DK146325B - BETA-GALACTOSIDASE, METHOD OF PREPARING IT AND USING THEREOF - Google Patents

Description

148325

Whey solids contain, as a main component, the disaccharide lactose, as well as smaller amounts of proteins, vitamins and salts.

5 Attempts have been made to convert whey into more product by hydrolysis of the lactose to glucose and galactose by microbial pathway-produced beta-galactosidases, but no satisfactory commercial process has so far been developed. One of the major 10 reasons why the enzymatic conversion of lactose to glucose and galactose has not led to any commercially satisfactory process is the fact that no beta-galactosidase with satisfactory properties is known. If the conversion of lactose is to be carried out satisfactorily, it is necessary that the beta-galactosidase must simultaneously exhibit both of the following two characteristics: 1) the beta-galactosidase should be stable at a temperature sufficiently high to prevent microbial contamination, and 2) the inhibition of beta-galactosidase by 20 lactose or by the products resulting from the lactose hydrolysis, i.e., galactose and glucose, should be minimal to cause the hydrolysis to terminate without using unreasonably high amounts of the enzyme.

It has been disclosed that acidic beta-galactosidases can be prepared by fungi, e.g. of the Aspergillus genus, especially Aspergillus oryzae and Aspergillus niger, citing U.S. Patent No. 3,629,073 and Journal of Food Science, Vol. 37 (1972), pages 619 - 623. These enzymes exhibit reasonably good thermostability. , but they are strongly inhibited by the hydrolysis products of lactose. On the other hand, beta-galactosidases from certain bacteria, such as Lactobacilli and Enterobacteria, are only slightly inhibited by the hydrolysis products of lactose, but their thermostability is very poor; This has been known for many years. Beta-galactosidases in thermophilic Bacilli, cf. RE

Goodman and D.M. Pederson, as in the Canadian Journal of Microbiology, Vol. 22 (1976), pages 817-825, discloses a beta 2 146326 galactosidase derived from B. stearothermophilus with relatively good thermostability, however strongly inhibited by the lactose hydrolysis products. In English patent no.

1,493,452 discloses a beta-galactosidase derived from B. coagulans and this enzyme has properties similar to the enzyme derived from B. stearothermophilus.

It is an object of the invention to provide a beta-galactosidase which simultaneously has good thermostability and low inhibition of the products resulting from the lactose hydrolysis and the lactose itself.

The beta-galactosidase is peculiar to that of the characterizing part of claim 1. Unsurprisingly, the new beta-galactosidase simultaneously exhibits good thermal stability and low inhibition of the products resulting from the lactose hydrolysis and the lactose itself.

The properties of the Bacillus NRRL B-20 11, 229 microorganism are as follows. Morphology: Spells 2-5μχ0.7-1μ.

Spores 1.6-1.8 µ x 0.8 - 1 µ appear on Schaeffer's agar, located subterminally to terminal. Slightly swollen sporangium that only prolongs the vegetative cell.

Physiology: Positive for catalase; no anaerobic growth; acid, but no gas, is formed on the basis of glucose, xylose, trehalose, lactose, arabinose or mannitol. Growth between 40 and 70 ° C and pH 6.0 and 8.5. No growth in 3% NaCl, but growth in 1% NaCl. The egg yolk and casein protein substrates as substrates were negative and the starch degradation was weak.

30 NO and reduced to NC ^. No dihydroxyacetone is formed from glycerol. The microorganism appears to be related to B. stearothermophilus.

A preferred embodiment of the enzyme of the invention is characterized by the method of claim 2.

The immunological properties of the enzyme are determined as described below. Soluble purified enzyme (the preparation of which will be described later in this specification) was used to prepare antiserum in rabbits in known manner.

5

The two-dimensional immunoelectrophoretic assay was performed in the form of duplicate experiments as follows.

10 First dimension: 10 µΐ of the sample containing 100 µg of purified enzyme / 1 is applied to a 1.5 mm agarose gel at 20 ° C and electrophoresis for 90 minutes with a gradient in the electric field of 7 volts / cm and in a buffer system consisting of 11 g of barbituric acid, 65 g of Na-barital, 15 28 g of glycine, 23 g of TRIS (TRIS is an abbreviation for tris- (hydroxy-methyl) -aminomethane) and water of 25 liters. Second dimension, perpendicular to first dimension: 1.0 ml of antiserum prepared as described above is dissolved in 10.8 ml of agarose gel and the agarose gel strip resulting from the first dimension electrophoresis is combined with the agarose gel with antiserum. Immune electrophoresis in the second dimension is carried out for 16 hours with a gradient in the electric field of 1 Volt / cm. The gel from one of the two experiments is pressed, washed and dried, and proteins are developed with 25 Coomasie brilliant blue, and the gel from the second experiment is directly reacted with 0.1% ONPG (o-nitro-phenylgalactoside) in 1/15 molar KNaHPC> 4 (pH 6.5). If lactase is present, ONPG will be cleaved to galactose and o-nitro-phenol, which exhibit a strong yellow color. A yellow color of o-nitro-phenol 30 became visible on the gel after 30 minutes. In this system, the lactase moved 51 mm in the direction of the anode, and an arc formed from the sediment formed by lactase and antiserum in the two-dimensional experiment.

After settling, the lactase was still active, which indicated that it could react with ONPG. Referring to FIG. First

FIG. 1 a shows the appearance of the gel in the first. Protein-induced experiments. It is seen that there are at least three different proteins, corresponding to arcs 1, 2 and 3. Figs. Figure 1 b shows the appearance of the gel in the second experiment in which a reaction was performed with ONPG. The shaded area of FIG. 1b was colored deep yellow, showing that the precipitate which lies within the arc 2 of FIG. 1a is similar to beta-galactosidase, whereas the precipitate of FIG. 1a, located within arcs 1 and 3, corresponds to proteins that are not beta-galactosidase.

10

Using tandem cross-linked immunoelectrophoresis (e.g., according to NH Axelsen et al., A Manual of Quantitative Immunoelectrophoresis, 1977), it is possible to determine whether the beta-galactosidase of the invention is identical to or different from a sample of any unknown beta-galactosidase. Similar quantitative tests can be performed by other immunological methods, e.g. immunodiffusion or direct immunoprecipitation followed by gel chromatography with sodium dodecyl sulfate.

20

Among the enzyme chemical properties, the thermal stability and inhibition of the products resulting from lactose hydrolysis are the most important.

The activity of the beta-galactosidase is determined as follows. A crude cell lysate is prepared by treating for 15 minutes at 37 ° C with lysozyme at a concentration of 2 mg / ml in milk buffer (pH 6.5, salt composition as in milk) followed by two cycles of freezing and thawing.

The lysate containing 2-10 lactase activity units (which will be defined later) per liter is incubated for one hour in milk buffer with 2% lactose, then quenched by boiling, settling in ice water and centrifuging. The supernatants are assayed for glucose using the glucose oxidase peroxidase method (Tech. Bull. No. 510, Sigma

Chemical Co.). 1 lactase activity unit is defined as the amount of enzyme which, under standard conditions including pH 6.5 and 60 ° C, releases 1 μπιοί glucose per minute.

B 146325 5

The thermal stability is measured as follows. The activity is determined as indicated above, except that different incubation temperatures are used. Glucose assay samples are taken every 30 minutes for 2 hours.

5

The inhibition derived from galactose is measured as follows. In the standard assay for galactose inhibition, the incubation mixture contains 2.0% lactose and 2.0% galactose; otherwise, the conditions as stated above are in connection with determining the activity. However, other combinations of glucose and galactose, e.g. 2% lactose and 10% galactose, corresponding to 90% hydrolysis of 5 times concentrated milk.

The pH-activity curve is another enzyme chemical property that is important because the pH optimum of the beta-galactosidase should not be too far from the pH of the whey to be treated, otherwise an unreasonable 20 large amount of beta-galactosidase. The pH optimum of the beta-galacto sidase of the invention is about 6.5 - 7.0 at 60 ° C when the activity measurements are performed according to the above method for determining beta-galactosidase activity. Likewise, the activity of the beta-galactosidase is above 25% of the maximum activity in the pH range 6.1 - 8.0.

The molecular weight of the beta-galactosidase is determined as follows. First, the enzyme is highly purified by gel electrophoresis on polyacrylamide. Then, the molecular weight of the beta-galactosidase is determined by gel electrophoresis, thereby causing the beta-galactosidase to run parallel to other proteins whose molecular weight is known, namely phosphorylase A of molecular weight 94,000 and subunits of E. coli beta-galactosidase (inactive). molecular weight 35 116,000. The molecular weight of the beta-galactosidase of the invention, which is still active, was approx. 116,000 in this system.

Surprisingly, there was a beta-galactosidase which simultaneously exhibited a high thermal stability corresponding to a half-life of over 500 minutes at 65 ° C and a low galactose inhibition of ca. 10% according to the standard provision stated above. With 10% galactose in the incubation mixture, the galactose inhibition is below 50%. Km was also very low (Si 5 mM) and no lactose inhibition was observed even at very high lactose concentrations.

The process of the invention for the preparation of the microbial beta-galactosidase product is characterized by the method of claim 4.

Enzyme formation is induced by lactose and galactose 15 and follows growth. However, the strain can be made constitutive using conventional mutation technique. The organism does not exhibit any growth requirements for particular C or N sources, but growth and enzyme formation are enhanced if the minimal substrate is enriched by organic N sources such as peptone 20 or tryptone, yeast extract or maize cast water. The organism is also very versatile in terms of utilization of carbon source, utilizing e.g. glucose, galactose, lactose, fructose, glycerol, glutaminates, succinates, lactates and acetates. Growth on mono- and disaccharides results in very strong acid production, which interrupts growth if the pH drops below approx. 6.0.

The beta-galactosidase is produced intracellularly, while being readily released from the cells using conventional methods such as e.g. by treatment with toluene, butanol or lysozyme, ultrasonic treatment or mechanical disintegration, after which the cell debris is removed and the enzyme purified using conventional techniques, e.g. salt precipitation or precipitation with organic solvents.

35

Whey, milk and other lactose-containing liquids, for the sake of brevity hereinafter referred to as whey, can be satisfactorily converted into a syrup containing 7,632,325 glucose and galactose and virtually no lactose by the beta-galactosidase of the invention. This conversion can be efficiently carried out discontinuously by the soluble beta-galactosidase. However, in order to implement this conversion in an economically more attractive and industrial scale, it is highly advisable to use an immobilized beta-galactosidase product.

Immobilization methods for enzymes are e.g. be written in the publications listed below:

Patent Immobilization of cells, isolated 15 including enzyme, whole cells including pure enzyme 20 English patent no.

1,444,539 x x US Pat.

4,001,085 x 25_________ US Pat.

4,025,391 x US Pat.

30 4,033,822 x US Pat.

4,038,140 x 35

It also appears from the above list that immobilization of both whole cells and pure enzyme is described. The beta-galactosidase product of the invention can also be immobilized both in whole cell form and as pure enzyme. Any of the above immobilization methods can be used for the beta-galactosidase product of the invention.

5

According to one embodiment of the invention, the beta-galactosidase is thus immobilized. This immobilized beta-galactosidase product of the invention has the excellent advantage of being capable of converting a liquid with a very high lactose concentration completely into galactose and glucose in a commercially attractive manner due to the lack of substrate inhibition and the lack of inhibition from the lactose hydrolysis products.

The use of the beta-galactosidase according to the invention is carried out in the form of a method for converting a lactose-containing liquid, preferably 20 whey, into a syrup containing glucose, galactose and a small amount of lactose, which is characterized by bringing the lactose-containing liquid in connection with the beta-galactosidase of the invention.

25

This use can be made discontinuously, in which case a preferred embodiment of the process is characterized by introducing the beta-galactosidase of the invention in soluble form 30 into the lactose-containing liquid at a pH of between ca. 5.5 and 8.5 and at a temperature between 55 and 75 ° C.

The use can also be made continuously, in which case a preferred embodiment of the method is characterized in that the lactose-containing liquid is introduced into a column packed with the beta-galactosidase according to the invention in immobilized form at a pH between about . 5.5 to 8.5, at a temperature between 55 ° C and 75 ° C and at a rate which gives rise to a lactose concentration in the columnar material of less than 10%, preferably less than 1% of the lactose concentration in the material entering the column.

The initial beta-galactosidase activity in the lactose-containing liquid, expressed in lactase activity units / 1, varies considerably, mainly due to 10 variations in pH, temperature, converter instinct and initial lactose concentration.

The invention will be illustrated by the following examples.

15

Examples 1 and 2 illustrate the preparation of the beta-galactosidase, Example 3 illustrates the discontinuous use of the soluble beta-galactosidase, Examples 4 and 5 illustrate the immobilization of the 20-galactosidase and Example 6 illustrates the continuous use of the immobilized beta-galacto. -sidase.

Example 1

The enzyme was prepared by submers batch fermentation in a 400 L conventional fermentation tank with stirrer, temperature control (external water jacket) and air inlet tube, whereby the aspect ratio was 2: 1.

The fermentation was carried out under the following conditions:

Temperature: 50 ° C

Air flow rate: 120 Nl / minute

Stirring rate: 200 rpm.

minute.

146325 ίο

The substrate consisted of the following components:

Whey powder 2.0 kg

Maize casting water 8.0 - (nh4) 2so4 0.4 - 5 MgSO4 · 7Η20 0.2 - k2hpo4 0.1 -

Pluronic® 0.2 - ®

Pluronic is a liquid, non-ionic surfactant.

The pH was adjusted to 7.2 before sterilization.

15 Waterworks water was added to a total volume of 400 l.

The fermentation liquid was seeded with a glass agar slurry, where the previous night at 50 ° 20 C there had been growth of NRRL B-11, 229, and 24 hours later the fermentation was completed and the culture was rapidly cooled to 10 ° C and the enzyme recovered. The cells were separated on a centrifuge and opened by passage through a Manton-Gaulin homogenizer of type SP 15 M 8 TA 25 with a single stage homogenization valve system, at a pressure drop of 300 - 350 kg / cm 2. The enzyme was extracted from the disrupted cells with a solution of a phosphate buffer (pH 6.5, 0.1 M) and the cell debris removed by centrifugation. The soluble enzyme fraction 30 was further concentrated by ultrafiltration (DDS membrane GR 6p, where DDS is an abbreviation of the Danish Sugar factories).

Yield: Fermentation liquid 1.0 unit / ml 410 1 35 Sludge 11/3 unit / ml 30.5 1

Concentrate 121 units / ml 1.7 1 λ1 Η632Β

Example 2

Microorganism NRRL B-11,229 was grown in a laboratory type chemostat at a dilution rate of 0.15 hours

Volume: 350 ml

Air flow rate: 0.5 Nl / min.

Temperature: 50 ° C

10 Stirring speed: 500 rpm pH: 7-8, adjusted with dilute acetic acid

Substrate: As described in Example 1

Yield: 2.1 units / ml 15

Example 3

Discontinuous hydrolysis with the soluble 20 lactase product, the preparation of which is described in Example 1, was carried out in a pH state under nitrogen at 60 ° C and pH 6.5 using as a substrate a deionized whey permeate to which was added 0.5 g of KCl / 1 as activator.

25

The whey permeate was deionized, i.e. both cations and anions were removed from the whey permeate. With an enzyme dosage of 100 units per 100% conversion was achieved over 45 hours and with an enzyme dose of 50 units per liter. a liter of 96.5% achieved over 71 hours.

Example 4 35

Immobilization of beta-galactosidase. The pH of fermentation liquid with beta-galactosidase containing cells prepared as described in Example 1 is adjusted to 7.5 and the cells are recovered from the fermentation liquid by centrifugation. An assessment shows that the concentrate contains approx. 10% dry matter and approx. 50% intact cells.

The slurry was then pumped through a Manton tank.

Goulin homogenizer of type SP 15 M - 8 TA with a single stage homogenization valve system where the pressure drop was 300 - 350 kg / cm2.

To 1 liter of the homogenized concentrate at a pH of 7.5 and 25 ° C, 50% glutaraldehyde solution was added and a concentration of 0.5% glutaraldehyde was obtained in the solution. After 30 minutes of reaction time, the gel was broken by mechanical means and diluted with 1 volume of water. 100 ml of a 20% flocculant Superfloc C 521 was added to give a clear aqueous phase in the solution. The mixture was filtered and the filter cake was granulated and air dried.

Yield: 15%

Specific activity: 100 units / g

Example 5 25

To 20 ml of the concentrate of Example 1, 16 g of acid precipitated casein and 4 g of egg albumen are added.

The mixture is stirred until a homogeneous paste is formed. 50% glutaraldehyde is added to produce a concentration of 1.5% glutaraldehyde in the paste. The moist product is left to itself for 30 minutes to complete the cross-linking process.

The product was granulated and air dried.

Yield: 10%

Specific activity: 25 units / g

Example 6 13 146325

Continuous hydrolysis of lactose by immobilized beta-galactosidase. In a fixed column laboratory column 5, hydrolysis of a lactose syrup was performed by the immobilized beta-galactosidase prepared according to Example 4.

The following test conditions were used: 10 Introduced material: 5.5% w / w lactose, 60 ° C, pH 6.7.

Preservatives: 50 mg NaN 2 and 150 mg chloramphenicol per ml. liter of lactose syrup Column dimension: Diameter 2.5 cm; height 40 cm 15 Amount of immobilized beta-galactosidase: 15 g

Time Liquid hash - Contact% Convert (days) density time call 20 (ml / hour) (minutes) monosaccharides 0 300 TO 9875 25 I 300 TO 9471 2 300 10 8172 3 120 ““ 26 9878 30 6 120 26 93.0 7 75 42 9871 35 8 75 42 9271

Claims (6)

14 146325 5 1. Beta-galactosidase, characterized in that it is identical to the beta-galactosidase formed by cultivation of the strain Bacillus NRRL B-11,229 10 under aerobic conditions and using a culture medium comprising a nitrogen source and small amounts of inorganic salts. The beta-galactosidase according to claim 1, characterized in that it is in soluble form. The beta-galactosidase according to claim 1, characterized in that it is in immobilized form. 20 Process for preparing the beta-galactosidase according to claim 1, characterized in that the strain Bacillus NRRL B-11,229 is grown under aerobic conditions on a growth medium containing 25 assimilable nitrogen, carbon and oxygen sources and small amounts of inorganic salts, after which the cells are harvested and the intracellular beta-galactosidase is isolated. A method of converting a lactose-containing liquid, preferably whey, into a syrup containing glucose, galactose and a minor amount of lactose, characterized by bringing the lactose-containing liquid into contact with the beta-galactosidase according to claim 1. 35 The method of claim 5, wherein the conversion is discontinuous, characterized in that the beta-galactosidase of claim 2 is introduced into the