Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1997-04-16
1999-04-27
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435 14, 435 27, 435134, 435190, C12P 764, C12P 758, C12N 996, C12N 904
Patent
active
058979956
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the enzymatic production of gluconic acid. More specifically, the invention provides a method for the conversion of glucose into gluconic acid using the enzymes glucose oxidase and catalase.
BACKGROUND OF THE INVENTION
Gluconic acid is a mild acid with many applications which comprise: its technical use as a complexing agent in industrial cleaning of metal surfaces; its use in the textile industry, in detergents and in concrete; as a food additive in beverages and also in bread and feed; and in pharmaceutical preparations as a chelating agent for ions like Fe. The gluconic acid used in food applications and especially in pharmaceutical preparations must be very pure.
Up until now the production of gluconic acid at industrial scale has been carried out using a fermentation process. Selected microorganisms such as e.g. Aspergillus or Gluconobacter species are grown in a fermenter which is at least equipped with air supply, pH control and temperature control. Optimal conditions are chosen for good growth of the microorganism and optimal development of the enzyme complex capable of converting glucose into gluconic acid/gluconate. At the end of the growth phase, a glucose solution is added to the fermentation broth and aeration is continued. The glucose is converted by the enzymatic complex into gluconic acid. Usually the pH is controlled by the addition of alkali, in which case the majority of gluconic acid will be present as a gluconate salt.
In fungi, glucose is transformed into gluconic acid by an enzymatic complex consisting of glucose oxidase (Gox) and catalase. Glucose oxidase catalyses the reaction:
The conversion of gluconolactone into giuconic acid can be catalysed by the enzyme gluconolactonase but can also occur spontaneously:
glucose+1/2O.sub.2 .fwdarw.gluconic acid
The enzymatic complex glucose oxidase/catalase is present in several micro-organisms belonging e.g. to the classes Aspergillus and Penicillium. Also bacteria like Acetobacter and Gluconobacter are capable of converting glucose into gluconic acid but the bacteria use a different mechanism: Gluconobacter oxydans contains two types of glucose dehydrogenases which convert glucose into gluconolactone without the formation of hydrogen peroxide. At this moment, industrial processes for the production of gluconic acid use almost exclusively selected strains of Aspergillus niger or Gluconobacter oxydans in a fermentation process.
The enzymes glucose oxidase and catalase are rather well characterized. The glucose oxidase of A.niger is a glycoprotein with a molecular weight of about 150 kDa and contains prosthetic groups of FAD; the enzyme is active between pH 4 and 7 with an optimal activity at pH 5,5; the enzyme is optimally active at temperatures between 20.degree. and 40.degree. C.; the isoelectric point is about 4,2; the K.sub.m for glucose is 0,11M at 27.degree. C. and the K.sub.m for oxygen is 0.48 mM. The catalase of A.niger has a molecular weight of 250 kDa; the enzyme is active between pH 2 and 7; the optimal temperature is 25.degree. C.; the isoelectric point is about 5,4; the K.sub.m for hydrogen peroxide is about 1,1M or about 37 g/l. The latter means that the affinity of the enzyme catalase for its substrate is very poor.
Taking into account the applications of gluconic acid, the existing fermentative production processes have the following drawbacks. Complex production media are used which, besides glucose, must contain all kinds of nutrients for the growth of the microorganisms, which are costly and give impurities. At the end of the bioconversion the broth contains many byproducts formed during the fermentation such as coloured substances and organic acids other than gluconic acid, such as e.g. citric acid, oxalic acid and 5-keto-gluconic acid. The overall production time includes both the growth time and the bioconversion time and usually requires several days. With fungi, the yield based on glucose is significantly lower than the theoretical maximum yield of 100% becaus
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Beverini Marc
Vroemen Albert J.
Gist-Brocades B.V.
Lilling Herbert J.
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