A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase

Tyrosinase efficiently catalyzes the ortho-hydroxylation of monophenols and the oxidation of diphenols without any additional cofactors. Although it is of significant interest for the biosynthesis of catechol derivatives, the rapid catechol oxidase activity and inactivation of tyrosinase have hampered its practical utilization as a monophenol monooxygenase. Here, we prepared a functional tyrosinase that exhibited a distinguished monophenolase/diphenolase activity ratio (V max mono/ V max di = 3.83) and enhanced catalytic efficiency against L-tyrosine (k cat  = 3.33 ± 0.18 s−1, K m  = 2.12 ± 0.14 mM at 20 °C and pH 6.0). This enzyme was still highly active in ice water (>80%), and its activity was well conserved below 30 °C. In vitro DOPA modification, with a remarkably high yield as a monophenol monooxygenase, was achieved by the enzyme taking advantage of these biocatalytic properties. These results demonstrate the strong potential for this enzyme’s use as a monophenol monooxygenase in biomedical and industrial applications.113Nsciescopu

Oriented in situ immobilization of a functional tyrosinase on microcrystalline cellulose effectively incorporates DOPA residues in bioengineered mussel adhesive protein

Background Catechol-containing polymers such as mussel adhesive proteins (MAPs) are attractive as biocompatible adhesive biomaterials, and the catecholic amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) is considered a key molecule in underwater mussel adhesion. Tyrosinases can specifically convert tyrosine to DOPA without any cofactors. However, their catalytic properties still need to be adjusted to minimize unwanted DOPA oxidation via their diphenolase activity and catechol instability at neutral and basic pH values in the reaction products. Methods and Results In this work, we constructed a novel functional tyrosinase, mTyr-CNK_CBM, by fusion of mTyr-CNK with a cellulose-binding motif (CBM) for oriented in situ immobilization on microcrystalline cellulose via the C-terminal CBM without any additional purification steps. mTyr-CNK_CBM showed optimal catalytic activity at pH 4.5–6.5 and room temperature and had a high monophenolase/diphenolase activity ratio (Vmaxmono/Vmaxdi = 2.08 at pH 6 and 25°C). mTyr-CNK_CBM exhibited 2.17-fold higher (as a unimmobilized free enzyme) and similarly high (upon immobilization) in vitro DOPA modification of a bioengineered MAP compared to a commercially available mushroom tyrosinase. Moreover, the immobilized mTyr-CNK_CBM showed long-term storability and improved reusability. Conclusions These results clearly demonstrate a strong potential for practical use of immobilized mTyr-CNK_CBM as a monophenol monooxygenase in preparing biocompatible DOPA-tethered biomaterials and other catechol-containing polymers.11Nsciescopu

Cellulose nanocrystals coated with a tannic acid-Fe3+ complex as a significant medium for efficient CH4 microbial biotransformation

Microbial biotransformation of CH4 gas has been attractive for the production of energy and high-value chemicals. However, insufficient supply of CH4 in a culture medium needs to be overcome for the efficient utilization of CH4. Here, we utilized cellulose nanocrystals coated with a tannic acid-Fe3+ complex (TA-Fe3+CNCs) as a medium component to enhance the gas-liquid mass-transfer performance. TA-Fe3+CNCs were well suspended in water without agglomeration, stabilized gas bubbles without coalescence, and increased the gas solubility by 20 % and the k(L)(a) value at a rapid inlet gas flow rate. Remarkably, the cell growth rate of Methylomonas sp. DH-1 as model CH4-utilizing bacteria improved with TA-Fe3+CNC concentration without any cytotoxic or antibacterial properties, resulting in higher metabolite production ability such as methanol, pyruvate, formate, and succinate. These results showed that TA-Fe3+CNCs could be utilized as a significant component in the culture medium applicable as a promising nanofluid for efficient CH4 microbial biotransformation.11Nsciescopu