U.S. patent application number 09/135436 was filed with the patent office on 2002-01-17 for purified proteolytic enzyme and method of purification.
Invention is credited to BRAUN, MARCEL, NEUMANN, FRED.
Application Number | 20020006655 09/135436 |
Document ID | / |
Family ID | 8228665 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006655 |
Kind Code |
A1 |
BRAUN, MARCEL ; et
al. |
January 17, 2002 |
PURIFIED PROTEOLYTIC ENZYME AND METHOD OF PURIFICATION
Abstract
A purified protease is prepared by adjusting the pH of a
protease solution to a value of between 6 and 9 and by keeping the
solution at this pH and at 20-35.degree. 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
phospholipases in the reaction medium and the pH of the solution is
reduced to a value of less than or equal to 3.5. The purified
protease allows in particular the manufacture of infant formulae
containing lecithin which are stable during storage, that is to say
which do not exhibit significant degradation of the added
lecithin.
Inventors: |
BRAUN, MARCEL; (KONOLFINGEN,
CH) ; NEUMANN, FRED; (STEFFISBURG, CH) |
Correspondence
Address: |
WINSTON & STRAWN
200 PARK AVENUE
NEW YORK
NY
10166-4193
US
|
Family ID: |
8228665 |
Appl. No.: |
09/135436 |
Filed: |
August 17, 1998 |
Current U.S.
Class: |
435/219 ;
435/212 |
Current CPC
Class: |
C12N 9/6427 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/219 ;
435/212 |
International
Class: |
C12N 009/50; C12N
009/48 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 1997 |
EP |
97202591.0 |
Claims
1. Purified proteolytic enzymatic preparation, characterized in
that it possesses a residual phospholipase A.sub.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.sub.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.
2. Preparation according to claim 1, characterized in that the
enzyme is porcine trypsin.
3. Method of purifying a proteolytic enzyme, in particular trypsin,
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.degree. 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.
4. Method according to claim 3, characterized in that it comprises
a final heat treatment step, preferably by UHT, which makes it
possible to also remove traces of residual lipases other than
phospholipase A.sub.2.
5. Method according to claim 3, characterized in that the
adjustment of the pH between 6 and 9 takes place before the
reduction of the pH to a value of less than or equal to 3.5.
6. Method according to claim 3, characterized in that there is
added to the reaction medium a magnesium salt which is soluble in
the latter, preferably at the beginning of the reactor, which makes
it possible to stabilize the proteases while promoting the
degradation of the phospholipases.
7. Method according to claim 6, characterized in that magnesium
chloride is added in an amount of 10 to 200 mM/l and preferably in
an amount of 50 to 100 mM/l of reaction medium.
8. Method according to claim 3, characterized in that the pure
protease concentration in the solution before treatment is between
0.5 and 6%, and is in particular about 2.5% by weight.
9. 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 according to claim 1, in
that the hydrolysate is treated at 75-85.degree. C./3-5 min, in
that liquid fat and minerals are added thereto, in that a UHT
treatment is carried out at 125-135.degree. 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.
10. Method according to claim 9, characterized in that after the
UHT sterilization treatment, the liquid is dried, in particular
spray-dried.
11. Use of a purified enzymatic preparation according to claim 1,
in the preparation of a nutritional, cosmetic or pharmaceutical
composition.
Description
[0001] The invention relates to a purified proteolytic enzyme and
to a method of purifying a proteolytic enzyme, in particular
trypsin.
[0002] 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.sub.2, which is particularly resistant to the heat
deactivation to which protease is subjected after its use in a
hydrolysis process.
[0003] 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.
[0004] 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.sub.2, which is very heat-resistant,
requires a prolonged heat treatment which unfortunately also
affects the protease.
[0005] 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.sub.2, by a simple and inexpensive
method.
[0006] 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.
[0007] 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.
[0008] The invention relates to a purified proteolytic enzymatic
preparation, characterized in that it possesses a residual
phospholipase A.sub.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.sub.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.
[0009] The measurements of the enzymatic activities are detailed in
the examples hereinafter. In particular, "nondetectable" is
understood to mean a residual phospholipase A.sub.2 activity <6
mU/g of enzyme.
[0010] 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.
[0011] The method according to the invention is characterized in
that:
[0012] 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.degree. 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
[0013] 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.
[0014] In a preferred embodiment which makes it possible to also
remove traces of residual lipases other than phospholipase A.sub.2,
the method comprises a final heat treatment step, preferably by
UHT. Traces of heat-sensitive lipases are thus removed.
[0015] 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.
[0016] 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.
[0017] 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.degree.
C./3-5 min, in that liquid fat and minerals are added thereto, in
that a UHT treatment is carried out at 125-135.degree. 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.
[0018] According to a variant of this method, the liquid is dried,
in particular spray-dried, after UHT sterilization treatment.
[0019] 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.
[0020] 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.
[0021] The examples below illustrate the invention. In these
examples, the parts and percentages are by weight, unless otherwise
stated.
EXAMPLE 1
[0022] 1 kg of commercially available porcine trypsin 6.0 S (Novo,
Denmark) is dissolved in 10 kg of demineralized water at 25.degree.
C., with stirring, in a vessel, the protease concentration being
9.1% and the initial pH 5. To ensure that undissolved protease is
not carried over to the next step, the solution is transferred to a
new vessel.
[0023] A dilute aqueous NaOH solution at 1 M/l is added to adjust
the pH of the solution to 8. The pH is then kept constant for 15
min by addition, as required, of the aqueous NaOH solution above,
for example by means of a pH-stat, with stirring, so as to
hydrolyse the phospholipases.
[0024] After this treatment, the proteases are stabilized, that is
to say trypsin and chymotrypsin, by reducing the pH of the reaction
medium to 3 by addition of an aqueous HCl solution at 1 M/l. The
solution of proteases may be used immediately in a hydrolysis
reaction or stored, for example, at -25.degree. C. for a deferred
use.
[0025] The analyses below show the enzymatic activity of the
purified proteolytic enzyme obtained according to the invention
compared with that of the original commercially available
crystallized enzyme (trypsin PTN 6.0 S, Novo, Denmark) which has
not been subjected to the treatment according to the invention.
[0026] 1. Determination of phospholipases:
[0027] 1.1 Determination of phospholipase A.sub.2 by a radioisotope
method.
[0028] The method is based on the cleavage of phosphatidylcholine
(C14-dioleyl) by phospholipase A.sub.2 and the punctual radiometric
detection of the labelled fractions after chromatographic
separation.
[0029] 1.2 Determination of the total phospholipases by
titrimetry.
[0030] The method is not specific to phospholipase A.sub.2 and
detects all phospholioases. It is based on the titration of fatty
acids released from egg yolk phospholipids (Fluka, Buchs,
Switzerland) by phospholipases at pH 8 (maintained by a pH-stat)
and at a constant temperature of 40.degree. C. with 1.4 mM of
sodium deoxycholate and 3 mM CaCl.sub.2. 2 g of purified egg yolk
phospholipid are used with addition of 250 mg of turkey egg white
trypsin inhibitor (Sigma, St. Louis, USA) per 1 g of trypsin.
[0031] 2. Determination of lipase and esterase:
[0032] 2.1 The activity of the lipases is determined by titrimetry
using olive oil as substrate in an amount of 100 g/l at a constant
pH of 8.9 (pH-stat) in the presence of 1.25 g/l of taurocholate and
82.5 g/l of gum arabic.
[0033] 2.2 The activity of the esterases is determined using the
above method (2.1) but taking medium-chain triglycerides (MCT) as
substrate.
[0034] 3. Determination of proteases:
[0035] 3.1. For trypsin, the method described by Erlanger et al. in
Arch. Biochem. Biophys. 95, 271-278 is used.
[0036] 3.2. For chymotrypsin, the US Pharmacopoeia XXI (1985)
method is used.
[0037] The results of the analyses of activities in the enzyme
preparation are indicated in Table 1 below:
1TABLE 1 Phos- Tryp- Chymo- Enzyme Phospho- pholi- Lip- Ester- sin
trypsin prepara- lipase A.sub.2 pases ases ase (g/ (USP/ tion
(U/g), 1.1 (U/g) (U/g) (U/g) kg) mg) Purified <0.0022 0.79 0.21
0.22 223 41 trypsin PTN 6.0 S according to the invention Original
87 14 0.34 0.39 213 42 trypsin PTN 6.0 S
[0038] It is observed that the treatment makes it possible to
considerably reduce the activity of enzymes other than proteases,
in particular to remove that of phospholipase A.sub.2, while
maintaining intact the proteolytic activity in terms of quality and
quantity, in particular the equilibrium between trypsin (93%
activity relative to the untreated enzyme) and chymotrypsin (86%
activity relative to the untreated enzyme).
EXAMPLE 2
[0039] 1 kg of commercially available porcine trypsin 6.0 S (Novo,
Denmark) is dissolved in 10 kg of demineralized water at 25.degree.
C., with stirring, in a vessel, the protease concentration being
9.1% and the initial pH 5. To ensure that undissolved protease is
not carried over to the next step, the solution is transferred to a
new vessel.
[0040] 224 g of magnesium chloride (MgCl.sub.2.6 H.sub.2O) are
added, the pH is adjusted to 8.5 over 15 min with a dilute aqueous
NaOH solution at 1 M/l. The medium is allowed to react or 120 min
at 25.degree. C. without controlling the pH, so as to hydrolyse the
phospholipases.
[0041] After this treatment, the proteases, that is to say trypsin
and chymotrypsin, are stabilized by reducing the pH of the reaction
medium to 3 by addition of an aqueous HCl solution at 1 M/1 and the
solution is allowed to stand for 16 h at 4.degree. C. The purified
trypsin solution is then ready for use.
[0042] It is observed that the relative activity of the trypsin is
93% of that of the original trypsin and that the relative activity
of the chymotrysin is 86% of that of the original chymotrypsin.
EXAMPLE 3
[0043] The purified enzymatic preparation of Example 2 is used to
prepare a hypoallergenic infant formula. Whey proteins are
hydrolysed and then the hydrolysate is treated at 75-85.degree.
C./3-5 min, fat and minerals are added thereto, a UHT treatment is
carried out at 125.degree. C./2 min, then maltodextrin and vitamins
are added, the liquid product is sterilized by UHT at 148.degree.
C./5 s and aseptically packaged.
[0044] To test the residual enzymatic activity, 10 ml of purified
trypsin solution according to Example 1 are added to 100 ml of the
liquid infant formula above whose phospholipase A.sub.2 activity is
<6 mU/g trypsin. After mixing, the activities of the lipase and
esterase are reduced by a heat treatment at 75-80.degree. C./3-5
min on a water bath. After cooling to room temperature, 55 mg of
sodium azide are added and the solution is incubated at 40.degree.
C./4 d. After incubation, the phospholipid composition is analysed
by high-performance liquid chromatography (HPLC) and the
phospholipase A.sub.2 activity calculated, expressed in mU/100 g of
product (mU -PL-A.sub.2), from the differences in the concentration
of the lysophospholipids according to the formula:
mU PL-A.sub.2=(LPC+LPE) at time T2 minus (LPC+LPE) at time t1 in
mg/100 g of product 540 (molecular mass of egg
lysolecithins).times.(t1-t2)
[0045] with LPC=lysophosphatidylcholine and
LPE=lysophosphatidylethanolami- ne,
[0046] as well as this value expressed in terms of the
concentration of pure trypsin g/100 g, that is to say mU
PL-A.sub.2/g of pure trypsin.
[0047] The degradation of trypsin and or chymotrypsin is also
evaluated in % relative to the initial activity, as well as the
degradation of the phospholipids after 9 months of storage at
20.degree. C. in % of the original phospholipids.
[0048] The results are indicated in Table 2 below:
2 TABLE 2 Degrada- PL-A.sub.2 tion of (mU/g of Chymo- the phos-
Liquid pure Trypsin trypsin pholipids infant trypsin), (% of (% of
(% of formula HPLC initial) initial) initial) Hydro- 16 94 75 <1
lyzed by purified trypsin PTN 6.0 S according to the invention
Hydro- 349000 100 100 100 lyzed by the original trypsin PTN 6.0
S
EXAMPLE 4
[0049] An infant formula is prepared as in Example 3, except that a
phospholipid is added to the liquid mixture before spray-drying it.
It observed that the activity of the phospholipases is strongly
linked to the degradation of the phospholipids in the product.
Large load volumes, an interrupted production, associated with a
high phospholipase activity degrades the phospholipids to a certain
degree before the product is dried. The products containing the
purified trypsin according to the invention show a considerably
lower degradation of the added phospholipids, depending on the
total quantity of phospholipids added to the formula, <1%,
whereas it represents 20 to 90% when the same trypsin is used which
has not been subjected to the purification treatment according to
the invention.
[0050] Furthermore, SDS-PAGE analysis of the residual proteins and
analysis of the immunologic ally active antigens by ELISA did not
show significant differences using purified trypsin according to
the invention compared with production with the unpurified
trypsin.
* * * * *