Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase.

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Cyclohexanone monooxygenase (CHMO) is a valuable biocatalyst for industrial applications due to its selectivity.
• Low enzyme stability hinders the industrial use of many Baeyer-Villiger monooxygenases.
• There is a need for robust and stable CHMOs for biotechnological processes.

Purpose of the Study:

• To characterize a novel, robust cyclohexanone monooxygenase (CHMO) from Thermocrispum municipale.
• To determine the crystal structure of the CHMO and understand its substrate-binding properties.
• To evaluate the enzyme's potential for industrial applications in biotechnology.

Main Methods:

• Enzyme purification and characterization.
• Crystal structure determination of CHMO.
• Substrate conversion assays with various ketones and sulfides.
• Evaluation of enzyme stability in different solvents and temperatures.
• Whole-cell biotransformation experiments.

Main Results:

• A robust CHMO from Thermocrispum municipale was identified and characterized.
• The enzyme demonstrated efficient conversion of diverse substrates, including aliphatic, aromatic, and cyclic ketones, and prochiral sulfides.
• Crystal structure revealed a compact substrate-binding cavity, explaining its preference for smaller substrates.
• The CHMO exhibited exceptional solvent tolerance and thermostability.
• Small-scale conversions using purified enzyme and whole cells confirmed its practical utility.

Conclusions:

• The newly discovered CHMO from Thermocrispum municipale possesses superior stability and a broad substrate range.
• Its compact active site influences substrate specificity, favoring smaller molecules.
• The enzyme's remarkable solvent tolerance and thermostability make it a highly attractive biocatalyst for industrial biotechnology.
• This robust CHMO offers significant potential for developing efficient and sustainable biotransformation processes.