Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

Abstract

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…The stabilization of dexOx-Cysteine-modified papaya proteinase was explained by two reasons: firstly, the rigidification of the biocatalyst structure by the crosslinking produced an increased stability; secondly, the enzyme was stabilized by the prevention of protein association via thioldisulfide exchange reaction. In another research effort, glucoamylase from Aspergillus niger was modified with dexOx, trying to get a high crosslinking degree that permitted enhancing the enzyme stability [151]. In order to increase the dexOx crosslinking of the enzyme, it was aminated (aminating both the sugar chains of the protein and the carboxylic residues of the enzyme), promoting a further increase in enzyme stability and activity at high temperatures.…”

Covalently Bound Antibody on Polystyrene Latex Beads: Formation, Stability, and Use in Analyses of White Blood Cell Populations

“…Examples are cited in the following references: urease on phospholipid-bound silica (21), chymotrypsin on aldehyde/agarose gels (22), glucoamylase on various periodateoxidized polysaccharides (23,24), and trypsin on cyanogen bromide activated dextran (25). For example, urease on a phospholipid-coated silica surface showed about 80-90% activity after being treated at 100…”

Fluorescent Neoglycoproteins: Antibody-Aminodextran-Phycobiliprotein Conjugates

“…It has been shown that enzymes possess enhanced thermal stability, when they have been immobilized by covalent attachment to polymers. Examples include glucoamylase on various periodate oxidized polysaccharides (15,16) and trypsin on cyanogen-bromideactivated dextran (12). Other noted effects of protein conjugation to polysaccharides were improved emulsifying properties (17), improved solubility of insoluble gluten (18), enhanced antioxidant effect of ovalbumin (19), and broadened bactericidal effect of lysozyme (20).…”