Freeze‐drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability

Abstract

The effect of different lyoprotectants (sucrose, dimethyl-succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran, mw 60 kDa) on the stability of the enzyme lignin peroxidase (LiP, EC number: 1.11.1.-), both during the freeze-drying process and storage were investigated. The shelf stability tests were performed at 4°C and 27°C. Both DMS buffer and sucrose showed a good protective action: the former was particularly effective during the process, while the latter improved the stability during storage. In contrast, mannitol and dextran had negative effects, reducing the activity also in the lignin peroxidase solution. BSA was discarded because, in the range of compatible concentrations with LiP, it does not confer a consistent structure to the freeze-dried product.

Freeze-drying of enzymes in case of water-binding and non-water-binding substrates

“…However, as the objective of this study is mainly to investigate the ability of different additives to preserve the enzyme activity during secondary drying, here primary drying was carried out at the same product temperature for all the formulations. In this way, similar values for enzyme activity should be observed at the end of primary drying between the various formulations since, as shown by Capolongo et al [19] for lignin peroxidase, during primary drying, the reduction in enzyme activity is not correlated with the type of additive used. In this study, the limit temperature was set to 252 K, that is, a few degrees lower than the collapse temperature of formulation D as this product was the most heat sensitive (see Table 1).…”

Clover Species Specific Influence on Microbial Abundance and Associated Enzyme Activities in Rhizosphere and Non-Rhizosphere Soils

“…Each soil sample was homogenized (bigger particles were crushed) by sieving through a 2 mm mesh and cooled down (4 • C). The samples for the enzyme activity assays and for the soil DNA isolation (and qPCR analysis) were freeze-dried [34].…”

Lyoprotection and stabilization of laccase extract from Coriolus hirsutus, using selected additives

“…Hall et al ( 2008 ) indicated that the residual activity of lipoxygenase (LOX) and hydroperoxide lyase (HPL) of the enzyme extract, obtained from Penicillium camemberti , was 55.0 and 29.7%, respectively, when 60% (w/w) dextran 72.2 kDa was added to the enzymatic extract before its lyophilization; these authors also reported that the addition of 86% (w/w) of KCl maintained 92.9% of the LOX residual activity and resulted by 2.25 times of enhancement of the HPL activity. In contrast, Capolongo et al ( 2003 ) findings indicated that the addition of mannitol and dextran to the lignin peroxidase extract was not suitable to protect the enzymatic extract during its lyophilization, since their interactions with the protein destabilize it by decreasing its unfolding temperature; these authors also indicated that although the sucrose had a stabilizing effect on the lignin peroxidase, the sucrose containing-solutions were difficult to be lyophilized since the glass transition was at − 33 °C, which resulted in a rubbery product after their lyophilization. The stabilization of enzyme activity, obtained by the addition of sugar and poly-alcohol, could be related to their ability to replace the water molecule by involving the hydrogen bonding with polar groups on the protein (Crowe et al 1993 ; Allison et al 1998 ).…”