Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-08-17
2002-07-16
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06420156
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a purified proteolytic enzyme and to a method of purifying a proteolytic enzyme, in particular trypsin.
BACKGROUND ART
Commercial proteases, in particular commercial trypsin, even after purification by a special treatment, for example by double crystallization, contains residual lipases, in particular phospholipase A
2
, which is particularly resistant to the heat deactivation to which protease is subjected after its use in a hydrolysis process.
Trypsin is commonly used in the manufacture of protein hydrolysates intended in particular to enter into the composition of infant products. To incorporate the protein portion into a finished product, for example an infant milk, any residual lipolytic enzymatic activity resulting from the protein hydrolysate must be removed. This is necessary in order to avoid the appearance of products of degradation of lecithin which is added to the final formula for technological reasons, for example to enhance the wettability of powders, into lysolecithin, in particular during storage. Such breakdown products may manifest themselves both in liquid products and in powders by the appearance of stability or organoleptic defects, for example spots, poor taste, or by their toxicity leading to side effects, for example of an inflammatory type in breastfeeding infants.
However, it is the case that the complete removal of phospholipases in particular is difficult to achieve. The complete purification of proteases generally requires various precipitation steps, chromatographic separations, heat treatments under well-defined conditions or chemical inactivations. The complete removal of phospholipase A
2
, which is very heat-resistant, requires a prolonged heat treatment which unfortunately also affects the protease.
The aim of the invention is the preparation of a purified protease whose proteolytic activity is quantitatively and qualitatively preserved, but which is free of lipolytic activity, in particular of phospholipase A
2
, by a simple and inexpensive method.
A method of preparing purified trypsin, described for example in U.S. Pat. No. 3,886,043, is known in which a buffer solution of crystallized trypsin is chromatographed by passing over a resin consisting of a dextran gel with grafted sulphonic groups, with the aim of separating the various active forms of porcine trypsin.
It is also known to prepare a lipase-free microbial rennet, for example by the method described in U.S. Pat. No. 4,136,201, by culturing Mucor miehei on an appropriate nutrient medium. The processes of these patents do not achieve the aim of the present invention.
SUMMARY OF THE INVENTION
The invention relates to a purified proteolytic enzymatic preparation and methods for producing the same, characterized in that it possesses a residual phospholipase A
2
activity of at most 20 mU/g of pure enzyme detectable by high performance chromatography analysis of phospholipids after incubating with an infant formula whose phospholipase A
2
activity is not detectable, and in that its protease activity is maintained at not less than 75% of the initial activity of the enzyme.
DETAILED DESCRIPTION OF THE INVENTION
The measurements of the enzymatic activities are detailed in the examples hereinafter. In particular, “nondetectable” is understood to mean a residual phospholipase A
2
activity of <6 mU/g of enzyme.
The enzyme may be any protease of plant, microbial or animal origin or of biogenetic origin. It is preferably a protease of animal origin, such as pancreatin, particularly trypsin of porcine origin.
The method according to the invention is characterized in that:
1) the pH of a solution of the protease is adjusted to a value of between 6 and 9 and in that the solution is kept at this pH and at 20-35° C. for at least 15 min and at most 120 min, so as to use the proteolytic activity of the protease to destroy the lipolytic activity of the lipases and of the phospholipases of the reaction medium, and
2) the pH of the solution is reduced to a value of less than or equal to 3.5, it being possible to reverse the order of steps 1) and 2) above.
In a preferred embodiment which makes it possible to also remove traces of residual lipases other than phospholipase A
2
, the method comprises a final heat treatment step, preferably by ultra high temperature (UHT), as is known in the art and is shown in the examples herein. Traces of heat-sensitive lipases are thus removed.
Preferably, the adjustment of the pH to the alkaline region takes place before the reduction of the pH to the acidic region, since it is thus possible to defer the use of the protease. In the variant where the two steps are reversed, the protease should be used immediately after the treatment.
In a preferred embodiment, there is added to the reaction medium a magnesium salt which is soluble in the latter, preferably at the beginning of the reaction, which makes it possible to stabilize the proteases while promoting the degradation of the phospholipases. Magnesium chloride is preferably added in an amount of 10 to 200 mM/l of the reaction medium, for example 50 to 100 mM/l of reaction medium.
The pure protease concentration in the solution before treatment may be between 0.5 and 6%, and is preferably about 2.5% by weight. The invention also relates to a method of preparing an infant formula based on protein hydrolysate, characterized in that a whey product is enzymatically hydrolysed by means of a purified protease above, in that the hydrolysate is treated at 75-85° C./3-5 min, in that liquid fat and minerals are added thereto, in that a UHT treatment is carried out at 125-135° C./2-3 min, then in that carbohydrates, vitamins and trace elements are added thereto, in that the liquid product is sterilized by UHT and in that it is aseptically packaged.
According to a variant of this method, the liquid is dried, in particular spray-dried, after UHT sterilization treatment.
The purified enzyme according to the invention may be used outside the food area in the applications of proteases, for example in the preparation of a nutritional, cosmetic or pharmaceutical composition.
There may be mentioned, in this regard, anti-inflammatory applications, the treatment of digestive disorders, the treatment of thromboses, the treatment of injuries and wounds and the elimination of necrosed tissues for example.
REFERENCES:
patent: 3886043 (1975-05-01), Walker et al.
patent: 4136201 (1979-01-01), Feldman
patent: 6704373 (1968-09-01), None
patent: WO 09/13638 (1990-11-01), None
patent: WO-9700316 (1997-01-01), None
Hazlett et al., “Proteinase K DecreasesPseudomonas auruginosaAdhesion to Wounded Cornea”, Exp. Eye Res., 55(4), pp. 579-587, in CAPLUS AN:1993:20366,1992.*
Somkuti, G.A., “Control of Lipase Content in Mucor Rennet Preparations”, J. Dairy Sci., 57(8), pp. 898-899, in CAPLUS AN: 1974:118816.*
Tietz et al., “A Specific Method for Serum Lipase Determination” (Mar., 1966) Clin. Chim. Acta, 13(3), pp. 352-358.*
Lee et al., “Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures inEscherichia coli” (1993) Biotech. Bioeng., 41(8), 781-790.*
Ausubel F.M.: “Current Protocols in Molecular Biology”, 1995, Wiley & Sons, N.Y., USA, p. .A.3F.12.
Braun Marcel
Neumann Fred
Nestec S.A.
Weber Jon P.
Winston & Strawn
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