Study On Roles Of Hydrophobic Residues Of The Lid Domain In Interfacial Binding And Activity Of Lipase T1 From Geobacillus Zalihae At The Lipid-water Interface

Lipase T1,a thermostable lipase from Geobacillus zalihae,is potential for various industrial processes.They are interfacial enzymes that catalyze various reactions,such as hydrolysis,esterification,acidolysis,alcoholysis and transesterification at the lipid-water interface.The overall reaction requires the adsorption of lipase from the aqueous phase to the interface before catalytic action.The process of lipase adsorption to the lipid-water interface involves a conformational change of the lid domain,called ‘interfacial activation'.At the same time,a serine residue located on the underside of the lid faces outward and the substrate gets access to the active site.The activated conformation of lipase presumably interacts favorably with the lipid interface and generates a large hydrophobic surface surrounding the catalytic cleft and part of the interfacial recognition site.Thus,efficient hydrolysis depends on structural changes in the enzyme upon activation and the physical state of the lipid.The amphipathic ?6 helix has been reported to function as the lid.During the activation process,the lid opening is triggered by the hydrophobic interaction with the lipid interface.In previous studies,it had been demonstrated that the hydrophobic residues have effects on the substrate specificity and enzymatic kinetics.However,the mechanism of the interfacial adsorption and activity of lipases T1 still needs further experimental investigation.(1)The cloning and expression of lipase T1 from Geobacillus zalihae in the E.coli system.The gene of lipase T1 was obtained from NCBI data bank and synthesized by the company.The pET23a-CBD-T1 plasmid was transformed into E.coli BL21 and expressed after 12 h.The protein was purified by CBD affinity tag.The purified proteins were collected and analyzed by SDS-PAGE,demosrated that the molecular weigh was 43 kDa.(2)The kinetic measurement was established based on the monolayer film and with four common lipases as modern lipases.The kinetic measured by the monolayer techinique was compared with the bulky method.The results showed that lipase T1?PLA1?RML and HIL displayed high hydrolytic activity on the triolein film,but only the lipase PLA1 showed high hydrolytic activity on the egg-PC film.The monolayer techinique was in good agreement with bulky method.(3)Sequence alignment and structural analysis of the closed conformation of liapse T1 with other member of lipases in I.5 family.The results showed that the primary sequence of lipase T1 with Geobacillus thermocatenulatus lipase(BTL2)and G.stearothermophilus lipase(L1 and P1)showed high sequence identities/similaritie.Moreover,the amino acid sequence of the lid regions(helix ?6 and helix ?7)had high sequence identities with those of the lid regions in lipases of the I.5 family).The helix ?6 has undergone structural rearrangement.The site-mutagenesis of hydrophobic amino acids into hydrophilic amino acids was performed in the lid region by QuikChange Site-directed Mutagenesis Kit with pET23a-CBD-T1 as the template.The protein was purified using affinity chromatography combined with anion exchange chromatography.(4)Investigation of interfacial adsorption behavior of lipase T1 and the mutants.The adsorption curves were fitted into the Lagmiur equation.The results showed that the kinetic constants,including the kinetic adsorption(ka),the desorption rate(kd)and the adsorption equilibrium coefficient(KAds)decreased significantly compared to T1-wt.The KAds values of the mutants were in according with ka values,which confirmed that the hydrophobic interaction is the mainly force between the lipid-water interface and hydrophobic residues of ?6 in lid.Furthermore,it revealed that the interaction with the lipid interface and/or the nature of the substrate as the main causes of the activation mechanism.Furthermore,GE*(%)of wide type T1 decreased compared with F181 Y mutants.The surface areas occupied by a single molecule of the adsorbed protein(AE*)were then found to be significantly increased.As expect,the amount of the lipase amount(?E*,GE*)are in good accordance with the adsorption kinetic parameters of T1-wt and its mutants onto egg-PC monolayer.Specifically,the residues(F181,L183,A186 and V187)are presumed to be the key amino acid involved in interfacial activation.(5)Further studies on the effect of hydrophobic amino acids on substrate binding in the process of hydrolysis.The results showed that lipase T1 and the mutants showed classical “interfacial activation”.The open conformation of lipase T1 constructed by homologous modeling was analyzed.The interaction for stabilizing the protein did not be interrupted by the site-mutagenesis.The molecular docking showed that the Leu183 and Val187 was involved in binding with substrate the dilaurin.The hydrolytic activities of the mutant L183 N and V187 A on the the dilaurin were decreaed,which were ascribed to the L183 and V187 contributed to both the adsorption and substrate binding.