DK149335B - α-acetolactate decarboxylase and a process for its preparation - Google Patents

Description

in 149335

This invention relates to a novel α-acetolactate decarboxylase and to a process for its preparation.

In the fermentation of alcoholic products, e.g. beer or wine, small amounts of diacetyl are often formed.

The formation of diacetyl is highly disadvantageous because its strong and unpleasant odor, and in the case of beer even has small amounts of diacetyl of approx. 0.10 - 0.15 mg / liter adversely affects the beer's aroma and taste. Diacetyl in beer is caused partly by an infection with a Pediococeus strain that directly produces diacetyl (E. Geiger, Diacetyl in Bier, Brauwelt 46, 1680 - 1692, 1980), and partly by the fact that beer yeast from pyruvate forms α -acetolactate which, in a non-enzymatic but temperature dependent reaction, is converted to diacetyl. During the maturation of beer, diacetyl is converted to 15 acetoin by reductases in the yeast cells. In terms of taste and aroma, acetoin is acceptable in beer at much higher concentrations than diacetyl.

Another possibility of reducing the amount of diacetyl in beer is to directly convert α-acetolactate to acetoin by acetolactate decarboxylase, cf. Danish submission no. 145.502. The acetolactate decarboxylase can be added during the main fermentation of the beer or during the ripening process.

The enzyme, which is an intracellular enzyme, is preferably recovered by the prior art from the microorganism Klebsiella pneumonia (June E., J. Biol. Chem. 195, 715- 726, 1952). However, since Klebsiella pneumonia is a pathogenic microorganism, it is not suitable for industrial use.

In addition, the acetolactate decarboxylase produced by this microorganism has poor stability under the conditions of its intended use, which in the case of beer is a pH of approx. 4.3 and a temperature of approx. 10 ° C.

The object of the present invention is to provide a novel α-acetolactate decarboxylase enzyme which has a better stability under the above conditions and which can be further recovered from non-pathogenic microorganisms in improved yield.

2 149335

The invention is based on the surprising realization that a novel α-acetolactate decarboxylase with such properties is produced in high yields of microorganisms belonging to the species Bacillus brevis. It is suggested in 5 Danish Patent Specification No. 145,502 that α-acetolactate cleavage enzymes can be obtained from sources other than Klebsiella pneumonia. However, this is not documented and it is not apparent from the disclosure that an α-acetolactate decarboxylase having the beneficial properties of the enzyme of the present invention can be prepared in high yields from Bacillus brevis.

In its first aspect, the present invention provides a novel, stable α-acetolactate decarboxylase which is characterized in that it has a pH optimum in the range of 5 to 7 and a temperature optimum in the range of 35 ° to 55 ° C and is identical to the α-acetolactate decarboxylase formed by growing Bacillus brevis ATCC 11031 in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts.

The alpha-acetolactate decarboxylase may be in solid or liquid form and usually has in solid form an activity in the range of 0.1 to 10 NE (as defined below) per mg protein.

According to a further aspect, the present invention provides a process for the preparation of this α-acetolactate decarboxylase, characterized in that it comprises culturing an α-acetolactate decarboxylase-producing Bacillus brevis strain in a suitable nutrient medium containing carbon. and nitrogen sources and inorganic salts upon which the α-acetolactate decarboxylase is recovered from the cells in the culture fluid.

According to a preferred embodiment of the method of the present invention, the Bacillus brevis strain is ATCC 11031 or a mutant or variant thereof having substantially the same properties as this one. The preferred microorganism capable of producing the α-acetolactate decarboxylase of the invention, 149335, was selected based on its ability to convert α-acetolactate to acetoin, which is then detected.

More than 300 Bacillus strains have been tested according to the following procedure: 5 The strains were grown in shake flasks at 30 ° C and 37 ° C for 24 hours on the following BCM substrate: K2hpo4 1 g

NaH 2 PO 4, 2H 2 O 1 g (NH 4) 2 SO 4 2.5 g NaCl 0.25 g

MgSO4.7H2 O 0.2 g

FeCl3.6H2 O 0.04 g

MnSO 4, H 2 O 0.25 mg

Glucose 10 g 15 Yeast extract 3 g

Water up to 1000 g

After culture, the cells were harvested by centrifugation, washed in 0.03 M phosphate / citrate buffer (pH 6.0) and resuspended in the same buffer until OD (450) 40. Samples of 2 20 ml were then subjected to ultrasound or 1 mg of lysozyme per ml at 37 ° C for 30 min. and then centrifuged.

The alpha-acetolactate decarboxylase activity of the supernatants was determined according to the procedure described below.

The screening procedure revealed that only a few of the approx. 300 microorganisms investigated produce α-acetolacetate decarboxylase in measurable amounts. These microorganisms are listed in the following Table I together with the enzyme activity determined according to the above procedure.

4 149335

Table I

Result of the screening procedure

Strain Strain Activity Protein (NOVO Collection (NE) per mg / ml 5 no) mg protein B. brevis A 303 0.13 3 "B. coagulans A 345 0.007 3 B. pumilus A 185 0.008 2 10 B. pumilus' A 328 0.011 3 B. pumilus A 329 0.004 2 B. subtilis A 518 0.005 3 B. subtilis C 601 0.06 1 B. licheniformx A 446 0.11 3 B. licheniformis C 600 0.19 2 B licheniformis A 88 0.15 2 20 B. licheniformis A 105 0.13 2 B. licheniformis A 106 0.15 2 B. Ixcheniformis A 200 0.15 2 B. licheniformis A 203 0.25 2 B. licheniformis A 244 0.11 2 30 B. licheniformis A 248 0.14 2 B. licheniformis A 1240 0.13 2

Strains C 600 and C 601 were deposited 35 by the applicants at the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland on May 27, 1983 and were given reference numbers NCIB 11868 and NCIB 11869.

The remaining strains were obtained from different cultural collections, as shown in the following list: 5 149335 NOVO no.

A 303 ATCC 11031 A 345 NRS 83 A 185 ATCC 71 5 A 328 ATCC 4520 (B ^ Mesentericus was flavus) A 329 ATCC 7065 A 518 IAM 1109 (B ^ natto) A 446 NRRL B-3751 (Determined by applicants to be and B ^ licheniformis) 10 A 88 ATCC 12759 A 105 ATCC 12713 = NRRL B-1001 A 106 ATCC 11946 A 200 NCIB 6816 A 203 NCIB 8537 15 A 244 NCTC 2120 A 248 NCTC 8721 A 1240 ATCC 27326

According to the prior art (June E., ibid.), Cell-free extracts of Bacillus subtilis are capable of decarboxylating α-acetolactate to form acetoin. However, the prior art does not disclose the use of such extracts. The inventors have examined 69 different Bacillus subtilis strains. Of these, only 5 α-aceto-lactate decarboxylase showed activity, and these 5 strains were also 25 very poor enzyme producers. Bacillus coagulans and Bacillus pumilus strains also proved to be too poor enzyme producers for industrial use.

Of the Bacillus licheniformis strains studied, 13 did not produce α-acetolactate decarboxylase in measurable 30 amounts, 21 strains produced the enzyme in very low amounts, and 11 strains produced the enzyme in high amounts.

Upon tandem cross-immunoelectrophoresis, it was found that all of the Bacillus licheniformis strains mentioned produce an α-acetolactate decarboxylase enzyme which is immunologically identical to the enzyme produced by Bacillus licheniformis strain C 600.

The Bacillus brevis strains were grown on three different media in shake flasks. Only one strain, A 303 (ATCC 11031), exhibited α-acetolactate decarboxylase activity on all 40 three media. The remaining 11 strains did not exhibit α-acetolactate decarboxylase activity in measurable amounts on any of the three media used, the α-acetolactate decarboxylase enzyme produced by Bacillus brevis A 303 was found to be m.h. immunochemical properties, not being identical to the enzyme produced by Bacillus licheniformis strain C 5 600. However, the fact that the α-acetolactate decarboxylase enzymes from the various Bacillus licheniformis strains exhibit immunochemical identity gives reason to believe that others α-acetolactate decarboxylase-producing Bacillus brevis strains than those studied will produce essentially the same enzyme as Bacillus brevis A 303. For "immunochemical identity", reference is made to NH. Axelsen et al., "A Manual of Quantitative Immunoelectrophoresis" (Oslo 1973) Chapter 10.

The Bacillus brevis strain is distinguished in relation to Bacillus licheniformis in that the α-acetolactate decarboxylase is formed from Bacillus brevis without undesirable bi-activities, especially ferrulinic acid decarboxylase activity produced by all the Bacillus licheniformis strains examined. Ferrulin acid decarboxylase decarboxylates ferrulinic acid, found in 20 fermented beers, to the very bad-tasting compound 4- vinylguajacol. Ferrulinic acid decarboxylase must therefore be removed from the culture medium of Bacillus licheniformis during the work-up procedure.

The Bacillus brevis strain also stands out in relation to Bacillus licheniformis because Bacillus brevis produces an enzyme that converts the normal precursors of 2,3-pentanedione in beer into 3-hydroxy-2-pentanone, thereby reducing the amount of poor-tasting pentanedions to an acceptable low level. Bacillus licheniformis strains 30 do not produce such an enzyme.

Finally, the Bacillus brevis strain provides better overall yields in the purification procedure due to less autolytic activity compared to Bacillus licheniformis breastfeeding.

35 A NOVO unit (NE) is defined as the amount of enzyme that produces one pmol of acetoin per liter. per minute at 20 ° C and pH 6.0 (Buffer O, 05 M MES (2 (N-morpholino) -ethanesulfonic acid), 0.05 mM MgCl 2 and 0.86 M NaCl) by incubating an enzyme-containing assay with an α-acetolactate substrate in the following assay:

substrate

The alpha-acetolactate substrate was prepared immediately before use by hydrolysis of o-acetoxy-α-methyl-acetoacetic acid ethyl ester. 30 μΐ of the diester (0.165 m mol), 240 μΐ of water and 330 μΐ of 1 M NaOH were mixed and stored on ice for 15 min. Then 5.4 ml of water was added and the resulting hydrolyzate was used for the sample.

Assay

Buffer and test sample with an enzyme activity of approx. 0.006 - 0.3 NE was mixed and tempered at 20 ° C for a few minutes (volume 10.0 ml). At t = 0, 400 μΐ of hydrolyzate was added and samples of 1 ml were taken at 15 t = 3, 6, 9, 20 and 50 minutes. The enzyme reaction was stopped by placing the samples on ice and adding 200 μΐ 1M NaOH.

The amount of acetoin formed is measured according to W.W.

Westerfeld (A Colorimetric Determination of Blood Acetoin, 20 J. Biol. Chem. 161, 495-502, 1945) by adding 0.2 ml of 0.5% creatine and 0.2 ml of 5% α-naphthol in 2.5 N NaOH (freshly prepared) to 1.2 ml of the above samples. After 60 minutes reaction time, E (524) was measured. As a blank, buffer was used instead of cell extract. A standard curve is plotted with 0, 0.5, 2 and 4 μg / ml acetoin.

A Bacillus strain capable of producing the α-acetolactate decarboxylase enzyme of the present invention is usually propagated in a suitable solid nutritional medium at about 100%. 30 ° C before growing under aerobic conditions in a suitable culture medium. The culture medium contains assimilable carbon sources (e.g. glucose) and a basal salt mixture comprising e.g. ammonium sulphate as a source of nitrogen. The cultivation is carried out at approx. 30 ° C and at pH approx.

7, held approximately constant by means of automatic aids. Aeration and agitation are adjusted to obtain a positive oxygen pressure. After culture, cells 8 are harvested by centrifugation and washing in a suitable buffer. The cells are lysed by ultrasound treatment, treatment with a French press, lysozyme treatment, Manton-Gaulin treatment or a combination thereof.

After centrifugation, a crude extract is obtained which can be subjected to a further purification step as described below.

The alpha-acetolactate decarboxylase enzyme of the present invention can be purified from the crude extract by a combination of one or more of the following steps: a) ammonium sulfate precipitation b) polyethylene glycol precipitation c) DE 52 anion exchange chromatography and dialysis, d) acid precipitation and dialysis, e) hydroxy and dialysis f) lyophilization.

g) phenyl-sepharose chromatography h) chromatofocusing The pH and temperature dependence of activity 20 of the Bacillus brevis α-acetolactate decarboxylase were determined on extracts of Bacillus brevis strain A 303 at various pH values and temperatures by the method of determining α-acetolactate decarboxylase activity. described above, using the following buffer system: 0.005 M MES (2 (N-morpholino) ethanesulfonic acid 0.5 mM MgCl 2 0.85 M NaCl pH 6.0) In the drawing, Figure 1 graphically shows the relative activity depicted at 30 ° C. versus pH, and Figure 2 graphically shows the relative activity at pH 6 plotted against the temperature.

The pH optimum of the Bacillus brevis enzyme was found to be in the range of 5 to 7, and the temperature optimum was found to be in the range of 35 to 55 ° C. 1

The stability of a crude extract of the Bacillus brevis enzyme was investigated under the conditions of use, i.e.

9 149335 at 10 ° C and in fermented but not ripened beer. The stability is shown in the following table.

Table II

Stability test 5 Strain Half-life in days A 303 11

Since the Klebsiella pneumonia enzyme has a half-life of only 2-3 hours, the enzyme of the present invention exhibits a significantly improved stability under the conditions of use for beer brewing relative to the known enzyme.

Furthermore, the stability of the Bacillus brevis A 303 enzyme is shown in the following Tables III and IV:

Table III

Residual activity (NE / ml) after incubation at different temperatures __

Incubation Residual activity (NE / ml) time (hours) Temperature

20 _ 30 ° C 40 ° C 50 ° C

0.05 M MES, pH 6.4 0 5.3 + 10 “3M Mg ++ 1 5.2 4.8 4.1 2 4.8 5.3 3.4 _4_4.7 4.0 0.2 25 0 5.1 0.05 M acetate, 4.9 4.9 3.6 3.6 pH 4.8 + 10 "3M Mg ++ 2 5.5 3.7 3.9 4 5.8 3.3 3.7 ellο 149335

Table IV

Residual activity in NE / ml after incubation at 50 ° C with -H- varying Mg concentrations_

Incubation Residual Activity (NOW / ml) 5 Time (Hours) None 10 µM 10 µM 10 Add-Mg ++ Mg ++ EDTA __of Mg + * ___ 0 5.7 6.2 5.8 5.8 10 0.05 M MES pH 6.5 1 4.8 5.2 5.0 1.4 2 4.2 4.6 3.3 0.3 _4_0 ^ 5_0.4 0.2 0 0 6.0 6.2 6.5 5.8 0.05 M acetate, pH 4.5 1 5.3 5.6 5.8 0.1-15.2 5.2 5.0 5.3 0 4 4.9 5.1 5.8 0

It is evident from the above that the Bacillus brevis enzyme retains from ca. 60 - 90% of its activity after 2 hours, the enzyme being more stable at a pH of 4.5. In addition, Mg ++ ions have a stabilizing effect on the activity while the enzyme is inactivated by EDTA.

pi determination pi of the Bacillus brevis A 303 enzyme was found to be 7.6 and 7.0 determined by the chromatofocusing technique 25 described by PHARMACIA (PHARMACIA TECHNICAL BULLETIN:

TM TM

CHROMATOFOCUSING with POLYBUFFER and PBE; PHARMACIA FINE CHEMICALS) .. The activity is eluted as two peaks, indicating that the? -Acetolactate decarboxylase activity may consist of two proteins with different pI.

30 Km determination

Km was determined for A 303 on α-acetolactate (DL) in unripened beer at 10 ° C to be 3.8 mM.

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Immunological properties

A purified fraction of the α-acetolactate decarboxylase from strain C 600 (Bacillus licheniformis) was used to immunize rabbits according to the procedure described by Harboe and Ingild in: N.H. Axelsen: Handbook of Immunoprecipitation-in-Gel Techniques, Blackwell Scientific Publications. London 1983.

The antibody produced in this way. against the Bacillus licheniformis enzyme was used to perform cross immunoelectrophoresis and tandem cross immunoelectrophoresis as described by A.O. Grubb and J. Krøll, in the above publication.

Since the antigen is inhomogeneous, the antibody formed is polyspecific and produces up to 15 bands at 15 crossed immunoelectrophoresis, one of which is the α-acetolacetate decarboxylase precipitation band. To identify this band, a coating technique based on enzyme activity was used: after the immunoelectrophoresis, the plate was not fixed and stained as usual, but incubated for 20 5 minutes in 30 ml of the following mixture in the lid of a petri dish of 14 cm diameter : 150 μΐ ethyl 2-acetoxy-2-methyl-acetoacetate, 12 00 μΐ and 1650 μΐ IN NaOH were mixed and incubated at room temperature for 15 minutes. Then, water was made up to 30 ml.

After incubation, the mixture was allowed to drip off the plate, which was twisted in a Vita-Wrap plastic film and in aluminum foil and incubated for 30 minutes at room temperature.

The plate was then placed in the lid of a petri dish 30 14 cm in diameter and covered with the following mixture: 30 ml of 2% LSA agarose (H 2 O) 3 ml of 1% creatine (H 2 O) 6 ml of 5% naphthen in 2, 5 N NaOH (mixed at 55 ° C) The covered plate was incubated at room temperature for approx. 1 hour. The band of α-acetolactate decarboxylase precipitates showed a red color and could be identified.

12 149335

As mentioned above, tandem cross-linked immunoelectrophoresis of the enzyme from all of the mentioned Bacillus licheniform strains against the C 600 α-acetolactate decarboxylase antibody showed that the Bacillus licheniform enzymes are immunochemically identical, while the -acetolactate decarboxylase antibody, showing that the Bacillus licheniformis and Bacillus brevis strains produce different types of the α-acetolactate decarboxylase enzyme.

The invention will be explained in more detail by means of the following examples.

Example 1

Strain A 303 (Bacillus brevis, ATCC 11031) was propagated on a TY medium (20 g trypticase, 5 g yeast extract, 7 mg FeCl 3, 6h 2 O, 1 mg MnCl 2, 4 H 2 O, 15 mg MgSO 4, 7 H 2 O and distilled water up to 1000 ml) in shake flasks at 30 ° C until the cells were in the middle logarithmic phase. 600 ml of this culture was transferred to a Biotec fermentation container containing 8 liters of the following medium: 20 (NH 4) 2 SO 4 2.5 g / liter k2hpo4 1

NaH 2 PO 4,2H 2 O 1

NaCl 0.25 -

0.125 - MgSO4.7H2 O 0.125 -

Spormetalopl. * 0.7 ml / liter Yeast extract 6 g / liter

Pluronic 0.13 ml / liter

Dextrose 31.25 g / liter 30 * Trace metal solution: H3B03 500 mg / 100 ml

CuSO 4, 5H 2 O 63 mg / 100 ml

At 100 mg / 100 ml

FeCl 3,6H 2 O 333 mg / 100 ml 35 MnSO 4, H 2 O 448 mg / 100 ml

NaMoO4.2H2O 200 mg / 100 ml

712 mg / 100 ml 13 149335

During cultivation, the pH was maintained at 7.0 by the addition of NaOH, the temperature was 35 ° C and stirring and aeration were adjusted to obtain a positive oxygen pressure.

At the optimum time, the cells were harvested by centrifugation.

The process of fermentation is shown in the following table V.

Table V

Activity measured during fermentation

Fermentation time in hours_NE / ml culture_OD45Q

10 1 0.005 2.4 2 0.01 4.1 3 0.02 7.5 4 0.18 15.3 5 0.47 17.5 15 6 0.41 22.2 7 0.17 24.0 8 0.13 24.0

Example 2

Strain A 303 (Bacillus brevis) was propagated and cultured as described in Example 1. The cells were harvested at OD 45 0 = 8 °

The cells were suspended in 20 mM K-phosphate buffer, pH 6.8 and passed through a Manton-Gaulin-type homogenizer at a pressure of approx. 400 bar. A fractionated precipitation of the activity with ammonium sulfate was carried out as described in Example 1.

The activity-containing precipitate was dissolved in the above K-phosphate buffer, dialyzed against the same buffer, and finally concentrated in an Amicon pressure dialyzing cell 30 to a protein concentration of ca. 10 mg / ml.

150 ml of this extract was the starting material for the following purification procedure, as shown in Table IX.

150 ml of DE 52 pre-circulated ion exchanger equilibrated in 20 mM K-phosphate buffer, pH 6.8 was added to the extract.

The slurry was filtered on a Buchner funnel.

The activity was found in the filtrate dialyzed against 25 mM histidine HCl buffer, pH 6.2.

14 149335

To this extract was added 40 ml of PBE94 (Pharmacia chromatofocusing agent) equilibrated in the above histidine buffer. The slurry was filtered on a Buchner funnel.

The activity was found in the filtrate, which was dialyzed against 20 mM K-phosphate, pH 6.8, 20% saturated with ammonium sulphate. A column was packed with 200 ml of phenylsepharose equilibrated in this buffer. The activity of the dialyzed filtrate was charged to the column and eluted with a 10 gradient in 20 mM K-phosphate, pH 6.8 ranging from 20% saturation to 0% saturation with ammonium sulfate and from 0% ethylene glycol to 50% ethylene glycol.

The peak of activity was found, pooled, concentrated and dialyzed against 20 mM K-phosphate, pH 6.8, and stored in the frozen state.

Table VI

149335 15

Fraction Volume Protein Activity NE per Purification Yield ml mg NE per ml. NE mg of percent (to total total factor of 5)

Extract 150 1890 2.4 365 0.19 1 100

Filtrate after 140 510 1.9 262 0.51 2.7 72 DE52 treatment

Filtrate after 135 263 2.02 273 1.04 5.4 75 PBE94 treatment 15 Pool after phenylse 165 25 0.95 157 6.3 32 43 Pharose treatment

Pool after 20 concentrations 34 4.8 4.7 158 33 171 43 ring and dialysis