Studies On Immobilization And Structural Biology Of Ligusticum Chuanxiong Caffeic Acid-3-O-Methyltransferase(LCCOMT)

Chuanxiong Rhizome,the dried rhizome of Ligusticum chuanxiong Hort,is a famous geo-authentic medicine distributed in Sichuan Province.It is believed to have beneficial effects on migraine,rheumatic arthralgia,menstrual disorders,bruises,and cardiovascular diseases.Its indicator ingredient is ferulic acid.Modern studies have demonstrated that ferulic acid and its derivatives have several pharmacological effects,including neurological protection,analgesia,anti-oxidation,anti-thrombosis,anti-atherosclerosis,and anti-cancer.L.chuanxiong caffeic acid-3-O-methyltransferase?LCCOMT?can catalyze caffeic acid to produce ferulic acid with S-adenosine-L-methionine as the donor of methyl group.In the previous study,recombinant plasmid LCCOMT-pET28a was constructed and transferred into the BL21 strain.The expression and purification conditions of the recombinant strain were optimized,and LCCOMT activity was detected by high performance liquid chromatography.Firstly,recombinant protein LCCOMT was successfully expressed under optimum conditions,and its concentration was determined by modified BCA protein assay kit.LiCl,NaCl,and KCl were selected as the effectors demonstrated that alkali metal ions Li⁺,Na⁺,and K⁺had little effect on the activity of LCCOMT in the concentration range of 0-1.0 mM.CoCl₂·6H₂O,CuSO₄·5H₂O,ZnSO₄·7H₂O,Pb?NO₃?₂,NiSO₄·6H₂O,CdCl₂,FeSO₄·7H₂O,CaCl?·2H₂O,BaCl₂·2H₂O and MnCl₂·4H₂O were selected as the effectors proved Co²⁺,Cu²⁺,Zn²⁺,Pb²⁺,Ni²⁺and Cd²⁺could reduce the activity of LCCOMT in the concentration range of 0-1.0 mM,with Cu²⁺being the most obvious,while Fe²⁺,Ca²⁺,Ba²⁺and Mn²⁺had little effect.Bi?NO₃?₃·5H₂O,Al?NO₃?₃·9H₂O,Fe Cl₃·6H₂O and CrCl₃·6H₂O were selected as the effectors proved that Bi³⁺could reduce the activity of LCCOMT in the concentration range of 0-1.0 mM;while Al³⁺,Fe³⁺and Cr³⁺had little effect.Study on the effects of Cu²⁺and Zn²⁺on the UV-visible absorption spectra and fluorescence spectra of LCCOMT found:Cu²⁺and Zn²⁺could change the three-dimensional structure of LCCOMT.These results provided guidances for LCCOMT's subsequent immobilization study and industrial use.Secondly,the experiment adopted embedding immobilized method and selected sodium alginate as carrier to immobilize LCCOMT.Immobilization efficiency was measured by the relative enzyme activity of the immobilized enzyme.Through the single-factor experiments and the orthogonal tests,the optimum process conditions were:the sodium alginate mass fraction was 2.0%,the CaCl₂ concentration was 2.5 g/L,the immobilization time was 1 h,and the mass ratio of sodium alginate solution to enzyme was 35000:1,and the immobilization efficiency could reach 75.43%.The optimum reaction pH,optimum reaction temperature,pH stability,thermal stability and Michaelis constant of free LCCOMT and immobilized LCCOMT were compared.The results showed that the optimum reaction pH of free LCCOMT and immobilized LCCOMT were 7.0 and 7.5,respectively.The optimum reaction pH of the immobilized LCCOMT increased by 0.5.And the optimum reaction temperature of free LCCOMT and immobilized LCCOMT both were 37°C.The immobilized LCCOMT improved the thermal stability,although it failed to the pH stability and the affinity to the substrate.In the operation stability test,it was found that the immobilized LCCOMT was continuously reacted 6 times,the relative enzyme activity of the immobilized LCCOMT could retain 50%;and after being used 12 times,the relative enzyme activity still retained around 30%.It showed that the prepared immobilized enzyme had good operational stability.Finally,polyethylene glycol 4000 was adopted to prepare the immobilized enzyme with porous structure.The relative enzyme activity of the immobilized LCCOMT was about 33%higher than that of the control group.The experiment achieved the purposes of easily separating the immobilized enzyme from the reaction products,facilitating automation in industrial production and reducing the cost of the enzyme.Finally,LCCOMT-pET28TEV-pccdB recombinant plasmid was constructed and transferred into E.coli Rosetta.The target protein LCCOMT with His-tag and TEV protease cleavage site was successfully expressed and purified by Ni²⁺affinity chromatography and molecular sieve gel filtration chromatography.The purity of LCCOMT was greater than 95%.The crystallization conditions which were obtained by systematically screen with commercial kits?Index,Screen,PACT,WIZARD,JCSG I,JCSG II,JCSG III,JCSG IV?were optimized.The crystal structures were obtained by the molecular replacement method using the structure of alfalfa Caffeic Acid/5-hydroxyferulic acid 3/5-O-methyltransferase?PDB ID:1KYW?as a model.The highest resolution of crystal diffraction of LCCOMT-SAM and LCCOMT-SAH composites were 1.8?and 1.95?,respectively.And the space group was P 2₁.There were 2 molecules in the asymmetric unit.Each molecule was composed of 18?-helices and 9?-sheets and loops connecting these structures,and binded a SAM or SAH at the C-terminus.The amino acid residues forming hydrogen bonds between LCCOMT and SAM were:D230,D250,M251,and K264.The amino acid residues forming hydrogen bonds between LCCOMT and SAH were:S183,G207,D230,D250,M251,and K264.By aligning with other homologous sequences,these residues were found to be very conservative.The study could explain the catalytic mechanism of LCCOMT more accurately.