The objective of the project complex is to develop oxidative enzymes (oxidases) for food with the special aim to target application in foods with a high content of liquids (oils, etc).
Oxidative enzymes (oxidases) are being developed for foods with a high content of liquids (oils, etc).
Research involved the optimization of the simultaneous production of glucose oxidase (GOX), superoxide dismutase (SOD) and catalase (CAT) from the strain BTL of Aspergillus niger on low cost carbon sources (molasses) and purification of extracellular and cell bound GOX.

For maximum total GOX activity the medium should contain (g/l): molasses sucrose, 20; peptone, 2; calcium carbonate, 50. The overall optimization procedure yielded a 5-fold increase in the activity of cell-bound GOX and a 10-fold increase in the activity of extracellular enzyme.
Optimum SOD production was achieved with a medium containing (g/l): molasses sucrose, 80; peptone, 5.
The production of CAT was unaffected by the optimization process.
A purification scheme for both GOX forms was employed to produce significant amounts of purified enzymes in order to study their kinetic characteristics.
2 purified GOX forms were examined. Apart from some significant differences in thermal stability of the 2 forms, the kinetic characteristics of these forms were not very different indicating similarities in their molecular structure.

Investigations on the stability of copper, zinc superoxide dismutase from Saccharomyces cerevisiae towards lipids showed that the enzyme activity declined with increased oil fractions in oil water emulsions caused by lipid hydroperoxides extracted from the oil phase to the aqueous phase.

The structure of glucose oxidase has been determined through development of crystals of the purified enzyme and collection of X-ray diffraction data and growth experiments of the bacterium ATCC 25589 resulted in the conclusion that the bacterium had lost the ability to synthesize thiamine oxidase. The Aspergillus niger gene encoding glucose oxidase has been isolated and the deoxyribonucleic acid (DNA) sequence has been determined (Scottish strain). The gene was then inserted into a bacterial expression vector.
Furthermore, a purification procedure has been established for the enzyme hexose oxidase from the red algae Chondrus crispus. Analysis of the substrates and products of this enzyme indicate that hexose oxidase could be a candidate as an antioxidant.
A number of problems and questions are addressed:

1.Production methods;

2.Development of production methods for genetically engineered enzymes;

3.Application of protein engineering in order to target oxidases to the lipid phase of food;

4.Development of new enzyme substrate combinations;

5.Applicability of enzymes through application studies.

1.Production methods

Use of specific enzymes in relation to foods introduces a number of problems. The enzyme must be cheap and pure. We address these two problems by analysis and optimization of the fermentation process and the purification process.

2.Genetic engineering

Genetic engineering may in many instances be used to improve production yields of microbial enzymes. Information about the gene and genetic structure of the particular gene corresponding to a protein is a prerequisite for introduction of changes in the protein according to the findings of protein engineering. The genetic structure of two of the enzymes is studied, glucose oxidase and hexose oxidase. The original plan was to study thiamine oxidase, but since the enzyme cannot be identified as originally described hexose oxidase has been substituted for it. We expect to find significant differences in the molecular nature of these two enzymes, since one originates from A. niger and the other from the red alga Chondrus crispus.

3.Protein engineering

Protein engineering is used to determine which changes in enzyme structure are necessary to obtain desired functional changes. A basic knowledge is the tertiary enzyme structure, and two such structures will be determined.

4.New substrate enzyme combinations

Glucose oxidase (with glucose as a substrate) has already been used as an antioxidant in fruit juice. Glucose is in certain respects an undesirable food component. It has therefore been of importance for the project to include an enzyme with a different substrate. Thiamine oxidase was given that part, but as mentioned above it had to be excluded from the project. Instead we have included hexose oxidase which can use various hexoses and di-hexoses as a substrate and thus be able to have antioxidative effect in almost all foods without addition of substrate.