| Lipoxygenases (EC1.13.11.12, LOX) are nonhaem, iron-containing dioxygenases thatcatalyze the formation of (Z, E)-conjugated hydroperoxides from polyunsaturated fatty acids(PUFAs), or any other molecules containing one or more (Z, Z)-1,4-pentadiene structures.Because of the specific enzyme reaction, LOX has been widely used in food industry,pharmaceutical industry and chemical industry, suggesting a huge market potential. Therefore,the production of LOX has been highlighted by the researchers at home and abroad. Althoughmany researches have been done about the eukaryotic LOX, the production of the enzymewas still unavailable due to the failure of high efficient secretion of LOX and the low thermalstability. To date, the investigation on the secretary expression and molecular modification ofbacterial LOX was rare. In this study, LOX from Pseudomonas aeruginosa wasextracellularly expressed in Escherichia coli Rosetta (DE3), and the recombinant enzyme wasfurther characterized and modified for enhanced enzymatic properties.(1) Secretory expression of the P. aeruginosa LOX in E. coli and fermentationoptimizationBased on the DNA information from NCBI database, the LOX gene from P. aeruginosawas cloned by PCR. The native LOX gene containing its endogenous signal peptide wascloned to the expression vector pET-22b(+) and expressed in E. coli extracelluarly. Whencultured in shaker flasks with TB medium and induced with1mmol/L IPTG and0.5%(w/v)Tween-20at20oC for50h, the yield of LOX reached the maximal vaule (8.5U/mL). Whencultured in3L fermenter with TB medium, induced at OD600=8with1mmol/L IPTG and0.5%(w/v) SDS at20oC for48h, the yield of LOX was up to8.0U/mL. This is the highestextracellular production of the recombinant LOX among all the publications.(2) Purification and characterization of the recombinant LOXThe recombinant LOX from E. coli Rosetta (DE3)(pET-22b(+)/lox1) was purified byammonium sulfate precipitation, dialysis, anion-exchange chromatography (Q SepharoseHigh Performance and Mono Q). The specific activity of the purified LOX was increasedfrom2.1U/mg to28.3U/mg. The purification fold and recovery yield were13.5and10%,respectively. The optimal temperature and pH of the purified LOX were25oC and7.5,respectively. The half-life times of the purified LOX at50was4.5while that at60oC was10min. The activity of purified LOX in pH6.5-7.5maintained over80%. The Kmand Kcatvalueof the purified LOX were48.9μmol/L and23.51/s, respectively. The frequently mentionedmetal ions with divalent ion and EDTA inhibited the enzyme activity to different extents, andthe eicosatetraynoic acid (ETYA) has no effect on the activity. The major part of the linoleicacid hydroperoxides (HPOD) produced by the recombinant LOX was13(S)-HPOD.(3) Enhanced thermostability of LOX by N-terminal loop deletionThe thermostability of LOX was increased through partial deletion of the high flexibleregion without compromising the catalytic efficiency. Deletion of the first10residues of theloop hardly affected the catalytic properties of the LOX while truncations of the first20and30residues increased the thermal stability of the LOX by1.3and2.1fold, respectively. Further deletion led to the formation of inclusion bodies of the enzyme in Escherichia coli. Tobe noted, over90%of the specific activity was maintained in the two LOX mutants withenhanced thermal stability. Circular dichroism analysis showed that the α-helix and β-sheetcontent was not changed by these modifications. Fluorescence spectra analysis indicated thatthe thermally stabilized mutants exhibited a more compact structure and a lower surfacehydrophobicity in contrast to the wild-type LOX.(4) Enhanced thermostability of LOX by modification of the inner high flexible loop (L6loop)The thermostability of LOX was increased through reducing the flexibility of hinge loop(L6) by deletion, mutation and replacement. Although deletion of the L6loop (DL6) led to asharp reduction of both thermal stability and catalytic activity of the enzyme, the residuesubstitutions with proline (G204P, G206P, and G204P/G206P) or even proline-rich linker(L6/PT) in L6loop increased the thermal stability of the LOX by values ranging from0.46to3.45fold. To be noted, over85%of the specific activity was maintained in all thermallystabilized LOX mutants. Circular dichroism analysis showed that the α-helix and β-sheetcontent was not changed by these modificatisons. Fluorescence spectra analysis indicated thatthe thermally stabilized mutants exhibited a more compact structure. ANS binding alalysisexhibited that the surface hydrophobicity of DL6was2.8-folder of the wild-type LOX.However, the surface hydrophobicities of other mutants were slightly lower that the wild-typeLOX. This result indicated that the substrate binding cavity of mutant DL6may be exposed tothe solvent.(5) Enhanced thermal stability and specific activity of LOX by fusing withself-assembling amphipathic peptidesSix SAPs were individually fused to the N-terminus of the LOX, that resulted theSAP-LOX fusions with approximately2.3to4.5-fold enhanced thermal stability at50oC. Thespecific activities of the SAP-LOX fusions were also increased by1.0to2.8-fold in ascompared with the wild-type LOX. Circular dichroism analysis showed that the α-helix andβ-sheet content was not changed by these modifications. Fluorescence spectra analysisindicated fusion with SAPs did not remarkably affect the tertiary structure of the LOX, but thesurface hydrophobicities of the LOX were increased. Dynamic laser scattering analysisexhibited that the aggregation of the LOX was enhanced by fusing with the SAPs. Thisoligomerization enhanced the intermolecular interaction of the LOX monomer and thenincreased the thermostability of the LOX. Meanwhile, the flexiblity of the SAPs may alsoimpact the thermostability of SAP-LOX fusions. This is the first report on the improvement ofthe thermal stability and specific activity of an enzyme by the fused SAPs, suggesting asimple technique to improve the catalytic properties of the recombinant enzymes byfusion-expression. |
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