A highly active esterase from Lactobacillus helveticus hydrolyzes chlorogenic acid in sunflower meal to prevent chlorogenic acid induced greening in sunflower protein isolates.

Chlorogenic acid (CGA) is an ester between caffeic and quinic acid. It is found in many foods and reacts with free amino groups in proteins at alkaline pH, leading to the formation of an undesirable green pigment in sunflower seed-derived ingredients. This paper presents the biochemical characterization and application of a highly active chlorogenic acid esterase from Lactobacillus helveticus. The enzyme is one of the most active CGA esterases known to date with a Km of 0.090 mM and a kcat of 82.1 s-1. The CGA esterase is easily expressed recombinantly in E. coli in large yields and is stable over a wide range of pH and temperatures. We characterized CGA esterase's kinetic properties in sunflower meal and demonstrated that the enzyme completely hydrolyzes CGA in the meal. Finally, we showed that CGA esterase treatment of sunflower seed meal enables the production of pale brown sunflower protein isolates using alkaline extraction. This work will allow for more widespread use of sunflower-derived products in applications where neutrally-colored food products are desired.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide.

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.