Biofuel cell is high-stability and easy to reduce weight and size. So, it is attracting attention as one of next generation energy sources. However, there are problems with the stability of the enzyme and electron transfer efficiency, so it has not been put to practical use. Multicopper oxidase (MCO) is often used as the enzyme for the cathode of biofuel cell. MCO is a group of enzymes that has four copper cofactors, catalyzes the reaction of copper to accept four electrons by oxidizing the substrate and using that electron to produce water from protons and oxygen. While other enzymes catalyze the transfer of 1, 2 electrons, MCO catalyzes the transfer of 4 electrons.
Enzymes derived from hyperthermophilic bacteria are known to have high long-term stability and thermal stability. MCO derived from hyperthermophilic archaea Pyrobaculum aerophilim (McoP) has been found and functional analysis has already been performed. Although this enzyme has high stability, it is difficult to apply it to a bio cell due to its low catalytic activity. Therefore, an enzyme showing high catalytic activity was searched by introducing random mutation into the gene encoding McoP. As a result, a mutant McoP showing a specific activity 9 times higher than the wild type McoP was found in the crude enzyme solution. It was confirmed that four amino acid mutations were introduced into mutant McoP. These mutations were introduced on the surface of the enzyme which is distant from the substrate binding site and the copper binding site. It was revealed that it is a site different from the mutant type McoP exhibiting high catalytic activity so far reported. Therefore, in this study, I aimed to elucidate the mechanism of high catalytic activation of mutant McoP.
Ecoli BL21-IRLP was transformed with a plasmid vector (pET15b) designed to express His-tag added to wild-type Mcop and McoP which one of four mutations was introduced. Pre-culture and main culture were performed on the cells. The bacterial cells were harvested and sonicated, and the supernatant was recovered by centrifugation to obtain a crude enzyme solution. Crude enzyme solution was added to the nickel chelating column, copper sulfate was added to a final concentration of 1 mM, and it was left to stand at 4 ºC for 16 hours to allow uptake of Cu atoms into McoP. Each activity of eluted protein solution was recovered and heat treated. The supernatant was recovered by centrifugation, concentrated and buffer replaced, and purified enzyme was obtained.
Next, the activity of the purified enzyme was measured using ABTS as a substrate. As a result of the measurement, F262I showed a remarkably high catalytic activity of specific activity of 10.71 Units /mg against the specific activity of WT of 0.81 Units/mg. From these results, it was revealed that the mutation enhancing the catalytic activity of mutant McoP is F262I in which phenylalanine at position 262 is substituted with isoleucine.
Enzymes derived from hyperthermophilic bacteria are known to have high long-term stability and thermal stability. MCO derived from hyperthermophilic archaea Pyrobaculum aerophilim (McoP) has been found and functional analysis has already been performed. Although this enzyme has high stability, it is difficult to apply it to a bio cell due to its low catalytic activity. Therefore, an enzyme showing high catalytic activity was searched by introducing random mutation into the gene encoding McoP. As a result, a mutant McoP showing a specific activity 9 times higher than the wild type McoP was found in the crude enzyme solution. It was confirmed that four amino acid mutations were introduced into mutant McoP. These mutations were introduced on the surface of the enzyme which is distant from the substrate binding site and the copper binding site. It was revealed that it is a site different from the mutant type McoP exhibiting high catalytic activity so far reported. Therefore, in this study, I aimed to elucidate the mechanism of high catalytic activation of mutant McoP.
Ecoli BL21-IRLP was transformed with a plasmid vector (pET15b) designed to express His-tag added to wild-type Mcop and McoP which one of four mutations was introduced. Pre-culture and main culture were performed on the cells. The bacterial cells were harvested and sonicated, and the supernatant was recovered by centrifugation to obtain a crude enzyme solution. Crude enzyme solution was added to the nickel chelating column, copper sulfate was added to a final concentration of 1 mM, and it was left to stand at 4 ºC for 16 hours to allow uptake of Cu atoms into McoP. Each activity of eluted protein solution was recovered and heat treated. The supernatant was recovered by centrifugation, concentrated and buffer replaced, and purified enzyme was obtained.
Next, the activity of the purified enzyme was measured using ABTS as a substrate. As a result of the measurement, F262I showed a remarkably high catalytic activity of specific activity of 10.71 Units /mg against the specific activity of WT of 0.81 Units/mg. From these results, it was revealed that the mutation enhancing the catalytic activity of mutant McoP is F262I in which phenylalanine at position 262 is substituted with isoleucine.