Rational Design And Amphiphilic Polymer Modification To Improve The Properties Of Rhizopus Chinensis Lipase

The microorganism is one of the major sources of lipase,among which Rhizopus is an important production strain of microbial lipases.Most Rhizopus lipases show good stability and high catalysis efficiency for applications in oil and fat industry such as trans-esterification,hydrolysis,and oil refining.Unfortunately,Rhizopus lipase is meso-thermal enzyme with short half-life and low acid-alkali tolerance,leading to the easy inactivation in its application process.Therefore,the tolerance and catalytic activity of lipase need to be improved.In order to improve the thermostability,alkali tolerance and catalytic activity of r27RCL?from Rhizopus Chinensis CCTCCM201021?,disulfide bond design,rational design based on??G,and molecular dynamics simulation were used to create a“small-but-excellent”mutant pool,from which qualified mutants with highly enhanced thermostability,improved activity at alkali environment and better alkaline tolerance were screened out.Subsequently,the chemical modification was used to enhance lipase activity.The results were as follows:?1?Improving lipase thermostability by introducing extra disulfide bonds.The Disulfide by Design software was used to predict potentially surface or buried disulfide bonds of lipase.Seven pairs of surface bonds and five pairs of buried bonds were finally selected for further experiments.A surface bond m9/10?S85C/Q145C?and a buried bond m17/18?F223C/G247C?were screened out with improved properties.The mutant lipase m9/10 and m17/18 processed improved T₅₀³⁰ by 4.2?and 8.5?,and lengthened their half-lives at60°C by 4.5 folds and 19 folds than that of wild-type.The optimum p H value of m17/18shifted from 8.0 to 9.0 and showed more resistance to the alkaline environment.?2?Improving lipase thermostability by rational design of Fold X5.Based on??G and flexibility,19 sites?containing 30 mutations?were selected for experimental verification and four mutant lipases m22?S142A?,m26?S250Y?,m28?Q239F?,and m29?D217V?with significantly improved thermostability were screened out.The T_optof m29 was improved to45?.Their T₅₀³⁰ were 4.1?5.5?6.0?and 7.2?higher than that of r27RCL and their half-lives at 60?were prolonged by 4.6-,5.3-,7.6-and 14.5-folds.Their simulated structural analysis revealed that the enhancement of interior hydrophobic packing in lipase was the major factor in improving lipase's thermostability.The molecular dynamics?MD?simulation results showed that the restricted flexibility within the region of the mutations led to the improvement of thermostability.?3?The thermal mutations screened out in previous work was combined for further thermostability improvement.The variants m30?F223C/G247C/S85C/Q145C?,m31?S142A/S250Y/Q239F/D217V?and m32?F223C/G247C/S85C/Q145C/S142A/S250Y/Q239F/D217V?were produced.Their optimum temperatures lifted to 45?,45?,and 50?,respectively.Their T₅₀³⁰ were improved by 14.2?15.8?,and 21.2?.Their half-lives at 60?were prolonged by 34.4-,41.8-,and 74.7-folds.The p H_opt of m30 and m32 changed from 8.0to 9.0,and their alkali-tolerance were significantly enhanced.?4?The MD simulations of m32 indicated that the RMSF values of m32 were much higher than those of r27RCL,indicating that the mutations reduced the flexibility of structure of m32,and made the lipase more stable.The solvent-accessible-surface-area?SASA?,radius of gyration?Rg?and gibbs free energy change???G?of m32 were decreased by 7 nm²,0.03?and 25-50 kcal/mol than those of r27RCL,indicating the rigidity improvement of m32.The newly formed salt bridge?Glu292-His171?also led to the improvement of thermostability of m32.?5?Enhancing lipase catalytic activity through chemical modification by amphiphilic polymer.Benzene-1,3,5-tricarbaldehyde,poly?ethyleneglycol?-bis-?3-aminopropyl?-terminated and 1,6-hexanediamine or 1,12-dodecanediamine have been used as starting meterials to synthesize the imine-liked amphiphilic polymers of P1 and P2.They self-assemble in water as nanoparticles,which can further encapsulate lipase,to form effective nano-sacled catalytic complex with diameter around 150-600 nm,showing enhancement effects on the catalytic activity of lipase by 2.75-and 3-fold with the addition of 0.04 m M P1 and P2.Additianally,they also demonstrated the ability to promote the reversibly-refolding process of the partially-and completely denaturated lipase.The above results showed that polymers could activate lipase catalytic ability and improve refolding process by the following reasons:1)the interfacial activation of lipase 2)the H-binding formed through EG backbond and amino acids of lipase.