The method of producing complex amylolytic and proteolytic enzymes

 

(57) Abstract:

The use of biotechnology. The invention is: to obtain a complex enzyme preparation containing an acidic protease and a-amylase, use the producer strain f-55, which are grown on agar soybean-glucose medium with sodium sulfate and cobalt chloride. After 5 to 7 days. conidia of the fungus inoculated liquid fermentation medium of the following composition, wt. %: glucose 3,0 - 3,2; starch 3,0 - 3,2; corn steep 1,2 - 1,25 (dry residue); soy flour to 2.0 - 2.2; ammonium sulphate 0,20 - 0,25; calcium carbonate 0,25 - 0,30; tap water the rest up to 100%. Deep cultivation should be performed within 3 days. At the end of fermentation proteolytic activity of the culture liquid at pH 3.0 is 10 - 12 GE/ml, amylolytic - 7 - 8 AU/ml of the Selection of the enzyme complex is conducted from the filtrate of the culture fluid by precipitation with ethyl alcohol at a temperature of 5 1oWith subsequent lyophilization of a solution of the enzyme. 3 table.

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

A method of obtaining complex protem submerged culture at a temperature of 27-28aboutC for 4-5 days. In this way not specified, the complex of any of the protease producing strain of Aspergillus oryzae F-369, at what pH will determine complex proteolytic activity [2] the article of the same authors, published in 1989 in the journal of Microbiology" (T. 58, B. 3, C. 475-479), indicated that the total proteolytic activity of different populations of Aspergillus oryzae, including population N 32 [2] was determined in the culture fluid by the modified method of the memory of hemoglobin at pH 4.7 and expressed in mg of tyrosine in ml.

Complex protease with a pH optimum of 4,7-known method can be used in the leather industry or in agriculture.

A method of obtaining enzyme preparation-amylase amiloride P10 X [1] including the cultivation of a strain of Aspergillus oryzae 3 in the surface conditions, the extraction of the enzyme with water, followed by its precipitation from the cell extract with ethyl alcohol and lyophilization wet sediment. Based Anileridine P10 X WASPP produces drug "oraz". In this way, using surface culture producer Aspergillus oryzae-3 is able to receive only one enzyme amylase, it does not synthesize acidic protease required for Rass the IOM method of obtaining the complex enzyme preparation, including cultivation producer Aspergillus oryzae c followed by separation of the enzyme by precipitation with ethyl alcohol and lyophilization, as a producer uses a strain of Aspergillus oryzae BKMF-55, grown on agar medium composition, soy flour 1,0-1,2; glucose 2,0-2,2; sulphate sodium 0,05-0,06; cobalt chloride 0,00010-0,00015; calcium carbonate 0,20-0,25; agar-agar 2,0; tap water to 100 at a temperature of (27 1)aboutC for 5-7 days.

Conidia of the fungus contribute in a fermentation medium composition, glucose 3,0-3,2; starch 3,0-3,2; corn extract 1,20-1,25; soy flour to 2.0-2.2; ammonium sulphate 0,20-0,25; calcium carbonate 0,25-0,30; tap water to 100% and hold deep cultivation at a temperature of (27 1)aboutC in Erlenmeyer flasks on a shaker (200 rpm) for 3 days.

A strain of Aspergillus oryzae KMF-55 obtained from the all-Union collection of microorganisms, Institute of biochemistry and physiology of microorganisms, USSR Academy of Sciences.

In submerged cultivation on a fermentation medium of the above composition a strain of Aspergillus oryzae BKMF-55 secretes into the culture fluid two enzyme is an acidic protease and a-amylase. At the end of fermentation proteolytic activity of the culture liquid (hemoglobin) at pH 3.0 accounted for are:

the use of a new strain of Aspergillus oryzae BKMF-55, producing under certain conditions of cultivation 2 enzyme is an acidic protease and a-amylase;

growing inoculum on agar medium composition, soy flour 1,0-1,2; glucose 2,0-2,2; sulphate sodium 0,05-0,06; cobalt chloride 0,00010-0,00015; calcium carbonate 0,20-0,25; agar-agar 2,0; tap water to 100% at a temperature of (27 1)aboutC for 5-7 days.

the cultivation of the producer in deep conditions on the medium composition, glucose 3,0-3,2; starch 3,0-3,2; corn extract 1,20-1,25; soy flour to 2.0-2.2; ammonium sulphate 0,20-0,25; calcium carbonate 0,25-0,30; tap water to 100% at a temperature of (27 1)aboutC for 3 days.

These differences allowed for 3 days. cultivation producer Aspergillus oryzae BKMF-55 to carry out the biosynthesis of two enzymes: amylase, acid protease with optimum action at pH 3.0. Contained in the product acid protease with a pH of 3.0 is required for the breakdown of food protein in various gastrointestinal diseases. Such pH is necessary for the digestion of protein in the stomach. Content-amylase in the resulting preparation is sufficient for the manifestation of its therapeutic action.

P R I m e R 1.

aboutC for 5 days. on agar medium composition, Soy flour 1,0 Glucose 2,0 Sulfate sodium 0,05 Chloride cobalt 0,0001 calcium Carbonate 0.2 Agar-agar 2,0 Tap water To 100

Conidia of the fungus in an amount of 5 ml of 1-billion-suspended in saline solution added to a flask with 100 ml fermentation medium composition, Glucose 3.0 Starch 3,0 Corn extract 1,2

(dry

residue) Soy flour 2,0 ammonium Sulphate 0,20 calcium Carbonate 0,25 Tap water To 100 pH before sterilization 6,7-6,8

Cultivation of the microorganism is carried out in-depth conditions in Erlenmeyer flasks with a capacity of 750 ml shaker (200 rpm) for 3 days.

The culture fluid (1100 ml), filtered through filter paper and receive 1000 ml native solution of proteolytic activity 10 GE/ml and amylolytic activity 8 AU/ml

Proteolytic activity determined using as substrate a 2% solution of hemoglobin (pH 3.0) according to the modified method of memory (Biochemistry 37, vol. 1, 1972, S. 198-208). The amylolytic activity determined using as substrate 1% starch solution (pH 5.0) at FS 2639-89.

Stage 2. The allocation of the enzyme preparation.

the new alcohol. All operations is carried out at a temperature of (5 1)aboutC, because at higher temperatures decreases the release of enzymes, the temperature drops below the 4aboutTo not give a positive effect.

To 1000 ml native solution add 500 ml of ethanol to a final concentration of 30-35% and incubated for 2 h

The mixture was centrifuged at 6000 rpm, sediment ballast proteins cast, and to the supernatant add 1500 ml of ethanol to a final concentration of 60-70% and leave for 10-12 h for the formation of a precipitate. The precipitate was separated by centrifugation at 6000 rpm, washed with 100 ml of ethanol, dissolved in 60 ml of water, again centrifuged, the precipitate drop, a solution of enzymes lyophilizer. Get 3 g of the drug with the specific proteolytic activity of 2.0 GE/mg and amylolytic activity of 1.5 AE/mg.

The output acidic protease 60%- amylase 50%

Specific activity (acid protease) received medication more than 4 times higher than the specific activity of similar foreign drug "Louisem" having the activity of 0.48 GE/mg.

Specific amylase activity of the drug received in 8-9 times higher than the specific activity of the drug "Louisem" with accentes enzyme complex.

Biosynthesis enzymes strain of Aspergillus oryzae BKMF-55 carried out analogously to example 1. The component composition of the agar medium, Soy flour 1,2 Glucose 2,2 Sulfate sodium 0,06 Chloride cobalt 0,00015 calcium Carbonate and 0.2-0.25 Agar-agar 2,0 Tap water To 100.

The composition of fermentation medium, Glucose 3.2 Starch 3,2 Corn extract 1,25 Soy flour 2,2 ammonium Sulphate 0,25 calcium Carbonate 0,3 Tap water To 100.

Receive 1000 ml native solution of proteolytic activity 8 AU/ml

Stage 2. The allocation of the enzyme preparation. The allocation of the enzyme preparation from native solution is performed analogously to example 1. Obtain 3.2 g of the preparation of proteolytic activity of 2.2 GE/mg and amylolytic activity of 1.4 AE/mg.

The output acidic protease 58.7%- amylase 56,0%

Variants 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 agar and liquid fermentation media achieved the highest level of education enzymes.

The data presented in the table. 3 suggests that the optimal temperaturerise ENZYMES providing for the preparation of mycelial inoculum producer Aspergillus oryzae on a medium containing soybean flour, tap water, and then deep cultivation received seed on the enzymatic environment containing soy flour, tap water, up to the maximum activity, characterized in that as a producer uses a strain of the fungus Aspergillus oryzae VKM F-55, and the cultivation of spore material are on medium, additionally containing glucose, sodium sulfate, cobalt chloride, calcium carbonate and agar-agar, in the following ratio, wt.%:

Soy flour - 1,0 - 1,2

Glucose - 2,0 - 2,2

Sulfate sodium - 0,05 - 0,06

Chloride cobalt - 0,00010 - 0,00015

Calcium carbonate - 0,20 - 0,25

Agar-agar - 2,0

Tap water - the Rest

at a temperature of 26 - 28oC for 5 to 7 days, the cultivation of seed are on the enzymatic environment, optionally containing glucose, starch, corn extract, ammonium sulphate, calcium carbonate, in the following ratio, wt.%:

Glucose - 3,0 - 3,2

Starch - 3,0 - 3,2

Corn extract - 1,20 - 1,25

Soy flour - 2,0 - 2,2

is the temperature 26 - 28oC for 3 days.

 

Same patents:
The invention relates to biotechnology, in particular to the production of complex enzyme preparations enriched-glucanase used in agriculture in feed barley feed in the food industry in brewing, and in all other cases associated with the processing of barley

The invention relates to the microbiological industry and genetic engineering and the receipt of a new strain, producing site-specific endonuclease, are able to recognize and cleave the nucleotide sequence 5'-GGTACC-3'

The invention relates to the production of enzymes, namely the method of production of hydrolytic enzymes from animal products, for example, proteases, nucleases
The invention relates to biotechnology and genetic engineering, in particular the production strain used to highlight the restriction enzyme (restrictase), recognize and cleave the nucleotide sequence 5'-G(A/T)GC(A/T)C-3'

FIELD: biotechnology, production of polygalacturonase enzyme preparation useful in wine production and hydrolysis industry.

SUBSTANCE: claimed method includes the first concentration of cultural liquid or solution of crude enzyme preparation and solution demineralization. Second enzyme concentration is carried out directly in column by solution passing through column filled with hydroxyapatite with granulation of 100-250 mum at 3-5°C. Columns are washed with 10 mM phosphate buffer with pH 6.0-7.0 followed by fraction elution by phosphate buffer gradient. Active fraction is subjected to gel chromatography and enzyme preparation is lyophilized.

EFFECT: method with increased yield, purification ratio, productivity and decreased dilution of active fraction.

2 cl, 1 tbl, 3 ex

FIELD: biotechnology, in particular clarifying of enzyme solutions.

SUBSTANCE: claimed method includes slurry coagulation at pH 6.0-7.0 by addition calcium chloride in amount of 0.015-0.025 mol/dm3 and equimolar amount of sodium or potassium phosphate into enzyme solution.

EFFECT: accelerated process for clarifying of hydrolase enzyme solutions; increased yield of target product; hydrolase enzyme preparations with increased specific activity.

1 tbl, 4 ex

FIELD: biotechnology, light industry.

SUBSTANCE: invention relates to protease variants obtained by substitution in subtilisin amino acid sequence from Bacillus amyloliquefaciens, Baccilus subtilus, Baccilus licheniformis, and Baccilus lentus of amino acid residues in 103, 236 and/or 245 sites and in one ore more other sites followed by selection of mutants having improved washing action. DNA fragments encoding said protease variants and expression vector also are disclosed.

EFFECT: detergent compositions with improved washing action.

24 cl, 7 dwg, 5 tbl, 3 ex

FIELD: biotechnology, biochemistry, enzymology, genetic engineering.

SUBSTANCE: invention relates to preparing new strain used for isolation of the new restriction endonuclease Bis I that can be used in detection of the modified DNA. The strain Bacillus subtilis 230 isolated from soil as result of search of producers of restriction endonucleases provides preparing the restriction endonuclease Bis I recognizing and cleaving both chain of DNA nucleotide sequence comprising at least one C5-methylcytosine base in the recognition site 5'-GCNGC-3'.

EFFECT: valuable properties of strain, new enzyme.

1 tbl, 3 dwg, 2 ex

FIELD: biotechnology, microbiological industry, biochemistry.

SUBSTANCE: invention relates to producing enzymes and represents a new strain of Bacillus macerans BS-04 as a producer of pectinases, protease, xylanase and amylase. This strain provides preparing the broad complex of enzymes and characterized by short cycle of growth in combination with enhanced accumulation of biomass.

EFFECT: valuable properties of strain.

1 tbl, 4 ex

FIELD: biotechnology, feedstuff production.

SUBSTANCE: method for phytase-containing aqueous liquid includes cultivation of genus Aspergillus microorganism transformed with expression vector, containing phytase gene of said microorganism bond to promoter and/or signal sequence of amyloglucosidase gene. Cultivation is carried out in aqueous medium containing carbon and nitrogen source under conditions, which allow recombinant phytase expression. Cultural liquid is filtered, cation exchange chromatography, anion exchange chromatography, and ultrafiltration are carried out to produce phytase-containing aqueous liquid having activity at least of 14000 U/g. Said aqueous liquid with phytase activity is useful in production of granulated material, animal feed, premix or semi-finished animal feed.

EFFECT: granulated material and animal feed for animal growth stimulation.

24 cl, 2 tbl, 10 ex

FIELD: biotechnology, genetic engineering, biochemistry, enzymes.

SUBSTANCE: invention relates to preparing a novel strain of microorganism used for isolation of the novel restriction endonuclease Gla I. The strain Glacial ice bacterium I is isolated from a reservoir as result of the search of producers of restrictases and provides preparing the restriction endonuclease Gla I recognizing and cleaving both chains of DNA nucleotide sequence comprising C5-methylcytosine bases in the recognition site 5'-GCGC-3'. Invention provides preparing the yield of enzyme 10 U/g of crude biomass with specific activity of the end enzyme 200 U/ml. This novel restriction endonuclease can be used for detection of methylated DNA.

EFFECT: valuable biological and biochemical properties of enzyme.

1 tbl, 3 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to Bacillus subtilis 1719 bacterium strain isolated under natural conditions in microbiological laboratory.

EFFECT: strain of wide spectrum of antagonistic activity, low adhesive activity, immunomodulating activity and producing complex of hydrolytic enzymes.

6 tbl, 1 ex

FIELD: biology.

SUBSTANCE: claimed strain provides synthesis of extracellular alpha-cyclodextrin glucanotransferase, forming of alpha-cyclodextrin. Activity of said enzyme in cultural liquid is 43.5 U/cm, wherein activity unit represents amount of cyclodextrin glucanotransferase which catalyzes of 1 muM alpha-cyclodextrin formation for 1 min from 3 % starch solution at pH 6.0 and 40°C. Part of alpha-cyclodextrin and other cyclic products is 74.7 %.

EFFECT: improved producer of alpha-cyclodextrin glucanotransferase.

3 tbl, 2 ex

FIELD: gene engineering.

SUBSTANCE: invention relates to introducing in plant polynucleotides encoding mutant polypeptides of ADP-glucose nitrophosphorylase from maize endosperm and soluble starch syntase to produce plant with increased yield and heat stability in heat stress conditions.

EFFECT: plants with increased yield and heat stability.

86 cl, 9 dwg, 4 tbl, 2 ex

FIELD: biotechnology, microbiology, organic chemistry.

SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

EFFECT: improved preparing method.

1 tbl, 6 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-4150 obtained by selection from the known strain Aspergillus oryzae-387 (VKPM F-683) by multistep selection using effective methods of mutagenesis. The strain is stored as lyophilic dried culture and on slants with wort-agar in section of biotechnology of enzyme preparation, in food processing industry department of the State Scientific Institute of VNII food processing technology in Moscow. Invention provides preparing acid α-amylase showing high activity that exceeds activity in analogue by 2-3 times and reducing the process culturing time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

Up!