| Conjugated linoleic acid(CLA)is a general term for the location and geometric isomers of octadecarbodienoic acid with conjugated double bonds.It has anti-cancer,anti-atherosclerosis,lipid-lowering and other physiological effects.The CLA synthesised by biological method has strong biological activity and safe to eat.Linoleic acid(LA)can be converted to CLA by a linoleic acid isomerase system of lactic acid bacteria consisting of Conjugated linoleic acid hydratase(CLA-HY),Hydroxy fatty acid dehydrogenase(CLA-DH)and CLA acetoacetate decarboxylase(CLA-DC),and the lactic acid bacteria has the advantages of easy large-scale cultivation and high safety.Therefore,the development of lactic acid bacteria that can synthesize CLA,the study of the method of synthesizing CLA,the exploration of the relationship between the structure and function of linoleic acid isomerase system,and the search for ways to improve the yield of CLA have great significance for the industrial production of biosynthesis of CLA.Lactobacillus plantarum p-8 was isolated from traditional fermented yogurt unique to Inner Mongolia,which is a probiotic strain and can isomerize LA into CLA.In this study,the products of fermentation of Lactobacillus plantarum p-8 for catalyzing LA in vivo and the products of its natural enzymes and recombinant enzymes catalyze LA in vitro were analyzed by gas chromatography.Furthermore,the relationship between structure and function of the heterogenously expressed CLA-HY and CLA-DH from linoleic acid isomerase system of Lactobacillus plantarum p-8 was studied by using homologous modeling,molecular docking and amino acid residue mutation.Main results were described as follows:(1)After Lactobacillus plantarum p-8 was cultured in MRS containing LA,the fatty acid analysis of supernatant solution and after LA was catalyzed by bacteria crushing liquid,it was found that a small amount of cis9,transll-CLA(c9,tll-CLA)?trans10,cisl2-CLA(t10,c12-CLA)and trans9,trans11-CLA(t9,t11-CLA)were found;fatty acid analysis of the bacteria revealed a very small amount of t10,c12-CLA.After heterologously expression of CLA-HY,CLA-DH and CLA-DC by E.coli,three recombinant enzymes were used for co-catalysis and fractional catalysis of LA in vitro,then the composition of fatty acids in the reaction products of each step was analyzed.The results showed that LA could be converted into three CLA isomers only with the existence of all three recombinant enzymes and the assistance of Flavin denine dinucleotide(FAD)?Nicotinamide adenine dinucleotide hydrogen(NADH)and Nicotinamide adenine dinucleotide(NAD+);the results of catalysis of LA by fractional step method showed that a small amount of 10-hydroxy-cis-12-octadecarenoic acid(10-HOE)and t10,c12-CLA appeared in the products of CLA-HY and FAD added,the product appeared 10-oxy-cis-12-octadecarenoic acid after the addition of CLA-DH and NAD+,then the c9,t11-CLA,t10,c12-CLA and t9,tll-CLA were detected when addition CLA-DC and NADH,the three isomers of CLA still appeared when CLA-DH and CLA-HY were added step by step,it was concluded that the pathway of LA converted into CLA of Lactobacillus plantarum p-8 was consistent with that of Lactobacillus plantarum AKU 1009a.(2)Homologous modeling was used to analyze the three-dimensional structure of CLA-HY with the crystal structure of Rhodococcus erythropolis oleate hydratase(OhyRe)of Rhodococcus erythroposus as the template.CLA-HY belongs to the Myosin cross reactive antigen(MCRA)protein superfamily,and its three-dimensional structure contains three structural domains.The N-terminal is the Rossman folding for bounding with FAD,corresponding to the amino acid sequence is GAGLSN(X)23D(10-45)Analysis of the results of the docking of CLA-HY and the Lactobacillus plantarum hydratase(LPH)of Lactobacillus plantarum ATCC8014 with the LA molecule respectively,it revealed that 20 amino acid residues at 2 sites binding with LA,and 28 amino acid residues are related to FAD binding.Based on this,the recombinant CLA-HY and its 22 site-directed mutants and 2 deletion mutants were obtained by genetic engineering,of which 4 mutants were in the form of inclusion bodies,indicating that these 4 mutation sites Met 201,Leu 144,Ile 148 and Met 149 are related to the solubility of CLA-HY.The recombinant CLA-HY could convert LA into 10-hydroxy-cis 12-octadecenoic acid,with the optimal reaction temperature of 35? and the optimal response buffer system of KPB buffer(20 mM,pH 6.0).Enzyme activity analysis showed that CLA-HY-Y82F,?TGYVARGG and ?SBS had completely lost their activity,indicating that Tyr 82 is a catalytic group,and these two peptides are essential for the activity of CLA-HY.Combining with the results of kinetic parameters,three-dimensional structure and molecular docking,further analysis of CLA-HY and the remaining 17 site-directed mutants showed that Met 204,Gly 74,Arg 75,Met 76,Thr185,Phe 186,His 399,His 403 and Trp 415 are closely related to the binding of LA,Gly 74 is related to the binding of FAD,and Met 76 is related to the binding with FAD and fixing of LA.In addition,CLA-HY-M204A can significantly improve the catalytic efficiency.(3)Using the short-chain dehydrogenases/reductase(SDR)protein crystal structure of Burkholderia nodules as a template,the three-dimensional structure of CLA-DH was obtained by homology modeling.CLA-DH belongs to the SDR proteins superfamily.The N-terminus of the CLA-DH monomer has a highly conserved Rossman fold that binds to NAD+/NADH in this family.The C-terminus is a specific substrate channel entrance and also has a highly conserved catalytic tetrad of this family.The analysis of molecular docking of 10-HOE and ricinoleic acid with CLA-DH showed that there is a site composed of 26 amino acid residues that interacts with 10-HOE,and there is a site composed of 27 amino acid residues that interacts with ricinoleic acid.Based on this,recombinant CLA-DH and its 28 site-directed point mutants were obtained by genetic engineering,of which 13 mutants were in the form of inclusion bodies,indicating that the 13 mutation sites of CLA-DH were Pro 188,Thr 12,Gly 13,His 16,Phe 18,Asp 37,Asp 63,Ala 91,Gly 92,Ile 113,Val 141,Lys160 and Phe 190 are related to the solubility of CLA-DH.When ricinoleic acid is used as the substrate,the optimal reaction temperature of recombinant CLA-DH is 45?,and the optimal reaction buffer system is KPB buffer(20 mM,pH 6.0).Combining the results of kinetic parameters,three-dimensional structure and molecular docking,further analysis of CLA-DH and the remaining 15 site-directed mutants showed that Ser 143 of CLA-DH is an amino acid residue related to CLA-DH catalysis,Ile 38,Asn 90,Ile 93,Thr 193 and Val 229 of CLA-DH are related to the binding of ricinoleic acid,Tyr 156 is related to substrate binding and enzyme catalysis.The catalytic efficiency of CLA-DH-N90G,CLA-DH-I93S,CLA-DH-Y156G,CLA-DH-T193A and CLA-DH-V229D was significantly improved,while the catalytic efficiency and specific activity of CLA-DH-I38S and CLA-DH-S143A decreased significantly. |
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