Study On Function Of Amino Acids In Substrate Binding Domain In Thermus Thermophiles SG0.5JP17-16 Laccase

Laccase?EC1.10.3.2? is a copper blue oxidase that exists in the form of monomeric glycoproteins in nature.It can catalyze the oxidation of phenols and degrade lignin.There are four copper ions in the active center of the laccase,which can be divided into three types according to the characteristics of visible light spectrum and electron paramagnetic resonance spectrum:T1Cu,T2Cu and T3Cu.In addition,laccase is widely used in industry and biotechnology,and it is a protease with great industrial application value.The lac TT derived from thermus thermophiles SG0.5JP17-16 has the characteristics of high temperature resistance,high concentration salt resistance and wide p H range.This study mainly explores the amino acid residues in the lac TT that would affect its catalytic function.This topic studies the role of the loop around T1Cu in catalytic reactions.By mutating the acidic and basic amino acids in the loop into non-polar methionine to study its function.UV-Vis spectroscopy and enzymatic reaction kinetics data indicate that the substitution of M397 for H397 will result in a reduction of Cu²⁺loading at the T1Cu site and a decrease in the catalytic efficiency of the enzyme.The amino acid residue at position 397 serves as the coordination amino acid of T1Cu and plays an important role in stabilizing the structure of T1Cu.After the mutation of D193 to Met,the specific enzyme activity of the mutant protein D193M is increased by 30%compared with the wild type.After mutation of the residues of D357,E390,K428 and K430 to methionine,the catalytic efficiency of the mutants is improved compared to the wild type.D193M,D357M,E390M,K428M and K430M are all positive mutants found in this study.Compared with the wild-type lac TT,the decolorization efficiency of D193M,D357M,E390M,K428M and K430M for Reactive Brilliant Blue is increased by 14%,22%,19%,13%and 21%,respectively.These positive mutants are superior to lac TT in decolorizing anthraquinone dyes.The study finds that after mutating D357 and K428 to methionine,the catalytic efficiency of D357M and K428M is improved by 92%and 154%,respectively,compared with the wild type.The residues of D357 and K428 are mutated to methionine through site-directed mutatgenesis to obtain the D357M-K428M.The absorbance of the D357M-K428M at 608nm is reduced by 89%compared with lac TT,indicating that the structure of T1Cu is disturbed.The decolorization efficiency of the D357M-K428M for Reactive Black WNN,Reactive Black B,and Congo Red is similar to lac TT,and the decolorization efficiency of Reactive Brilliant Blue is 16%lower than the wild type.In summary,the residues of D357 and K428 may play an important role in maintaining the catalytic function of lac TT.