Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Patent
1993-02-01
1994-04-26
Housel, James C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
426 12, 435 93, 435161, 435188, G12N 988, G12N 996, C12C 100, C12G 100
Patent
active
053066340
DESCRIPTION:
BRIEF SUMMARY
The invention comprises a soluble ALDC derivative and a use thereof. ALDC is an abbreviation for acetolactate decarboxylase.
By fermentation of carbohydrate containing substances, e.g. wort or grape juice, various by-products may be formed by processes other than the wanted alcoholic fermentation. Thus, an unwanted by-product is diacetyl.
It appears from EP 46066 that ALDC can be used as an enzyme, which prevents the formation of diacetyl. However, the pH optimum of most naturally occurring ALDC's is around 6, and the activity at pH 3.8-4.7, which is the pH of the fermenting wort, is too low for practical purposes, especially at pH below 4, which is a usual pH of fermenting worts with low malt content. For that reason, the process of preventing the formation of diacetyl from fermenting beer or wine has not yet found its way into the practical industrial production on any larger scale. In EP 46066 it is stated that ALDC may be chemically modified to shift the optimum activity to lower pH values, reference being made to Biochem., vol. 11, No. 22, 1972 (p. 4022-4084). The modification methods mentioned in the Biochem. article comprise growing poly(ornithyl) side chains on chymotrypsin; the Biochem. article thus does not offer any suggestions for modification of ALDC. The prior art modification method for shifting the optimum activity to lower pH values is not suitable for industrial application, and furthermore it is not known, if the poly(ornithyl) method can be transferred from chymotrypsin to ALDC. Finally, it does not appear from the Biochem. article, if the stability at low pH of the prior art modified chymotrypsin is satisfactory.
Thus, the purpose of the invention is the provision of an ALDC derivative which can be used with advantage in industry, and which exhibits a satisfactory stability at low pH.
Surprisingly, it has been found that treatment of ALDC with glutaraldehyde provides a soluble modified ALDC, which in the first place exhibits the wanted pH profile, which in the second place is cheap and easily manufactured, and which in the third place exhibits a satisfactory stability.
Thus, the soluble ALDC derivative according to the invention is characterized by the fact that ALDC in an aqueous medium is treated with glutaraldehyde in a concentration corresponding to between 0.1 and 5 g of glutaraldehyde per g of pure ALDC protein, preferably corresponding to between 0,25 and 2 g of glutaraldehyde per g of pure ALDC protein. It has been found that this ALDC derivative is soluble, if not further treated and that it can be produced in a high activity yield.
In Biotechnology Letters Vol. 10 No. 5 325-330 (1988) it is described that crosslinking of .beta.-glucosidase with glutaraldehyde provides a derivative with improved thermal stability. In Adv. Biochem. Eng. 12, p. 41-118, 1979, Rolf D. Schmid describes that crosslinking of different enzymes, e.g. papain, glucose oxidase, catalase and uricase, but not ALDC, provides enzyme derivatives with improved thermal stability. Thus, the prior art does not point to glutaraldehyde as an agent which could fulfil the purpose of the invention.
It is to be understood that all ALDC enzymes, i.e. ALDCs produced from any microorganism, can be used according to the invention. Preferred ALDC's are from Bacillus brevis and Bacillus licheniformis.
Reference can be made to EP 131251, which describes a special inorganic carrier, on which enzymes, e.g. ALDC, are immobilized by adsorption and crosslinking with glutaraldehyde. The invention, in contradistinction thereto, comprises a soluble ALDC derivative exclusively. Also this EP, does not describe the shift of the pH of the pH-activity curve of the ALDC, which is one of the main features of the present invention. Another main feature of the present invention, which is not described in the EP, is the treatment of the ALDC with glutaraldehyde in a defined small concentration.
Also, the invention comprises a use of the soluble ALDC derivative according to the invention in beer fermentation.
A preferred embodiment of the use ac
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Baker et al., Biotech. Letts, vol. 10, No. 5, pp. 325-330 (1988).
Carpenter Robert
Housel James C.
Lowney Karen A.
Novo Nordisk A S
Zelson Steve T.
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