CS276695B6 - Method of producing cortalcerone antibiotic by enzymatic transformation of D-glucose or D-glucosone - Google Patents

Abstract

Method for producing the antibacterial antibiotic cortalcerone, which is effective especially against gram-positive bacteria, by enzymatic transformation of D-glucose or D-glucosone using enzymes produced by cultures of some basidiomycetes, preferably the species Phanerochaete chrysosporium. Transformation can be performed using washed intact or immobilized mycelium from submerged cultures of P. chrysosporium and/or free enzymes pyranose-2-oxidase and pyranosone hydratase isolated from the same source to varying degrees of purity. Transformation can also be performed using an immobilized disintegrated cell preparation and/or an immobilized water-insoluble enzyme preparation based on cross-linked enzymes pyranose-2-oxidase and pyranosone hydratase isolated from P. chrysosporium. The stability of these preparations can be increased by co-immobilization of the enzyme catalase. The solution also includes a method for isolating the antibiotic cortalcerone, which involves chromatography on an ion exchanger in the Ca2+ cycle, with water used for elution.

Description

The invention relates to a process for the production of the antibiotic cortalcerone by enzymatic transformation of β-glucose or D-glucosone by intact or immobilized disintegrated cells of higher fungi or by co-immobilized enzymes and to a process for the isolation of this antibiotic.

Cortalcerone is an antibacterial antibiotic of fungal origin, effective especially against gram-positive bacteria isolated from the fermentation fluid of a culture of the basidiomycet Corticium caeruleum, exposed to stress factors (temperature rise to 42 ° C, exposure to vapors of toxic solvents such as chloroform, ether, acetone). , ethyl acetate or contact of mycelium with a mixture of water-toluene at room temperature (Baute et al., Phytochemistry 15, 1753-55 (1976)). -carboxaldehyde hydrate, the structure of which is shown in Figure 1, of formula I.

Baute et al. also found a direct biogenetic relationship of cortalcerone to D-glucose, or to the intermediate D-glucosone (D-arabino-2-hexosulose) (Baute et al., Phytochemistry 16, 1895-97 (1977)). They also proposed a scheme for the production of this antibiotic, involving the oxidation of D-glucose to D-glucosone by the enzyme pyranose 2-oxidase (EC 1.1.3.10) and subsequent double enzymatic dehydration of D-glucosone to an intermediate whose spontaneous cyclization produces cortalcerone (Baute et al. Phytochemistry 25; 1395-97 (1987). This scheme is also used by U.S. authors (U.S. Pat. No. 4,569,910) to optimize the process for preparing pure pyranose-2-oxidase preparations and their use in the production of pyranoeones. In some cultures of some basidiomycetes, they found the presence of the enzyme pyranosone dehydratase, which catalyzes the above-mentioned double dehydration of D-glucosone to cortalcerone. They purified and partially characterized this enzyme.

Current information on the antibiotic activity of cortalcerone is limited to stating that it is effective against gram-positive bacteria, including some pathogenic species, such as Staphylococcus aureus or Staphylococcus pyogenes (Berdy et al., CRC Handbook of Antibiotic Compounds, vol. V, p. 395/1981 /). More detailed data is still missing. Due to its structural similarity to microthecin, Fig. 1, Formula II (Japan, Patent No. 79,122,796), a possible application perspective of cortalcerone can be inferred. However, this possibility has so far been hindered by the low level of production of this antibiotic by lengthy stationary cultivation of the basidiomycet Corticium caeruleum (Baute et al., Phytochemistry 25, 1472-73 (1986)) or some related macromycetes. Incubation of the mycelium with a solution of D-glucose and plasmolytic agents (eg 1% toluene) induces the production of D-glucosone and its subsequent conversion to cortalcerone. The culture times used are between 15 and 20 days and the yield of antibiotic is around 200 mg per 1 liter of submerged fungal culture. The procedure for isolating cortalcerone from fermentation broth described by Baut et al. (Phytochemistry 15, 1753-55 (1976)) involves the extraction of the product with a chloroform-methanol mixture 93: 7 and repeated chromatography on a silica gel column. The fermentation fluid contains a number of ballast substances; which must be precipitated with methanol and propanol before the actual extraction.

The above-mentioned method, based on the principle of enzymatic transformation of D-glucose or alternatively D-glucosone, eliminates the stated disadvantages of the prior art process of lengthy and low yielding preparation of cortalcerone. It uses - in a suitable sequence or combination - crude or partially purified enzyme preparations of fungal origin, containing free or immobilized enzymes involved in the formation of cortalcerone (ie pyranose-2-oxidase and pyranosone dehydratase). The advantages of the proposed process are speed, high yield and easy isolation of the product (without the use of organic solvents).

The essence of the method for the production of the antibacterial antibiotic cortalcerone by the enzymatic transformation of D-glucose or D-glucosone is that the transformation is performed by means of enzymes produced by mycelial cultures of some basidiomycetes. It is advantageous to use certain species of basidiomycetes for this transformation, in particular the species Phanerochaete chrysosporium. Preferably, the present method of transforming D-glucose or D-glucosone to the antibiotic cortalcerone is performed using an intact mycelium from submerged cultures of the basidiomycet Phanerochaete

CS 276 695 B6 2 chrysosporium. The transformation of D-glucose or D-glucosone to the antibiotic cortalcerone can also be carried out according to the present method by means of disintegrated or disintegrated and mobilized mycelia of this type of basidiomycetes or co-immobilized enzymes isolated to different degrees of purity from this or other types of basidiomycetes. co-immobilization of the catalase, which allows the multiple use of mobilized preparations for the preparation of this antibiotic from said substrates. Isolation of the antibiotic cortalcerone from the reaction mixture is performed according to the present method by chromatography on an ion exchanger in a Ca cycle with water as eluent.

D-glucosone is prepared by oxidation of D-glucose (eg according to Czechoslovak author's certificate No. 175897). Only the D-form of glucose or glucosone can be used for biotransformation; Neither L-glucose nor L-glucosone react.

In the case of G-glucose (1 to 10% w / v), washed mycelium from a submerged culture of Phanerochaete chrysosporium containing both pyranose-2-oxidase enzyme, oxidizing D-glucose to D-glucosone, can be used as the enzyme active preparation. as well as pyranosone dehydratase, catalyzing the subsequent double dehydration of the D-glucosone to form a hitherto precisely unidentified intermediate (presumably 4-deoxy-aldehyde-D-glycero-2,3-hexodilose and their tautomers). The efficiency of the biotransformation process can be increased by the addition of a hydrogen peroxide decomposing catalyst; released in the reaction mixture during the oxidation of D-glucose. Dinak accumulates hydrogen peroxide, which denatures both enzymes involved.

3e Of course, instead of the enzymatically active mycelium Phanerochaste chrysosporium, it is possible to use a partially purified preparation containing free pyranose-2-oxidase and pyranosone dehydrate, prepared, for example, according to the method of Eriksson et al. (Appl. Microbiol. Biotechnol., 23; 257-262 (1986)). Particularly preferred is the use of a biocatalyst with both enzymes koimobilizovanými along with catalase cell fragments disintegrated mycelium production mushroom, enzyme active mycelial biomass can be obtained from 10 to lldenní submerged cultivation respective fungi in flasks on a shaker or in a fermenter at 28 ° C, _{N, and} nutrient medium composed D-glucose 20 g, corn extract 15 g, MgSO ₄ .7H ₂ 0 1.5 g in 1 liter of a solution adjusted to pH 5.5 by the addition of 6M NaOH. The pH of the fermentation broth is decisive for the end of the fermentation: the maximum content of both enzymes involved in the biosynthesis of cortalcerone was found in the mycelium harvested at pH 6.3 to 6.8, which is documented in the following examples.

In the case of D-glucoeone as a substrate (prepared, for example, according to the procedure according to Czechoslovak author's certificate No. 175897), washed Phanerochaete chrysosporium mycelium or the partially purified enzyme preparation prepared therefrom can also be used as a source of pyranosone dehydratase, in this case the only required enzyme. , or biocatalyst based on immobilized enzyme. The preparation of this antibiotic using D-glucose as a substrate is of course simpler and considerably more economical.

For the immobilization of the disintegrated mycelia of the production cells of basidiomycetes containing the respective enzymes, or for the co-immobilization of the catalase enzyme and / or the co-immobilization of these enzymes isolated to different purity, the principle of immobilization according to MS No. 231458 and / or No. 231656, which consist of the reaction of glutaraldehyde, polyethyleneimine and whole cells or disintegrated cell mixtures containing different enzymes in different species of microorganisms, including basidiomycetes and / or different crude or to different degrees of purity isolated enzymes from different microbial, plant or animal sources to form chemically bound water-insoluble cellular or enzyme aggregates used for discontinuous or continuous enzyme biotransformations.

Isolation of cortalcerone from the reaction mixture of the present method involves the chromatographic separation of cortalcerone from sugar intermediates and from 2-furylglyoxal, identified as a spontaneous degradation product of cortalcerone. This can be done according to the present invention

CS 276 695 B6 is performed on a column packed with ion exchangers in a Ca cycle. The advantage of this procedure over the purification used by Baut et al. (Phytochemistry 15, 1753-55 (1976)) that the substance does not need to be converted into an organic solvent during isolation; Only distilled water is required for isolation, and the eluate obtained from the column can be directly lyophilized, as also demonstrated in the following examples of the present invention, without being limited thereto.

The following are examples of embodiments of the process for the preparation of the antibiotic cortalcerone according to the invention.

Example 1

Phanerochaete chrysosporium basidiomycetus mycelium was cultured on a reciprocating shaker (150 rpm) in 500 ml flasks containing 80 ml of liquid medium consisting of: D-glucose 20 g, corn extract 15 g, MgSO 4 .THgO 1.5 g in 1 liter of tap water . PH, biomass production and pyranose-2-oxidase and pyranosone dehydratase activities were monitored during cultivation. The respective values were obtained by measuring average samples from three connected parallel flasks taken at 2-day intervals. (One unit (U) of activity pyranose-2-oxidase (P-2-0) corresponds to the oxidation of 1 / umol of D-glucose per 1 min. At 25 ° C in the applied reaction system (Eriksson et al., ^R Appl. Microbiol. Biotechnol., 23, 257-262 (1986), one unit of (U) pyranosone dehydratase (PO) activity is defined as the amount of enzyme causing absorbance at 265 nm per unit (△ Aggg = 1) per minute in the closed system used. 25 ° C.

Figure 2 in the appendix of the present invention graphically depicts the production of the enzymes pyranose-2-oxidase (P-2-0), pyranosone dehydratase (PD), pH and biomass during submerged cultivation of the basidiomycet Phanerochaete chrysosporium. The x-axis shows the culture days (d). The course of the production of P-2-0 is shown by filled circles (units are U / g dry ^mycelium: the values are on the y axis on the left), the PD output is represented by open circles (units are U / g dry weight of mycelium, the values are on the y axis on the left ). Squares show pH values (y-axis on the right), triangles show biomass production values (units are mg dry matter / ml culture, values are on the y-axis on the right). These courses indicate that the optimal culture time for the production of active mycelium (due to the transformation of D-glucose to cortalcerone) is 10 to 11 days. The time suitable for harvesting the cultures is signaled by an increase in pH above pH 6.3. At pH values of the fermentation broth above 6.8, there is a significant decrease in both enzyme activities. The active mycelium harvested at the appropriate time is separated by filtration on a filter cloth, washed with water and either immediately or after storage in a frozen state (-20 ° C) for transformation as follows.

10 g of moist enzyme-active mycelium Phanerochaete chrysosporium were weighed into two 250 ml flasks and 50 ml of 2% D-glucosone solution was added. The mixture was incubated at 20 ° C on a reciprocating shaker (150 rpm). After 24 hours, the active mycelium was filtered off, washed and stored in aqueous suspension for further use. The cortalcero2 + nu solution was concentrated on a rotary evaporator and purified on a Dowsx 50 column in a Ca cycle. The combined fractions containing pure cortalcerone were frozen and lyophilized. The yield of the process was 0.654 g of cortalcerone (i.e. 41% with respect to the substrate concentration used).

Example 2 g of Phanerochaete chrysosporium wet mycelium containing pyranose-2-oxidase and pyranosone dehydratase enzymes prepared according to Example 1, after washing with water, are suspended in 120 ml of distilled water and homogenized 5 times for 0.5 minutes at maximum speed with an Ultra-Turrax homogenizer. . Ibomolization of disintegrated mycelium and co-immobilization of catalase is performed according to MS. Author's Certificate No. 231458 suitably modified for a biocatalyst used for the preparation of the antibiotic cortalcerone as follows:

. 0.6 g of technical branched polyethyleneimine (Sedipur CL-930, BASF, Germany) is added to the indicated amount of disintegrated mycelium with vigorous stirring, followed by 10 ml of co-suspension

CS 276 695 B6 of the measuring enzyme catalase (Reanal, Hungary) and the pH of the reaction mixture was adjusted to 7.8 with aqueous 0.5 M NaOH solution under vigorous cooling. A total of 4.8 ml of a 25% glutaraldehyde solution is then added dropwise with stirring. After a further 5 minutes of vigorous stirring, the suspension is poured into a thin layer in a photomix and stored in a freezer at -15 ° C. After 24 hours, thaw by pouring distilled water. The immobilized preparation is filtered and washed with distilled water until the filtrate does not contain residues of used reagents (check eg by scanning UV spectra). The enzyme preparation thus prepared can be stored as a suspension in distilled water in the cold for about 4 months without significant loss of activity. A total of 60 g of wet mass of the mixed immobilized cell and enzyme preparation in the form of aggregates with a total dry matter content of 22% was obtained from 75 g of wet mycelium (dry matter content 6%).

Example 3

10 g of moist enzyme-active mycelium of the Phanerochaete chrysosporium basidiomycet prepared according to Example 1 were weighed into two 250 ml flasks and 50 ml of 2% D-glucose solution were added. The mixture was incubated on a reciprocating shaker (150 rpm) at 20 ° C. Fluid samples (0.1 mL) were taken at various time intervals to determine cortalcerone content by high performance liquid chromatography on a Separon SGX _{NH 2 column.} The mobile phase consisted of 70% aqueous acetonitrile pH 5.0 (H ₃ PO ₄ ), detection by UV detector at 225 nm.

Figure 3 of the appendix of the present invention shows the time course of D-glucose biotransformation to the antibiotic cortalcerone by the active mycelium of the basidiomycet Phanerochaete chrysosporium.

The x-axis shows the incubation time in hours, the y-axis the cortalcerone content in g / l of the reaction mixture. The figure shows that the maximum conversion was reached at 26 hours of incubation. After completion of the transformation, the active mycelium was filtered off, washed and kept in aqueous suspension for further transformations. The cortalcerone solution was concentrated on a rotary evaporator, purified on a Dowex 50 column in a Ca cycle, and the combined fractions containing pure cortalcerone were frozen and lyophilized. The yield of the process was 0.567 g of cortalcerone (i.e. 32% with respect to the substrate concentration used - D-glucose).

Example 4

7 g of the immolized mixed enzyme preparation prepared as described in Example 2 were weighed into five 250 ml flasks and 50 ml of 2% D-glucose solution was added. The flasks were incubated on a reciprocating shaker (150 rpm) at 20 ° C. Fluid samples (0.1 mL) were taken at various time intervals to determine cortalcerone content by high performance liquid chromatography on a Separon SGX _{NH 2 column.} The mobile phase consisted of 70% aqueous acetonitrile at pH 50 (H ₃ PO ₄ ), detection by a UV detector at 225 nm.

Figure 4, in the appendix to the present invention, shows the increase in cortalcerone as a function of transformation time. The x-axis shows the time of transformation of D-glucose to the antibiotic cortalcerone in hours, the y-axis the content of cortalcerone in g / l of the reaction mixture. The values shown in this figure show that the maximum conversion was reached at 22 hours of enzyme transformation. After completion of the transformation, the immobilized enzyme preparation was filtered off, washed and stored for further reuse. The cortalcerone solution was lyophilized after concentration and purification as described in the following example. Process yield was 3.73 g cortalcerone (i.e. 84%, owing to the concentration of substrate D-glucose) J ^J which is approximately three times higher than an intact mycelium in Example 3.

Example 5

The cortalcerone solution obtained as in Example 4, after filtering off the immobilized enzyme preparation, was concentrated on a rotary evaporator at a temperature not exceeding 40 ° C or 40 ° C to a volume of 50 ml. 2.5 ml of concentrated cortalcerone solution was loaded onto a column (30 cm x 4 cm) of a Dowex 50 ion exchanger in a Ca ^{2+ cycle.} Distilled water was used as the mobile phase and 10 ml fractions were collected. The absorbance at 225 nm was measured in each fraction.

Figure 5j, annexed to the present invention, shows the elution profile of cortalcerone on Dowex 50 in the Ca cycle. The x-axis shows the volume of eluate in ml (V / ml), the y-axis the absorbance values at 225 nm. The figure shows that the cortalcerone peak (1) corresponds to an elution volume of about 180 ml, the 2-furylglyoxal peak (2) to an elution volume of about 430 ml. The peaks of the sugar intermediates (3; 4) follow. Fractions containing cortalcerone were pooled and lyophilized. The ion exchange column was washed thoroughly with distilled water to prepare for further use. At a dosage of 63.4 mg of crude cortalcerone in 2 ^J 5 ml sample was found after passage through the column of 54.4 mg of the pure cortalcerone 50 ml; the yield of the described isolation and purification procedure thus reaches 86%.

Claims (5)

PATENT CLAIMS A process for the production of the antibiotic cortalcerone by enzymatic transformation of D-glucose or D-glucosone, characterized in that this transformation is carried out by means of mycelial cultures of basidiomycetes, preferably of the species Phanerochaete chrysosporium. 2. The production method according to item 1, characterized in that the enzymatic transformation of D-glucose or D-glucosone to the antibiotic cortalcerone is performed by washing intact or permeabilized mycelia from submerged cultures of Phanerochaete chrysosporium and / or free enzymes pyrariasis-2-oxidase and pyranosone dehydratase isolated from the same source. 3. The production method according to item 1, characterized in that the enzymatic transformation is performed by an immobilized disintegrated cell preparation based on aggregates of cross-linked cell fragments and soluble components of enzymatically active mycelia of Phanerochaete chrysosporium basidiomycetus and / or immobilized, water-insoluble enzyme preparation based on cross-linked enzymes. -2-oxidases and pyranosone dehydratases isolated from P. chrysosporium. 4. The production method according to items 1 and 3, characterized in that the immobilized disintegrated cell preparation and / or the immobilized enzyme preparation preferably contain a co-immobilized enzyme catalase. 5. The production method according to items 1, 2, 3 and 4, characterized in that the isolation of the antibiotic cortalce2 + ron from the concentrated reaction mixture is carried out by chromatography on an ion exchanger in a Ca cycle with water as eluent.