Study On The Function Of Amino Acids Related To The Catalysis In Thermus Thermophiles SG0.5JP17-16 Laccase

Laccase is a multicopper oxidase,widely distributed in nature,such as fungi,bacteria,insects and plants.Substrates are oxidized at the T1 Cu,the electrons are rapidly transferred to the trinuclear copper cluster which is composed of one T2 Cu and two T3 Cu.Dioxygen is reduced to water at the trinuclear copper cluster.Laccase can oxidize a broad range of substrates,and the by-products are nontoxic,so it is considered to be a green catalyst with the application value.Laccase from Thermus thermophiles SG0.5JP17-16(lacTT)is a bacterial laccase,tolerable for the high temperature,high salt,and a wide range of pH.In this study,lacTT was used as the model system.The functions of several amino acids related to the catalysis in lacTT were exploredFirst,the role of axial amino acid residue M460 at the T1 Cu site in lacTT was studied The M460 residue was mutated into alanine,phenylalanine,leucine,histidine and glutamine by site-directed mutagenesis,respectively.UV-Vis spectroscopy and kinetic data showed that the substitution of axial residue M460 by non-coordination residue resulted in a decrease in copper ion loading at the T1 Cu site and an increase in catalytic efficiency,while the substitution of axial residue M460 by strongly coordination residue led to an increased in copper ion loading at T1 Cu site and a decrease in enzyme catalytic efficiency.The mutation of this site also affects the redox potential of T1 Cu site.With the enhancement of the metal-ligand interaction in mutants,the redox potential of T1 Cu site decreased.In summary,the type of the axial residue at the T1 Cu site is an important factor affecting the redox potential and catalytic behavior of lacTTThe function of the Lys428 residue,near the substrate-binding pocket of lacTT,was analyzed.The Lys428 residue was mutated into leucine,glutamic acid,arginine and methionine by site-directed mutagenesis.Spectroscopic characteristics showed that the mutation of the K428 residue to glutamic acid or arginine had little effect on the copper center and secondary structure of the enzyme.The reduction of catalytic efficiency was mainly related to the change of the substrate affinity.When the K428 residue was mutated to leucine or methionine,the secondary structure of K428L and K428M mutants was affected,and the copper centers were disturbed.Two mutants had lower copper ion loading at the T1 Cu site and higher catalytic efficiency as compared with those of the wild type enzyme.The specific activity of the K428L mutant decreased due to the reduction of copper ion loading at the T1 Cu site.The K428M mutant was used to decolorize four synthetic dyes,reactive black B,reactive black WNN,Congo red and remazol brilliant blue R,the results indicated that the K428M mutant had similar efficiency to lacTT for reactive black B and reactive black WNN,however,for Congo red and remazol brilliant blue R,showed higher decolorization efficiency than lacTT.The K428M variant may be a positive mutant found in this study.