Enzymatic modification of vegetable protein: Mechanism,kinetics, and production of soluble and partially soluble protein in a batch reactor

Enzymatic hydrolysis of insoluble soybean protein by a protease enzyme produced by Penicillium duponti K 1104, was investigated in a batch reactor. The reaction conditions were 30–55°C and pH 3.4–3.7. The mechanism of solubilization of the insoluble protein by the Penicillium duponti enzyme was deduced from a series of experiments. Kinetic models were developed that involved adsorption followed by peptic digestion of protein, inhibition of low-molecular-weight peptides, and enzyme deactivation. The uncoupled kinetic parameters were estimated using the Marquardt nonlinear parameter estimation algorithm. A bang–bang production of soluble and partially soluble protein is suggested for higher productivity. The essential amino acids pattern of the enzyme-Hydrolyzed soy protein was comparable with the unhydrolyzed protein isolate. Aggregation of the soluble protein for an extended time was observable. The low-molecular-weight soluble protein was incorporated into noncarbonated beverages. The amount of protein that could be incorporated into a can of 355 ml noncarbonated beverage, without observable changes in the optical density and also aggregation of the protein, was 2.5 g soluble protein. Beverages with caramel color showed excessive decrease in optical density and precipitation. The kinetics and diffusion in a multipore immobilized-enzyme recycle reactor will be considered in part II of this series.     

Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.     

Characterization of glucose oxidase immobilized on collagen

Summary The enzyme glucose oxidase (E.C. 1.1.3.4) was immobilized on collagen — a proteinaceous material found in biological systems as a structural material for a wide variety of cells and membranes. The novel technique of electrocodeposition, which utilizes the principles of electrophoresis, was used to deposit the enzyme-collagen complex on stainless steel helical supports. This technique has been developed in our laboratory. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds and van der Waals interactions.As a first step toward examining its feasible technical use, the kinetic behavior of the collagen-supported glucose oxidase was studied in a batch recycle type reactor and was compared with that for the soluble form. A novel reactor configuration consisting of multiple concentric electrocodeposited helical coils was used. The reactor was found to attain a stable level of activity which was maintained for several months under cyclic testing. The optimum levels of pH and temperature for the immobilized form of the enzyme were the same as those of the soluble enzyme, but the immobilized enzyme was more active than the soluble form at higher temperatures and pH. The values of the Michaelis-Menten parameters indicate that the overall reaction rate of the immobilized enzyme may be partially restricted by bulk and matrix diffusion.