Glassy State and Thermal Inactivation of Invertase and Lactase in Dried Amorphous Matrices

Abstract

The thermal stability of enzymes lactase and invertase in dried, amorphous matrices of sugars (trehalose, maltose, lactose, sucrose, raffinose) and some other selected systems (casein, PVP, milk) was studied. The glass transition temperature (Tg) was limited as a threshold parameter for predicting enzyme inactivation because (a) enzyme inactivation was observed in glassy matrices, (b) a specific effect of enzyme stabilization by certain matrices particularly trehalose was observed, and (c) enzyme stability appeared to depend on heating temperature (T) "per se" rather than (T-Tg). For these reasons, a protective mechanism by sugars related to the maintenance of the tertiary structure of the enzyme was favored. A rapid loss of enzyme (lactase) activity was observed in heated sucrose systems at T > Tg, and this was attributed to sucrose crystallization since it is known that upon crystallization the protective effect of sugars is lost. Thus, the stabilizing effect could be indirectly affected by the Tg of the matrix, since crystallization of sugars only occurs above Tg. Trehalose model systems (with added invertase) showed an exceptional stability toward "darkening" (e.g., non-enzymatic browning) when heated in the dried state to elevated temperatures and for long periods of time.

Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

“…A non-enzymatic browning reaction, which is a chemical reaction between carbonyl compounds (for example, reducing sugar such as maltose and lactose) and amino compounds, results in the formation of browning pigments. It is known that the progress of a non-enzymatic browning reaction causes destabilization of a protein (9,19), and that this reaction can progress even at a temperature much lower than T g within an hour to a day (42,43). In the fully dried trehalose and sucrose formulations which exhibited no browning change, there was a gradual loss of the relative LDH activity at the storage temperature of 60-C.…”

Improvement in the remaining activity of freeze-dried xanthine oxidase with the addition of a disaccharide–polymer mixture

“…The water substitution hypothesis involves the native-like structure of protein being maintained by the formation of hydrogen bonds between dried protein and stabilizers in place of the removal of water molecules (Arakawa et al, 2001;Imamura et al, 2001;Kreilgaard, Frokjaer, Flink, Randolph, & Carpenter, 1998;Schebor, Burin, Buera, Aguilera, & Chirife, 1997;Suzuki et al, 1997;Wang, 2000). The interrelated stabilization mechanism, ''glass transition", is intrinsically similar to the ''freeze-concentrated glass transition" mechanism, involving embedding of the protein molecules in a glassy matrix.…”

Stabilizing effects of sucrose–polymer formulations on the activities of freeze-dried enzyme mixtures of alkaline phosphatase, nucleoside phosphorylase and xanthine oxidase

“…Preferential exclusion involves the preferential exclusion of additives from the enzyme surface, and conformation of the enzymes is thermodynamically stabilized in the liquid and/or freezing state (Arakawa, Prestrelski, Kenney, & Carpenter, 2001;Arakawa & Timasheff, 1982;Carpenter & Crowe, 1988;Nema & Avis, 1992;Wang, 2000). Water replacement involves hydrogen bonding of the enzyme with additives instead of the removed water molecules, and native-like conformation of the enzyme is maintained in the dried state (Allison et al, 1999;Arakawa et al, 2001;Kawai & Suzuki, 2007;Kreilgaard et al, 1998;Prestrelski et al, 1993;Schebor et al, 1997;Wang, 2000). Glass transition leads to the prevention of physical and chemical enzyme degradation, due to the slowing down of conformational changes in freeze-concentrated and/or freeze-dried glassy matrices (Anchordoquy et al, 2001;Franks, 1993;Kawai & Suzuki, 2007;Prestrelski, Pikal, & Arakawa, 1995;Schebor et al, 1996Schebor et al, , 1997.…”

“…Many efforts have been devoted to improving the stability of freeze-dried enzymes using additives (Allison, Chang, Randolph, & Carpenter, 1999;Anchordoquy & Carpenter, 1996;Anchordoquy et al, 2001;Izutsu, Yoshioka, & Kojima, 1995;Izutsu, Yoshioka, & Takeda, 1991;Kawai & Suzuki, 2007;Prestrelski et al, 1993;Schebor, Buera, & Chirife, 1996;Schebor, Burin, Buera, Aguilera, & Chirife, 1997;Srirangsan et al, 2010). Among the additives, sugar and polymer are usually effective (Allison et al, 1999;Anchordoquy et al, 2001;Chang, Beauvais, Dong, & Carpenter, 1996;Kreilgaard, Frokjaer, Flink, Randolph, & Carpenter, 1998;Prestrelski et al, 1993;Sampedro, Guerra, Pardo, & Uribe, 1998;Schebor et al, 1997;Wang, 2000).…”

“…Among the additives, sugar and polymer are usually effective (Allison et al, 1999;Anchordoquy et al, 2001;Chang, Beauvais, Dong, & Carpenter, 1996;Kreilgaard, Frokjaer, Flink, Randolph, & Carpenter, 1998;Prestrelski et al, 1993;Sampedro, Guerra, Pardo, & Uribe, 1998;Schebor et al, 1997;Wang, 2000). For example, Anchordoquy et al (2001) investigated the activity of reconstituted freeze-dried lactate dehydro-0308-8146/$ -see front matter Ó 2010 Elsevier Ltd. All rights reserved.…”