the lipase Lip catalytic substrate p-NPP and tributyrin was speculated,and it was found that Ser77 in the catalytic triplet formed a complex with the carbonyl carbon atom of the substrate ester group,and Asp129 played a role in stabilizing the position of His152 through hydrogen bonding.（5）To further improve the activity and stability of the lipase Lip and explore the role of key residues in conserved regions,the lipase Lip was modified by point-directed mutagenesis,and a mutant enzyme,which was expressed in E.coli BL21（DE3）,was constructed.After purification and characterization of enzyme activity,only A75C and A75G retained their activities and were selected as study subjects.Compared with those of wild-type lipase and A75G,A75C activity significantly increased,and A75C showed greater thermal stability at 40℃and better alkali resistance at p H 9.0～10.0.The secondary structure of the lipase did not change significantly before and after mutation,while the activity,thermal stability,and hydrophobicity of the mutant were enhanced.Finally,molecular dynamics simulations showed that the more stable and tighter binding conformation between A75C and p-NPP was a potential reason for the enhanced activity and performance and proved that the presence of a mutant Ala residue at position 1 of the pentapeptide could significantly affect the functional properties of the enzyme.