Ross Eppler.

Graduate Student
University of California, Berkeley

Sc.B. Chemical Engineering, 2001
Brown University

Co-Advisor: Jeffrey Reimer, Ph.D.

Office Location: 497A Tan Hall
Office Telephone: 643-8340
Office Fax: 643-1228

Salt-Activated Enzymes

The exquisite selectivity and high catalytic rate of enzymes under ambient conditions confer a unique position to enzymes in the realms of biotechnology and synthetic chemistry. Unfortunately, the wide spread applicability of enzymes in these industries has been hindered by the narrow range of environments in which they exhibit these desirable features (ie aqueous environments). A necessary feature of commercially viable bio-catalysts would be broad utility in a variety of environments, chief among which are organic solvents. With the goal of increasing the utility of enzymes in organic media the Clark group has focused in recent years on activating enzymes through a lyophilization process in which the enzyme is combined with small amounts of excipient molecules. These excipient molecules include salts, sugars, and polymers. Salts in particular have been shown to increase the activity of enzymes suspensions in organic solvents by nearly 35,000 fold over the corresponding non-salt suspensions. In particular, the classification of salts using the Jones-Dole viscosity B coefficient has been helpful in providing a quantitative measure to the existing data of the salt interaction with the enzyme/water matrix. It has been shown that the greatest affect on enzyme kinetics occur with chaotropic anions and kosmotropic cations.

To help elucidate the mechanisms of enzyme activation in organic solvents a class of experiments will be aimed at probing the mobility and possible confinement of residual water molecules retained during the lyophilization process. Using O[17] enriched water to prepare the enzyme samples affords the opportunity to use NMR as a probe of water mobility. The unique relaxation times of the residual water in the enzyme/water/excipient complex will provide a concise method to determine where the water is located, such as confinement to the active site or the presence of a monolayer coating, and how this locale is correlated to % (w/w) excipient and Jones-Dole viscosity parameter. To increase the sensitivity of our samples experiments will be performed using 30% O[17] enriched water. In summary, these experiments will help to answer the following.

Exactly how the structure and dynamics of enzyme-bound water vary with the properties of the organic solvents, i.e. hydrophobicity, dielectric constant, and polarity?

How are the catalytic properties of the enzyme affected by the structure of water?

Do the hydration layers of highly activated enzyme preparations exhibit unique structural properties, and can the extraordinary activities of these preparations be explained by the water structure?