Improving enzymes by error

29 January 2008

Mutant enzymes could make for greener chemical synthesis, say UK scientists.

Luet-Lok Wong's group from the University of Oxford have grown variant forms of a cytochrome P450 monooxygenase (P450) enzyme which can oxidise hydrocarbons under ambient conditions with greatly improved efficiency and selectivity over the natural enzyme.

'Biocatalysis is an increasingly important branch of chemical synthesis which avoids the use of high temperatures and pressures and is typically pollution-free,' explained Wong. P450 enzymes are metabolic enzymes found in all types of life. They typically catalyse the insertion of an oxygen atom into a C-H bond. 'Potential applications for the new variants being explored include the production of fine chemicals, flavours and fragrances, the degradation of environmental contaminants such as dioxins, and the preparation of drug metabolites from pharmaceuticals,' said Wong.

Working with the soil bacterium Bacillus megaterium, Wong's team used a variation of the PCR (polymerase chain reaction) technique to copy the DNA from which the enzyme is made. Using an "error-prone" PCR introduced changes in the DNA to create several new artificial P450 enzymes. A simple screening test - can this variant turn indole into indigo? - identified promising variants. The team went on to pick out variants which efficiently oxidised naphthalene, propylbenzene and pentane.

"Using an "error-prone" PCR introduced changes in the DNA to create several new artificial P450 enzymes"'It is intriguing that preselection with one small substrate, indole, allowed isolation of efficient catalysts for several different reactions. This seems to contradict the accepted principle that "you get what you screen for",' said Elizabeth Gillam, an expert on P450 enzymes from the University of Queensland, Brisbane, Australia.

Wong accepts that there is a way to go before commercial biosynthesis is practical. 'Scaling up P450 reactions is the greatest single challenge. Control over the regio- and stereo-selectivity of oxidation also needs to be considerably enhanced before chemicals can be manipulated to order,' he said.

Clare Boothby

Link to journal articleEvolved CYP102A1 (P450BM3) variants oxidise a range of non-natural substrates and offer new selectivity options
Christopher J. C. Whitehouse, Stephen G. Bell, Henry G. Tufton, Richard J. P. Kenny, Lydia C. I. Ogilvie and Luet-Lok Wong, Chem. Commun., 2008
DOI: 10.1039/b718124h

Also of interestArtificial enzymes that outdo nature?

A highly selective catalyst that works like an artificial enzyme has been made using the molecule-targeting system that nature uses to combat infection.

Emotional enzymes

Chemists in the US have created fluorescent probes that can detect enzymes affecting our emotions.

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Biocatalysis can be defined as the application of a biocatalyst to achieve a desired conversion under controlled conditions in a bioreactor. A biocatalyst can be an enzyme, an enzyme complex, a cell organelle or a whole cell. Over recent years there has been an exponential increase in the production of high-value specialty chemicals using either isolated enzymes, especially hydrolases, to catalyze single-step transformations or whole cells to catalyze multi-step
reactions. This increase is motivated by the fact that enzymes, whether isolated or contained within whole cells, can catalyze a broad range of reactions, at times with high levels of chemoselectivity, regioselectivity and stereoselectivity. In fact, biocatalysts in the form of isolated enzymes, or enzymes contained in whole cells, can often catalyze reactions with a specificity that is difficult to obtain by classical chemical routes. Further, they act under mild conditions of pH and temperature and biocatalytic processes generate fewer residues than chemical processes do, such that biocatalysis is often referred to as »green chemistry«.