Biocatalytic Systems Development

Problem/Opportunity

Biocatalytic systems represent a major opportunity for the development of chemicals from biobased materials. If these systems prove feasible, it may be possible to produce chemicals by design in relatively small modular systems. Estimates from industrial partners have indicated that biocatalytic systems have the potential to reduce costs to about 10-15% of the costs for conventional fermentation/separation systems because of the reduced separations requirements and lower capital costs. There are, however, significant technical hurdles, including the costs of enzymes and cofactors and the ability to carry out reactions with multiple enzymes.

Approach

Argonne has initiated a Laboratory Directed Research and Development (LDRD) project focused on the limitations of continuous biocatalytic systems related to the cost of cofactors and the inability to carry out multistep reactions. The purposes of this project are to

• Develop systems to carry out key classes of reactions that include immobilized cofactors,
• Generalize these reactions to other substrates,
• Immobilize enzymes to membranes to stabilize them and remove products of reaction, and
• Explore the use of new materials capable of concentrating and immobilizing enzymes while retaining activity.

The final goal is to put together systems that can carry out multiple reactions. This project puts together unique capabilities at Argonne, including membrane separations, bioprocessing, structural biology, nanoscience, structural biology, and bioinformatics. We will also use techniques developed at Argonne for extracting genes for specific protein variants from unculturable microorganisms that may have broader specificity for the reactions of interest and draw on our ability to conduct rapid, high-resolution structural analysis of proteins to attempt rational design.

Structure of an enzyme being used as one of the model
systems in Argonne biocatalysis studies.

Results

Argonne has identified an enzyme that can carry out a certain class of reactions and that contains immobilized cofactor. This enzyme has been purified, cloned, and expressed. Mutants of this enzyme have also been obtained. Initial structural analysis suggests that current theories regarding the mechanism of immobilization are incorrect. Argonne has also been shown, in a related enzyme system, that this reaction can be immobilized on a membrane surface with simultaneous removal of product and reduced product inhibition. In parallel work, the Chemistry Division researchers have demonstrated that certain clays with engineered pore sizes or secondary structures can absorb about 100 times the amount of enzyme (based on activity) than molecular sieve materials used in previous studies. Furthermore, both water-soluble and lipid-soluble proteins were absorbable and retained significant enzyme activity.

Future Plans

During the next year, we will identify the mechanisms of cofactor retention, broaden specificities through rational design and recruitment from unculturable microbes, and begin putting together bioreactor systems capable of multistep enzyme reactions. Intellectual property will also be identified and new project proposals will be prepared.