DE3940431C1 - Fructose-1,6-di:phosphate aldolase used in prepn. of carbohydrate(s) - is obtd. from fermentation of Staphylococcus carnosus by fractional pptn. - Google Patents

Abstract

Fructose-1,6-diphosphate aldolase (I) obtd. from Staphylococcus carnosus has the following characteristics: (a) at 60-100 deg.C the purified form (25 U/mg) has a stability at pH 7.5 in 0.06M Tris-HcL buffer soln. in an Eppendorf vessel corresp. to a residual activity after 30 mins. between 70 and 80% (60 deg.C) and 20-30% (100 deg.C); and (b) a diactivation rate under synthesis conditions at 25 deg.C, pH 6.5 in 20 mM DHAP (dihydroxyacetone phosphate) 75mM glyoxyllic acid, enzyme activity at 2.9 U/ml to 25 U/ml, of up to 1%/d. (I) is prepd. from a ferment of Staphyloccus carnosus, by fractional pptn. with ammonium sulphate, pH fractionation and ion-exchange chromatography. USE/ADVANTAGE - (I) is used to prepare carbohydrates and their derivs. by the enzymatic reaction with DHAP (claimed).

Description

The invention relates to a fructose-1,6-diphosphate Aldolase (F-1,6-DP aldolase), and it includes a method for the production thereof and their use.

The current interest in targeted aldol reactions and at the synthesis chemically difficult to access or in non-naturally occurring monosaccharides led to that the aldolases in this regard more and more Attention was paid. For organic synthesis of these biologically important substances, which also for the medicine can be of use, the use provides of biocatalysts an effective alternative. The F-1,6-DP aldolase of the rabbit muscle is so far the only enzyme used in the synthesis (A.E. Seriani et al., Meth. Enzymol., 89 (1982) 83-92). This rabbit muscle-isolated aldolase is expensive and relatively unstable. On the other hand, already one Class 1 F-1,6-DP aldolase from Staphylococcus aureus isolated, which has similar enzymological properties which has aldolase isolated from rabbit muscle, However, it is significantly more thermostable (F. Götz et al. j. Biochem. 108 (1980) 293-301). Because of its pathogenic However, Staphylococcus aureus appears properties as enzyme producer little suitable.

The aim of the invention is therefore an aldolase with improved Accessibility compared to the previously known F  1,6-DP aldolases from rabbit muscle or staphylococcus aureus having a satisfactorily enzyme-catalyzed process Stability.

This object is achieved with the fructose-1,6-diphosphate aldolase (F-1,6-DP-aldolase) according to the invention from Staphylococcus carnosus with the following parameters:
a 60 ° C or 100 ° C stability in purified form (≈ 25 U / mg) at pH 7.5 in 0.06 M Tris-HCl buffer solution in Eppendorf tubes corresponding to a residual activity after 30 minutes between 70 and 80% or 20 and 30%, in particular of 76% and 26%, respectively, and a deactivation rate under synthesis conditions at T = 25 ° C; pH 6.5; 20 mM DHAP; 75 mM glyoxylic acid; Enzyme activity 2.9 U / ml and 25 U / mg of 1% / d, respectively.

This aldolase stands out in comparison to the "Rabbit Muscle Aldolase" significantly improved stability and it has a slightly lower thermal Stability as the aldolase from Staphylococcus aureus (The latter shows after 90 min at 100 ° C no noticeable Loss of activity), without their serious material opportunistic pathogenic origin.

The enzyme used as enzyme producer Staphylococcus carnosus is from natural sources, especially from dry ones Raw sausage readily available (see K. H. Schleifer u. a., boarding school. J. System. Bacterial. 32 (1982) 153- 156) and commercially available in the form of starter cultures. In the context of the present invention has been particular the under number DSM 20 501 at the German Collection for microorganisms in Göttingen deposited Staphylococcus carnosus strain used.

The cultivation of the trunk takes place in shake cultures in  500 ml Erlenmeyer flask with 200 ml medium at 110 rpm or in the fermenter with a working volume between 5 and 10 l. The culture medium (M 1) had the following composition:

1.0% tryptone
0.5% yeast extract
0.5% NaCl
0.1% Na₂HPO₄
1.0% glucose
pH 7.2

To correct the pH were 3M NaOH and 3M H₃PO₄ used. Unless otherwise stated, during the fermentation follows the following parameters:

Speed | 100 rpm temperature 37 ° C ventilation 5% PO₂ broth M 1 pH 7.2 Crop time 8-13 h

The cells were at the end of the logarithmic phase harvested by centrifugation at 8000 rpm for 10 minutes. The cell digestion was carried out by wet grinding with Glass beads.

The wet weight of the biomass from 9 l fermentation volume was 121 g. The following cleaning steps were between 1 and 6 ° C in 0.06 M tris HCl buffer at pH 7.5.

For purification of the enzyme was a fractionated Ammonium sulfate precipitation, pH fractionation and ion exchange chromatography performed:  

Fractionated ammonium sulphate precipitation

195 ml of cell-free crude extract were mixed with solid ammonium sulfate up to a saturation of the solution of 40%. The pH was adjusted to 7.5 with 3 M ammonia and the precipitation was carried out for two hours in an ice bath. The precipitated components were dissolved by 20 minutes Centrifuged at 20 000 rpm separated. Gradually then the (NH₄) ₂SO₄ concentration was on 60% or 80% increase in saturation, again in an ice bath precipitated and centrifuged. For the precipitation of aldolase the supernatant was brought to 100% (NH₄) ₂SO₄ saturation and the pH lowered to 5.0 with 7 M acetic acid. The precipitate was dissolved in 50 ml digestion buffer (0.06 M Tris-HCl pH 7.5 with 6 mM 2-mercaptoethanol).

pH fractionation

The pH of 50 ml of the 100% (NH₄) ₂SO₄ fraction was added dropwise with vigorous stirring 7 M acetic acid first to 4.0, then with 6 M HCl to 3.5 and finally lowered to 3.0. The formed precipitate was centrifuged off at 20,000 rpm for 20 min. the aldolase was in the supernatant, which by means of Diafiltration with an ultrafiltration membrane Amicon® YM 10 was desalted.

Ion Exchange

30 ml of the desalted enzyme solution was added to a 160 ml DEAE-Sephadex® A 25 column applied previously with 0.1 M NaCl containing 0.06 M Tris-HCl buffer of pH 7.5 had been rinsed. The enzyme was characterized by a linear Salt gradients of 0.1-0.5 M NaCl in 0.06 M Tris-HCl Buffer pH 7.5 eluted. The flow rate was 30 ml / h. The active fractions were pooled and freeze-dried.  

The attached diagram ( Figure 1) shows curves for growth and enzyme production as a function of the culture time in the fermenter (5% pO₂).

Table 1 below shows an overview of the Results of the purification.

Table 1

enzyme characterization

For the characterization of the Enyzms 40 μg / ml of the purified aldolase used for molecular weight determination 120 μg / ml were used.

Classification as class I aldolase

Due to the following characteristics, the F-1,6-DP Aldolase from S. carnosus the class I type of aldolases be assigned.

• - The activity against F-1,6-DP remains unimpaired in the presence of different concentrations EDTA.
• - S. carnosus aldolase is in the presence of DHAP and NaBH₄ completely inhibited.  
• - Mono- and divalent cations have no influence on aldolase activity.

Furthermore, the S. carnosus aldolase behaves very well specific to the natural substrate.

The molecular weight of the aldolase was determined by means of SDS PAGE determined and ~33,000 daltons were determined.

K _m values according to Michaelis-Menten

The K _m values for the substrates F-1,6-DP and F-1-P were determined via the enzyme activities at different substrate concentrations. Fig. 2 shows the Michaelis-Menten plot for the determination of the K _m value of F-1,6-DP.

This was determined to be 0.022 [mmol / min / mg], that for F-1-P was 18.8 [mmol / min / mg]. It is clear that the aldolase behaves very specific to the natural substrate. The K _m value for F-1-P is greater by a factor of 1000 than that for 1-1,6-DP.

pH optimum

To determine the pH optimum ( FIG. 3), the enzyme activity of the aldolase was measured at 37 ° C. between pH 3 and pH 10 in the following buffers:

0.06 M citrate buffer pH 3.0-6.0 0.06 M KP _i buffer pH 6.0-7.5 0.06 M Tris-HCl buffer pH 7.5-9.0 0.06 M glycine buffer 9.0-10.0

From Fig. 3, two things can be seen:

• The pH optimum of the aldolase comprises the pH range 6.5 9.0 with the peak at pH 7.5.
• - The enzyme shows different activities in Presence of the different buffers. sodium citrate Buffer shows the strongest inhibition of the enzyme.

Stability of aldolase pH stability

The pH stability of the enzyme was determined by incubation of the aldolase at room temperature (RT) between pH 1 and pH 12 for 10 minutes ( FIG. 4). It was worked only in 0.06 M Tris-HCl buffer, the respective pH was adjusted with 2 N NaOH or 2 N HCl. After the incubation period, the sample solutions were brought back to pH 7.5 and measured the enzyme activities.

As shown in Fig. 4, the S. carnosus aldolase has a very good pH stability. In a further test it was found that the aldolase activity was still 100% of the initial activity even after 72 h storage at pH 2 and 4 ° C.

Temperature stability

To study the temperature stability of aldolase the following measurements were carried out:

1) The enzyme was incubated for 5 min in 0.06 M Tris-HCl buffer of pH 7.5 at temperatures between 40 ° C and 100 ° C and thus determines the dependence of the enzyme activity on the temperature ( Fig. 5).
2) In a further series of tests, samples of the purified aldolase were incubated for different times at 60 ° C. and 100 ° C., thereby determining the dependence of the enzyme activity on the time at two different temperatures ( FIG. 6).

Both Fig. 5 and Fig. 6 show that the F-1,6-DP aldolase from S. carnosus is a relatively thermostable enzyme. Although it does not have the heat stability described for S. aureus aldolase (no loss of activity after 90 min at 100 ° C), it far exceeds the temperature stability of rabbit muscle aldolase.

Storage stability of aldolase

To be able to make statements about the storage stability, were samples of purified aldolase with different Reagents are added and incubated at RT for 5 days. -20 ° C stored. Tab 2 shows the result:

Table 2

Stability comparison with rabbit muscle aldolase

The stability of the aldolase according to the invention was with compared to that of rabbit muscle aldolase. For this purpose, both enzymes were below those in Table 3 tested conditions:

Table 3

For both experiments, an enzyme concentration of comparable starting activity is set. It was 3.4 U / ml (9 U / mg) for rabbit muscle aldolase and 2.9 U / ml (25 U / mg) for S. carnosus aldolase. Fig. 7 shows the stability of both aldolases over a period of 300 hours. To determine the deactivation rate, a first-order exponential deactivation was assumed, the deactivation rate being based on the exponential factor (1 = 100% / time unit). From Fig. 7 takes a deactivation rate of 0.77% / d for the aldolase of the invention (compared to 59.3% / d for the "rabbit muscle aldolase").

substrate spectrum

To determine the substrate spectrum were exemplary tested a number of aldehydes. The following became Approach chosen:  

20 mM DHAP 200 mM aldehyde 1-4 U / ml aldolase

After incubation of aldolase was discontinuous TLC control. In the approaches below indicated Aldehydes could no longer produce DHAP after 5-8 h be detected.

Aldolase substrates aldehyde R _{p of} the product Glyceraldehyde-3-phosphate 0.36 methylglyoxal 0.37 D (+) - glyceraldehyde 0.44 L (-) - glyceraldehyde 0.44 D, L-glyceraldehyde 0.44 glycolaldehyde 0.66 phthalaldehyde 0.69 formaldehyde 0.71 acetaldehyde 0.93 chloroacetaldehyde 1.08 propionaldehyde 1.20 3-methylmercaptopropionaldehyde 1.24 isobutyraldehyde 1.36 trimethylacetaldehyde 1.41 butyraldehyde 1.40 3-Ketobutyraldehyd 1.48 isovaleraldehyde 1.49 malondialdehyde 1.49 Pyridine (2) carbaldehyde 1.50 Pyridine (4) carbaldehyde 1.50 phenylacetaldehyde 1.53 2-methylacetaldehyde 1.54 valeraldehyde 1.60

Catalysed as an example of an F-1,5-DP aldolase Reaction will be the synthesis of 5,6-dideoxyhexulose below described:

Dihydroxyacetone phosphate (DHAP) was used in the preparative Scale with propionaldehyde in the presence of F-1,6-DP- Aldolase reacted at 25 ° C.

Approach:
50 mM (5 mmol) DHAP
500 mM (50 mmol) of propionaldehyde
40 U aldolase

The propionaldehyde was freshly distilled before the reaction and stored under argon. The course of the reaction was both polarimetric at one wavelength of 595 nm as well as the determination of the residual DHAP Salary observed. After 6.5 h, the reaction was complete, and the product was isolated as a cyclohexylammonium salt.

To elucidate the structure, 250 mg of the substance was dephosphorylated with acid phosphatase. The peracetylation of the free sugar (P _p = 1.56) was carried out with acetic anhydride in pyridine. With the help of the 1 H and 13 C NMR spectra, the isolated compound was clearly identified as the expected product.

The aldolase of the invention is for the synthesis of Carbohydrates or carbohydrate derivatives extraordinarily In particular, it can be useful for making of 6-deoxy-fructose-1-phosphate, a precursor of the Flavoring Furaneol (Wong et al., Z. Org. Chem (1983) 3493-3497). Furthermore, can using aldolase higher sugar with C₈, C₉ etc. (see Bednarski et al., Tetrahedron Lett. (1986) 27 5807-5810). Complicated amino sugars such as 1-deoxymannojirimicin, 1-deoxynojirimicin and  1,4-dideoxy-1,4-imino-D-arabinitol, which are considered to be effective Glucosidase inhibitors are important (Ziegler et al. Angew. Chemie 100 (1988) 737-738), are with the help of the aldolase according to the invention more accessible; these are partially endowed with HIV virus inhibitory activity (see Fleet et al., FEBS Letters 237 (1988) 128-132).

Claims (5)

1. fructose-1,6-diphosphate aldolase (F-1,6-DP-aldolase) from Staphylococcus carnosus and having the following parameters:
a 60 ° C or 100 ° C stability in purified form (≈25 U / mg) at pH 7.5 in 0.06 M Tris-HCl buffer solution in Eppendorf tubes corresponding to a residual activity after 30 minutes between 70 and 80% or 20 and 30%, in particular of 76% or 26%, and a deactivation rate under synthesis conditions at T = 25 ° C, pH 6.5; 20 mM DHAP; 75 mM glyoxylic acid; Enzyme activity 2.9 U / ml and 25 U / mg of 1% / d, respectively. 2. aldolase according to claim 1, characterized by the following parameters:

• - Class I aldolases (formation of protonated Schiff base with dihydroxyacetone phosphate, DHAP, no effect on F-1,6-DP activity by EDTA, complete inhibition by DHAP and NaBH₄);
• K _M values for cleavage of F-1,6-DP to DHAP and glyceraldehyde-3-phosphate and of F-1-P to DHAP and glyceraldehyde at 37 ° C. and pH 7.5 of 0.022 mM, respectively 18.9 mM;
• - specific activity (purified) of ≈25 U / mg protein (protein determination according to Bradford);
• a molecular weight of ≈33,000 daltons (no subunit breakdown);
• - pH optimum over the range of pH 6.5 to pH 9;
• - pH stability: residual activity after 10 min at pH 1-12 (25 ° C) and after 72 h at pH 2 (4 ° C) 100%;
• T stability: residual activity after 5 min at 100 ° C. (pH 7.5) and after 90 min at 60 ° C. (pH 7.5) 70%;
• Storage stability: Residual activity of the purified lyophilisate after 5 days
  a) RT: 100%
  b) -20 ° C: 100%.
• - Substrate spectrum: particularly good conversion of short-chain aliphatic aldehydes with additional increase in the turnover rate for molecules with functional groups with -I effect.

3. Method for recovering F-1,6-DP-aldolase Claim 1 or 2, characterized, that in a fermenter grown cell mass of Staphylococcus carnosus from the culture fluid separates and the enzyme after cell disruption wins. 4. The method according to claim 3, characterized, that the enzyme was fractionated by cell disruption Ammonium sulphate precipitation, pH fractionation and Ionenaustauscherchromatograhie isolated.   5. Use of F-1,6-DP-aldolase according to claim 1 or 2 for the synthesis of carbohydrates or Derivatives thereof by enzymatic reaction of aldehydes with DHAP.