RU2054479C1 - Method of preparing amylolytic and proteolytic enzyme complex - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: for preparing complex enzymatic preparation containing acid protease and α-amylase the strain-producer BKMF-55 is used. Strain is grown on agarized soybean-glucose medium with sodium sulfate and cobalt chloride. After 5-7 days liquid fermentation medium is sown with fungus conidia. Medium has the following composition, wt.-%: glucose 3.0-3.2; starch 3.0-3.2; corn extract 1.2-1.25 (as dry matter); soybean meal 2.0-2.2; ammonium sulfate 0.20-0.25; calcium carbonate 0.25-0.30, and tap water - the rest up to 100%. Subsurface cultivation is carried out for 3 days. After fermentation end proteolytic activity of cultural fluid at pH 3.0 is 10-12 U/ml, amylolytic activity - 7-8 U/ml. Isolation of enzyme complex from cultural fluid filtrate is carried out by precipitation with ethanol at 5 ± 1 C followed by lyophilization of enzyme solution. EFFECT: improved method of enzyme complex preparing. 3 tbl

Description

 The invention relates to the medical industry, in particular to the microbiological synthesis of enzymes for medical purposes.

A known method of producing a complex of proteases and α-amylase using Aspergillus oryzae VKPM F-369 by obtaining first mycelial seed, then deep cultivation at a temperature of 27-28 ^about C for 4-5 days. In this method, it is not indicated which complex of proteases produces the Aspergillus oryzae F-369 strain, at which pH the complex proteolytic activity is determined [2] In an article by the same authors published in 1989 in the journal Microbiology (vol. 58, c. 3, pp. 475-479), it is indicated that the total proteolytic activity of various populations of Aspergillus oryzae, including population N 32 [2], was determined in the culture fluid using the modified Anson method with hemoglobin at pH 4.7 and expressed in mg tyrosine in ml

 The complex of proteases with a pH optimum of 4.7 known method can be used in the leather industry or in agriculture.

 A known method for producing the enzyme preparation of amylorisin α-amylase P10 X [1] comprising culturing the strain Aspergillus oryzae 3 under surface conditions, extracting the enzyme with water, followed by its precipitation from the cell extract with ethyl alcohol and lyophilization of the wet cake. Based on amylorizin P10 X VVEZFP produces the medical preparation Oraza. In this method, using the surface cultivation of the producer Aspergillus oryzae-3, it is possible to obtain only one enzyme, α-amylase, while the acidic protease necessary for the breakdown of food protein in the stomach during digestive disorders (putrefactive dyspepsia) is not synthesized.

In the proposed method for producing a complex enzyme preparation, including culturing the producer Aspergillus oryzae followed by isolation of the enzymes by ethanol precipitation and lyophilization, the producer uses the Aspergillus oryzae BKMF-55 strain grown on an agarized medium of the composition, soy flour 1.0-1.2; glucose 2.0-2.2; sodium sulfate 0.05-0.06; cobalt chloride 0.00010-0.00015; calcium carbonate 0.20-0.25; agar agar 2.0; tap water up to 100 at a temperature of (27 ± 1) ^о С for 5-7 days.

Conidia of the fungus contribute to the fermentation medium of the composition, glucose 3.0-3.2; starch 3.0-3.2; corn extract 1.20-1.25; soy flour 2.0-2.2; ammonium sulfate 0.20-0.25; calcium carbonate 0.25-0.30; tap water up to 100% and deep culturing is carried out at a temperature of (27 ± 1) ^{° C} in Erlenmeyer flasks on a shaker (200 rev / min) for 3 days.

 The strain Aspergillus oryzae BKMF-55 was obtained from the All-Union Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms of the USSR Academy of Sciences.

 When deep cultured on a fermentation medium of the above composition, the strain Aspergillus oryzae BKMF-55 releases two enzymes, acid protease and α-amylase, into the culture fluid. At the end of the fermentation, the proteolytic activity of the culture fluid (for hemoglobin) at pH 3.0 is 10-12 GE / ml, amylolytic 8 AE / ml.

The differences of the proposed method in comparison with the known are:
the use of a new strain of Aspergillus oryzae BKMF-55, producing under certain conditions of cultivation of the 2 enzyme acid protease and α-amylase;
growing seed on an agarized medium composition, soy flour 1.0-1.2; glucose 2.0-2.2; sodium sulfate 0.05-0.06; cobalt chloride 0.00010-0.00015; calcium carbonate 0.20-0.25; agar agar 2.0; tap water up to 100% at a temperature of (27 ± 1) ^о С for 5-7 days.

cultivation of the producer in deep conditions on the composition medium, glucose 3.0-3.2; starch 3.0-3.2; corn extract 1.20-1.25; soy flour 2.0-2.2; ammonium sulfate 0.20-0.25; calcium carbonate 0.25-0.30; tap water up to 100% at a temperature of (27 ± 1) ^о С for 3 days.

 These differences allowed for 3 days. cultivation of the producer Aspergillus oryzae BKMF-55 to carry out the biosynthesis of two enzymes: α-amylase and acidic protease with optimum action at pH 3.0. The acidic protease contained in the preparation with a pH of 3.0 is necessary for the breakdown of food protein in various gastrointestinal diseases. It is this pH that is needed to digest protein foods in the stomach. The content of α-amylase in the resulting preparation is sufficient for the manifestation of its therapeutic effect.

 PRI me R 1.

 Stage I. Biosynthesis of the enzyme complex.

Fungus Aspergillus oryzae BKMF-55 is grown at a temperature (27 ± 1) ^{° C} for 5 days. on an agarized composition medium, Soya flour 1.0 Glucose 2.0 Sodium sulfate 0.05 Cobalt chloride 0.0001 Calcium carbonate 0.2 Agar-agar 2.0 Tap water Up to 100
Conidia of the fungus in the amount of 5 ml of the 1 billionth suspension in physiological saline is added to the flasks with 100 ml of the fermentation medium of the composition, Glucose 3.0 Starch 3.0 Corn extract 1.2
(dry
residue) Soy flour 2.0 Ammonium sulfate 0.20 Calcium carbonate 0.25 Tap water Up to 100 pH of the medium before sterilization 6.7-6.8
The microorganism is cultured under deep conditions in 750 ml Erlenmeyer flasks on a shaker (200 rpm) for 3 days.

 The culture fluid (1100 ml) is filtered through a paper filter and get 1000 ml of native solution with proteolytic activity of 10 GE / ml and amylolytic activity of 8 AU / ml.

 Proteolytic activity is determined using a 2% hemoglobin solution (pH 3.0) as a substrate according to the modified Anson method (Biochemistry 37, issue 1, 1972, p. 198-208). Amylolytic activity is determined using 1% starch solution (pH 5.0) according to FS 2639-89 as a substrate.

 Stage 2. Isolation of the enzyme preparation.

Isolation of the enzyme preparation from the native Aspergillus oryzae BKMF-55 solution is carried out by precipitation with 95% ethanol. All operations are performed at a temperature (5 ± 1) ^{° C,} since at higher temperatures decreases yield of enzymes, reducing the temperature below 4 ^{° C} did not give a positive effect.

 To 1000 ml of the native solution add 500 ml of ethyl alcohol to a final concentration of 30-35% and incubated for 2 hours

 The mixture is centrifuged at 6000 rpm, the ballast protein precipitate is discarded, and 1500 ml of ethanol are added to the supernatant to a final concentration of 60-70% and left for 10-12 hours to form a precipitate. The precipitate was separated by centrifugation at 6000 rpm, washed with 100 ml of ethyl alcohol, dissolved in 60 ml of water, centrifuged again, the precipitate was discarded, the enzyme solution was lyophilized. Get 3 g of the drug with a specific proteolytic activity of 2.0 GE / mg and amylolytic activity of 1.5 AU / mg.

Acid protease yield 60% for α-amylase 50%
The specific activity (acidic protease) of the obtained preparation is more than 4 times higher than the specific activity of the analogue of the foreign drug “Louiseme”, which has an activity of 0.48 GE / mg.

The specific amylolytic activity of the obtained preparation is 8–9 times higher than the specific activity of the Luizim preparation, which has an activity of 0.17 AU / mg, and corresponds to the amylolytic activity of P10X [1]
PRI me R 2.

 Stage 1. Biosynthesis of the enzyme complex.

 The enzyme biosynthesis with the strain Aspergillus oryzae BKMF-55 is carried out analogously to example 1. The composition of the agar medium, Soy flour 1.2 Glucose 2.2 Sodium sulfate 0.06 Cobalt chloride 0.00015 Calcium carbonate 0.2-0.25 Agar-agar 2 0 Tap water Up to 100.

 The composition of the fermentation medium, Glucose 3.2 Starch 3.2 Corn extract 1.25 Soy flour 2.2 Ammonium sulfate 0.25 Calcium carbonate 0.3 Tap water Up to 100.

 Obtain 1000 ml of native solution with proteolytic activity of 8 AU / ml.

 Stage 2. Isolation of the enzyme preparation. The selection of the enzyme preparation from the native solution is carried out analogously to example 1. Obtain 3.2 g of the drug with a proteolytic activity of 2.2 GE / mg and amylolytic activity of 1.4 AU / mg.

Acid protease yield 58.7% for α-amylase 56.0%
Embodiments of the proposed method are given in table. 1 and 2, 3.

 As can be seen from the data presented in table. 1 and 2, when using the II, III and IV variants of agarized and liquid fermentation media, the maximum level of enzyme formation is achieved.

The data presented in table. 3, indicate that the optimal cultivation temperature is (27 ± 1) ^o C.

Claims (1)

A METHOD FOR PRODUCING A COMPLEX OF AMYOLYTIC AND PROTEOLYTIC ENZYMES, which involves preparing the mycelial seed material of the producer Aspergillus oryzae on a medium containing soy flour, tap water, and subsequent deep cultivation of the obtained seed material in an enzymatic medium that reaches its maximum water level until the enzymatic activity reaches the fact that the producer uses the strain of the fungus Aspergillus oryzae VKM F-55, and the spore material is grown on a medium supplemented with containing glucose, sodium sulfate, cobalt chloride, calcium carbonate and agar-agar, in the following ratio, wt.%:
Soya flour - 1.0 - 1.2
Glucose - 2.0 - 2.2
Sodium sulfate - 0.05 - 0.06
Cobalt Chloride - 0.00010 - 0.00015
Calcium carbonate - 0.20 - 0.25
Agar Agar - 2.0
Tap Water - Else
at a temperature of 26 - 28 ^o C for 5 to 7 days, the cultivation of seed is carried out on an enzymatic medium additionally containing glucose, starch, corn extract, ammonium sulfate, calcium carbonate, in the following ratio, wt.%:
Glucose - 3.0 - 3.2
Starch - 3.0 - 3.2
Corn Extract - 1.20 - 1.25
Soya flour - 2.0 - 2.2
Ammonium sulfate - 0.20 - 0.25
Calcium carbonate - 0.25 - 0.30
Tap Water - Else
at a temperature of 26 - 28 ^o C for 3 days.

Images