Gelatin and Starch: What Better Stabilizes the Enzyme Activity?

The regularities of the functioning of a number of enzymes in a viscous environment created by natural polymers, starch and gelatin are examined. Based on the analysis of kinetic curves of thermal inactivation, mechanisms of thermal inactivation of enzymes in a viscous microenvironment are proposed. Using the example of butyrylcholinesterase, NAD(P)H:FMN oxidoreductase, and coupled system of the luminous bacteria (NAD(P)H:FMN oxidoreductase + luciferase), the conditions, under which starch and gelatin have a stabilizing effect on enzyme activity during storage and exposure to various physical and chemical environmental factors, were found. A significant increase in the stabilizing effect is achieved by eliminating water during drying the enzyme preparations immobilized in starch and gelatin polymer gels.

Enzyme-support interactions and inactivation conditions determine Thermomyces lanuginosus lipase inactivation pathways: Functional and florescence studies.

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

Co-localization of oxidase and catalase inside a porous support to improve the elimination of hydrogen peroxide: Oxidation of biogenic amines by amino oxidase from Pisum sativum.

Diamine oxidase (DAO) from Pisum sativum is an enzyme that catalyzes the degradation of biogenic amines (BA) present in wine, producing harmless aldehydes and hydrogen peroxide (H2O2). H2O2 promotes a rapid inactivation of the immobilized enzyme. At first glance, co-immobilization of DAO and catalase (CAT) could improve the elimination of the released hydrogen peroxide. Two different co-immobilized derivatives were prepared: (a) both enzymes co-localized and homogeneously distributed across the whole structure of a porous support, and (b) both enzymes we de-localized inside the porous support: DAO immobilized on the outer part of the porous support and catalase immobilized in the inner part. Co-localized derivatives were seven-fold more effective than de-localized ones for the elimination of hydrogen peroxide inside the porous support. In addition to that, the degradation of putrescine by DAO was three-fold more rapid when using both co-localized enzymes. The optimal co-localized derivative (containing 1.25 mg of DAO plus 25 mg of CAT per g of support) promoted the instantaneous elimination of 91% H2O2 released inside the porous support during putrescine oxidation. This optimal derivative preserves 92% of activity after three reaction cycles and DAO immobilized without catalase only preserves 41% of activity. Co-localization seems to be the key strategy to immobilize two sequential enzymes. When enzymes are immobilized in close proximity to each other in a co-localized pattern, the generation of byproducts as H2O2 is strongly reduced.