Oxygenases play a central role in degrading/interpreting exogenous compounds in the environment. Without the activity of these oxygenases, the mineralization of these compounds would not be possible. With its advanced protein engineering platform established over the years, Creative BioMart has successfully developed engineered oxygenases with stability, substrate range and catalytic activity. Our scientists are committed to providing fully customized engineered oxygenases production solutions for environment to customers around the world.

Introduction of Oxygenases Production

Extensive urbanization and rapid industrialization over the past few decades have resulted in a large amount of exogenous compounds entering the environment. Organic (aromatic/non-aromatic) compounds constitute one of the environmental pollutants. Oxygenases are enzymes that oxidize substrates by transferring oxygen from molecular oxygen. They are attractive for chemical synthesis and bioremediation because they play key roles in the metabolism of organic compounds by increasing their reactivity or water solubility or by causing cleavage of aromatic rings. Recently oxygenases are being developed in various environmental applications. Although oxygenases play such an important role, their practical application is limited due to their complexity, poor stability, and generally low catalytic rates. Based on a growing understanding of structure-function relationships, scientists are paving the way for the use of oxygenases in chemical synthesis and bioremediation through improved protein engineering methods. The study found that P450s have been designed to oxidize new substrates and function without the need for biological cofactors, expanding the substrate range of dioxygenases and enhancing the peroxygenase activity of chloroperoxidase.

Fig 1. Schematic diagram showing the role of aromatic dioxygenases in the bacterial degradation of aromatic compounds. (Arora P K, et al., 2009)

Solutions

Oxygenases are widely used in environmental remediation and play an important role in improving environmental quality. Creative BioMart has successfully utilized a variety of protein engineering techniques to produce engineered oxygenases with stability, substrate range and catalytic activity. In order to provide customized high-quality oxygenases to global customers, we perform protein engineering on the following oxygenases, including but not limited to:

• Cytochrome P450s
  Cytochrome P450s form a broad group of monooxygenases that catalyze the oxidation of a wide range of substrates. We use protein engineering methods to design or improve the properties of these heme-containing enzymes to degrade chlorinated hydrocarbons in the enviroment.
• Dioxygenases
  Biphenyl dioxygenase is an important enzyme in the degradation of polychlorinated biphenyls (PCB). We used protein engineering methods to design biphenyl dioxygenase mutants to oxidize a wider range of PCBs and other biphenyl-related compounds in the enviroment.
• Chloroperoxidase
  Due to the inability of chloroperoxidase to express fungal enzymes in suitable microbial hosts, protein engineering has been hindered. We used protein engineering methods to express and screen chloroperoxidase mutants in yeast to increase their activity for synthesis.

Applications of Protein Engineering in Oxygenases Production

The need for clean, economical oxidation processes, as well as the need for increasingly complex and specific oxidation products, provides a strong impetus to consider biocatalytic pathways. We are learning through protein engineering to develop and refine the capabilities of oxygenases in the environment. Our protein engineers employ the following protein engineering strategies to support the above solutions:

• Modification of cytochrome P450 activity by combining molecular dynamics and site-directed mutagenesis.
• Site-directed mutagenesis of several residues in the active site of naphthalene dioxygenase.
• Design of biphenyl dioxygenase mutants by a combination of directed evolution and rational design.
• Two methods, random mutagenesis and saturation mutagenesis, were used to try to expand the substrate range of toluene dioxygenase.

Creative BioMart has in-depth knowledge and experience of the tools and processes involved in engineered oxygenases development, and provides professional oxygenases production solutions for the environment. Whether your oxygenases are in the discovery and screening stages, or are planning engineered oxygenases production, please contact us to discuss further details to ensure your next success.

References

1. Cirino P C, Arnold F H. (2002) Protein engineering of oxygenases for biocatalysis[J]. Current Opinion in Chemical Biology. 6(2): 130-135.
2. Arora P K, Kumar M, Chauhan A, et al.. (2009) OxDBase: a database of oxygenases involved in biodegradation[J]. BMC research notes. 2(1): 1-8.

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