Comparison of yeast biomass production in five wood aqueous extracts

A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.

Immobilization and Utilization of the Recombinant Fusion Proteins Trypsin−Streptavidin and Streptavidin−Transglutaminase for Modification of Whey Protein Isolate Functionality

Wilcox

Clare

Valentine

et al. 2002

Modification of the Rheological Properties of Whey Protein Isolate through the Use of an Immobilized Microbial Transglutaminase

2002

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.

Effects of Ca²⁺ and Sulfhydryl Reductant on the Polymerization of Soybean Glycinin Catalyzed by Mammalian and Microbial Transglutaminases

Zhang

Matsumura

Matsumoto

et al. 2002

Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.