| Lipoxygenase?LOX?can catalyze the 1-cis,4-cis-pentadiene structure of polyunsaturated fatty acid dioxygenase reactions,and which will form hydrogen peroxide with a conjugated double bond that is an important chemical reaction intermediates,and have wide range of application in the food,chemical,pharmaceutical and other fields.The lipoxygenase from Anabaena sp.PCC 7120?Ana-LOX?will be limited in application of industrial production,because of its poor thermostability.Therefore,improving the thermostability of the Ana-LOX has important significance and value.In this paper,the appropriate point of mutations was choosed in the molecular study,based of three-dimensional structure of Ana-LOX by simulation analysis.The mutant enzymes improved thermostability was obtained by site-directed mutagenesis.To analyze the 3D structure of the mutant enzymes,the molecular mechanism of the improved thermostability was parsed.The mutant enzymes were determined the enzyme properties,optimized the fermentation conditions,and applied in decolorization of triphenylmethane dyes.The results are as follows:1.Simulation of the 3D structure and selection of the mutation sites.The homology alignment analyses of Ana-LOX gene sequences were performed with the help of BLAST.The amino acid sequences of Ana-LOX enzymes were inputted into the SWISS-MODEL database and a template was selected to construct homology models of their 3D structure.With model for quality assessment,90.8%of the residues was distributed in the best reasonable area,while only 1.7%of the residues appeared in the unreasonable area.The B-factors of all the amino acids in the structure of the LOX from Anabaena sp.PCC 7120 were extracted from PDB file of 3D homology model structure built by SWISS-MODEL with the computer program B-FITTER.Through calculation by B-FITTER,20 residues of highest B-factor was obtained.The analysis revealed that a high flexibility sites of Ana-LOX?the top ten amino acid residues of B-factor?was mainly concentrated in the 422 area and 140 area,and which were respectively located in the Loop and C-terminal of Ana-LOX structure.After analysis of the structure,N419H,N419Q,N419A,V421S,V421A,V421G were selected as mutation points for 422 area,and G139D,Q155E,N156D were selected for mutation in 140 area.2.Improving the thermostability of Ana-LOX by site-directed mutagenesis.According to the mutation points by forecast,primers were designed to obtain mutants by site-directed mutagenesis.After fermentation expression of the mutant strains,the necessary mutants?V421A,V40A and V421A/V40A?were obtained.The mutant V421A/V40A was constructed using pET-32a?+?/Ana-LOX-V421A as a template,with the primer of V40A.The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40.Compared to the wild-type enzyme which has a half-life?T1/2?of inactivation of 3.8 min at 50 ?,the T1/a of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min,respectively.The mutant V421A/V40A showed a synergistic effect with a T1/2 value of 8.3 min,resulting in a 1.18-fold improvement compared to the original Ana-LOX.V421A,V40A and V421A/V40A also obtained 4.83%,41.58%and 80.07%increase in specific activity,respectively.The optimal temperature of three mutants was increased by 5? as compared to wild-type Ana-LOX.The optimum pH of wild and mutant Ana-LOX was 9.0.The Km of V421A/V40A mutant was significantly decreased while Km of two mutants V421A and V40A did not markedly increase in comparison with Km of the wild-type Ana-LOX.Lower Km of enzyme indicated higher affinity to the substrate.In other words,the V421A/V40A mutant had a higher affinity to the substrate than wild-type Ana-LOX.The kcat values of the mutant enzymes were higher than the wild-type Ana-LOX,and the V421A mutant had the highest catalytic rate?kcat?,reached to 1.65 times of the wild-type.The affect of the metal ions on the wild and mutant enzymes was not significantly different.Fe2+ had a strong activating effect to recombinant Ana-LOX,increasing by about 30%compared with the control group.Fe3+ and Cu2+ had an obvious inhibitory effect,completely inhibiting the activity of Ana-LOX.3.Analysis of the mutant enzyme structure and resolution of molecular mechanism improving thermostability.Based on the data from CD spectra,the percentage of each secondary structure was estimated by online program.It was shown that the percentage of ?-sheet of the Ana-LOX was not significantly changed.The a-helix percentage of wild-type Ana-LOX was 29.3%,while the ?-helix percentage of V421/V40,V421 and V40 was 38.7%,33.9%and 33.1%,respectively.Compared to the wild-type Ana-LOX,the a-helix percentage of V421A and V421A/V40A were increased by 4.6%and 9.4%,respectively.The changes in surface hydrophobicity of the mutants were determined by fluorescence assay.V40A showed a decrease in the surface hydrophobicity,while the surface hydrophobicity of other mutants and wild-type enzyme was similar.As shown in the 3D structure model of wild-type Ana-LOX,valine at 421 site undermined the integrity of the whole a-helix that V421 located in.Therefore,replacing large ?-side chains of amino acid by the small conformation Ala in the a-helix of Ana-LOX could improve the stability of the enzyme.Interestingly,a new a-helix at the chain?55 to 70?near 421 was generated after replacing valine with alanine,which could generate a new stable secondary structure for the Ana-LOX enzyme.The substitution by V40A could reduce the hydrophobicity of amino-acid residues at the surface of proteins and finally improve the thermostability of Ana-LOX.4.Fermentation conditions optimization of the mutant enzyme and decolorization of triphenylmethane dye.Through optimizing the fermentation conditions of Ana-LOX-V421/V40,the TY medium was found that had the best expression effect for the mutant enzyme V421/V40,and the enzyme activity reached 8836 U/mL.In the aspect of culture conditions,the enzyme activity reached 7902 U/mL after inducting for 12h with IPTG.When OD600 was 0.4,the mutant enzyme V421/V40 activity reached the highest,8453 U/mL.And when the IPTG final concentration was 0.1 g/L,the mutant enzyme V421/V40 activity is up to highest 8587 U/mL.In decolorization of triphenylmethane dyes,the experimental group C reaction system mixed of Ana-LOX and linoleic acid had remarkable decoloration effect.The mutant enzyme V421/V40A at 45 ? had 85.34%catalytic efficiency for aniline blue and 74.51%for light green.While the catalytic efficiency of the wild enzyme at 45 ?for the two dyes were 71.11%and 35.52%.It showed the mutant enzyme maintained higher decolorization ability than the wild enzyme in a certain temperature,and it also proved that the thermostability of the mutants was better than the wild Ana-LOX.To explore the HPLC mobile phase of aniline blue and bright green:Aniline blue selected acetonitrile-acetate/ammonium acetate?pH 4.5,50 mM?buffer as mobile phase,and Light green chose acetonitrile-acetate/ammonium acetate?pH 4.5,20 mM?buffer.Using HPLC analysis,the decolorization ability of aniline blue solution was 0.0827 mg·U-1·h-1,and the decolorization ability of light green solution reached 0.1609 mg·U-1·h-1. |
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