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Substrate Construes the Copper and Nickel Ions Impacts on the Mushroom Tyrosinase Activities
Gheibi, N.,Saboury, A.A.,Haghbeen, K. Korean Chemical Society 2006 Bulletin of the Korean Chemical Society Vol.27 No.5
Mushroom tyrosinase (MT) structural changes in the presence of $Cu ^{2+}$ and $Ni ^{2+}$ were studied separately. Far-UV CD spectra of the incubated MT with the either of the metal ions indicated reduction of the well-ordered secondary structure of the enzyme. Increasing in the maximum fluorescence emission of anilinonaphthalene-8-sulfonic acid (ANS) was also revealing partial unfolding caused by the conformational changes in the tertiary structure of MT. Thermodynamic studies on the chemical denaturation of MT by dodecyl trimethylammonium bromide (DTAB) showed decrease in the stability of MT in the presence of $Cu ^{2+}$ or $Ni ^{2+}$ using their activation concentrations. Both activities of MT were also assessed in the presence of different concentrations of these ions, separately, with various monophenols and their corresponding diphenols. Kinetic studies revealed that cresolase activity on p-coumaric acid was boosted in the presence of either of the metal ions, but inhibited when phenol, L-tyrosine, or 4-[(4-methylphenyl)azo]-phenol was substrate. Similarly, catecholase activity on caffeic acid was enhanced in the presence of $Cu ^{2+}$ or $Ni ^{2+}$, but inhibited when catechol, L-DOPA, or 4-[(4-methylbenzo)azo]-1,2-benzenediol was substrate. Results of this study suggest that both cations make MT more fragile and less active. However, the effect of the substrate structure on the MT allosteric behavior can not be ignored.
Reyhane Zamani,Sayyed Shahryar Rahpeyma,Moein Aliakbari,Mousa Naderi,Mohsen Yazdanei,Saeed Aminzadeh,Jafar Khezri,Kamahldin Haghbeen,Ali Asghar Karkhane 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.4
Improving the thermal stability of enzymes is an essential factor in the industrial applications of enzymes. Many methods related to increased thermal stability were explained, and increasing salt bridges is one of the strategies for improving the thermal stability of enzymes. In this study, mutations T59E, I145R, N149R, V219D, and A262R are introduced into the native cellulase gene to produce the mutant 5M-cel5E cellulase. In silico results showed that the mutation increased the salt bridges from 15 to 28. Root mean square fluctuation (RMSF) calculations confirmed that the mutation increased protein stability. Furthermore, the docking results showed that the affinity of cellobiose for the 5M-cel5E active site (-122.759) was slightly decreased compared to native cellulase (-130.93). No enzymatic activity was found in 5M-cel5E cellulase after cloning, expression and purification. Activated the enzyme with a back mutation of R149N, the result of which was named 4M-cel5E. The last mutation increases the salt bridges from 15 to 22, creating 4 salt bridge networks. The 4M-cel5E enzyme exhibited a maximum activity of 463 U/mg at pH 6.0 and 45°C. The mutations also increased the enzyme thermal stability up to 1.5 and 3.4-fold at temperatures of 65 and 67oC, respectively. These mutations made the Clostridium thermocellum cellulase suitable for various industries such the biofuel and paper.