A strain of penicillium funiculosum km msu 433-producer of glucose oxidase and method for producing the oxidase

 

(57) Abstract:

The invention relates to the field of biotechnology. Get the glucose oxidase is used in medical diagnostics in the food industry. A strain of Penicillium funiculosum KM MSU 433 has a high glucose oxydase activity. Extracellular oxidase receive, by growing the strain in a medium containing as the sole source of nitrogen and potassium nitrate. The environment also includes sucrose, phosphate, magnesium sulfate and sources of trace elements. The proposed strain and method provide the output of extracellular glucose oxidase 140 - 150 u/ml of medium. 2 S. p. f-crystals, 2 tab.

The invention relates to the field of biotechnology, medicine and food industry, namely the method of producing the enzyme glucose oxidase. Glucose oxidase is widely used in medical diagnostics for the determination of glucose in blood, as well as in the food industry to remove residual oxygen in the juices, beer and hermetically packaged food products. The presence of impurities of oxygen causes damage to them when stored.

Renowned producers of extracellular glucose oxidase from fungi of the genus Penicillium.

In the author's certificate N 237 080 (1969) described studerade 0.5 to 0.7% of corn extract, 2% sucrose and 0.2% potassium nitrate. The obtained yield was 80 - 100 u/ml

Because of the great practical importance of glucose oxidase in different countries are searching for producers of glucose oxidase. Thus, the Italian researchers as a promising producer oxidase selected strain of Penicillium variabile P16 and developed a method of producing glucose oxidase by culturing it on selected authors medium with 8% glucose, 0.3% of peptone, 0.5% sodium nitrate, phosphate and chalk. Received oxidase 15 IU/ml (J. Appl. Bacter. 1993. V. 75, P. 369-372; Biotechnol. Appl. Biochem. 1995. V. 22. P. 169 - 178).

As the prototype should take the strain of Penicillium funiculosum G-15 and a method of producing glucose oxidase by culturing it on a medium containing (%): sucrose - 6,0, KNO3TO 0.8, KH2PO4- 0,1, MgSO4to 0.05, KCl - 0,05, FeSO47H2O - 0,00005, MnSO45H2O - 0,00026, CaCO3- 2,0, twin 85 - 0.3, gidrol - 0.1, extract of malt sprouts - 1,0 for obtaining glucose oxidase (20 u/ml (Mikhailova R. C., Szyszko J. F., Jasenko M. I., "Influence of nutrient medium components on the formation of extracellular glucose oxidase from Penicillium funiculosum G-15". Proc of the Academy of Sciences of Belarus. Ser. biologist. 1998. N 2. S. 57-60).

The present invention is to increase the output of the glucose oxidase and the development of standard conditions for achieving it.

The problem is solved

1) using the newly selected by the authors of the strain Penicillium funiculosum KM MSU 433 - producer of glucose oxidase, capable of synthesizing glucose oxidase in the absence of complex organic substances uncertain;

2) the development of a new method of producing extracellular glucose oxidase, involving the cultivation of producing microorganism Penicillium funiculosum on a nutrient medium containing sucrose, potassium nitrate, potassium dihydrophosphate, magnesium sulfate, sources of trace elements such as manganese sulfate, and distilled water, characterized in that environment further added potassium phosphate, from sources of trace elements using the sulphates of zinc and copper (II), cobalt chloride, sodium molybdate and boric acid, with the following ratio of components, wt.% (in terms of crystallohydrate form of salts):

Sucrose - 8,0-12,0

Potassium nitrate - 1,0-4,0

Potassium dihydrophosphate - 0,15-0,4

Phosphate potassium - 0,15-0,4

Magnesium sulfate - 0,05

Distilled water - Rest

with the addition of Mick the edit (II) - 0,02-0,1

Manganese sulfate - 0,5-3,0

The cobalt chloride - 0,04-0,1

Molybdate sodium - 0,02-0,1

Boric acid is 0.4-1.0

The selection of the glucose oxidase from the culture fluid are known methods by precipitation of the enzyme with ammonium sulfate, further purification on Sephadex and liofilizatsii coolant. Go active. beach. and the microbe. 1978, so-14, N 3, S. 377-382; Biotechnol. Appl. Siochem. 1995, v. 22, p. 169-178).

A strain of Penicillium funiculosum KM MSU 433 acselectionsetall M. B. Kupletskaya as more active synthesis of glucose oxidase clone from the original strain of Penicillium funiculosum, which was outlined in A. C. Karakulam in 1988 from infected mushrooms paint.

A strain of Penicillium funiculosum KM MSU 433 belongs to section Biverticillat symmetrica. Conidia are on the air vultures, short, bear bunk symmetric brush. Conidia are oval. Is an aerobic organism is not pathogenic. To maintain the strain in the best environment is wort agar 4-5oThe balling with an initial pH of 6-6,2. The temperature of cultivation 25-26oC. On wort-agar forms a dense bloom of white mycelium, the surface of which at least sporoobrazovanie becomes gray-green in color. For the best sporoobrazovanie necessary diffused light.

A strain of Penicillium funiculosum KM MSU 433 I had food, does not contain complex organic substances directly composition.

A strain of Penicillium funiculosum KM MSU 433 deposited in the collection of microorganisms of the Department of Microbiology, biological faculty, Moscow state University under the number 433.

Example 1

A strain of Penicillium funiculosum KM MSU 433 grown on nutrient composition (%):

Sucrose - 12,0

KHO3- 3,0

KH2PO4- 0,15

K2HPO43H2O - 0,25

MgSO47H2O - 0,05

Tap water - the Rest

The initial pH of 6.9

Wednesday poured into 50 ml conical flask with a volume of 750 ml.

Sterilized at 1 ATM.

Seed grown in test tubes beveled wort-agar 4-5oOf balling. Sowing produce spores of the fungus, using well sysparavalues culture 3-8 weeks of age. Spores washed from the surface of the agar with sterile 0.1 M phosphate buffer, prepared with distilled water. Planted bulb is grown on the rocking chair with 180 rpm or when 15-26oC.

After 5 days the mycelium is separated from the medium by filtration through a paper filter. The filtrate determine the amount of glucose oxidase colorimetric method with on-DIAA 1 unit of glucose oxidase is assumed that the amount of enzyme, which decomposes 1 micromoles of D-glucose in 1 min at pH 7 and 25oC.

In the culture fluid detect the glucose oxidase in the number of 30 - 35 u/ml

When replacing in the above environment tap water to distilled yield of glucose oxidase is only 1 - 2 u/ml. It proves necessary trace elements for the synthesis of glucose oxidase.

Example 2

A strain of Penicillium funiculosum KM MSU 433 grow in example 1, but using a nutrient medium prepared with distilled water, the following composition (in %):

Sucrose - 8,0

KNO3- 3,0

KH2PO4- 0,15

K2HPO43H2O - 0,25

MgSO47H2O - 0,05

Distilled water

The initial pH of 6.9

The environment is used with the addition of trace elements in various combinations with or without them.

After 5 days of cultivation in various embodiments, find the following quantities of extracellular glucose oxidase (PL. 1).

Presented in table. 1 shows the necessity of the six trace elements for the synthesis of glucose oxidase.

The dramatic effect of trace elements affects the synthesis of glucose oxidase and does not affect roam Penicillium funiculosum KM MSU 433 grow in example 2, but in a nutrient medium prepared with distilled water and containing the following amounts of trace elements (mg %): 0,5 ZnS047H2O; 0,05 CuSO45H2O; 1,0 MnS047H2O; 0,04 CoCl26H2O; 0,02 Na2MoO42H2O; 0,4 H3IN3- contribute different amounts of potassium nitrate. By increasing the concentration of potassium nitrate yield of extracellular glucose oxidase increases (table. 2).

Source of sucrose in the media served food sugar.

The method can be used for other strains of Penicillium funiculosum, we have the producers of the oxidase of the species Penicillium funiculosum show the following activity on the proposed method:

Pen. funiculosum C-140 u/ml Pen. funiculosum 50K - 70 u/ml Pen. funiculosum 238K - 60 u/ml Pen. funiculosum C - 65 u/ml Pen. funiculosum 476K - 65 u/ml

All strains with different degrees of activity yielded by the proposed method of glucose oxidase in several times more than the entrance of glucose oxidase in the prototype (20 u/ml). The maximum activity is observed within the proposed method.

Thus, the proposed method can improve the output glucotoxicity and standardize the conditions of its implementation.

1. Stasi, providing for the cultivation of producing microorganism Penicillium funiculosum on a nutrient medium containing sucrose, potassium nitrate, magnesium sulfate, sources of trace elements such as manganese sulfate, and distilled water, characterized in that environment further added potassium phosphate, from sources of trace elements using zinc sulfate and copper (II), cobalt chloride, sodium molybdate and boric acid, in the following ratio, wt.% (in terms of crystallohydrate form salts:

Sucrose - 8,0 - 12,0

Potassium nitrate - 1,0 - 4,0

Potassium dihydrophosphate - 0,15 - 0,4

Potassium dihydrophosphate - 0,15 - 0,4

Magnesium sulfate - 0,05

Distilled water - Rest

with the addition of sources of trace elements, mg wt.% (in terms of crystallohydrate form of salts):

Zinc sulfate - 0,4 - 1,0

Sulphate of copper (II) - 0,02 - 0,1

Manganese sulfate - 0,5 - 5,0

The cobalt chloride - 0,04 - 0,1

Molybdate sodium - 0,02 - 0,1

Boric acid - 0,4 - 1,0

 

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FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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