Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Abstract

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzyme's thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three b-galactosidases from Escherichia coli , Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

“…The other group used to attain an oriented immobilization of enzymes is the thiol group of Cys, immobilizing the enzyme via thiol/disulfide exchange, a very specific reaction that cannot be produced by any other group on the enzyme (Figure ). ,− Cysteine groups are quite scarce on the protein surface, and when needed, if the native enzyme has some external Cys, it may be transformed into Ser via site-directed mutagenesis, as the physical properties of both lateral chains are somehow similar. To achieve an immobilization fully directed by the Cys location, there are two possibilities: to use thiol reactive disulfide groups on the support , or to generate it on the enzyme (e.g., by modification of the exposed Cys of the enzyme with 2,2-dipyridyl disulfide) (Figure ). , …”

Enzymatic Generation of Chitooligosaccharides from Chitosan Using Soluble and Immobilized Glycosyltransferase (Branchzyme)

“…Another possible interpretation for this behavior is that the enzyme was attached to the support through a “critical area” for enzyme stability . It has been reported that denaturation of a protein begins in a defined region of the protein called the “critical area”, so when enzymes are immobilized through this area, their stability will be improved …”

“…39 It has been reported that denaturation of a protein begins in a defined region of the protein called the "critical area", so when enzymes are immobilized through this area, their stability will be improved. 40 Enzyme Reuses. One of the advantages of enzyme immobilization onto an insoluble support is the possibility of reuse of the enzymatic derivative for consecutive cycles of chitosan hydrolysis.…”

“…39 It has been reported that denaturation of a protein begins in a defined region of the protein called the "critical area", so when enzymes are immobilized through this area, their stability will be improved. 40 Enzyme re-uses One of the advantages of enzyme immobilization onto an insoluble support is the possibility of re-use of the enzymatic derivative for consecutive cycles of chitosan hydrolysis. In order to characterize the operational stability of the enzymatic derivative, the capacity to generate reducing sugars from chitosan was evaluated in a repeated batch process as detailed in the Material and Methods section.…”

Covalent immobilization of tobacco‐etch‐virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins

“…It has been reported that denaturation of a protein begins in a defined region of the protein called ‘critical area’. So when enzymes are immobilized through this area, their stability will be improved [44].…”