Optimization of 2,3-dihydroxybiphenyl 1,2-dioxygenase expression and its application for biosensor

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biosensor Development

Background:

• 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC_LA-4) is crucial for catechol degradation.
• Enhancing BphC_LA-4 expression is key for developing sensitive catechol biosensors.
• Understanding enzyme-substrate interactions aids in biosensor design.

Purpose of the Study:

• To optimize fermentation conditions for enhanced BphC_LA-4 expression using statistical designs.
• To investigate the binding affinities of BphC_LA-4 with various catecholic substrates.
• To construct and characterize a catechol biosensor utilizing immobilized BphC_LA-4.

Main Methods:

• Plackett-Burman design (PBD) for initial factor screening.
• Response surface methodology (RSM) for parameter optimization.
• Homology modeling and molecular docking for interaction analysis.
• SiO2 sol-gel method for enzyme immobilization.

Main Results:

• Optimized fermentation conditions yielded a maximal specific activity of 0.58 U/mg for BphC_LA-4 with catechol.
• Molecular modeling revealed lower binding affinity of BphC_LA-4 to 4-methylcatechol compared to catechol and 3-methylcatechol.
• The developed SiO2 sol-gel immobilized enzyme electrode showed good response to catechol, 3-methylcatechol, and 4-methylcatechol.
• Differences in selectivity for 4-methylcatechol between free and immobilized enzymes suggest electro-catalysis influence.

Conclusions:

• Statistical experimental designs effectively enhanced BphC_LA-4 expression and activity.
• BphC_LA-4 exhibits differential substrate binding, impacting biosensor selectivity.
• The developed catechol biosensor demonstrates broad responsiveness to related catecholic compounds.
• Further research into electro-catalysis effects on enzyme-substrate interactions is warranted.