CS239033B1 - Method of fermentation production of thiaminase I-thiamine: base of 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2 - Google Patents

Abstract

The invention relates to aerobic cultivation of the microorganism Bacillus Tbiominolyticus Kuno, which is carried out in a fermentation medium containing, as a stimulator of thiaminase I production, yeast autolysate in an amount of 0.2 to 30 g/1. The production of thiaminase I can be further increased by adding calcium ions in an amount of 0.01 to 50 g/1. The obtained thiaminase I can be used even in its raw state for the determination of thiamine.

Description

The invention relates to a fermentation preparation of the enzyme thiaminase

I-thiamine base 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2., which enzyme can be advantageously used for the determination of vitamin B^ in biological or clinical material, in food or pharmaceutical products or for scientific purposes.

Vitamin B^, i.e. thiamine and some of its derivatives, plays a very important role in human nutrition and its deficiency leads to serious nervous disorders (for example, beriberi disease caused by severe vitamin B^ deficiency or Wernicke's encephalopathy caused by thiamine deficiency in alcoholics). Food technologies used at present lead to suboptimal concentrations of vitamin B^ in human nutrition in most countries, and therefore in many countries the dose of this vitamin is supplemented with synthetic thiamine added to flour, pastries, milk, etc. For these reasons, the determination of total thiamine in individual food raw materials and products as well as in clinical material is very important and is receiving increasing attention. The concentrations of this vitamin determined are mostly in fractions of mg/g of the analyzed material, and therefore the fluorometric thiochrome method, which achieves this sensitivity, is generally used for the determination of thiamine. However, this method requires laborious and lengthy isolation of thiamine, which is fraught with considerable error. Also, the preparation and activation of Dekals, i.e. the most used sorbent for this isolation, is quite laborious and time-consuming. Microbiological methods for determining this vitamin are burdened with even greater error and fail completely in some materials.

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- 2 The laboriousness of the thiochrome method can be reduced by the use of the enzyme thiaminase I (thiamine:base 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2.), which specifically cleaves thiamine and binds its pyrimidine component to a base (e.g. pyridine) added to the reaction medium. This makes it possible to detect non-thiochrome fluorescence in the analyzed samples, so that the thiochrome analytical method does not require the laborious and lengthy isolation of thiamine from these samples. C.A.Kennedy and B.V.McCleary (Analyst 106« 344, 1981) recommend for this purpose thiaminase I isolated from the leaves of carp plants found in Australia (e.g. Marsilea drummondii, Marsilea augustifolia, Marsilea mutica, Pteridium asculentum, Cheilanthes siebri). For these purposes, this enzyme must be obtained from a plant extract by adsorption onto DEAE-cellulose and the eluate concentrated by vacuum distillation and purified by repeated ultrafiltration and adsorption.

Another source of thiaminase I may be the bacterium Bacillus thiaminolyticus Kuno, which produces it in a fermentation medium. H.A. Douthit and R.L. Airth (Arch. Biochem. Biophys.

113. 331, 1966) propose for the production and isolation of thiaminase I for scientific purposes an inoculum of Bacillus thiaminolyticus from a rich nutrient medium (infusion from brain and heart) and a fermentation medium containing, in addition to the usual sources of carbon, nitrogen and minerals, a significant amount of glutamic acid and thiamine hydrochloride in a pure state. Bacterial thiaminase is isolated for analytical purposes by precipitating the enzyme from the culture liquid using solid ammonium sulfate with subsequent purification. The obtained enzyme preparation is stored at -20 °C.

The disadvantages of the expensive isolation of thiaminase from plant material and the expensive nutrient medium used for the production of bacterial thiaminase are eliminated in the method for the production of thiaminase according to the invention. The essence of the invention lies in the fact that the production strain Bacillus thiaminolyticus Kuno, preferably the strain CCM 3599 of the Czechoslovak Collection of Microorganisms in Brno, is multiplied in a medium containing as production stimulants

- 5 thiaminase dry yeast autolysate as a source of thiamine and additional nutrients in amounts of Ófl to $ g/1, leading to a further increase in yield

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thiaminase I. Cultivation was carried out under aerobic conditions for 12 to 46 h and this culture is used as an inoculum for aerobic production cultivation in liquid medium with the above composition at 50 to 58 °C for 12 to 72 h. Then the bacterial cells are separated from the fermentation liquid, preferably by centrifugation, and the clear liquid, which can be used as a crude preparation of thiaminase I for the determination of thiamine and its derivatives, is filled into suitable containers and stored at -15 to -20 °C. Enzyme activity remains preserved under these conditions for more than 6 months. To preserve this preparation, so that it can be stored even at temperatures above 0 °C /up to 50 °C/, common preservatives can be used, for example sodium benzoate to a final concentration of 0.2 to 0.5% benzoic acid in the enzyme preparation, in which the pH is then adjusted to 4.0 to 5.0 with hydrochloric or sulfuric acid. The stability of the preparation preserved in this way is more than 4 weeks at a temperature of 20 to 50 °C. To extend the stability, the crude enzyme preparation can be dried by lyophilization.

For special purposes, the crude enzyme preparation can be purified by isolating the enzyme from the clear liquid and purifying it by one of the common isolation procedures, for example, fractional ion precipitation with ammonium sulfate in combination with gel filtration, ion exchange chromatography and dialysis.

The yield of thiamine I using yeast autolysate and CaCl2 is increased approximately threefold compared to the medium described by H.A. Doutnit and R.L. Airth /Arch. Biochem. Biophys. 115, 351, 1966/, as is evident from comparative fermentation experiments carried out using the methodology given in Example 1 using different mediums /Tab. 1/.

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Table 1.

Thiaminase I activity in fermented centrifuged medium used soil Thiaminase activity ^+// % activity mg of decomposed thiamine/1 ml of enzyme preparation/10 minutes

Synthetic according to H.A.

Douthita, R.L. Airxna 1.4 100%

Synthetic e with the addition of dry M-Vasničný autolysate /5g/l/ 5.1 226% Synthetic e with addition dry autolysate /5g/l/ 4.0 293% and CaCl ₂ .6H ₂ 0 /2g/l/

+/ determined according to E.E.Edwin /Methode in isniymology/ /D.B.McCormick, L.D.Wrigirt eda./ 62, 113» 1979/ using radioactive /^C/ thiamine /4.48 μβ with a total activity of 332 nCi per 1 μΐ of centrifuged fermented medium· The reaction took place for 10 minutes at 40 °C and pH 6.4·

Example 1

A one-liter flask is inoculated with 150 ml of sterile nutrient medium containing per liter 18.0 g glycerol, 3.0 g urea, 5.0 g sodium citrate .2H ₂ 0, 3.0 g dry yeast autolysate, 1.0 g KH ₂ PO ₄ , 2.0 g T7a ₂ HPO ₄ .12H ₂ O, 2.0 g CaCl ₂ .6H ₂ 0, 1.1 g NaCl, 0.7 g MgSO ₄ .7H ₂ O, 0.03 g FeSO ₄ .7H2O and having a pH adjusted to 7.2 and 1.0 ml of a 24-hour shaking culture of Bacillus thiaminolyticue Euno CCM 3599 grown at 33 ± 2 °C on a medium of the same composition. Incubate with shaking for 66 h at 33 * 2 °C. Then the culture is cooled, the bacterial cells are separated by centrifugation, solid sodium benzoate is added to the clear supernatant to a final concentration of 0.2% benzoic acid /weight/volume/ and the pH of the supernatant is adjusted with HCl at a concentration of 1 mol/1 to 4.5 to 3.0. The crude enzyme preparation thus obtained is filled into suitable containers and stored in the cold, preferably at -13 to -20 °C. The activity of this- 5 239 033 ►

The activity of this preparation at pH 6.4 and a temperature of 40 °C in the presence of pyridine /with a final concentration of 14.1 (imol/ml/ is under optimal conditions, i.e. at zero concentration of the reaction product, 5 to 4 ucatal/mg of protein, while 1 ml of centrifuged fermented medium contains about 0.7 mg of proteins. The activity of this preparation remains preserved when stored at temperatures lower than -10 °C for more than 6 months at temperatures up to 50 °C for more than 4 weeks. For the determination of thiamxine with a concentration of 0.2 to 5.0 ug/g of diluted sample using the thiochrome method, it is most appropriate to use 0.2 ml of this preparation in a total volume of 4.2 ml of the reaction mixture and an incubation time of 50 minutes at 40 °C at pH 6.4 /buffered with citrate-phosphate buffer with a concentration of 0.05 mol/l/

Example 2

Similar fermentation broth as in Example 1 with a content of 1 g/l yeast autolysate and 4 £ CaC^/l is fermented and the cells are separated as in Example 1. Thiaminase I is isolated from the supernatant obtained from 10 to 15 culture flasks by the method described by J.L. Wittliff and R.L. Airthe /Method· in Enzymology /D.B.McCormick, L.D.Wright, eda./ 18 A, 254, 1970/ and the final enzyme solution is stored at -20 °C or dried by lyophilization and stored in the cold. The stability of the solution under these conditions is more than 6 months, the lyophilized preparation is stable for several years.

Example 5 ml of the nutrient medium with the composition given in Example 1 and inoculated with a culture of Bacillus thxaminolyticus CCM 5599 from a slant agar and incubated with shaking for 24 h at 55 - 2 °C. Pa* the entire volume is transferred to a laboratory fermenter containing 1,500 ml of sterile medium with the composition given in Example 1 and cultivated with aeration for 24 to 56 h at 54 ± 1 °C, after which the entire volume of the fermentation liquid is transferred to a fermenter containing 50 liters of nutrient medium with the same composition as in the previous stage and cultivated for 48 h at 54 - 1 °C with intensive

- 6 239 033 aeration. Then the fermentation liquid is cooled, the cells are separated in a flow centrifuge and the clear liquid is processed as described in Example 1 or 2 or dried by lyophilization. The activity of the fermented medium after centrifugation is

3.5 ukatal/mg protein, with the protein content in this medium being 0.6 mg protein.

Claims (2)

Process for the production of thiaminase I-thiamine: 2-methyl-4-aminopyrimidine-5-methenyltranferase base, EG 2.5,1.2, by aerobic cultivation of the production microorganism Bacillus thiaminolytieue KUno, characterized in that the cultivation is carried out in a fermentation medium containing as a thiaminase I stimulant, and qaan autolysate in an amount of 0.2 to 50 g / l, whereupon. the fermentation liquid is centrifuged and the eupernatant is preserved by a chemical or lyophilized. 2. A method according to claim 1, characterized in that: The method of claim 1, wherein the cultivation is carried out in a fermentation medium containing calcium ions in an amount of 0.0 / 5.0 to 5.0 g / l.