CS245343B1 - Process for preparing p-diphenol oxidase - Google Patents

Abstract

The invention relates to a method of preparing the p-diphenol oxidase enzyme. The invention solves a method of preparing p-diphenol oxidase by cultivating the wood-destroying fungus Fomes fomentarus. The stated purpose is achieved by cultivating the isolated Fomes fomentarus strain submerged on a substrate with D-glucose, casein minerals, thiamine, xylidine or o-toluidine in a temperature range of 20 to 40°C. The invention is used in medicine, clinical biochemistry and toxicology, and in analytical chemistry for the identification of pollutants in the environment.

Description

(54) Preparation of p-diphenol oxidase 2

The invention relates to a process for preparing the enzyme β-diphenol oxidase.

The invention solves a process for the preparation of p-diphenol oxidase by cultivating the wood-destroying fungus Fomes fomentarus. This is accomplished by culturing the isolated Fomes fomentarus strain submerged on a D-glucose, casein mineral, thiamine, xylidine or o-toluidine pod in a temperature range of 20 to 40 ° C.

The invention has applications in medicine, clinical biochemistry and toxicology, and in analytical chemistry to identify pollutants in the environment.

The present invention relates to a process for the preparation of p-diphenol oxidase (p-diphenol oxigen oxidoreductase EC 1.10,3.2).

The preparation of p-diphenol oxidase is usually based on microscopic fungi, e.g. from Aureobasidium pullulans (Risssch R., Liese W., Berndt H .: Arch. Microbiol. 67, 28 (1969)) or Schizophyllum commune (Leonard P.J., Phillips LE: J. Bacteriol. 7, (1973)) . In isolation and purification of the enzyme, the proteins obtained from the fermentation broth are usually fractionated by ammonium sulfate precipitation, organic solvents or by affinity techniques. The disadvantages of these processes are their aggressiveness, the difficulty of the materials used, especially in the case of affinity preparation.

These drawbacks are solved by the process according to the invention which uses wood-destroying fungi of the Fomes species of Basidiomycetes to prepare p-diphenol oxidase. The principle of the invention is that the wood decaying fungus Fomes fomentar (Linne ex Fries) Kickx is cultivated submerged on a base composed of 10 to 30% by weight. parts of D-glucose, 1 to 3 parts of casein, 0.5 to 12 parts of KH 2 PO 4, 0.1 to 0.3 parts of NaH 2 PO 4. 12 H2O, 0.1 to 1 part MgSO3, 0.001 to 0.003 part CuSO4, 0.0005 to 0.002 part ZnSO4, 0.0005 to 0.002 part MnSOa, 0.005 to 0.015 part FeSO4, 0.005 to 0.015 part CaCl2, 0.00002 to 0 00008 parts of tlamine, 0.1 to 0.001 parts of xylidine or o-toluidine in 1000 parts of water, where the cultivation is carried out at 20 to 40 ° C for 250 to 750 hours while aerating by blowing air at a rate of 0.05 to 0.2 volume per minute and subsequent isolation of the released β-diphenol oxidase as the only extracellular protein by salting out by precipitation with organic solvents or other known methods.

The advantage of the proposed process is the ease of preparation of β-diphenol oxidase as well as the fact that on the culture medium of the above composition, the fungus Fomes fomentarus releases the subject enzyme into the extracellular environment as the only enzymatically active protein.

Example 1

The fungus Fomes fomentarus (Linne ex Fries) Kickx strain 191 deposited at the Wood Decay fungus culture collection of the State Wood Research Institute in Bratislava was cultivated in ten Erlenmayer flasks (2 L volume) containing 400 ml of culture medium composed of 10 g D-glucose, 1 g casein, 0.5 g KH2PO4, 0.1 g NaH2PO4.

. 12 H2O, 0.1 g MgSO4, 1 mg CuSO4, 5 mg ZnSO4, 0.5 mg MnSO4, 5 mg FeSO4, 5 mg CaCl2, 0.02 mg thiamine and 0.001 g o-toluidine in 1,000 ml water for 750 hours at 20 ° C on a 20 rpm cultivator at 25 cm amplitude.

After separation of the mycelium by centrifugation at

500 g for 15 minutes the supernatant obtained was precipitated with ammonium sulfate until the solution was saturated. The precipitate obtained, corresponding to 4 L of culture broth, contained 1.2 g of 24-ukat p-diphenol oxidase protein. Example 2

The procedure is as in Example 1 except that the strain Fomes fomentarus 22 of the same collection as in Example 1, which was cultivated submerged in a 20 L fermenter, was used on a culture medium of 30 g D-glucose. g casein, g KH2PO4, 0.3 g Na2HPO4.12 H2O, 1 g MgSO4, 3 mg CuSO2, 2 mg ZnSO2, 2 mg MnSO4, 15 mg FeSO4, 15 mg CaCl2, 0.08 mg thiamine and 0.1 g xylidine in 1 liter of soil at 40 ° C, for 250 hours under aeration at a rate of 0.05 l of air per minute per liter of culture medium.

After removal of the biomass, the supernatant obtained in 20 l was precipitated with 96% ethanol in a 1: 5 by volume ratio. The obtained precipitate contained 6.5 g of protein containing exclusively 200 .mu.l of p-diphenol oxidase.

Example 3

The procedure is as in Example 2 except that the culture medium is aerated with sterile air at a rate of 0.2 L per liter of culture medium per minute. After treatment as in Example 2, 6.4 g of a protein containing exclusively p-dlphenol oxidase with an activity of 216 µg were obtained.

The invention can find wide application in medicine, clinical biochemistry and toxicology and in analytical chemistry to identify pollutants in the environment.

Claims (1)

SUBJECT Process for the preparation of β-diphenol oxidase, characterized in that the wood decaying fungus Fomes fomentarus (Linne ex Fries) Kickx is cultivated submerged on a base composed of 10 to 30 wt. parts of D-glucose, 1 to 3 parts of casein, 0.5 to 2 parts of KH 2 PO 4, 0.1 to 0.3 parts of NaHPCU. . 12 H2O, 0.1 to 1 part MgSCU, 0.001 to 0.003 part CuSO3, 0.0005 to 0.002 part ZnSOd, 0.0005 to 0.002 part MnSCU, 0.005 to 0.015 part FeSCH, 0.005 to 0.015 part CaCl2, 0.00002 to 0 00008 parts of thiamine and 0.001 to INVENTION 0.1 parts by volume of xylidine or o-toluidine in 1000 parts of water, cultivating at 20 to 40 ° C for 250 to 750 hours, while aerating by blowing air at a rate of 0.05 to 0.2 parts by volume per minute and subsequent isolation released β-diphenol oxidase, as the only extracellular protein, by salting out, precipitation with organic solvents, or other conventional procedures.