Substrate-specific Modification And Immobilization Of Lipase From Thermomicrobium Roseum

Microbial lipases have been widely used in food,medicine,detergent and other industries due to its advantages of high catalytic specificity,few by-products and environmental friendliness.Thermostability is one of the most important properties of lipase regardless of the actual process conditions on industrial production.However,most of the current research have been mainly focused on mesophilic lipases,which have poor heat-resistance and are difficult to meet the requirements of large-scale industrial applications.In our previous studies,the lipase from Thermomicrobium roseum DSM 5159（Tr Lip）has been expressed successfully and characterized.It has outstanding thermostability,p H stability and organic solvent tolerance.However,the low catalytic efficiencies of Tr Lip on mid-long or long chain substrates have greatly limited its industrial application.In this study,multiple strategies were employed for Tr Lip modification to improve its affinity and catalytic efficiency on substrates with longer chain-length,and then the most efficient mutant was subsequently immobilized in inorganic hybrid nanoflowers to enhance the reusability.The following are the main research contents and results.（1）Three mutation libraies were constructed based on the evolutionary trends of the catalytic center and the substrate pocket,respectively.All mutants were expressed and purified in E.coli expression system,and the kinetic parameters of each mutant on mid-long and long substrates were determined.Among all single mutants,W219G was the most efficient mutant with K_m values against p NP-C12,-C16,and-C18 reduced by nearly 40%.Moreover,the catalytic efficiency of W219G against the three substrates increased to 15336 L·mmol^-1·min^-1、4870 L·mmol^-1·min^-1 and 4441 L·mmol^-1·min^-1,respectively,which was separately 4.09,2.81and 2.93 times higher than that of the WT.In addition,the catalytic efficiencies of F265M,I192V,P19A,R40A and M97A were also improved with the high stability maintained.（2）The multiple mutants were constructed based on the six single mutants screened above.Finally,the triple mutant W219G/F265M/R40A（V3）was screened out.The specific enzyme activity of V3 against p NP-C12,-C16,and-C18 was increased by 3.05,2.68,and 3.12-folds when compared with that of the WT,respectively.The optimum temperature of V3 shifted from85°C to 80°C as well as the optimum p H changed from 8.5 to 9.0.In addition,V3 still maintained strong thermal stability with the Tm value up to 96.41°C.The catalytic efficiency of V3 against the three substrates was further improved to 25488 L·mmol^-1·min^-1、8651 L·mmol^-1·min^-1 and 8746 L·mmol^-1·min^-1,respectively.The mechanism analysis of V3showed that the reduction of steric hindrance was the main reason for the improvement of catalytic efficiency,while the alteration of hydrophobic force,hydrogen bonding and Pi-Pi interaction led to the changes in thermal stability.（3）The best mutant V3 screened above was immobilized with inorganic hybrid nanoflowers,and the immobilization conditions were optimized based on the encapsulation rate and the recovery rate of enzyme activities.Two metal ions,Cu²⁺and Ni²⁺,were screened out.The best immobilization performance was obtained when 0.05 mg·m L^-1 and 0.02 mg·m L^-1 of enzymes were separately reacted with 3 mmol·L^-1 metal ions.The surface characteristics of the immobilized enzymes were studied by scanning electron microscopy,Fourier transform infrared spectroscopy and X-ray diffraction.Moreover,it was found that the storage-stability of two immobilized enzymes was better than the free enzyme.In addition,Ni-V3 hybrid nanoflowers possessed good reusability that they still had nearly 60%of initial enzymatic activity after being reused five times.