Efficient Immobilization Strategies For Pseudomonas Fluorescens Lipase And Their Application Research

Currently,immobilized lipase has been widely used in the industrial catalysis field.However,there are still some urgent problems in the traditional immobilization strategies,such as the inability of carriers to reuse,time-consuming and low efficiency of grafting dendrimers from carriers,monotonous strategy for immobilization and so on,which can not meet the demands of rapid development of green manufacturing in the new era,and further greatly limits the large-scale application of the immobilized lipase.For this purpose,a typical bacterial lipase,Pseudomonas fluorescens lipase（PFL）,was selected as the research object to overall design three new immobilization strategies,and in consideration of the functional specificity of the immobilized PFL to achieve efficient application in biodiesel production,enantioseparation and high value added lipid preparation.The main research contents and results are summarized below:1.In order to determine the immobilization strategy for PFL,Swiss-Model automatic homology modeling was employed to predict the active sites and three-dimensional spatial structure of PFL.Subsequently,the relative location of PFL active sites to its surface amino acid residues was analyzed by Py Mo L software,which indicated that carboxyl,?-amino and imidazolyl were away from the active sites,and their amount were also moderate,so these amino acid residues could be used as the metal chelated or covalent immobilized sites.Next,on the basis of these results of bioinformatics analyses for PFL,three immobilized strategies were designed for preparation of the immobilized enzyme:Co²⁺chelated support for adsorption immobilization of PFL,new dendrimer for covalent binding PFL and coordinated self-assembling system for embedding two enzymes including PFL.Finally,according to their characteristics of the immobilized enzymes,they were respectively applied in the fields of biodiesel preparation,chiral pharmaceutical resolution and high value added lipid production.2.Based on immobilized metal-chelate affinity chromatography,5-aminoisophthalic acid,as a novel ligand,was employed to prepare Co²⁺-chelated magnetic responsive nanocarriers(AGMNP-Co²⁺)for reversible immobilization of PFL.The result revealed that PFL loading of AGMNP-Co²⁺was 136.9 mg_protein/g_support,and 2125%of activity recovery of the immobilized PFL(AGMNP-Co²⁺-PFL)was tested via esterification.Subsequently,AGMNP-Co²⁺-PFL was employed to transform waste cooking oil into biodiesel,and 95%of conversion was achieved.Biodiesel yield could still yield 83%after 10 cycles of usage,displaying a good operational stability of AGMNP-Co²⁺-PFL.Moreover,AGMNP-Co²⁺could be easily regenerated and reused via a simple desorption procedure.After 5 batches of running,AGMNP-Co²⁺could effectively adsorb PFL,reaching 116.7 mg_protein/g_support,and the activity recovery of the regenerative AGMNP-Co²⁺-PFL was insignificantly changed.Therefore,the strategy achieves efficient carrier regeneration and recycling.3.The interfacial polymerization process was introduced to efficiently and quickly graft a rigid-flexible dendrimer from the magnetic nano-flower(PFMMs-G_3.0)for covalent immobilization of PFL.The transesterification activity recovery of the immobilized PFL reached 864%at p H 9.0.Subsequently,the immobilized PFL(PFMMs-G_3.0-PFL)was used for the enantioseparation of（R,S）-1-phenylethanol,and 50%of conversion together with99.0%of enantiomeric excess of the substrates（ee_s）could be obtained within 1.5 h.In addition,49.2%of conversion and 96.9%of ee_s were still retained after PFMMs-G_3.0-PFL running 10 consecutive batches.The improved reusability of the immobilized PFL might be attributed to the moderate rigidity/flexibility of the chain in dendrimer.Therefore,the strategy can greatly save time of constructing support to efficiently prepare immobilized PFL and keep a good catalytic activity.4.Inspired by the self-assembly of small biological molecules in nature,self-assembly of Mn²⁺induced L-cystine generating crystals show a twin lotus flowers-like architecture.Morphology of model protein directed self-assembled system reveals that protein amino acid residues directly get involved in coordinated self-assembly,further controlling crystal growth to adjust their size.Subsequently,embedding PFL and soybean lipoxygenase（type I）into the self-assembled system was employed to imitate lipid metabolism of soybean toward synthesis of fatty acid hydroperoxides（HPOs）,valuable intermediates with potential use in anti-cancer medicine.When soybean oil was used as raw material,the yield was1527.0μmol·HPOs/m L·oil upon the cascade reaction initiating within 3 h.The result of high performance liquid chromatography-mass spectrometry analysis revealed that the major component of HPOs was 13-hydroperoxides of linoleic acid.84.7%of the relative yield was still obtained after 9 consecutive batches of running,which implies a good catalytic performance and operation stability for the bi-enzyme self-assembled system.To sum up,three newly designed strategies have solved some important problems in the traditional immobilized lipase,and synchronously achieved successful application of PFL in different field.Meanwhile,the three kinds of immobilized PFL show excellent catalytic performance and good operational stability,which lay a good foundation for further large-scale application.Additionally,focusing on key problems in enzyme engineering,integrating multidisciplinary approaches will certainly open up a new window for efficiently preparing immobilized enzymes and creating their new applications.