DE2605921C3 - Process for the enzymatic production of L-tartaric acid from cis-epoxysuccinic acid - Google Patents

Description

The subject matter of the invention is a process for the enzymatic production of L-tartaric acid from cis-epoxysuccinic acid according to the claims.

An advantage of this process is that L-tartaric acid is of the same type as naturally occurring Tartaric acid can be obtained from the cheaper and more readily available cis-epoxysuccinic acid or its salts will.

The term L-tartaric acid as used in the present Registration is used to denote the dextrorotatory (+) tartaric acid, which is the natural one Is tartaric acid.

Previously, L-tartaric acid was made using techniques that used tartar Ίη wine dregs as the raw material. The delivery of the raw material was therefore discontinuous ^and: limits, and it has been desired to develop a process which can be produced by the L-tartaric acid from an inexpensive and readily available raw material. In an attempt to meet these needs, methods of producing tartaric acid from maleic acid as a raw material have been developed. However, the tartaric acid obtained by such methods has a racemic form (DL form). The DL-form has the disadvantage of a considerably lower solubility than the L-form and this disadvantage restricted its usability in many respects. On the other hand, it is very disadvantageous from the point of view of operation and costly to convert the DL shape into an L shape by complex formation and the disadvantageous optical resolution or optical resolution. Transfer separation technology and racemization technology.

It has also been recommended to use tartaric acid through a fermentation technique or technique of microorganisms. For example, US Pat. No. 2,947,665 teaches that mesotartaric acid by hydrolysis of trans-epoxysuccinic acid using a microorganism Technology can be produced. However, this US patent does not teach the production of L-tartaric acid nor any technique that uses microorganisms Used to produce L-tartaric acid from cis-epoxysuccinic acid. Recently there was a technique for Manufacture of L-tartaric acid from cis-epoxysuccinic acid using a technique recommended that Microorganisms used (Japanese Patent Laid-Open Publication Nos. I 40 683/75 and 1 40 684/75 and DE-OS 25 17 941). These publications teach that microorganisms belonging to the genus Achromobacter or the genus Alcaligenes, have the ability to form an enzyme necessary for the enzymatic conversion of cis-epoxysuccinic acid This can be used in L-tartaric acid (referred to in these publications as "d-tartaric acid") However, references contain no reference to microorganisms belonging to the genus Nocardia, a completely different genus.

in It was now the existence of the microorganisms Genus Nocardia found, which have the ability to form an enzyme that enzymatically affects the convert cheap and readily available cis-epoxysuccinic acid to L-tartaric acid with superior activity can. It has also been found that the enzyme produced by the microorganisms of the genus Nocardia, e.g. B. from Nocardia tartaricans nov. sp, and cis-epoxysuccinic acid into L-tartaric acid by hydrolysis the epoxy ring of the former (to the as

2 » cis-epoxysuccinate hydrolase is referred to), is completely different from those in the above-mentioned publications with regard to some of its properties such as molecular weight, optimum pH or optimum temperature. It has also been found that, according to the process of the present invention, L-tartaric acid of the same type as natural tartaric acid can be produced with commercial benefit with markedly improved performance, up to 800 units (µ mol / hr) / ml broth or more

jo can by taking a microorganism of the genus Nocardia, which has the ability to produce cis-epoxysuccinate hydrolase, or a raw one from which Microorganism isolated enzyme with cis-epoxysuccinic acid or its salt in an aqueous medium

Vi in the presence of a surfactant, preferably a nonionic, anionic or cationic surfactant. The aforementioned publications, which describe the use of microorganisms belonging to the genus Achromobacter or Alcaligenes, give no indication of the use of surface-active agents.

The development Ί ;;, <. ■ in accordance with the method made it possible to lower L-tartaric acid

■ n cost and using more readily available Manufacture components and therefore makes a valuable contribution in the field of technology, to which the present invention pertains.

Accordingly, the aim of the invention is to provide a method

ίο for the production of L-tartaric acid of the same type how to make natural tartaric acid available with commercial benefit.

The above and other objects of the invention, along with their advantages, will be apparent from

Visible from the following description.

The cis-epoxysuccinic acid or its salts used in the practice of the invention can be easily, for example by epoxidizing maleic anhydride, maleic acid or a derivative of a

w) obtained these compounds with hydrogen peroxide will. Examples of inorganic salts of cis-epoxysuccinic acid are the sodium, potassium, calcium and ammonium salts of this acid, for organic salts primary, secondary and tertiary aliphatic amine,

h'i aromatic amine. Pyridine and quinoline salts of these Acid.

All salts which are soluble in an aqueous medium and which have the ability can be used.

the cis-epoxysuccinate hydrolase, the epoxy ring of the cis-epoxysuccinic acid does not affect hydrolyzing. The cis-epoxysuccinic acid or its Salts can be in the form of crystals or the reaction mixture, as indicated by a synthetic ι Process was obtained. However, the presence of impurities in amounts that the Slowing down the reaction, as from too much hydrogen peroxide, is undesirable

The concentration of the substrate in the method according to the invention can be within a wide range Range, generally from 0.1% by weight, as used initially, to very high concentrations. Is the cis-epoxysuccinic acid or its salt already more microbial at the time of growth If cells are present in the culture medium, the growth of the cells tends to be due to excessive concentrations of cis-epoxysuccinic acid to be inhibited. The concentration of the cis-epoxysuccinic acid or its salt, for example, to a value of not more than about 0, preferably be set of not more than 5 wt .-%. Higher concentrations can be used if the microorganism according to the invention or an enzyme which produces L-tartaric acid and which has been isolated therefrom, with cis-epoxysuccinic acid or its salt in a system for hydrolysis of cis-epoxysuccinic acid or their salt, in which there is no growth of microbial cells, are brought into contact.

A preferred species of the microorganism belonging to the genus Nocardia with the ability Forming cis-epoxysuccinate hydrolase is Nocardia tartaricans nov. sp, which is isolated from nature. Typical strains of Nocardia tataricans nov. sp. are in the Fermentation Research Institute, Agency of r> Science and Technology, Japan and American Type Culture Collection, USA deposited. The properties of these strains are described below.

Description of Nocardia tartaricans nov. sp.

I. strain ESl-T (FERM P 2882; ATCC 31191)

A. Morphological Properties ^.

Bars: 0.8-1, of times 5-30 "in young cultures, 0.8-1.0f times 1-5f in older cultures; in young Cultures, mycelial development, branching, and curvature is observed in older cultures small sticks of coconut; on none of the tested Agar slopes formed an aeral mycelium; not able to move; not acid-proof; gram positive; it will no spores formed.

B. Culture characteristics

1. Nutrient agar colonies: circular, pin head-shaped, smooth, complete, opaque, glossy, yellowish-orange to reddish-orange.

2. Glucose peptone agar colonies: circular, convex, smooth, wavy, opaque, pale yellowish-orange

ge

3. Glycerin-asparagine agar colonies: rhizoid, domed, opaque, glossy, pale orange.

4. Nutrient broth: slightly cloudy, membranes, sediment.

5. Pierced gelatin: good growth on the h ". Surface, no liquefaction.

6. Nutrient agar slope: abundant growth, spread out, shiny, buttery, initially pale yellowish brown and becoming yellowish-orange and reddish-orange as the culture ages.

7. sucrose nitrate agar slope; moderate growth, pale orange, no pigment formation with diffusivity.

8. Glucose-asparagine-agar slope: moderate growth, pale orange, no pigment formation with diffusivity.

9. Glycerine-asparagine-agar slope: moderate growth, pale orange, no formation of pigments with diffusivity.

10. Starch-agar slope: insufficient growth, colorless, no formation of pigments with diffusivity.

11. Tyrosine agar slope: moderate growth, pale orange, no formation of pigments with diffusivity.

12.Yeast-malt-agar slope: abundant growth, orange, no formation of pigments with diffusivity.

13. Oatmeal agar slope: moderate growth, pale orange, no formation of pigments with diffusivity.

14. Inorganic salt agar slope: insufficient growth on an inorganic salt agar that keiuc contains carbon sources, colorless, none Formation of pigments with diffusibility.

15. Potato plugs: abundant growth, yellowish orange to reddish orange, no formation of Pigments with diffusivity.

C. Physiological properties

Temperature for growth: 10-45 ° C
Optimal temperature for growth: 25-40 ° C pH range for growth: 5.5-11
Salt tolerance: is not inhibited by 10% NaCl

Resistant to 0.001% lysozyme
Penicillin sensitive (5 i. E. discs [iu discs])
Nitrate reduction: positive
Voges-Proskauer test: negative
Methyl red test: negative
I ndole formation: negative
Hydrogen sulfide formation: positive
Ammonia formation from peptone: negative
Gelatin liquefaction: negative
Casein peptonization: negative
Litmus milk: alkaline, slightly lightening with color, no coagulation
Starch hydrolysis: negative
Cellulose hydrolysis: negative
Catalase: positive
Cytochrome oxidase: negative
Urease: positive
DNA hydrolysis: negative
Ammoniumsai /:, nitrate and urea are used as nitrogen sources

Acetic acid, n-butyric acid, lactic acid, succinic acid, citric acid, malic acid and L-tartaric acid are assimilated

Splitting of carbohydrates: acid without gas from L-arabinose, D-xylose, D-glucose, DFiuctose, D-mannose, sucrose, maltose, trehalose, glycerin, ethanol, D-sorbitol, D-mannitol, inositol; neither Acid still made from galactose, lactose, raffinose, dextrin

Assimilation of various carbon sources (incubation on Pridham and Gottlieb agar and

9.10.11.12.13.14.15.

16. 17. 18. 19.? 0. 21-22

- Broth at 30 ⁰ C for 3 weeks) L-arabino se (+), D-xylose (+), D-glucose (+), D-fructose (-t), sucrose (+), inositol (+), L-rhamnose (+), raffinose (-t-), D-mannitol (+), ethano! (+), N-propanol (+), isopropanol (-I-), n-butanol (+), propylene glycol ( +), Glycerine (+), propylene oxide (+), n-paraffin (-f), acetamide (+), phenol {-), p-cresol (-), m-cresol (-), naphthalene (±)

D. Whole Cell Infrared Absorption Spectra

Infrared absorption spectra of whole cells were examined, which were obtained by means of Schiittel culture on a Glycerin-Kelner-Morton medium had been obtained by the method of Arai (J. General Applied Microbiology 9,119 [1963]).

The key absorption patterns were as follows: Area I (2800-3000 cm - '): Type D,
Area II (1650-1750 cm ->): Type D,
Area III (1370-1550 cm '): Type C.
Area IV (950-1250 cm- ¹ ): Type C.

II. ES3-T strain (FERM P-3374; ATCC 31190)

The organism is very similar to the strain ESl-T with the exception of the following Properties: no acid nor a gas from L-arabinose, D-xylose, D-mannose, maltose.

The investigation of EsI-T and ES3-T strains according to Bergey's Manual of Determinative Bacteriology. 8th. Ed. And Waksmann: "The Actinomycetes", Vol. 2, showed that these are new ones of the genus Nocardia belonging to tribes.

As strains analogous to the strains according to the present invention, there can be mentioned: Nocardia vaccinii, Novardia lutea, Nocardia corallina, Nocardia salmonicolor, Nocardia rubra and Nocardia opaca. The ES1-T strain and the ES3-T strain differ from Nocardia vaccinii and Nocardia lutea in terms of growth characteristics in a nutrient agar slope, the formation of the mycelium, the acid resistance, the pH range for growth and the hydrolyzability of deoxyribonucleic acid (DNA) as shown in Table I below is. Furthermore, as shown in Table 2, these strains differ from Nocardia corallina in terms of the growth properties in a nutrient agar slope, the optimal growth temperature, the tolerance against NaCl, the formation of acid from sucrose and the utilization of phenol and m-cresol; and from Nocardia salmonicolor regarding the growth properties in a nutrient agar slope, the growth properties in a glycerine-asparagine agar slope, the optimal growth temperature, the growth pH, tolerance to NaCl, the formation of acid from dextrin and the utilization of p-cresoi. as shown in Table J, the ESI-T strain and ES3-T strain are still different from Nocardia rubra regarding the growth properties in a nutrient agar slope, the optimal growth temperature, the tolerance to NaCl and the Reduction of nitrate; of Nocardia opaca regarding the growth characteristics in a nutrient agar slope, the optimal growth temperature, growth pH and salt resistance. Since the ESl-T strain and ES- 'T strain in an inorganic salt agar culture medium their microbiological properties were similar to those of Nocardia marina and Nocardia atlantica, which can utilize agar as a carbon source. Those given in Table 4 Results show that these strains differ from Nocardia marina and Nocardia atlantica in terms of liquefaction differ from gelatin and the decomposition of cellulose.

Table 1

Nocardia tartaricans nov. sp.
ESI-T fFERM P-2882: ATTCC 31191)
ES3-T (FERM P-3374; ATTCC 31190) Nocardia vaccinii Nocardia lutea 1. Nutrient agar colonies yellowish-orange to reddish
lich-orange, pin
head-shaped. smooth, complete reddish, flat,
uneven
Margins 2. Aeral mycelium on none of the examined
Agar slopes formed White White 3. Acid resistance negative positive partially positive 4. Temperature for that
growth 10-45 ⁰ C 10-35 ⁰ C 10-40 ⁰ C 5. pH range for that
growth 5.5-11 6-9 6-10 6. Hydrolysis of DNA negative positive positive Table 2 Nocardia tartaricans Nov. sp.
ESl-T (FERM P-2882; ATCC 31191)
ES3-T (FERM P-3374; ATCC 31190) Nocardia corallina Nocardia
salmonicolor 1. Nutrient agar colonies the same as in Table 1 coral red, raised,
filamentous or
tree-like branching
Margins salmon red,
raised, rough

I Ol tsct / IIMü

2. Glycerol-asparagine agar colonies

3. Temperature for that
growth

4. Optimal temperature
for growth

5. pH range for that
growth

Nocardia larlaricans Nov. sp. KSI-T (T'-RM P-2882: ΛΤ (C Jl 141) KSJ-T (KKRM I »- 5374: Λ IC (31190)

moderate growth, pale orange, rhizoid

IO-45 ° C 25-40 ⁰ C 5.5-11

Nocardia corallina

25-28 ° C

Nocardia
salmonicolor

well developed

Red orange

Colonies

10-40 ° C

28 ° C

5-10

6. Salt (NaCl) tolerance 10% 7% Phenol, m-cresol ESl-T (FERM P-2882; ATCC 31191) light lemon yellow, 5% 7. Breakdown of carbohydrate no acids from dextrin; no acid off Recovery ES3-T (FERM P-3374; ATCC 31190) flat, smooth, Acid off . Acid formation from sucrose sucrose the same as in Table 1 wavy edges Dextrin 8. Assimilation of Phenol, m-cresol, p-cresol positive p-cresol Carbon sources are the only coals Nocardia rubra negative Recovery Sources of material not recycled negative positive Table 3 positive positive Nocardia tartaricans nov. sp. negative Nocardia opaca ESl-T light red, flat, rough. negative (FERM P-2882; ATCC 31191) wavy edges (FKRM P-3374; ATCC 31190) 1. Nutrient agar colonies the same as in pale buff-pink. Table 1 25-28 ° C received, rough, filamentous to full 6-10 permanent margins 2. Optimal temperature 25-4O ⁰ C 7% 30 ° C for growth negative 3. pH range for growth 5.5-11 6-9 4. Salt (NaCl) tolerance 10% Nocardia marina 7% 5. Nitrate reduction positive positive Table 4 Nocardia tartaricans nov. sp. Nocardia atlantica 1. Nutrient agar colonies yellow to orange-yellow 2. Gelatin liquefaction positive 3. Nitrate reduction positive 4. Cellulose hydrolysis positive 5. hydrolysis of starch negative

Based on the above studies, ES1-T strain and ES3-T strain as new strains of the genus Nocardia with the name Nocardia tartaricans nov. sp. identified «>

According to the invention, a microorganism of the genus Nocardia tartaricans nov. sp. to form with the capability of cis-epoxysuccinate hydrolase, or an isolated microorganism of this enzyme with cis -epoxysuccinic acid or salts thereof housed in an aqueous medium in contact _b s

The cis-epoxysuccinate hydrolase used in the present invention is obtained by culturing the above

Microorganism obtained in the usual way. The cultivation of the above microorganism is usually carried out in an aqueous medium. It can also be performed on a solid surface will. Natural culture media such as nutrient broth and synthetic media can be used for cultivation Culture media composed of a suitable carbon source such as glucose, sucrose, Glycerine, ethanol, isopropanol or η-paraffin, a suitable nitrogen source such as ammonium salts, nitric acid salts and urea and other inorganic salts can be used. It is desirable.

cis-epuxybernsieic acid or its salts to the Add culture medium to obtain the desired I ji / y in / u induce.

The pH of the culture medium is adjusted to 5.5-11, preferably 6-10. The cultivation is aerobically b at about 10's about 45 ° C preferably about 25 to about 4O ⁰ C, performed. In batch operations, cultivation is usually carried out for half a day to 10 days.

When carrying out the method according to the invention, the microorganism which can form cis-epoxysuccinate hydrolase or the enzyme isolated from it can be brought into contact with cis-epoxysuccinic acid or its salts in various ways. For example, the contacting can be carried out while the microorganism in the form of a growing culture, a culture broth, in the form of live cells, dried 7PJJPn ₁ niilvprisjprtpr 7ρ11ρπ nc \ pr pines inches extract is present. It can also be in the form of a purified enzyme using means or measures for purification.

If the enzyme is used in a purified form, the following procedure for isolation and Purification of the enzyme can be applied. Cells obtained from a culture broth by centrifugation are suspended in a 0.05 M phosphate buffer (pH 7) and destroyed by sound oscillation. The so obtained crude cell-free extract is fractionated by precipitation with ammonium sulfate and a fraction with 50 to 60% saturation gained. The precipitate is dissolved in a small volume of 0.05 M phosphate buffer (pH 7) and dialyzed against a large volume of the same buffer and then by column chromatography with DEAE-Sephadex A-50 (three-dimensionally cross-linked polysaccharide with functional diethylaminoethyl groups) and gel filtration with a Sephadex G-200 column (three-dimensional cross-linked polysaccharide) cleaned.

From Nocardia tartaricans nov. sp. ES3-T (FERM P-3374; ATCC 31190) isolated cis-epoxysuccinate hydrolase, purified by the method described above has the following properties. (The elution pattern of the second gel filtration of Sephadex G-200 is shown in Fig. 1.)

1. Catalytic effect: hydrolysis of cis-epoxysuccinate, formation of L (+) -tartaric acid.

2. Molecular weight: 60,000 to 95,000 by the Andrew method.

3. Substrate specificity: no hydrolysis of d- or l-trans-epoxysuccinate.

4. pH optimum: 8-9.

5. Optimum temperature: 5O ⁰ C with an incubation of 20 to 80 minutes and 45 ° C when incubated for several hours.

6. pH stability: stable at pH 7.0, unstable at a pH below 5.0 and a pH of over 10.0.

7. Temperature stability: stable at temperatures below 40 ^° C. with an incubation of 10 minutes at a pH of 7.0; Loss of activity at temperatures greater than 60 ° C with a 10 minute incubation at pH 7.0.

8. Inhibitors and activators: they are given in Table 5 below.

Table 5

Inhibitors and Activators:

niM

None
EDTA

-la'-dipyridyl

o-phenanthroline

Ammonium oxalate

Mercaptoethinol

p-chloro mercury benzoate

Iodoacetamide

MgSO, ■ 7H ₂ O

Mp SO, · 7 H> O

ZnSO ₄ 7 HO

CuCl, · 2H, O

CoCI:

CaCl ₂ · 2 HO

Fumarate

d.l-trans-F.poxysuccinate

10

10

10 100

Remaining activity

100 88 84 88 39 45

109 89 86

106

115 55 5b 8b 56

IH 90

126

The pH optimum (4), the temperature optimum (5), the pH stability (6) and the temperature stability (7) are shown in FIGS. 2, 3, 4 and 5 shown. In these figures, TA is an abbreviation for L-tartaric acid and ESA for cis-epoxysuccinic acid.

The enzymatic activity, the amount of tartaric acid and the amount of protein that is used in determining the enzymatic properties of the invention cis-epoxysuccinate hydrolase used were determined by the following methods.

1. Determination of the enzymatic activity

1.0 ml of 1-M ESA Na: were added to 3.9 ml of 0.1 M phosphate buffer (pH 8.0) and the mixture was left at i / L for 5 minutes. An enzyme solution was then added and the mixture was left to stand for 20 minutes at 37 ° C. The amount of tartaric acid formed in the reaction mixture obtained was determined. The amount of enzyme which produces 1 μΜοΙ tartaric acid per hour under the above-mentioned reaction conditions is defined as one unit, and the number of such units per mg of protein is defined as specific activity.

2. Determination of the amount of tartaric acid

An enzyme reaction solution was added to 1 ml of 2% ammonium metavanadate reagent and diluted the mixture with water to 4 ml. Then 1 ml of 1N sulfuric acid was added and determined after 30 Minutes the absorption of the solution at 530 µm.

3. Determination of the amount of protein

The amount of protein was determined from the absorption at 280 nm using a setting of E [;: at 15 made.

The cis-epoxysuccinate hydrolase according to the invention differs from conventional ones in that disclosed Japanese Patent Publication No. i 40 684/75 and other publications mentioned above Enzymes produced by microorganisms of the genus

Achroniobacter or the genus Alcaligenes are formed and were used in the formation of L-Weinsäurc from cis-Epoxybe ^r nsteinsäure. For example, it is stated in the earlier patent application that the usual enzymes have a molecular weight of about 25,000 to about 45,000. However, the cis-epoxysuccinate hydrolase according to the invention has a molecular weight of approx. 60,000 to approx. 95,000. Furthermore, the pH optimum of the cis-epoxysuccinate hydrolase according to the invention is 8 to 9, while that of conventional enzymes is 7 to 8. The optimal temperature is 57 ° C for an incubation of 1 hour and 50-52 ° C for an incubation of 3 hours for the conventional enzymes, but for the cis-epoxysucciral hydrolasc according to the invention it is 50 ° C for an incubation of 1 hour and 45 ° C with an incubation of 3 hours.

According to the method according to the invention, particularly favorable results are achieved by one Microorganism which can produce cis-epoxysuccinate hydrolase, or a crude enzyme isolated therefrom with cis-epoxysuccinic acid or a salt thereof in an aqueous medium in the presence of a surfactant, preferably one selected from the group consisting of nonionic, anionic and canonical surfactants. The surface-active Agents can be used either alone or in combination of two or more.

Examples of suitable anionic surface-active agents Mfltel are alkyl polyoxyethylene phosphoric acid esters, in which the alkyl group preferably has 2 to 20 carbon atoms contains and the polyoxyethylene has a degree of polymerization of 2 to 20, such as Dipolyoxyethylene alkyl ether phosphoric acid ester DDP-8 and dipolyoxyethylene oleyl ether phosphoric acid ester DOP-8 and higher fatty acid salts, preferably containing 10 to 30 carbon atoms, such as sodium laurate or sodium oleate.

Examples of suitable nonionic surface-active agents are polyoxyethylene sorbitan fatty acid esters, wherein preferably the polyoxyethylene is a polymeric gray vun 2 to 50 owns and del Fcusäui caiucii Contains 2 to 20 carbon atoms, such as polyoxyethylene sorbitan monooleate Tween-80, a polyoxyethylene / polyoxypropylene block copolymer, such as Pluronic L-61, and polyoxyethylene alkyl ethers, in which preferably the alkyl group contains 2 to 20 carbon atoms and the polyoxyethylene has a degree of polymerization of 2 to 50, such as Polyoxyäthylenlauryläther BL-42 and BL-9EX, Polyoxyäthylencetyläther BC-IOTX and Polyoxyethylene oleyl ether BO-1OTX.

Examples of suitable cationic surfactants are quaternary ammonium salts with long Alkyl chains in which the long chain alkyl group preferably contains 10 to 30 carbon atoms, such as Cetyltrimethylammonium bromide and pyridinium salts with long-chain alkyl groups, including the long-chain Alkyl group preferably contains 10 to 30 carbon atoms, such as laurylpyridinium chloride.

In the present invention, tripolyoxyalkyl phosphoric acid esters can also be used TDP-8, polyoxyethylene sorbitan monolaurate Twccn-20 and polyoxyethylene trioleate Twecn-85 can be used.

The amount of the surface-active agent can optionally be varied according to its type, however, it is generally about 1 to about 0.001% by weight, based on the aqueous medium.

It is not clear by what mechanism the presence of a surfactant affects the rate the hydrolysis (hydration) of cis-hpoxysuccinic acid or the formation of L-tartaric acid from their derivatives by using νυη cis-epoxysuccinate hydrolase and thus the amount of L-tartaric acid formed. However how As can be seen from the following working examples, their effect is in terms of promoting education of L. tartaric acid clearly.

Research has shown that in the case of enzymes produced by those mentioned in the above Japanese Laid-Open Patent Publication No. I 40 684/75 and other mentioned microorganisms the addition of surfactants has no noticeable accelerating effect the cell suspension reaction results. The investigations carried out in this context are given below in part.

Using each Achromobacter tartarogenese nov. sp. TORAY 1246 (FERM P2507), Achromobacter acinus nov. sp. TORAY 1366 (FERM P2509) and Alcaligenes epoxylyticus nov. sp. TORAY 1128 (FERM P2511), which are mentioned in the above publications and Nocardia tartaricans nov. sp.

CSJ- ι ^ FERiVl r-337 * t; Αιυυ ~ ji ia \ jf gCiTtüu uCr invention, was cis-epoxysuccinic acid in Ϊ -tartaric acid (TA) under the same conditions by the same technique in the presence of polyoxyethylene alkyl ether (BL-9EX) as a nonionic surfactant, alkyl polyoxyethylene phosphoric acid ester

(DDP-8) carried out as an anionic surface-active agent and ι toluene. The results are in Table 6 specified.

Table 6

Microorganisms additions TA (g / l) TA (g / l) EinhVml- 10 hours 20 hours broth (μΜοΙ / hour ml broth) Achromobacter tartarogenes _ 17th 34 22.7 (FERM P-2507) BL-9EX 15.3 29 20.4 DDP-8 18th 36.2 24.0 Toluene 2 µ 18th 36.2 24.0 Achromobacter acinus - 21 43 28.0 (FERM P-2509) BL-9EX 19th 37 253 DDP-8 19.5 38.8 26.0 Toluene 2 u. 19.5 38.8 26.0

Korlsci / ung

Microorganisms additions TA (g / l) Day 1) EinhVml- 10 SuL 20h B rest (μΜοΙ / Sid. ml broth) Alcaligenes epoxylyticus 23.2 43.4 303 (FERM P-2511) BL-9EX 11th 134 14.7 DDP-8 23 44 30.7 Toluene 2 µ 223 444 29.7 Nocardia tartaricans ES3-T 14th 29 18.7 (FERN! P -3374; ATCC 31190) BL-9EX 116 150 (100%) 154.6 DDP-8 40 84 53.4 Toluene 2 µ 0 0 0

The reaction can be carried out by a batch method, a continuous method, or a semi-batch method in which the substrate and other necessary materials are added successively during the reaction. It is also possible to use a flow reaction technique in which the microorganism or enzyme is fixed on a suitable support and packed in a column. The reaction may be at a temperature of about 20 to about 60 ⁰ C, preferably about 25 to about 45 ° C, at a pH of about 5 to about 10, preferably about 6 to about 10, more preferably 7 to 9 can be carried out.

According to one embodiment of the invention, cis-epoxy succinic acids or one of their derivatives either partially or completely added to the liquid culture medium and then the above specified microorganism can be cultivated in this to produce L-tartaric acid in the culture broth.

The L-tartaric acid can be removed from the reaction mixture, for example by removing the cells Centrifugal separation and concentration of the filtrate. Another extraction method involves the addition of a calcium salt to precipitate the product as calcium tartrate tetrahydrate. Split off of the L-tartrate by filtration or any other desired means for separating solids and Liquids and converting them to L-tartaric acid in the usual way. The obtained L-tartaric acid was the same as natural tartaric acid because of its Rf value in paper chromatography, its degree of migration in filter paper electrophoresis, and also because of its IR spectrum, NMR spectrum, rotation value and elemental analysis values determined.

The following examples illustrate the invention. example 1

Each 500 ml shake flask was charged with 50 ml of a culture medium (pH 7.0) containing 1% meat extract, Contained 1% peptone and 03% sodium chloride, and that Culture medium was sterilized at 121 ° C. for 15 minutes. It was sterilized sodium cis-epoxysuccinate in a concentration of 1% is added to the culture medium and Nocardia tartaricans nov. sp. (Tribe ESl-T; FERMP-2882; ATCC 31191) and cultured with shaking at 30 ° C for 24 hours. the Cell concentration was 3.5 g / l. The cells were extracted from the obtained culture broth by centrifugation separated, washed once with a physiological saline solution and centrifuged again to collect the cells obtained.

The collected cells, the dry weight of 25 mg, a possessed were added to 25 ml of a 1 molprozentigen aqueous solution of sodium cis-epoxysuccinate (pH 7.0) was added and the reaction was carried out at 30 ⁰ C. The results are given in Table 7.

-n table 7

Time (Hours)

The maximum rate of tartaric acid formation was 2.04 g / l hour, corresponding to 2.04 g / g Cells per hour. This corresponded to 47.6 units of enzymatic activity for every t ml of the original Culture broth.

Example 2

A 10 liter fermentation vessel was charged with 5 liters of a culture medium of the following composition tongue:

Amount of Yield to formed tartaric acid Tartaric acid (g / l) (%) 394 263 90.0 60.0 139.1 92.7 145.7 97.1

Propylene Glycol Urea KH ₂ PO ₄ Na ₂ HPO _{4 MgSO 4} · 7 H ₂ O FeSO ₄ · 7 H ₂ O CaCl ₂ · 2 H ₂ O MnSO ₄ · 4 H ₂ O ZnSO ₄ · 7 H ₂ O Co (COs) ₂ 6 H ₂ O yeast extract pH

14%

03%

0.2%

0.2%

0.1%

0.001%

0.003%

0.0004%

O ₁ (XX) I%

0.00001%

0.05%

7.0.

The culture medium was prepared as follows: A culture medium containing the Containing the above ingredients with the exception of the urea, 15 minutes at 12PC and added one filtered and sterilized urea solution prepared separately. The pH of the mixture was adjusted to Sodium hydroxide and hydrochloric acid adjusted to ensure that the culture medium ultimately meets the above said composition and the above pH.

In this culture medium, Nocardia tartaricans nov.sp. (Strain ES3 T; FERM P-3374; ATCC 31190) was inoculated and cultured for 48 hours at 30 ⁰ C, while air was at a rate of 51 per minute (1 vv m) passed through and set the stirring speed to 600 revolutions per minute . The final cell concentration was 53 g / L. After the cultivation, 300 ml of the culture broth was taken out, and the cells were separated by centrifugation. The cells were dissolved in 300 ml of a culture broth of the same composition as above except that 0.2 mol / L of sodium cis-epoxysuccinate were used and propylene glycol was omitted, suspended. The suspension was kept at 30 ° C. for 20 hours in order to induce enzyme formation. To 5 ml of this solution, 5 ml of a solution of 1 mol / l sodium cis-epoxysuccinate was added, and the reaction was carried out at 37 ° C. 5 ml of a solution of 2 mol / l sodium cis-epoxysuccinate were successively after 4 hours , 22 hours and 45 hours, respectively. The results are given in Table 8.

22 45 67

15 20 25

95.4

143 212

1431 2860 5300 934 19.1 353 90.6 83.1 135

Example 3

50 ml of a culture medium having the following composition was placed in a 500 ml shake flask a:

Table 8 Response time 4

(Hours.)

Merge the rea- 10

tion mix

Concentration 20.8

of tartaric acid

(mg / nv)

Amount of billed 208

tartaric acid

Amount of billed 139

tartaric acid

(mmol)

Activity (a 693

units / ml broth)

The activity shown in the last horizontal column of Table 8 represents an average activity per ml of original culture broth for each reaction period from 0 to 4 hours, 4 to 22 hours, 22 to 45 hours Hours and 45 to 69 hours.

Propylene glycol 1% NH ₂ CONH ₂ 03% KH ₂ PO ₄ 0.15% Na ₂ HPO ₄ 0.15% FeSO ₄ · _{7H 2} O 0.001% CaCl ₂ -2H ₂ O 0.001% MnSO ₄ · 4 H ₂ O 0.0002% Yeast extract 0.02% pH 7.0.

The above culture medium was prepared as follows: A culture medium which containing the above ingredients with the exception of urea, 15 minutes at 121 ° C and added one

: n Add filtered and sterilized urea solution prepared separately. The pH of the mixture was adjusted to Sodium hydroxide adjusted to form a culture medium which ultimately has that given above Composition and the aforementioned pH having

Nocardia tartaricans nov.sp. was vaccinated. (ESI-T strain: FERM P-2882; ATCC 31191) on a platinum loop in the culture medium obtained and incubated 100 hours at 30 ° C. The final

The cell concentration was 4.8 g / l. The received Cells were collected by centrifugal separation and placed in a culture medium of the same composition except that it did not contain propylene glycol contained suspended, and sodium cis-epoxysuccinate

) 5 was added at a concentration of 0.2 mol / l. The mixture was kept for 20 hours at 3O ⁰ C, in order to induce enzyme production. The cells were again collected by centrifugal separation and at a concentration of 5 g / l as dry cells to 10 ml of a solution of sodium cis-epoxysuccinate of 0.7 mol / l (pH 7.0) was added and then each of the surface-active agents shown in Table 9 in the specified concentrations added. The reaction was carried out with shaking at 37 ° C. 5

4> hours later, the amount was obtained Tartaric acid determined. The results are given in Table 9. It can be seen that the addition of surfactants markedly accelerated the rate of reaction.

Table 9

Surface active agents concentration Educated L-Wine Average acid enzymatic activity (O / o by weight) (g / l) (Einhy mg Zsüen) No encore - 10.8 2.88 Alkyl polyoxyithylene phosphoric esters 0.1 34.4 9.17 (DDP-8) 0.05 44.0 11.7 0.025 64.0 17.1 0.0125 48.0 12.8 Sodium laurate 0.1 8.0 2.13 0.05 12.4 3.31 0.025 22.4 5.97 0.0125 11.2 2.99

\ 7

18th

Continuation

Surface active agents

concentration
(Wt .-%)

Formed L-tartaric acid

(g / l)

Average enzymatic activity
(EinhV
mg cells)

Cetyltrimethylammonium bromide

Lauryl pyridinium chloride

Polyoxyethylene sorbitan fatty acid ester (Tween-80)

Polyoxyethylene / polyoxypropylene Example 4 Propylene glycol 1% Block copolymer urea 03% (Pluronic L61) KH ₂ PO ₄ 0.2% Na ₂ HPO ₄ 0.2% MgSO ₄ · _{7H 2} O 0.1% FeSO _{4 7H 2} O 0.001% CaCl ₂ · 2 H ₂ O 0.003% MnSO ₄ · 4 H ₂ O 0.0004% Yeast extract 0.02% pH 7.0.

0.1 0.05 0.025 0.0125

0.1 0.05 0.025 0.0125

0.1 0.05 0.025 0.0125

A culture medium containing the above ingredients except for urea was used 15 Sterilized minutes at 121 ° C and a separately prepared filtered and sterilized urea solution admitted. The pH of the mixture was adjusted with sodium hydroxide to prepare a culture medium, which finally had the above composition and pH.

500 ml shake flasks were filled with 50 ml of the culture medium. Nocardia tartaricans nov.sp. was vaccinated. (Strain ESl-T; FERM P-2882; ATCC 31191) on a loop and cultivated for 75 hours at 30 ° C.

A 10-liter fermentation vessel was charged separately 10.4

8.0

84.4

28.0

62.8
58.4
55.2
57.4

22.4
21.2
20.8
21.6

19.6
21.2
26.0
36.0

2.77
2.13
22.5
7.47

16.7
15.6
14.7
153

537
5.63
5.55
5.76

5.23
5.65
6.93
9.60

with 5 1 of a sterilized culture medium of the same composition as above and added 0.1%

to Pluronic L-61 (manufactured by Nikko Chemicals) as a defoaming agent. Then 5 bottles of the culture broth were inoculated and cultivated at 30 ^° C., while air was passed through at a rate of 5 l / min and the stirring speed was set at 600

ji revolutions per minute has been set. One sat the cultivation continued for 17 hours and after the Cell concentration had reached 0.9 g / l, 100 ml of a filtered and sterilized 25% solution of Sodium cis-epoxysuccinate (pH 7.0) is added to the

to induce enzyme formation. The KutWierung was 24 Continued for hours and the final cell concentration reached 4.73 g / L.

Each of the surfactants shown in Table 10 was added to the one shown in Table 10

4) the indicated concentration to 10 ml of the culture broth obtained and furthermore 10 ml of a solution of 1.4 mol / l sodium cis-epoxysuccinate (pH 8.0). The reaction was then carried out ^{at 37 ° C. with shaking.} 5 hours later, the amount of L-tartaric acid obtained was determined. The results are given in Table 10. The addition of the surfactants markedly accelerated the rate of the reaction.

Table every 10 concentration Educated Average enzymatic Surface active agents Tartaric acid activity (Weight%) (g / l) (g tartaric acid / (Units/ Ii. g cells ■ hour) ml broth) *) 1 _ 6.48 1.10 34.7 No encore 0.1 20.6 3.48 110 Alkyl polyoxyethylene phosphorus 0.05 26.4 4.46 141 acid ester (DDP-8) 0.025 38.4 6.49 205 0.0125 28.8 4.87 154

20th

FortsL't / ung

Surface active agents

Concentration of tartaric acid formed

(% By weight) (g / l)

Average enzyme activity

(g tartaric acid / (units / g cells hour) ml broth) *)

Sodium laurate

Cetyltrimethylammonium bromide

Lauryl pyridinium chloride

Polyoxyethylene sorbitan fatty acid ester (Tween-80)

Polyoxyethylene / polyoxypropylene block copolymer (Pluronic L-61)

Polyoxyethylene alkyl ether) (Bl-9EX)

0.1

0.05

0.025

0.0125

0.1

0.05

0.025

0.0125

0.1 0.05 0.025 0.0125

0.1 0.05 0.025 0.125

0.1 0.05 0.025 0.0125

0.1 0.05 0.025 0.0125 4.8 7.4 13.4 6.7 6.2 4.8 50.6 16.8 37.7 35.0 33.1 34.3 13.4 12.7 12.5 13.0 11.8 12.7 15, o 21.6 71.7 60.3 53.6 51.8

0.81 1.25 2.26 1.13 1.05 0.81 8.55 2.84 6.37 5.92 5.59 5.80 2.26 2.15 2.11 2.20 1.99 2.15 2.64 3.65 12.12 10.19 9.06 8.75

25.5 39.4 71.3 35.6 33.1 25.5

270 89.6

201

187

176

183 71.3 67.8 66.5 69.4 62.8 68 ,! 83.2

115

382

321

286

276

*) The enzyme activity per ml of the culture broth over a period of 5 hours.

Example 5 Propylene glycol 1% NH ₂ CONH ₂ 03% KH ₂ PO ₄ 0.15% Na ₂ HPO ₄ 0.15% MgSO ₄ · _{7H 2} O 0.05% FeSO ₄ · _{7H 2} O 0.001% CaCl ₂ · _{2H 2} O 0.001% MnSO ₄ · 4 H ₂ O 0.0002% Yeast extract 0.02% pH '/, Ο.

Was sterilized, a culture medium containing the above ingredients with the exception of urea, for 15 minutes at ⁰ C and 12I added a filtered and sterilized urea solution separately prepared. The pH of the mixture was adjusted with sodium hydroxide to ensure that the culture medium finally contained the above composition and pH.

50 ml of the culture medium were charged to 50 ml shake flasks. Nocardia tartaricans nov.sp. was vaccinated. (ESI-T strain; FERM P-2882; ATCC 31191) on a loop ^! n the culture medium and incubated at 30 ° C for 100 hours.

After the cultivation, the cells were collected by centrifugal separation and suspended at a concentration of 5 g / l as dry cells in a culture medium of the same composition except that propylene glycol was not contained, and sodium cis-epoxysuccinate was added in one Add a concentration of 0.2 mol / l. The suspension was left at 30 ^° C. for 20 hours in order to induce an enzyme. The cells were again collected by centrifugal separation and suspended in a

■ to concentration of 10 g / l in the form of dry cells in ml of a solution of 0.7 mol / l sodium cis-epoxysuccina « The suspension was cooled to 0 to 5 ° C and subjected to ultrasound oscillation treatment (20 kHz, 60 W., Minutes).

After the ultrasonic oscillation treatment were the cell sediments were removed and each of those shown in Table 11 was added to the supernatant Surfactants added in the concentration given in the table. The addition

5 (i of the surfactants accelerated the reaction rate noticeably.

Table 11

5 surfactants Conc Educated tration L-wine acid (Wt .-%) (g / l) No encore 12.0 Sodium laurate 0.1 12.5 0.05 13.2 0.025 13.8 0.0125 20.5 i alkyl polyoxyethylene phosphor 0.1 49.6 phosphoric acid ester (DDI-3) 0.05 52.0 0.025 54.0 0.0125 28.0

21

Surface active agents Conc Educated tration !.-Wine acid (Wt%) (g / l) Alkyl phosphoric acid ester 0.1 13.2 sodium salt (SLP, a Pro 0.05 15.5 product of Nikko Chemikals) 0.025 20.8 0.0125 22.0 Polyoxyethylene sorbitan 0.1 46.8 fatty acid ester (Tween-80) 0.05 48.0 0.025 47.2 0.0125 25.6 Polyoxyethylene / polyoxy- 0.1 42.4 propylene block mixed poly 0.05 21.2 merisar (Plnrnnic 1.-61) 0.025 16.0 0.0125 14.8 Polyoxyethylene alkyl ethers 0.1 42.5 (BL-4.2) 0.05 40.5 0.025 40.0 0.0125 39.7

Example 6

Propylene glycol
Ammonium sulfate
KH ₂ PO ₄
Na ₂ HPO ₄
MgSO ₄ · _{7H 2} O
FeSO ₄ · _{7H 2} O
CaCl ₂ -2H ₂ O
MnSO ₄ · 4 H ₂ O
Yeast extract
pH

1%

0.3%

0.2%

0.2%

0.1%

0.001%

0.003 '/ ..

0.0004%

0.02%

7.0.

A culture medium of the above composition was sterilized at 121 ° C. for 15 minutes and brought 50 ml of the same in 500 ml shake flasks. Man vaccinated Nocardia tartaricans nov. sp. (ES3-T strain; FERM P-3374; ATCC 31190) on a loop in the Culture medium and cultivated for 24 hours at 30 ° C Then filtered and sterilized sodium cisepoxysuccinate was added until a concentration was achieved of 0.6%. The cultivation was carried out for another 24 hours and a cell concentration was reached of 6.93 g / l.

Separately, a 10-liter fermentation vessel was charged with a culture medium of the same composition as above, which had been sterilized at 121 ° C. for 15 minutes, and 0.1% of Pluronic L-61 as a defoaming agent was added. Seeding 5 bottles of the above culture broth and incubated at 30 ⁰ C, while air at a Geschwindigkeil of 5 i / iTiin. iimGurCiiiCiiCiC Una g; c ■ »jrirgcscrivviriGig speed set to 600 revolutions per minute. After continuing the cultivation for 7 hours and after the cell concentration had reached 1.15 g / l, a separately prepared, filtered and sterilized solution of sodium cis-epoxysuccinate was added until a concentration of 1.0% was achieved. Continued for 5 hours. The final cell concentration was 4.4 g / l.

Each of the surfactants shown in Table 12 was added to 10 ml of the culture broth in the concentration given in Table 12. A further 10 ml of a solution of 2 mol / l was added Sodium cis-epoxysuccinate (pH S, 0) was added and the reaction was carried out at 37 ° C. with shaking.

2.5 hours later, the amount of L (+) -tartaric acid formed was determined. The results are in Table 12 given. The addition of the surfactants showed a marked accelerating effect Effect on reaction speed.

Table 12

I -Wpin-

(Wt .-%)

acid

(mg)

riiirrhcrhn

enzymatic activity (unit / ml broth)

No encore
Alkyl polyoxyethylene phosphoric acid ester (DDP-8)

Sodium laurate

Cetyltrimethylammonium bromide

Lauryl pyridinium chloride

Polyoxyethylene sorbitan fatty acid ester
(Tween-80)

0.1

0.05

0.025

275 1155 1397 2024 1705

528

990 1122

935

275 220 1672 1430 1804 1694 1683 1694 1111 1122 1155 1144

73.3 308 373 540 455 141 264 299 249

733 58.7

446

381

481

452

449

452

296

299

308

305

I I'll ·, (. '! / Iini! Surface active agents

Polyoxyethylene / polyoxypropylene-Bi «., I.kmischpolymerisat (Pluronic L-61)

Polyoxyethylene alkyl ether (BL-9EX)

26 05 921 24 Average Educated I. Wine enzymatic Con / cntralion acid activity (Einh mi broth) (mg) 235 (Wt .-%) 880 241 0.1 902 252 0.05 946 373 0.025 1397 777 0.0125 2915 669 0.1 2508 461 0.05 1727 375 0.025 1408 0.0125

Example 7

Nocardia tartaricans nov. sp. (Mute _> < ES3-T; FERM 3374; ATCC 31190) in a 10 liter fermentation vessel at 30 ° C for 48 hours. The composition of the medium was as follows:

Propylene 1% urea 0.3% KH ₂ PO ₁ 0.2% Na ₂ HPO ₄ 0.2% MgSO, · 7H ₂ O 0.1% FeSO ₁ · _{7H 2} O 0.001% CaCl ₂ · 2 H ₂ O 0.003% MnSO ₄ · 4 H ₂ O 0.0004% Yeast extract 0.05% PH 7.0.

Cis-epoxysuccinate (0.5%) was added during the r> logarithmic growth phase. The cells were harvested and disrupted by ultrasonic oscillation. The obtained crude cell-free extract contained 11,300 mg of protein and had a total enzymatic activity of 200,000 units. The crude cell-free extract was treated with streptomycin sulfate and the same mixture was centrifuged. The ijKi »rcii» hAttrio Toil tiriit-rjo tv * ^ ^rnfr ! '"* N! ^Il ! T !? ¹¹ ! I5t fractionated and a fraction of 50 to 60% saturation was obtained. This fraction was against a 0.05 mol / l J ₁ phosphate buffer at a pH of 7.0, which contained 5 χ 10- ^J mol / l mercaptoethanol, dialyzed and placed in a column with three-dimensionally crosslinked polysaccharide with diethylaminoethyl groups (DEAE Sephadex A-50), which was previously with the same buffer The column was washed with the equilibrating buffer. The enzyme was treated with a 0.05 mol / l phosphate buffer (pH 7.0) containing 03 mol / l NaCl and 5 10 ^-3 mol / l The active fractions were collected, their total volume was 1250 ml. These fractions contained 832 mg of protein and had a total enzymatic activity of 65,400 units. This enzyme solution was concentrated by ammonium sulfate precipitation

The precipitate was dissolved in a small volume of a 0.05 mol / l phosphate buffer and against the dialyzed the same buffer. The dialysate was fractionated with ammonium sulfate. One collected Fraction from 50 to 55% saturation and subjected it to gel filtration on three-dimensionally crosslinked Polysaccharide (Sephadex G-200). The enzyme was eluted with a 0.05 mol / l phosphate buffer (pH 7.0)

The active fractions were collected and concentrated by ammonium sulfate precipitation. The precipitation was dissolved in a small volume of phosphate buffer and again in the one described above Way subjected to a Sephadex G-200 treatment.

F i g. 1 shows an elution pattern of the second gel filtration on Sephadex G-200. The factions were collected 36 to 42 until their total volume reached 37.0 ml. This purified enzyme solution contained 19.9 mg of protein and had a total enzymatic activity of 18,000 units.

A mixture consisting of 0.4 ml of the purified thus obtained was incubated for 16 hours at 37.degree Enzyme solution, 7.6 ml of a 0.05 mol / l tris-HCl buffer (pH 8.5) and 2.0 ml of 2 mol / l sodium cis-epoxysuccinate. 615 mg of L (+) -tartaric acid formed in the resulting reaction mixture in a yield of 100%.

Example 8

Nocardia tartaricans nov. sp. (ES3-T strain; FERM 3374; ATCC 31190) in a 10 liter fermentation vessel containing the medium described in Example 7, at 30 ° C. for 48 hours. After an ultrasonic oscillation treatment, the crude cellular »· ο | α PvtroLrt with F) FA.F- ^ AnhoHpv A-SO tiwz 7iivnr with 0.05 mol / l phosphate buffer containing 5-10" ³ mol / l mercaptoethanol This DEAE-Sephadex was put in a column (6.0 53 cm), the column was washed with the equilibrating buffer, and the enzyme was with the same buffer containing 0.3 mol / l An elution pattern is shown in Fig. 2. Fractions 10-20 were collected and concentrated by ammonium sulfate precipitation Protein and had a total enzymatic activity of 121,200 units, centrifuged

A mixture consisting of 10 ml of these was incubated for 15 hours at 37 ° C. with gentle stirring Enzyme solution, 50 ml of distilled water and 50 ml of 2 mol / l sodium cis-epoxysuccinate, its pH to 8.5 adjusted with 4N sodium hydroxide. There were 15.1 g of L (+) - tartaric acid in a yield of 100% obtained in the reaction mixture obtained.

Example 9

A 10 liter fermentation vessel was also charged 5 1 of a culture medium of the same composition

as in Example 1 and added 0.1% Pluronic L-61. Nocardia tartaricans nov. sp. (Strain ES3 T; FERM P-3374; ATCC 31190) into the culture medium and cultured at 30 ⁰ C, while air at a rate of 5 l / min, bubbling, and adjusting the stirring speed to 600 revolutions per minute. After reaching a cell concentration of 1.0 g / l, sodium cis-epoxysuccinate was added to achieve a concentration of 0.5% by weight. After culturing for 35 hours, the cell concentration reached 4.6 g / l.

The enzymatic activity of this culture broth was determined by the following method.

5 ml of a mixture consisting of 1.0 ml of the culture broth, 2.9 ml of phosphate buffer with a pH of 8.0, 0.1 ml of 5% BL-42 and 1.0 ml of 1 mol / l sodium cisepoxysuccinate were added with shaking at 37 ^° C. for one hour and determined the amount of tartaric acid obtained.

The enzymatic activity per ml of culture broth determined by this method was 613 Units.

41 of the culture broth were transferred to a separate 20 L reactor and 4 L of one was added Solution of 1 mol / l sodium cis-epoxysuccinate added and reacted at 37 ° C. with stirring. One added and according to the consumption of sodium c'sepoxysuccinate 2 mol / l cis-epoxysuccinate. the The total amount of 2 mol / l cis-epoxysuccinate was 8 1. After 20 hours the concentration reached the L-tartaric acid in the reaction mixture 184 g / l. the The amount of tartaric acid formed in If I reaction mixture was 2.94 kg.

Gypsum (CaSO ₄ · 2 H ₂ O) was added to the reaction mixture in an amount of 3.8 kg, and the resulting calcium tartrate slurry was thoroughly washed with water and then separated by filtration. The separated calcium tartrate was suspended in water and equimolar sulfuric acid was added. The resulting gypsum precipitate was separated off by filtration, and the precipitate was washed until it was completely free from V / anic acid. The tartaric acid solution thus obtained was concentrated by heating to precipitate the remaining calcium sulfate, and the gypsum was again separated by filtration. The concentration of tartaric acid in the solution was adjusted to about 50% by weight, and the solution was decolorized with activated charcoal. The solution was passed through a cation exchange column and an anion exchange column in turn. The purified aqueous tartaric acid solution was concentrated by heating to obtain 2.8 kg of purified L-tartaric acid. A value of + 12.81 ° was found as the rotation value for the purified aqueous tartaric acid solution [λ] (c = 21), H _{: O).} In contrast, tartaric acid with a special, commercially available reagent quality had a value for [λ] (C = 20. HiO) of + 12.84 ³ . The rotation value of tartaric acid, as found in the literature, is + 12.0 °.

Here

Claims (2)

Claims; 1. A process for the enzymatic production of L-tartaric acid from cis-epoxysuccinic acid, characterized in that a microorganism Nocardia tartaricans nov. sp. or the bringing of this isolated enzyme with cis -epoxysuccinic acid or their organic or inorganic salts in an aqueous medium at 20 to 60 ⁰ C and a pH value of 6 to 10 in contact 2. The method according to claim 1, characterized in that that the conversion is carried out in the presence of a surfactant