DE3000598A1 - MICROBIOLOGICAL ENZYMPRODUCT CONTAINING A BETA GALACTOSIDASE - Google Patents

Description

PATENT LAWYERS

WUESTHOFF - ν. PECHMANN - BEHFENS - GOF.TZ

PROFESSIONAL REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE MANDATAIRES AGREES PRES!. 'OFFICE EWROPEEN DES BREVETS

IMi -ING. FRAN2 * UlSTHOI 1 ·

OR. ^ HlL. FREDA TUEST1IO1F (1927-1956) IM "L.-ING. GERHARD FUIS (1952-I971) DIPL.-CHEM. DR. E. BARON OF TECHMANN DR.-ING. DIETER BEHRENS DIPL.-ING .; DIPL.-VIRTSCH .-ING. RUPERT GOBTZ

1-53-5

NOVO INDUSTRI A / S

D-8000 MÜNCHEN 90 SCHWEIGERSTRASSE 2 phone : (089) 6620 51 telegram: protectpatent telex: 514070

Patent application 1 du η g

Applicant;

NOVO ■ INDUSTRI A / S

Novo all

DK-2880 Bagsvaerd, Denmark

Title:

Microbiological enzyme product containing a ß-galactosidase

030031/0632

30th

I) K ₁ -INCPRANZ-SUESTHOFi

PATENT LAWYERS

-TR. THIL. FREDA WUKSTHOPF (1927-1956)

WUESTHOFF-v.PECHMANN-BEHRENS-GOETZ _Dltl ., _NC . _{GEllHAM PU1} , _(19Jz ., _97l)

DIFL.-CHEM. DR. E. BARBER OF PECHMANN PXOFESSIONAL REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE DR.-ING. DIETER BEHRENS

MANDATAIRES AGREiS PRES l'OFFICE EUROPEEN DES BREVETS DIPL.-ING .; DIPL.-VIRTSCH.-ING. RUPERT GOET2

NOVO INDUSTRI A / S D-8000 MUNICH 90

1A-53 105 SCHWEIGERSTRASSE 2

Telephone: (089) 66 20 $ 1 telegram: protectpatent telex: 524070

description

In addition to smaller amounts of proteins, vitamins and salts The main component of whey solids is the disaccharide lactose.

Attempts have been made to convert whey into more valuable products by hydrolyzing the lactose to glucose and galactose with the help of microbiologically produced (b-galactosidases. Although Numerous attempts have been made to solve this problem, which is reflected in the extensive References to literature, no successful commercially viable process has yet been developed. One of the main reasons why the enzymatic conversion of lactose to glucose and galactose has not become a commercially successful process Has led to the fact that there is no ß-galactosidase with satisfactory properties is known. When the conversion of lactose is carried out in a satisfactory manner is to be, it is necessary that the ß-galactosidase at the same time has the following properties: 1) The (b-galactosidase should be stable at a temperature high enough to avoid microbial contamination and 2) the inhibition of ß-galactosidase by lactose or the Galactose hydrolysis products, i.e. galactose and glucose, should be minimal in order for the hydrolysis to proceed to completion to leave without using too large amounts of enzyme.

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It is known that acidic p-galactosidases from fungi, e.g. B. Aspergillus species, in particular Aspergillus oryzae and Aspergillus niger, can be formed (US Pat. No. 3,629,073 and Journal of Food Science, 37, 619-623 (1972)). These enzymes have good thermal stability but they will strongly inhibited by products of the hydrolysis of lactose. On the other hand, fc-galactosidases are produced by certain types of bacteria like Lactobacilli and Entrobacteria only slightly inhibited by the products of the hydrolysis of lactose, but their thermal Stability is very low. This has been known for many years. I-galactosidases have also been found in thermofil bacilli (R.E. Goodman and D.M. Pederson in Canadian J. of Microbiology, Vol. 22 (1076), pp. 817-825). There is one there ß-galactosidase from B. stearothermophilus described with a relatively good thermal stability, which, however, is strongly inhibited by the hydrolysis products of lactose. Even a ß-galactosidase from B. coagulans is described (GB-PS

452) and this enzyme has properties similar to those of the enzyme _? derived from B. stearothermophilus.

It is the object of the present invention to provide a (b-galactosidase to make available, which at the same time has a good thermal stability and by the products that are in the hydrolysis caused by lactose and is only slightly inhibited by lactose itself.

This object is achieved by a microbiological enzyme product containing an fh-galactosidase, which is identical to β-galactosidase in terms of the chemical and immunological enzyme properties (as will be explained in more detail below) _; which is formed by cultivating the microorganism NRRL B-11, 229.

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It is surprising that the new ß-galactosidase simultaneously has good thermal stability and due to the products that occur during lactose hydrolysis, as well as due to Lactose itself is only slightly inhibited.

The characteristics of the microorganism NRRL B-11, 229 are as follows: Morphology: rods 2-5 / µm 0.7 - 1 / µm, spores 1.6 - 1.8 / µm χ 0.8 - 1 / µm Schaeffer's Agar subterminal to terminal. Slightly distended sporangium that only extends straight to the vegetative cell. Physiology Catalase positive, no anaerobic growth, formation of acid but no gas from glucose, xylose, trehalose, lactose, arabinose or mannitol. Growth between 40 and 70 ^° C. and pH 6.0 to 8.5. No growth in 3 % NaCl but growth in 1 % NaCl. The proteinase tests comprising egg yolk and casein as substrates were negative and the starch degradation was weak. NO ^ "is _{reduced to NO 2} ". No hydroxyacetone is formed from glycerine. The microorganism appears to be related to B. stearothermophilus.

The invention is not limited to the NRRL B-11,229 strain but includes the taxonomy group represented by the typical stem NRRL B-11, 229 and the members which have essentially the taxonomic characteristics of this strain as described above and which are capable of To form ß-galactosidase, which is identical to the enzyme-chemical and immunological properties β-galactosidase, which is produced by cultivating the microorganism NRRL B-11, 229.

A preferred microbiological enzyme product according to the invention comprises a microbiological enzyme which, as an ingredient of a microorganism - cell product occurs, whereby the microorganism belongs to the taxonomic group mentioned above, which is represented by the typical culture NRRL B-11,229. In this "context, the invention relates

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also fractions of such microorganisms.

The immunological properties of the enzyme are determined as detailed below. Soluble, purified Enzyme (the preparation of which is described below) has been used to generate antiserum in rabbits way known in and of itself.

The two-dimensional immunoelectrophoretic determination was carried out in duplicate in the following manner.

First direction: 10 / μl of a sample containing 100 / Ug of purified enzyme per ml were applied to a 1.5 mm agarose gel at 20 ° C. and the electrophoresis was carried out for 90 min with an electric field gradient of 7 volts / cm and in a buffer system, consisting of 11 g barbituric acid, 65 g sodium barbital, 28 g glycine, 23 g TRIS buffer (tris (hydroxymethyl) aminomethane) and HpO on 25 1. Second direction perpendicular to the first direction: 1.0 ml of antiserum, which had been prepared as above , was dissolved in 10.8 ml agarose gel and the agarose gel strip, which was formed during electrophoresis in the first direction, was combined with the serum agarose gel. Immunoelectrophoresis in the second direction was carried out for 16 hours with an electric field gradient of 1 volt / cm. The gel from one of the two runs was pressed, washed and dried and the protein developed with Coomasie brilliant blue. The gel from the other run was reacted directly with 0.1% ONPG (o-nitropheny ^ lactoside) in 1/15 m KNaHPO ^ (pH 6.5). If lactase is present, the ONPG will break down to galactose and o-nitrophenol, which is a strong yellow color. A yellow coloration of o-nitrophenol was visible on the gel after 30 minutes. In this system, the lactase had migrated 51 mm towards the anode and a precipitate of lactase and antiserum had formed

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when passing in the second direction. The lactase was still active after precipitation, as by the reaction could be shown with ONPG. Reference is made to FIG.

Figure 1a shows the appearance of the gel on the first run with the protein developed. It seems, that at least three different proteins are present, corresponding to precipitates 1, 2 and 3. Figure 1b shows the appearance of the gel in the second run in which the reaction was carried out with ONPG. The hatched area of Figure 1b was strongly colored yellow, which indicates that the precipitate 2 in Figure 1a corresponds to ß-galactosidase, while the precipitates 1 and 3 of Figure 1a correspond to proteins that do not are β-galactosidase.

When using tandem cross immunoelectrophoresis (e.g. according to N.H. Axelsen et al., A manual of quantitative immunoelectrophoresis, 1977) it is possible to determine whether an aulhenti see ß-galactosidas-e identical according to the invention or different from an unknown β-galactosidase sample. Similar qualitative studies can be carried out using other immunological methods e.g. immunodiffusion or direct immunoprecipitation and subsequent sodium dodecyl sulfate gel chromatography.

The enzyme-chemical properties include thermal stability and inhibition by products of lactose hydrolysis the most important.

The ß-galactosidase activity is determined as follows: A crude cell lysate is prepared by 15 niin long treatment at 37 ⁰ C with lysozyme at a concentration of 2 mg / ml in milk buffer (pH 6.5, salt composition as in milk) and subsequently twice Freezing and thawing.

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The lysate containing 2 to 10 units of lactase activity (explained in more detail below) per liter is treated for 1 h in milk buffer with 2.0% lactose, the reaction is then terminated by heating to boiling, precipitate in ice water and centrifugation. The supernatant fluids are glucose studied using the glucose oxidase-peroxidase method (Tech. Bull. No. 510, Sigma Chemical Co.) · A Lactaseeinheit is defined as the amount of enzyme which under standard conditions of pH 6.5 and 60 ⁰ C. , 1 / umol of glucose released per minute.

The thermal stability is measured as follows: The activity is determined as indicated above, with the exception that different incubation temperatures are used. Samples are used for glucose analysis • -ei3 _r e ~ -5Q-JiiiJa ^ _J2LÄ, ^

The inhibition of galactose is measured as follows: In the standard determination for galactose inhibition, the incubation mixture contains 2.0 % lactose and 2.0% galactose. Otherwise the conditions are the same as those given above for determining the activity. However, other combinations of glucose and galactose are being investigated, e.g. B. 2 % lactose and 10 % galactose corresponding to a 90% hydrolysis of 5-fold concentrated milk.

The pH-activity curve is a different enzyme chemical one Property which is important due to the fact that the pH optimum of the β-galactosidase is not too far from the pH the whey to be treated should be removed, otherwise an inappropriately large amount of ß-galactosidase would have to be applied. The pH optimum for the inventive ß-galactosidase is about 6.5 to 7.0 at 60 ° C, 'if the activity measurements according to the above

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given methods for the determination of ß-galactosidase activity can be carried out. Correspondingly, the ß-galactosidase activity is more than 75 % of the maximum activity in the pH range from 6.1 to 8.0.

The molecular weight of β-galactosidase becomes as follows determined: First, the enzyme is highly purified using gel electrophoresis on polyacrylamide. then the molecular weight of the ß-galactosidase is determined by gel electrophoresis, with the ß-galactosidase in parallel runs with other proteins whose molecular weight is known, i.e. phosphorylase A with a molecular weight of 94,000 and the subunit of E. coli β-galactosidase (inactive) with a molecular weight of 116,000. The molecular weight 'of the ß-galactosidase according to the invention, which still active is approximately 116,000 in this system.

It is surprising that there is a β-galactosidase which at the same time has a high thermal stability corresponding to a half-life of more than 500 min at 65 ° C. and a low lactose inhibition of approximately 10 % according to the standard determination method given above. With 10% galactose in the incubation mixture, the galactose inhibition is less than 50 %. The K value is also very low (approximately 5 mmol) and no lactose inhibition could be detected, even with very high concentrations of lactose.

The invention also relates to a method for producing a microbiological β-galactosidase product in which a microorganism belonging to the taxonomy group given above ^ typically represented by the typical Culture NRRL B-11, 229, grown in growth medium is containing assimilable nitrogen, carbon and oxygen sources, whereupon the cells are recovered and

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the intracellular β-galactosidase product isolated will.

Enzyme formation is induced by lactose and galactose and follows growth. However, the trunk can get through customary mutation processes are made constitutive. Of the The organism does not show any special growth requirements with regard to special carbon or nitrogen sources but growth and enzyme formation are improved if the minimum substrate is organic nitrogen sources such as peptone or tryptone, yeast extract or corn water is fortified. The organism is also very versatile in Relationship to the use of carbon sources, i.e. it uses e.g. glucose, galactose, lactose, fructose, glycerine, Glutaminates, succinates, lactates and acetates. The growth on the mono- and disaccharides is followed by strong acid formation, which interrupts growth when the pH drops below approximately 6.0.

The ß-galactosidase is produced intracellularly, but they can easily be released from the cells by conventional methods, e.g. B. by treatment with toluene, butanol, or lysozyme, sonication or mechanical destruction of the cells whereupon the cell debris is removed and the enzyme is purified in the usual way, e.g. B. by salt precipitation or precipitation with organic solvents.

Whey, milk and other liquids containing lactose which, for the sake of simplicity, are referred to as whey in the following, can be satisfactorily converted into a syrup of the Contains glucose and galactose and practically no more lactose, be converted using the invention β-galactosidase. This conversion can be effective when working in batches with the help of soluble ß-galactosidase be performed. To the transformation in industrial

* (i.e. increases with stronger growth of the microorganisms)

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To make the scale more economical, it is recommended to use an immobilized ß-galactosidase product.

Immobilization methods for enzymes are given in the following publications, among others:

Immobilization of isolated enzyme,
including rei
an enzyme. Cells, incl
whole cells X GB-PS 1,444,539 X X U.S. Patent 4,001,085 X U.S. Patent 4,025,391 X U.S. Patent 4,033,822 X U.S. Patent 4,038,140

This list also shows that both the immobilization of whole cells and of pure enzyme are described is. The β-galactosidase product according to the invention can also be in the form of whole cells as well as pure enzyme can be immobilized. Any of the immobilization methods given above can be used for the inventive β-galactosidase product can be applied.

The invention also relates to an immobilized β-galactosidase product. This immobilized according to the invention ß-galactosidase product possesses the extraordinary Advantage that it is able to produce a liquid with a very economical on an industrial scale to completely convert high lactose content into galactose and glucose, as there is no inhibition by the substrate and the

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Products of lactose hydrolysis occurs.

The invention further relates to a method for batchwise converting a lactose-containing liquid, preferably whey, into a syrup containing glucose and galactose and a smaller amount of lactose, a soluble microbiological ß-galacatosidase product according to the invention to the lactose-containing liquid at a pH between approximately 5.5 and 8.5 and a temperature between 55 and 75 ⁰ C is added.

The initial activity of ß-galactosidase in the lactose-containing liquid _; expressed in units of lactase activity per liter varies considerably, mainly due to changes in pH, temperature}, conversion time and initial lactose concentration.

The invention also relates to a continuous process for converting a lactose-containing liquid, preferably whey, into a syrup containing glucose and galactose and a smaller amount of lactose, the lactose-containing liquid being passed through a column which is immobilized with the microbiological β -Galactosidase product according to the invention is filled, at a pH between about 5.5 and 8.5 and a temperature between 55 and 75 ° C at a rate which leads to a lactose concentration in the liquid exiting the column of less than 10 % _{ preferably less than 1 % of the lactose concentration in the entering liquid.

The invention is further illustrated by the following examples explained. Examples 1 and 2 show the production of β-galactosidase, Example 3 the use of soluble ones ß-galactosidase when working in batches, the two

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games 4 and 5 immobilize ß-galactosidase and Example 6 the use of immobilized β-galactosidase for continuous work.

EXAMPLE 1

The enzyme was formed by submerged cultivation in a standard 400 l fermentor equipped with stirrers for temperature control (outer water jacket) and ventilation inlet pipe was provided, the ratio height to diameter was 2: 1.

The cultivation was carried out under the following conditions :

Temperature: 50 ⁰ C

Air flow rate: 120 l _s / min (normal liters / min). Stirring speed: 200 rpm

The substrate consisted of the following components:

Whey powder 2.0 kg Corn water 8.0 kg (NH ₄ ) ₂ S0 ₄ 0.4 kg MgSO ₄ -7H ₂ O 0.2 kg K ₂ HPO ₄ 0.1 kg surfactant * 0.2 kg (non-ionic)
each * PluronicvJy

The pH was adjusted to 7.2 prior to sterilization.

Tap water was added to a total volume of 400 liters.

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The nutrient broth was inoculated with a UbernacHkultur (5O ⁰ C) of NRRL B-11-229 on agar slants and after 24 h, the fermentation or growth was completely and the culture was rapidly cooled to 10 ⁰ C and then the enzyme recovered. The cells were centrifuged off and opened by passing them through a Manton-Gaulin homogenizer of the SP 15 M 8 TA type with a single-stage homogenization valve arrangement, the pressure drop being 300 to 350 kg / cm. The enzyme was extracted from the disrupted cells with phosphate buffer (pH 6.5, 0.1 m) and the cell debris was removed from the centrifuge. The soluble enzyme fraction was further concentrated by ultrafiltration (DDS membrane GR 6p, DDS is an abbreviation for De Danske Sukkerfabrikker).

Yield:

Culture broth

Sediment

concentrate

1.0 units per ml

11.3 units per ml

121 units per ml

410 1 30.5 1 1.7 1

EXAMPLE

The microorganism NRRL B-11, 229 was grown in a laboratory-scale chemostat at a dilution rate of 0.15 h ^-1 .

Volume:

Air flow speed:

Temperature:

Stirrer speed:

Substrate: Yield:

350 ml

0.5 l _s / min

50 ° C

500 rpm

7-8 (adjusted with dilute acetic acid)

as in Example 1 2.1 units per ml

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EXAMPLE 3

There was a batch hydrolysis with the soluble Lactase product according to Example 1 carried out using in a pH stat under nitrogen at 60 ° C and pH 6.5 of a deionized whey permeate to the 0.5 g KCl / 1 had been added as an activator. The whey permeate was deionized, i.e. both cations and anions were removed.

With an amount of enzyme of 100 units per liter, 100% conversion was achieved in 45 hours and with one Enzyme amount of 50 units per liter gives a 96.5% conversion in 71 hours.

EXAMPLE 4

Immobilization of β-galactosidase.

The fermentation broth with cells containing β-galactosidase, which had been obtained according to Example 1, was brought to a pH of 7.5 and the cells were centrifuged off from the fermentation broth. The concentrate was believed to contain approximately 10% solids and approximately 50 % intact cells.

The sediment was then passed through a Manton-Gaulin homogenizer Type SP 15 M - 8 TA pumped with a single-stage homogenization valve arrangement, whereby the pressure drop Was 300 to 350 kg / cm.

50% glutaraldehyde solution was added to 1 liter of homogenized concentrate at pH 7.5 and 25 ^° C. up to a concentration of 0.5% glutaraldehyde in the solution.

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After a reaction time of 30 minutes, the gel was mechanically broken up and diluted with 1 volume of water. There were 100 ml. 20% flocculant (Superfloc C 521) added, a clear phase was formed in the solution. The mixture was filtered and the filter cake granulated and on the Air dried.

Yield: 15%

Specific activity: 100 units per g

EXAMPLE 5

16 g of acid-precipitated casein and 4 g of egg albumin were added to 20 ml of the concentrate according to Example 1. That The mixture was stirred until a homogeneous paste was formed. 50% glutaraldehyde solution was used up to one concentration of 1.5% glutaraldehyde added in the paste. The moist product was left to stand for 30 minutes Complete networking. The product was granulated and air dried.

Yield: 10 %

Specific activity: 25 units per g

EXAMPLE 6

Continuous hydrolysis of lactose with the help of immobilized β-galactosidase.

In a laboratory-scale column with a fixed bed, the hydrolysis of lactose syrup was carried out using corresponding Example 4 produced immobilized ß-galactosidase carried out. The experimental conditions were as follows adhered to.

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30QO598

5.5% by weight lactose, 60 ^° C., pH 6.7

50 mg NaN ₃ and 150 mg chloramphenicol per liter of lactose syrup

Column size: 0 2.5 cm; Height 40 cm

immobilized β-galactosidase: 15 g

Set of test results

Time
(d) Flow rate
speed
(ml / h) Contact time
(min) Conversion to
Monosaccharides
00- 0 300 10 98.5 1 300 10 94.3 2 300 1O 89.2 3 120 26th 98.8 6th 120 26th 93.0 7th 75 42 98.3 8th 75 42 92.1

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EXAMPLE 7

The microorganism DSM 1687 was placed on a Petri dish inoculated, in which TY agar (R.E. Goodman and D.M. Pederson, Canadian Journal of Microbiology, Vol. 22, P. 817 (1976J) was included. The Petri dish was incubated at 50 ° C. for 2 days. The bacteria from the petri dish were then used to make a crude extract in the following manner: One volume that of the surface of the substrate lifted bacteria was suspended in 1.5 volumes of 0.1 M phosphate buffer at pH 6.5 and 4 ° C and the bacteria are destroyed by ultrasound treatment (frequency 20 KHz, 4x1 minute). The suspension with the broken Bacteria were centrifuged for 20 min at 4 ° C. at 16,000 g in an MSE 21 centrifuge. The centrifugate (supernatant liquid) represents the raw extract.

By tandem cross immunoelectrophoresis as described above is indicated, it was found that the ß-galactosidase formed by DSM 1687 is identical to the ß-galactosidase which has been formed by NFlRL B-11, 229. For this, see Referring to Figure 2, where the left circle corresponds to NRRL B-11, 229 and the right to DSM 1687.

Similar experiments were carried out with DSM 1688 and DSM 1689. Tandem cross immunoelectrophoresis performed as above showed that the β-galactosidases from DSM 1688 and DSM 1689 were immunologically identical to the β-galactosidase from NRRL B-11, 229.

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Claims (1)

Claims 1. Microbiological enzyme product containing a ß-galactosidase which is related to the enzyme chemistry and immunological properties is equivalent or identical to the ß-galactosidase, which is formed in Cultivation of the microorganism NRRL B-11, 229. 2. The microbiological enzyme product of claim 1 comprising a microbiological enzyme that occurs as a component of a microorganism cell product, the Microorganism belongs to the taxonomy group represented by the typical strain NRRL B-11,229 and its members are essentially the taxonomy properties own this tribe. J. Method for producing a microbiological ß-galactosidase product according to claim 1, characterized in that a microorganism the taxonomy group represented by the strain NRRL B-11, 229 in a culture medium with assimilable Nitrogen, carbon and oxygen sources grows and then wins the cells and the intracellular β-galactosidase product isolated. 030031/063 2 / 2 4. Microbiological ß-galactosidase product according to claim 1, characterized in that it is immobilized. 5. Use of the ß-galactosidase according to claim 1 to 4 for the cleavage of lactose in liquids, preferably in whey, at a pH between approximately 5.5 and 8.5 and a temperature between 55 and 75 C. 6. Use of the ß-galactosidase according to claim 4, for the continuous cleavage of the lactose according to claim 5, in that the lactose-containing liquid is introduced into a column which is filled with the immobilized ß-galactosidase product, at a rate that leads to a lactose concentration in the exiting liquid that is less than 10 % preferably less than 1 % of the lactose concentration in the entering liquid. Ö30031 / 0632