Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-02-09
2004-04-27
Hutson, Richard (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S267000, C435S252300, C435S320100, C530S350000, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06727084
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a purified &bgr;-galactosidase specific for lactose.
&bgr;-galactosidase catalyzes the hydrolysis of lactose disaccharide into its constituent monosaccharides, glucose and galactose.
DESCRIPTION OF THE RELATED ART
This enzyme is widely distributed in numerous micro-organisms, plant and animal tissues.
The ability of &bgr;-galactosidase to hydrolyse lactose into galactose is applied in food industry, particularly in the field of dairy products because of the nutritional (lactose intolerance), technological (crystallisation) and environmental (pollution) problems associated with lactose (Triveni P. S., 1975, CRC Critical Reviews in Food Technology 325-354). The added value gained by the hydrolysis of lactose, to its constituent monosaccharides glucose and galactose, lies in the increased usefulness of hydrolysed lactose as a food carbohydrate. Lactose itself has limited use in this respect because of its relatively low sweetness, solubility and digestibility, but the hydrolysis products of lactose, i.e. glucose and galactose, are superior in all of these respects. Increased sweetness and solubility improve the technical usefulness of whey products while the increased digestibility of hydrolysed lactose also offers the opportunity of supplying milk solids to populations which have hitherto been unable to consume milk products because of their inability to hydrolyse lactose in the digestive tract.
The &bgr;-galactosidase can be applied to the production of low-lactose milk and in the production of galactose or glucose from lactose contained in milk serum which is formed in large amount in the process of producing cheese.
The major applications for lactose hydrolysis are listed below.
a) Liquid milk. Lactose hydrolysis in liquid milk improves digestibility for lactose intolerant consumers. In flavoured milks, lactose hydrolysis increases sweetness and enhances flavours.
b) Milk powders. Lactose hydrolysed milk powders for dietetic uses, especially for infants with temporary &bgr;-galactosidase deficiency.
c) Fermented milk products. In some cases, lactose hydrolysis in milk used for the manufacture of cheese and yoghurt can increase the rate of acid development and thus reduce processing time.
d) Concentrated milk products. Lactose hydrolysis in concentrated milk products (e.g. sweetened condensed milk, ice cream) prevents crystallisation of lactose.
e) Whey for animal feed. Lactose hydrolysis in whey enables more whey solids to be fed to pigs and cattle and also prevents crystallisation in whey concentrate.
f) Whey. Lactose hydrolysed whey is concentrated to produce a syrup containing 70-75 per cent solids. This syrup provides a source of functional whey protein and sweet carbohydrate and is used as a food ingredient in ice cream, bakery and confectionery products.
The conventional approach in food processing is to carry out the hydrolysis of lactose at 40° C. during approximately four hours. (T. Godfrey and J. Reichelt in : “Industrial Enzymology: the application of enzymes in industry”; The Nature Press, Mac Millan Publishers Ltd, GB, 1983). However, milk or lactose solution as a raw material is a preferable nutrition source for bacteria. As the result, the putrefaction owing to the saprophyte contamination during the treatment is a serious problem in the food production. Thus, the fact is that the conventional &bgr;-galactosidase is not put into practical use.
Attempts to solve these problems consisted in using thermophilic enzymes as described in U.S. Pat. No. 4,237,230 and U.S. Pat. No. 4,007,283 but a problem of high energetic cost still remains.
On another hand, cold-adapted &bgr;-galactosidases have been studied (Trimbur D. E. and al., 1994, Appl. Environ. Microbiol. 60:4544-4552; Rahim K. A. A. and Leb B. H., 1991, Biotechnol and Appl. Biochem., 13, 246-256).
However, these &bgr;-galactosidases generally known in the prior art, when used for food processing, all have one or more disadvantages such as low enzyme activity and low stability at a temperature below 20° C., narrow range of optimum pH and the inhibition of enzymatic action by a reaction product, such galactose or others products particularly calcium.
SUMMARY OF THE INVENTION
The object of the present invention is to hydrolyse lactose by using a &bgr;-galactosidase, which could overcome the above-mentioned drawbacks which are usually associated to this process, while advantageously avoiding contamination problems during the hydrolysis process and lowering the energy consumption.
This problem is solved according to the present invention by a purified cold-active &bgr;-galactosidase, specific for lactose, having a stable enzymatic activity at temperatures up to below 8° C., preferably up to below 6° C., and specifically at 4° C., which corresponds to refrigerating conservation temperature for dairy products. This enzyme of the invention is consequently able to hydrolyse lactose in dairy products and stable enzymatic activity at temperatures up to below 8° C., preferably up to below 6° C., and specifically at 4° C., which corresponds to refrigerating conservation temperature for dairy products. This enzyme of the invention is consequently able to hydrolyse lactose in dairy products and milk processing at such a low temperature that saprophytes are hindered to proliferate. The hydrolysis of lactose can be carried out in these refrigeration conditions with no need of a particular treatment to the dairy product concerned.
According to the invention, an enzymatic activity is considered as stable when, in the concerned conditions, the enzyme is capable of lasting long enough to obtain the desired effect, for example, the hydrolysis of a substrate.
According to an embodiment of the invention, the cold-active &bgr;-galactosidase has a stable enzymatic activity between 0 and 50° C.
Advantageously, the cold-active &bgr;-galactosidase according to the invention has a stable enzymatic activity at a pH range from 6 to 10, preferably from 6 to 8.
Preferably, the cold-active &bgr;-galactosidase according to the invention has a stable enzymatic activity in presence of calcium and/or galactose, meaning that the activity of this enzyme is neither inhibited by its reaction product nor by products being present in milk. This property allows to use efficiently this enzyme in milk treatment.
Such a cold-adapted &bgr;-galactosidase according to the invention attains the level of practical application, having simultaneously the following properties:
(1) Having a sufficient stability in the neighbourhood of 0 to 10° C.
(2) Having a sufficient enzymatic activity at a pH range from 6 to 10
(3) Having an enzymatic activity non inhibited by reaction products or other products substantially present in milk, such calcium.
According to an advantageous embodiment of the invention, the enzyme can be inactivated at a pasteurisation temperature. This property of the enzyme according to the present invention allows to apply the &bgr;-galactosidase according to the invention and to stop the enzymatic reaction of lactose hydrolysis without any additional step during a current milk treatment.
Another object of the present invention is a strain of an isolated psychrophilic bacterium capable of producing a cold-active &bgr;-galactosidase according to the present invention. A preferable strain is
Pseudoalteromonas haloplanktis
deposited on the Nov. 4, 1999, under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms (BCCM™), Laboratorium voor Microbiologie—Bacteriënverzameling (BCCM™/LMG), Universiteit Gent, K. L. Ledeganckstraat 35, 9000 Gent, Belgium, with the Accession NO LMG P-19143 and variants and mutants derived therefrom.
To purify a cold-active &bgr;-galactosidase according to the invention, a bacterium living in the Antarctic area was isolated and characterised in order to study how its enzymes, and particularly, the &bgr;-galactosidase was adapted to cold. These studies led to the purification of the &bgr;-galactosidase, meaning that this protein was obtained substa
Baise Etienne
Dubois Phillip
François Jean-Marie
Genicot Sabine
Gerday Charles
Axelrod Nancy J.
Hutson Richard
Kinberg Robert
Universite de Liege
Venable LLP
LandOfFree
Cold-active beta-galactosidase, the process for its... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cold-active beta-galactosidase, the process for its..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cold-active beta-galactosidase, the process for its... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3241551