DE2554407A1 - PRODUCTION OF THERMOSTABLE LACTASE - Google Patents

Description

HOFFMANN <& EITLE - PATENT LAWYERS

D-SMO MÖNCHEN 81 - ARABELLASTRÄSSE 4 {STERNHAUS) · TELEPHONE (089) i! TOS7 · TELEX 05-29619 (PATHE)

27 470 i / ra

B. J. Reynolds Tobacco Company Winston Salem, N.C. (UNITED STATES)

Production of thermostable lactase

The present invention relates to a method for the production of laotase from a heat tolerant organism, belonging to the genus Bacillus.

There has been considerable interest in procedures for several years to develop, according to which the amounts of lactose in milk and products made from milk can be reduced. This interest was heightened as recent evidence became available that a large percentage of human Population suffers from a lactase deficiency, which is due to either

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inheritance or the aging process. Such lactase deficiencies lead to intestinal problems if, for example, the dietary amounts of lactose are high. A similar lactose intolerance has also been observed in certain domestic animals.

The hydrolysis of lactose in milk and in those made from milk Glucose and galactose products is a goal set, not just because it would solve the lactose intolerance problem, but also because it will increase the sweetness of the products and the so-called sandy textures in certain, made from milk Products caused by lactose crystallization. Those who work in this field appreciate it have long been using the Mutzen, which is made by hydrolysis could be caused by lactase. Despite the fact that lactase is quite widespread in nature and by many microorganisms are produced, the use of lactase has so far been involved. the commercial production of milk and Dairy products are only possible to a limited extent. One reason for the limited commercial use of lactase is that many of the commercial lactases, such as those made from yeast, gain their optimal enzymatic activity unfold at temperatures that also favor the growth of bacteria. Accordingly, there is a growing interest in it given to find a lactase that has a high degree of thermal stability. Such a thermostable lactase would enable the lactose hydrolysis to be carried out under conditions to carry out those responsible for the growth of certain bacteria, which are generally present in milk or dairy products are unfavorable. One such lactase that is made up of Streptomyces coelicolor has recently been described in US Pat. No. 5,816,259. At this point it is however, it is not clear whether lactase from S. coelicolor is safe can be used because certain members of these species Supposed to produce antibiotics.

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The production of lactase by individual members of the genus Bacillus has already been reported. PJ Anema has in Biochira. Biophys. Acta 89 (3), 495-502 (1964) describe the isolation of lactase from B. subtilis. Lactase from B. megaterium has already been described by SR Rohlfing and IP Crawford in J. Bacteriology 92 (4), I258-9 (1966). ■ None of these organisms is, however, regarded as a heat-tolerant, and the lactase produced from these strains must be vervendet at temperatures below about 50 ⁰ C to maintain an effective enzyme activity over a longer period of time in general.

According to the invention, a method for producing a Lactase, which has improved thermal stability, was created by an organism belonging to the genus Bacillus in a nutrient medium containing lactose is cultivated and the lactase produced thereby is obtained, whereby the organism belongs to those that have optimal growth at a temperature of at least about 45 ° C.

A preferred organism for connection according to the present invention Invention, was isolated from a soil sample.

The cultivation of the organism in a suitable nutrient medium which contains lactose results in the desired lactase. intracellular route. It was carried out to characterize this culture and it was found that it belongs to the species Bacillus coagulans, is given in Bergey's Manual of Determinative Bacteriology ^1, 7th Edition according to the classification. This organism has been deposited with the USDA Northern Regional Research Laboratory's culture collection under the designation NRRL B-8IOO. The classification properties of this strain are given in Table 1 below.

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- 4 - Table 1

A) Morphological characteristics

1. Vegetative rods: Less than 0.9 vl in diameter,

varying lengths generally ranging up to 5.0 to 6.0 w. Some Threads. Not in chains. Gram positive, uniform color. Versatile.

2. Spore carrier: Generally not swollen, but

occasionally swollen spore carriers can be found.

3. Spores: 0.9 to 1.2 to 1.5 κ, ellipsoidal, sub-

terminal to terminal.

B) Culture characteristics

1. Gelatin agar streak-inoculation plate - no hydrolysis.

2. Agar colonies - opaque, small, round. Not different.

3; Agar bevels - sparse to moderate growth. Flat,

smooth, opaque.

4. Glucose agar inclined surface - growth stronger than on nutrient

agar. Smooth white.

5. Glucose asparagine agar bevels - No growth after

24 hours. Moderate growth at 48 hours.

6. Proteose-Peptone-Acid Agar Bevels - Good Growth,

better than on agar nutrient medlura.

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7. Soybean Agar Slants - Moderate growth, a

- little stronger than on

Agar medium.

8. Stock culture agar inclined surfaces - growth sparse

24 hours, as good as agar medium after hours.

9 * Broth - growth poor after 24 hours.

10. Sodium Chloride Broth - no growth in Taigen Sodium Chloride

11. Milk Agar Streak Plate - No hydrolysis.

12. Potato - scanty, dry, wrinkled.

0) Physiological CharakerJstika

1. When using peptone as a nitrogen source, the organism produced acid but no gas from glucose, lactose, Arabinose, xylose, mannitol and maltose. There was a neutral reaction between sucrose and glycerin.

2. The pH of glucose broth was 5 * 0 or less in seven days.

5 · citruses were not used.

4. Tomatoes, yeast, milk curdled in 24 hours at 45 ° C.

5. Nitrites were not made from nitrates.

6. The Voges-Proskauer test was negative. The pH value of the Voges-Proskauer broth was 4.2.

7. The hydrolysis of starch - positive.

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8. Catalase - positive. .

9 · No growth in a nitrate medium under anaerobic conditions. Growth in glucose broth under anaerobic conditions gives a pH of less than 5.2 in 7 days.

10. Aerobic, optionally anaerobic.

11 »The minimum temperature for growth is 25 ° C The maximum temperature for growth is 60 ° C. Optimal growth occurs at 45-50 ° C.

As was performed under the physiological characteristics in Table 1, the herein disclosed Bazillusorganismus shows optimum growth at temperatures of about 45 to 5 ⁰ ° C'iund must therefore be regarded as a heat-tolerant organism in comparison with other bacilli, the optimum growth at show about 37 ° C. A heat-tolerant bacillus organism should therefore be one that has optimal growth at temperatures of around 45 ° C and above.

example

The optimum pH of the lactase produced with the aid of this organism was determined by taking whole cells in the presence of ο -nitrophenyl-ß-Glactoside (ONPG) were tested as substrate. The procedure was essentially that which was written by J. Lederberg in J. Bacteriology 60, 38I (1950). The cells were first treated with toluene; a phosphate buffer was used and the test temperature was 37 ° C. The lactase activity was observed in the range of about pH 4.5 to 8.0, with optimal activity occurring at about pH 6.0.

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The stability of the lactase enzyme produced by B. coagulans was checked using ONPG as a substrate in a modification of the Lederberg method. For this test, washed cells were suspended in 0.05 molar phosphate buffer at a pH of 7.0 in the presence of the ONPG substrate. The suspension was then left for 4 days at 6O ⁰ C, while samples were taken periodically to determine the remaining Laetaseaktivität. The temperature for this stability test was chosen to be at about the temperatures used for the low temperature passivation. The test results are shown in Table 2.

Table 2 Time in hours O 21 45 71 99

Lactase activity as a percentage of

STARTING ACTIVITY

100

56

20th

The lactase produced by Bacillus coagulans shows an effective enzyme activity up to about 70 ⁰ C. The optimum activity appears to occur at temperatures of 60 to 65 ⁰ C. The particular temperature selected for lactose hydrolysis using this enzyme will depend to some extent on the substrate medium used. In general, however, temperatures between approximately 4-5 and 65 ^° C. are preferred.

A typical medium for culturing Bacillus coagulans to produce lactase is as follows:

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Protepse peptone. 1.0 %

Yeast extract 1 * 0 ^

Potassium dihydrogen phosphate. 0.8 $ S>

Lactose (separately sterilized) 2.0 %

pH 6.0

Cultures are incubated on a circular shaker for 48 hours at 45 ° C. At the end of this incubation period, toluene is used broth (0, added -5 Volun $ £ per volume of base material} and the mixture stirred for 30 min vigorously. Cells are then by flocculation method, as described in U.S. Patent No. J) have been described 821,086 won and the resulting flocculated cell aggregate dried at 55 ° C. The lactase activity of the dried aggregate particles was 38.5 units per gram, one unit being defined as the amount of enzyme necessary to produce one micromole dextrose per minute under the test conditions. The test procedure used for this determination is that of Weetall et al as published in Biotechnology and Bioengineering 16, 295 (19720 ⁾ .

The effectiveness of the lactase produced with the help of Bacillus coagulans was determined by the hydrolysis of lactose in a sweet whey feed stock. The organism was cultured and the cells were obtained as described above ... The dried aggregate particles were sieved and 5% of the portion with a size of 1.19 mm to 0.84 mm (16 to 20 mesh) was in a 50 # solution of lactose buffered with a 0.05 molar phosphate buffer at pH 7.0. The hydra thai term particles were then packed into a small glass column which was maintained at a temperature of 60 ⁰ C. An aqueous solution containing 70 g of a commercially available dry, sweet whey powder per liter was passed continuously through this packed column, the solution containing 0.05 molar phosphate at a pH of 7.0

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was buffered «The feed solution contained about 5 wt .- ^ lactose, based on the lactose content of the sweet whey, with 100 mg per liter of methyl p-hydroxybenzoate as a preservative were admitted. The flow rate through the column was maintained at 375 rol per day and the degree of lactose hydrolysis was monitored daily by routine analysis. The initial degree of lactose hydrolysis was 90 #. Continuous after J5 weeks During operation, the degree of hydrolysis of the acetose had dropped to $ 80.

It is understood that the lactase produced by this invention can be used both for the batchwise _f as well as for continuous treatment of Lactosesubstratmedien. In addition, the lactase can be used using the cells directly or it can be used in the form of cell-free enzyme using methods known to those skilled in the art.

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Claims (7)

255U07 - ίο - Claims (l) Process for the production of lactase with improved Thermostability by placing an organism belonging to the genus Bacillus in a nutrient medium containing lactose, cultivated and the lactase produced thereby wins, characterized in that the organism is such that exhibits optimal growth at a temperature of at least about 45 ° C. 2. The method according to claim 1, characterized in that that the organism is cultivated in a nutrient medium at a temperature of at least 40 ° C. J5. Method according to one of Claims 1 or 2, characterized characterized in that the lactase obtained displays optimal enzyme activity at a pH of about 6.0. 4. A process for hydrolyzing lactose to produce glucose and galactose, thereby lehnet denoted, that lactose with any of the above claims 1 to J5 produced lactase is brought into contact. 5. The method according to claim 4, characterized in that the lactose is brought into contact with the lactase at a pH between 4.0 and 8.0 and at a temperature between approximately 45 and 65 ^{° C.} 6. The method according to any one of claims 1 to 5 *, characterized in that that the organism belongs to the species Bacillus coagulans. 7. The method according to claim 6, characterized in that that the organism is NRRL B-8IOO. 609824/0950