Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Development of a two phase aqueous/organic system for enhancing the activity of ethene monooxygenase (#285)

Samantha Cheung 1 , Peter J Rutledge 2 , Nicholas V Coleman 1
  1. School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia
  2. School of Chemistry, University of Sydney, Sydney, NSW, Australia

Bacteriathat grow on ethene contain a monooxygenase (MO) enzyme which has a broad substrate range, and can transform many alkenes into their corresponding epoxides1,2. Epoxides are versatile and widely-used intermediates in organic chemistry, and so the activity of MO enzymes is of interest for making these chemicals. This is an example of biocatalysis3. Unfortunately, the ethene MO cannot be functionally expressed in E.coli – this has made it difficult to do mutagenesis to understand or modify the enzyme.

We recently succeeded in expressing the ethene MO of Mycobacterium rhodesiae JS60 in the host Mycobacterium smegmatis 4, but the activity of the recombinant cells is low. Here, we aimed to improve this activity by using two-phase systems consisting of a cell-containing aqueous phase emulsified with an organic phase. The organic phase has multiple potential benefits; it acts as a solvent for the substrate and product (these can be toxic to the cells), it reduces epoxide hydrolysis, and it allows higher concentrations of poorly water-soluble substrates into the system.

Ethene oxidation by recombinant M.smegmatis cells was examined in the presence of hexadecane, glyceryl trioctanoate, glyceryl tributyrate, 1-decanol, trioctyl phthalate and dimethylpolysiloxane. The presence of glyceryl tributyrate or 1-decanol was inhibitory, reducing epoxidation activity by 80-90%. Addition of dimethypolysiloxane and trioctyl phthalate gave moderate (approx. 2-fold) improvement of activity. Addition of hexadecane or glyceryl trioctanoate gave the best increases in activity (3-fold, and 4-fold, respectively).

Addition of glyceryl trioctanoate or hexadecane to cell suspensions containing styrene raised the activity sufficiently to allow the detection of the product (styrene oxide) by a colorimetric assay. This has not been possible before with recombinant ethene MO-containing cells, due to their low activity. This is a crucial breakthrough that will enable us to perform high-throughput screening on ethene MO mutant clone libraries, using styrene and related larger alkenes as substrates.

  1. S. Cheung, V. McCarl, A.J. Holmes, N.V. Coleman, and P.J. Rutledge, Appl. Microbiol. Biotechnol., 2013, 97, 1131-40.
  2. C.R. Owens, J.K. Karceski, and T.E. Mattes, Appl. Microbiol. Biotechnol., 2009, 84, 685-92.
  3. N. Ran, L. Zhao, Z. Chen, and J. Tao, Green Chemistry, 2008, 10, 361-72.
  4. M.A. Ly, E.F. Liew, N.B. Le, and N.V. Coleman, J. Microbiol. Methods, 2011, 86, 320-6.