Fermentation medium and method of production of recombinant proteins

FIELD: biotechnology.

SUBSTANCE: fermentation medium for production of recombinant proteins using methanol inducible fungi of species Pichia pastoris is characterised by maintained concentration of urea or its derivatives in the range from about 0.3 M to about 1 M. The medium contains per liter of water basic salts in the following amounts: orthophosphoric acid (85%) from 2.67 to 133.5 ml, calcium sulfate from 0.093 to 4.65 g, potassium sulfate from 1.82 to 91 g, magnesium sulfate-7H2O from 1.49 to 74.5 g, potassium hydroxide from 0.413 to 20.65 g, glycerol from 4 to 200 g, and microelements per liter of water in the following amounts: copper sulfate-5H2O from 0.6 to 30 g, sodium iodide from 0.008 to 0.4 g, manganese sulfate-H2O from 0.3 to 15 g, sodium molybdate-H2O from 0.02 to 1 g, boric acid from 0.002 to 0.1 g, cobalt chloride from 0.05 to 2.5 g, zinc chloride from 2 to 100 g, sulphate divalent iron-7H2O from 6.5 to 325 g, biotin from 0.02 to 1 g, sulfuric acid from 0.5 to 25 ml. The method for production of recombinant proteins comprises the stage of reproduction of methanol inducible fungi of species Pichia pastoris, and the stage of recombinant protein expression using the said fermentation medium in which feeding with methanol is carried out at a rate of about 6 g/l/hr to about 20 g/l/hr.

EFFECT: increased yield of target products.

11 cl, 27 dwg, 16 ex

 

The technical field to which the invention relates.

The present invention relates to the use of amides of carboxylic acids, such as urea or its derivatives, carbamates, carbodiimide and thiocarbamide as nitrogen supplements in a fermentation medium for the production of recombinant proteins in order to achieve higher levels of bioconversion, and peptides such as insulin and insulin analogs, extending and enzymes, such as lipase, using induced by methanol mushroom expressing systems, such as Pichia. Significant aspects of the invention, in particular, relate to the experimental fermentation process using optimized parameters of nutrient media, responsible for higher yield over short periods of education. The principle of the present invention can also be applied for the production of a wide range of proteins and secondary metabolites by fermentation of a suitable expressing organism.

The level of technology

Expressing the system based on the yeast, such as Pichia pastoris, usually used for expression of recombinant proteins, see Cregg, J.. et al., in Dev. Ind. Environ. 29: 33-41; 1988. Expressing the system in P. pastoris uses induced by methanol promoter of alcoholiday (AO), which controls gene, which encodes expre is this alcoholiday, the enzyme that catalyzes the first stage of metabolism of methanol (see Cregg J M et al. in Bio/Technology 11: 905-910; 1993). P. pastoris has the potential for high level expression, efficient secretion of extracellular protein, post-translational modifications, such as glycosylation, and growth to high density of cells on minimal medium in bioreactor cultures.

Method periodic fermentation recharge using Pichia pastoris as described in "Pichia fermentation Process Guidelines" (guidelines for the process of fermentation of Pichia) company Invitrogen Co. (San Diego, CA), then it is designated as the control. To obtain recombinant proteins transformed Pichia pastoris grown to the desired biomass corresponding to the high density of cells on glycerol as a carbon source. Phase product education initiate a recharge of methanol, which serves as the inductor and the only carbon source for the culture. During the formation of biomass and phase product education ammonia, which serves as the source of nitrogen used for pH control.

Despite the various advantages given by yeast expressing systems, there is a need to optimize affected by the nutritional components of the physico-chemical environment for effective and maximum production of the protein in the bioreactors. Very necessary t is aetsa achieving high specific productivity. It can be obtained by optimizing the initial medium composition, strategies for feeding methanol and physico-chemical parameters of the process. There are publications in which ammonium sulfate, ammonium phosphate, diammonium phosphate, potassium nitrate, urea, corn syrup, soy flour, cottonseed flour, molasses sugar cane and beet, gelatin, flour hydrolysates and the like are used as source of nitrogen for growing bacteria, yeast and fungi. The use of different sources of carbon and nitrogen for growth of microorganisms represents the prior art.

However, the optimal combination of specifically identified sources of carbon and nitrogen for efficient production of recombinant proteins, peptides and enzymes has not been disclosed in the literature, the relevant prior art. For example, application WO/2007/005646 discloses the production of ethanol essentially by culturing the recombinant yeast in an integrated environment for growth, contain complex carbohydrates, as well as a number of cheaper sources of nitrogen, such as corn syrup, corn extract, yeast autolysate and urea. In addition, this method does not use induced by methanol mechanism for the growth or production in contrast to developed in the present invention method of producing recombinant proteins. Oppo is ichno U.S. patent 4288554 describes the continuous fermentation method only for the growth of non-GMO (phenotolamine) Candida species using urea in combination with other sources of nitrogen and the main salt environment. In this case, there is no assumption or a description of where the urea can be used during the fermentation process (periodic, periodic with water, continuous) using induced by methanol GMO Pichia pastoris for efficient production of recombinant proteins and peptides, for example, human insulin and its analogues, or enzymes such as lipase.

Unexpectedly discovered that the use described fermentation environment, characterized by controlled addition of some rich and easily soluble sources of nitrogen, such as urea, optionally in optimized concentrations relative residual concentrations of urea and residual concentrations of ammonia generated during the hydrolysis of urea, gives increased formation of product, productivity and, thus, reduced production time.

Production of recombinant proteins using E. coli have long been known, and expressing the system is well studied and understood. Expressing system based on E. coli is widely used to produce such molecules, such as G-CSF (granulocyte colony-stimulating factor), HGH (human growth hormone), streptokinase, and many other biological products. To obtain recombinant proteins transformed E. coli vyrashivaiut desired high biomass density of cells on the dextrose as carbon source. Phase product education initiated by induction using the required inductor and then the culture is only supported with a minimum of added nutrients until the end of fermentation.

For many years the culture of the fungi used for the production of valuable biomolecules, such as enzymes, and other useful molecules. Mushroom culture, for example, Rhizopus, Aspergillus, Penicillium, and the like, used in classical fermentation, designed to obtain a wide range of enzymes, such as lipases, amylases, dextrans, etc. that are used in the food, textile, leather and other similar industries. Actinobacteria, known as the basic components for the production of antibiotics, widely used for a number of secondary metabolites that are useful to humans. One of the key properties of fungi and actinomycetes is the property of bioconversion", for example, hydroxylation, esterification, etc. the Main advantage is that the bioconversion of specific targets, and you can get the products of relatively high purity. A classic example is the conversion compactin in pravastatin.

Methodological improvements, known in the art include measures related to fermentation technology, such as the stirring and supply of oxygen, or is tificatio, related to the composition of the nutrient media, such as modification of the concentrations of Sugars during fermentation, the change processing of the campaign process, or changes related to the natural properties of the microorganism, etc.

Unexpectedly discovered that the use of fermentation environment, characterized by controlled addition of some rich and easily soluble sources of nitrogen, such as urea, provides enhanced productivity and/or increased level of bioconversion and, thus, reduced production time.

Disclosure of inventions

The main purpose of the present invention to provide a fermentation medium for the production of recombinant proteins, their derivatives or analogues by fermentation using induced by methanol species of fungi.

Another main objective of the present invention to provide a fermentation medium for the production of recombinant proteins, their derivatives or analogues by fermentation using using microorganisms.

In addition, another main objective of the present invention to provide a fermentation medium for the production of secondary metabolites by fermentation using microorganisms.

Another main objective of the present invention is to develop a method of producing recombina is the shaft or non-recombinant products, their derivatives or analogues.

Another main objective of the present invention is to develop a method of producing secondary metabolites.

Another main objective of the present invention is to develop a method of bioconversion compactin in pravastatin.

Another main objective of the present invention is the production of recombinant protein product.

Accordingly, the present invention relates fermentational environment intended for the production of recombinant proteins, their derivatives and analogues by fermentation using induced by methanol species of fungi, and this environment differs in that it contains an effective concentration of the amide of carbonic acid; fermentation environment intended for the production of recombinant proteins, their derivatives and their analogs by fermentation using microorganisms, with the specified environment differs in that it contains an effective concentration of the amide of carbonic acid; fermentation environment designed to produce secondary metabolites by fermentation using microorganisms, with the specified environment differs in that it contains an effective concentration of amide carbonic acid; a method for producing recombinant proteins, their derivatives or their analogues, which provides the size of agenie induced by methanol species of fungi, expressing insulin, in a fermentation environment, and this environment differs in that it contains an effective concentration of the amide of carbonic acid; the method of obtaining a recombinant or non-recombinant protein products, their derivatives or their analogues, which predusmatrivaetsya induced or pendulumeca microorganism expressing the protein in a fermentation environment, and this environment differs in that it contains an effective concentration of the amide of carbonic acid; a method for production of secondary metabolites, which involves the multiplication of a microorganism in a fermentation environment, and this environment differs in that it contains an effective concentration of the amide of carbonic acid; the method of bioconversion compactin in pravastatin, and the specified conversion is carried out in environment, characterized in that the medium contains an effective concentration of the amide of carbonic acid; and the recombinant protein product obtained as claimed in any of the preceding claims.

The present invention relates to a fermentation medium for the production of recombinant proteins, their derivatives or their analogues via fermentation using induced by methanol species of fungi, and this environment differs in that it contains effective the th concentration of the amide of carbonic acid.

In another embodiment, the present invention amide of carbonic acid selected from the group consisting of urea or its derivatives, such as demethylation, determation, N-acetylphenylalanine, isopropylpyrimidine, prilocaine, or a combination of both.

In yet another embodiment, the present invention amide of carbonic acid is a urea.

In yet another embodiment, the present invention amide of carbonic acid is added in the form of a liquid, spray, powder or granules.

In yet another embodiment of the present invention the residual concentration of the amide of carbonic acid is up to 1 M

In yet another embodiment of the present invention the concentration of the basic salts and trace elements vary from 0,1X to 5X from the control environment, preserving the residual concentration of the amide of carbonic acid to 1 M

In yet another embodiment, the present invention improves the consumption of phosphate.

In yet another embodiment, the present invention induced by methanol species of fungi expressing recombinant product of insulin selected from the group comprising Pichia pastoris, Pichia sp., Saccharomyces sp., Saccharomyces cerevisiae, Kluyveromyces sp. and Hansenula polymorpha.

The present invention relates to a fermentation medium for the production of recombinant proteins, and the derivatives and their analogues by fermentation using microorganisms, moreover, this environment differs in that it contains an effective concentration of the amide of carbonic acid.

The present invention relates to a fermentation medium to produce secondary metabolites by fermentation using microorganisms, with the specified environment differs in that it contains an effective concentration of the amide of carbonic acid.

In another embodiment, the present invention amide of carbonic acid selected from the group comprising urea or its derivatives, such as demethylation, determation, N-acetylphenylalanine, isopropylpyrimidine, prilocaine, or a combination of both.

In yet another embodiment, the present invention amide of carbonic acid is a urea.

In yet another embodiment, the present invention amide of carbonic acid is added in the form of a liquid, spray, powder or granules.

In yet another embodiment of the present invention the residual concentration of the amide of carbonic acid is up to 10 g/L.

In yet another embodiment of the present invention the microorganism is selected from the group consisting of E. coli, Streptomyces sp, Aspergillus sp, Rhizopus sp, Penillium sp and Rhizomucor sp.

The present invention relates to a method for producing recombinant proteins, their derivatives or their analogues, which provides for RA is mnozenie induced by methanol expressing insulin species of fungi in a fermentation medium, moreover, this environment differs in that it contains an effective concentration of the amide of carbonic acid.

In another embodiment, the present invention amide of carbonic acid selected from the group comprising urea or its derivatives, such as demethylation, determation, N-acetylphenylalanine, isopropylpyrimidine, prilocaine, or a combination of both.

In yet another embodiment, the present invention amide of carbonic acid is a urea.

In yet another embodiment of the present invention obtained recombinant product of insulin is an IN-105.

In yet another embodiment of the present invention obtained recombinant product insulin is a precursor of insulin, insulin or its analogs, or its derivatives.

In yet another embodiment of the present invention the recombinant product of the insulin is an insulin glargine.

In yet another embodiment of the present invention obtained recombinant protein is a cyclic or acyclic peptide.

In yet another embodiment of the present invention the recombinant peptide is a basis.

In yet another embodiment of the present invention obtained recombinant the first protein is an enzyme.

In yet another embodiment of the present invention the product obtained recombinant enzyme is a lipase.

In yet another embodiment of the present invention obtained recombinant protein selected from the group consisting of a precursor of insulin, insulin or its analogs, or its derivatives, glargine, on the basis, carboxypeptidase and lipase.

In yet another embodiment, the present invention induced by methanol species of fungi expressing recombinant product of insulin selected from the group comprising Pichia pastoris, Pichia sp., Saccharomyces sp., Saccharomyces cerevisiae, Kluyveromyces sp. and Hansenula polymorpha.

In yet another embodiment, the present invention induced by methanol type of fungi is a Pichia pastoris.

In yet another embodiment of the present invention, the level of feeding methanol up to 20 g/liter of broth per hour.

In yet another embodiment of the present invention obtained the maximum titer of the product exceeds 0.1 g/l

The present invention relates to a method for producing a recombinant or non-recombinant protein products, their derivatives or their analogues, which predusmatrivaetsya induced or pendulumeca expressing the protein of the microorganism in a fermentation medium, and the specified environment Otley is highlighted by the fact that that contains an effective concentration of the amide of carbonic acid.

In another embodiment, the present invention amide of carbonic acid selected from the group comprising urea or its derivatives, such as demethylation, determation, N-acetylphenylalanine, isopropylpyrimidine, prilocaine, or a combination of both.

In yet another embodiment, the present invention amide of carbonic acid is a urea.

In yet another embodiment of the present invention obtained recombinant protein product is a G-CSF.

In yet another embodiment of the present invention obtained recombinant product is a streptokinase.

In yet another embodiment of the present invention the protein product is a lipase.

The present invention relates to a method for production of secondary metabolites, which involves the multiplication of a microorganism in a fermentation environment, and this environment differs in that it contains an effective concentration of the amide of carbonic acid.

In another embodiment of the present invention, the secondary metabolite is a pravastatin.

The present invention relates to a method of bioconversion compactin in pravastatin, and is nversio carried out in the environment, characterized in that the medium contains an effective concentration of the amide of carbonic acid.

In another embodiment, the present invention amide of carbonic acid selected from the group comprising urea or its derivatives, such as demethylation, determation, N-acetylphenylalanine, isopropylpyrimidine, prilocaine, or a combination of both.

In yet another embodiment, the present invention bioconversion compactin in pravastatin is at least 35%.

The present invention relates to a recombinant protein product obtained as described above.

In another embodiment, the present invention obtained recombinant protein product selected from the group comprising the precursor of insulin, insulin or its analogs, or its derivatives, glargine, on the basis, carboxypeptidase and lipase.

The invention provides for the composition of nutrients intended for use in the development of the fermentation medium, and the composition contains nitrogen components, such as amides of carbonic acid, for example, urea and the aforementioned related forms or derivatives, together with one or more other components of the fermentation medium, which is specially optimized to obtain increased output of insulin or the relationship of the different analogues derivatives for short periods of time education.

Unexpectedly discovered that the use of certain fermentation medium with the addition of some nitrogen-containing sources, such as amides of carbonic acid, for example, urea and related forms or derivatives in specific concentrations has no effect on the growth of yeast cells and contributes to productivity.

Additional nitrogen component, for example, urea can be added in the form of a liquid, spray, powder or granules.

The main problem of the invention consists in the fact that the productivity of the fermentation method of the insulin or insulin analogue, using Pichia sp. strongly influenced by the content of urea in the medium for cultivation. As a result, the product yield is significantly increased, especially in abbreviated time periods fermentation, adding nitrogen component such as urea, in the medium for cultivation.

According to a preferred variant of the invention, the addition of urea in a fermentation medium increases the level of consumption is the key ingredient "phosphate", which, in turn, increases productivity. Found that the faster the consumption of phosphate, the shorter the cycle time of fermentation and, therefore, higher productivity. Thus for the first time shows the metabolism of urea along with phosphate, which is ishaat the level of expression of a protein or peptide, without affecting the profile of growth and shortens the fermentation time.

According to another aspect of the invention, the addition of urea provides increased selection of product at the end of fermentation at any pH value.

Thus the present invention provides increased outputs of the protein product, reduced the cycle time of production, better utilization of nutrients introduced into the process, and in General reduces capital costs and production costs.

Suitable microbial strain for industrial fermentation method using a chemically defined medium may be a wild-type strain, producing a valuable connection, interest, provided that the wild-type strain has good growth characteristics.

Preferred yeast for use as a body-producer include, for example, Pichia pastoris, Pichia sp., Saccharomyces sp., Saccharomyces cerevisiae, Kluyveromycessp. Hansenula polymorpha.

In addition, suitable microbial strain for industrial fermentation method using a chemically defined medium may be a strain that receive and/or enhance the fact that the parent strain of interest is subjected to classical mutagenic processing or transformation of recombinant DNA, provided that the resulting the resulting mutant or transformed microbial strain has good features growth in chemically defined medium. Thus the characteristics of the growth of the parent strain in a chemically defined environment will depend on whether the resulting mutant or transformed strains have improved or similar characteristics of the growth in chemically defined medium compared with the characteristics of the parent strain.

As should be understood by a competent expert in the field of technology, the optimal concentration of additives amide of carbonic acid will vary from clone to clone, although in all cases the end result is a higher titer in less time.

The term "fermentation medium" or "fermentation medium" refers to the environment in which carry out the fermentation, which includes fermentation substrates and other raw materials used fermenting microorganisms for the formation of a specific medicinal product.

"Nitrogen components are substrates, raw materials or components that are sources of assimilated nitrogen in the fermentation medium.

According to an important aspect of the invention, the preferred nitrogen component in the fermentation medium are amides of carbonic acid, such as urea. These may include compounds containing N-CO-N, or related groups. The present invention provides used the e derivatives of urea, such as dimethyloxetane, determation, N-acetyl-N-phenyl urea, isopropylidenebis, N-phenylacetone and the like, or combinations thereof.

Used the term "effective amount" is a quantity of urea or its derivatives, the introduction of which, according to the invention, in a fermentation medium leads to the formation of a substantial quantity/yield of protein, in addition, for short periods of time without affecting the growth of yeast cells.

The term "fermenting organism" refers to any microorganism suitable for use in a desired fermentation process. Examples of fermenting organisms include fungal organisms, such as yeast. Examples of fermenting organisms in the context of the present invention are Pichia pastoris, Pichia sp., Saccharomyces sp., Saccharomyces cerevisiae, Kluyveromyces sp. or Hansenula polymorpha.

The invention may be suitable for expression of any recombinant peptide using induced by methanol species of fungi, but are not limited to recombinante expressed peptides, proteins, insulin, a precursor of insulin, insulin derivatives or analogues of insulin.

The term "recombinant"as used in this context to describe a protein or polypeptide means a polypeptide produced by expression of recombinant floor in the of cleotide. The term "recombinant"as used in this context in the cells means cells that can be or have been, used as recipients for recombinant vectors or other transferred DNA, and include the progeny of the original cell, which was transliterowany. It should be borne in mind that the offspring of a single parent cell may not be completely identical in morphology or in genomic complement or the total DNA of the original parent body due to random or directed mutation.

The term 'polypeptide', 'protein', 'peptide' refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term also does not apply to postexplosion modifications of the polypeptide, or exclude them, although chemical or postexplosion modifications of these polypeptides can be included or excluded as specific embodiments. In one embodiment, the molecule is a polypeptide or its related analogs or derivatives thereof. Preferably, when the polypeptide is a cyclic peptide. According to another preferred variant implementation, the polypeptide is a cyclic peptide. Another is predpochtitelno embodiment, the polypeptide selected from the group containing the basis, eptifibatide, atosiban, enzymes, such as lipase, carboxypeptidase, etc.

Insulin is a polypeptide of 51 amino acids, which are distributed between two chains of amino acids: A-chain of 21 amino acids and a B chain of 30 amino acids. The chains are connected to each other 2 disulfide bridges. This definition includes the use of natural insulin, but the insulin derivatives and analogues. The insulin connection may, for example, to provide a compound of insulin mammal, such as human insulin, or derivatives or analogs of the compounds of the insulin.

Insulin derivatives are derivatives of natural insulin, a human insulin or insulin animals, which differ from the corresponding in other respects identical to natural insulin replacement of at least one natural amino acid residue and/or by introducing at least one amino acid residue and/or organic residue. It should be borne in mind that the term insulin determines the polypeptide consisting of B - and A-chains. Insulin derivative may be at least 60% homologous to the natural insulin. Insulin derivative can be even more homologous, for example at least about 75%, or at least the least about 90% homologous to the natural insulin. As a rule, insulin derivatives have slightly modified the action than human insulin.

Upon receipt of the insulin and insulin derivatives by genetic engineering, the precursor of insulin, often Express "proinsulin", containing the B-, C - and A-chains. This proinsulin can be turned into insulin or insulin derived by enzymatic or chemical removal of the C-chain after proper and correct installation and formation of disulfide bridges. Proinsulin can be derived by at least 60% homologous, B - and A-chain natural proinsulin. However, connecting C-peptide can be selected as completely different from any known C-peptide. Proinsulin derived can be even more homologous, for example at least about 75%, or at least about 90% homologous to the natural proinsulin.

According to some embodiments of the invention the recombinant product of insulin is an IN-105. The resulting drug product specifically refers to a molecule IN-105. IN-105 is an insulin molecule, conjugate via e-the amino acid lysine in position B29 of the B-chain of insulin with amphiphilic oligomer of structural formula CH3O-(C4H2O)3 -CH2-CH2-COOH. The molecule can be nanoconjugates for A1, B1 and B29, deconjugating on various combinations of A1, B1 and B29 or trichohyalin with various combinations of A1, B1 and B29.

According to another aspect of the invention a recombinant protein obtained by fermentation using a fermentation medium corresponding to the present invention is a cyclic or acyclic peptide.

According to another aspect of the invention a recombinant protein obtained by fermentation using a fermentation medium corresponding to the present invention is an enzyme.

In one aspect of the invention, the Protocol fermentation may include three phases: loading, feeding (optional) and phase induction with methanol.

According to the most significant aspect of the invention the fermentation medium used in the context of this invention, includes the following components. Also included is a method of cooking environment.

The composition of the medium:
ComponentsQuantity (g/l)
CaSO4·2H2O0,93
MgSO4·7H2O 29,8
K2SO436,4
KOH4,13
Glycerin40
H3PO4(Density of 1.7%)22, 95mm
Urea6,0

The individual components are dissolved in a minimum volume of water in the above sequence, and sterilized at 121°C for 1 hour. Trace element solution and D-Biotin (previously sterilized by filtration) is aseptically added to the medium, each with a rate of 4.35 ml/l environment (density of the trace element solution is 1.05, and D-Biotin is 1.0.

The composition of trace element solution:
Components (salt)Quantity (g/l)
Copper sulphate, CuSO4·5H2O6,0
Sodium iodide, NaI0,08
Manganese sulfate, MnSO4·H2O3,0
Molybdate sodium, Na2MoO4·2H2O 0,20
Boric acid, H3BO30,02
The cobalt chloride, CoCl2·6H2O0,50
Zinc chloride, ZnCl220,0
Sulfate, ferrous iron, FeSO4·7H2O65,0
Sulphuric acid, H2SO45.0 ml

All salts are dissolved one by one in water and sterilized by filtration through a device for sterilizing filter.

Preparation of a solution of Biotin:

D-Biotin, 0.2 g/l

Biotin is dissolved in water and sterilized by filtration through a device for sterilizing filter.

Feeding yeast extract and soy peptone:

In addition, feeding yeast extract and soy peptone also introduced during fermentation. It should be prepared as follows:

ComponentsConcentration (g/l)
Soy peptone100
Yeast extract50

Component is s dissolve and using drinking water receive the necessary volume. Then the solution is sterilized at 121-123°C for 90 min. Density of feeding soy peptone is approximately 1,05.

Feeding methanol:

12.0 ml of trace element solution, 12 ml of a solution of D-Biotin and 40 g of urea is added per liter of methanol before the introduction of the feed.

The fermentation method:

The fermentation method includes the growth phase of the cells in the boot, optional phase feeding glycerin download and phase induction with methanol.

The growth phase of the cells in loading

Monitoring and control download

Product parameters of the fermenter specify the source and control as follows:

Temperature:30°±2°C
pH:5±0,2
DO:>10%
Date:22-24 hour.

Phase induction methanol (FIM)

Recharge of methanol begin immediately after the end of the loading phase. Methanol sterilized (in boot mode) by filtering using commercially available sterilizing filter.

At the beginning of the FIM, the pH down to a value of 4.0±0.1 or 6,0±0,1 or 6,3±0,1 depending on the expression of the protein in the medium (varies from product to product, but also by the clone to the clone) and the temperature adjusted to approximately 18-24°C (varies from product to product, and from clone to clone).

At the same time another recharge, recharge yeast extract and soy peptone also begin in the fermenter with a speed of 0.4 g/l/h. the original volume.

Monitoring and control phim

Temperature:18-30°C (varies from product to product and from clone to clone)
pH:3,0-7,0
DO:>1% (used to control the concentration of methanol in the broth)
Date:5-8 days (varies from clone to clone)
Analysis pH:1-9,5 (depending on the type of protein)

According to another aspect of the invention the seed material is prepared by culturing dried glycerol original culture on minimal glycerol medium (EASC). The main fermentation medium obtained from Control Pichia process guidelines" (guidelines for the control of processes using Pichia), contain phosphoric acid, digidrirovanny calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, glycerin, trace elements, and D-Biotin. Nutrient medium for cultivation the Oia should also contain known compounds in small or trace quantities, which is usually injected in a fermentation medium for cultivation, such as water-soluble Ca, Mg, Mn, Fe, K, Co, Cu, Zn, B, Mo, Br and I. May also be present other trace elements. Trace element solution corresponding to the present invention, in particular, includes copper sulfate pentahydrate, sodium iodide, manganese sulfate monohydrate, sodium molybdate dehydrate, boric acid, cobalt chloride, the uranyl chloride zinc sulfate heptahydrate ferrous iron. Although the concentration of each ingredient environment specially optimized for each product, the lower lead of the control environment:

Control environment

Fermentation is the main salt environment:

1 liter mix the following ingredients:

Phosphoric acid:85% (26,7 ml)
Calcium sulfate:0,93 g
Potassium sulfate:18,2 g
Magnesium sulfate-7H2O:14.9 g
Potassium hydroxide:4,13 g
Glycerin:40,0 g

Water to 1 liter.

Add in the fermenter with water until it matched the existing volume and sterilized.

PTM1 trace elements

Mix the following ingredients:

The copper sulfate 5H2O6.0 g
Sodium iodide0.08 g
Manganese sulfate-H2O3.0 g
Molybdate sodium-2H2O0.2 g
Boric acid0.02 g
The cobalt chloride0.5 g
Zinc chloride20,0 g
Sulfate ferrous iron-7H2O65.0 g
Biotin0.2 g
Sulfuric acid5.0 ml
Waterto a final volume of 1 L.

Filtered, sterilized and stored at room temperature.

When mixing these ingredients may appear muddy sediment. Environment can be filtered to sterilize and use.

In addition, the control environment include urea at different concentrations.

what according to another aspect of the invention the biomass formation during the growth phase of the booting until while in the original environment is glycerin. In addition, the biomass formation is not an important factor and it is carried out only in few cases.

According to further aspect of the invention, after reaching the desired biomass culture induce a constant recharge of methanol and urea. During recharging of methanol is performed also feed yeast extract and peptone solution.

According to another aspect of the speed of the feed methanol up to 20 g/l/h. How to understand any competent specialist, optimization speeds up to further improve levels of products provided by the present invention.

In addition, the invention will now be described in connection with certain preferred options for implementation in the following examples, in order to fully understand and appreciate aspects of it. To restrict the invention is data specific implementation options do not provide. On the contrary, intend to cover all alternatives, modifications and equivalent solutions, as they may be included in the scope of the invention as defined in the attached claims. Thus, the following examples which include preferred embodiments of will serve to illustrate the practical implementation of the present invention, and which should be borne in mind, that shows the details are given only as an example and with the purpose of illustrative discussion of preferred embodiments of the present invention and are present for the presentation of the material, which is considered to be the most useful and readily understood description of methods of fermentation, as well as of the principles and conceptual aspects of the invention.

The invention provides for a nutritional composition designed for use in the preparation of fermentation medium, and the composition contains nitrogen components such as urea, for example, urea and related forms or derivatives, for example, carbamates, carbodiimide, thiocarbamide, together with one or more other components of the fermentation media, which are specially optimized to obtain high yields of product for short periods of time products.

Thus the invention makes it possible to obtain high outputs of recombinant protein products, such as insulin glargine IN 105, on the basis, lipase and carboxypeptidase during the fermentation process (periodic, periodic with water, continuous) using induced by methanol GMO Pichia pastoris provided by adding urea without affecting the growth of yeast cells.

According to one aspect izobreteniyami component, which specifically affects the outputs and the production time, represents the amides of carbonic acid, such as urea and its derivatives and the above-mentioned related compounds.

According to another aspect of the invention a preferred yeast for use as a body-producer include, for example, Pichia pastoris, Pichia sp., Saccharomyces sp., Saccharomyces cerevisiae, Kluyveromyces sp. or Hansenula polymorpha.

The present invention demonstrates the use of amides of carbonic acid, such as urea or its derivatives, carbamates, carbodiimide and thiocarbamide as nitrogen supplements in a fermentation medium to obtain proteins with the aim of achieving high levels of bioconversion using E. coli, Actinomycetes and fungal cultures. Significant aspects of the invention, in particular, relate to the experimental fermentation process using optimized parameters of nutrient media, responsible for increased productivity. The principle of the present invention can be applied for the production of a wide range of proteins and secondary metabolites by fermentation of a suitable expressing organism.

The invention provides for a nutritional composition designed for use in the preparation of fermentation medium, and the composition contains nitrogen components such as carb the ministries of foreign Affairs, for example, urea and related forms or derivatives, for example, carbamates, carbodiimide and thiocarbamide, together with one or more other components of the fermentation medium, which are specially optimized to obtain high yields of product for short periods of time products.

Thus the invention makes it possible to obtain high outputs of protein products, such as G-CSF, streptokinase, human growth hormone, etc. during the fermentation process (periodic, periodic with water, continuous) using induced by methanol GMO Pichia pastoris using induced E. coli is carried out by adding urea without affecting the growth of yeast cells.

The invention also makes it possible to improve the bioconversion to obtain high yields of products, for example, a method of fermentation of pravastatin (periodic, periodic with water, continuous) using actinomycetes and/or fungal cultures

The invention, furthermore, enhances the production of enzymes, such as lipases, amylases, cellulases in periodic or periodic injection processes using fungal cultures.

According to one aspect of the invention the nitrogen component, which specifically affects the outputs and the production time are the Xia amides of carbonic acid, for example, urea or its derivatives and the above-mentioned related compounds.

According to another aspect of the invention, the preferred microorganisms are strains of the family Enterobacteriaceae, preferably, when used as a body-producer include, but are not limited to E. coli.

According to another aspect of the invention, the preferred microorganisms are strains of actinomycetes and/or collection of fungi, including, but without limitation, Streptomyces sp, Actinoplanes sp, Aspergillus sp, Rhizopus sp and Penicillium sp.

Other objects, features, advantages and aspects of the present invention will be obvious to a competent specialists from the following description. However, it should be borne in mind that the following description and specific examples, although they show preferred embodiments of the invention, given only as an illustration. Various changes and modifications are included in the nature and scope of the disclosed invention will readily become apparent for competent professionals in the field of machinery from reading the following description and from reading other sections of this description.

The invention provides for a nutritional composition designed for use in the preparation of fermentation medium, and the composition contains a nitrogen component the coefficients, such as amides of carbonic acid, for example, urea and related forms or derived above, together with one or more other components of the fermentation media, which are specially optimized to obtain the desired protein product, or secondary metabolites.

Unexpectedly discovered that the use of certain fermentation medium with the addition of some nitrogen-containing sources, such as amides of carbonic acid, for example, urea and related forms or derivatives, in specific concentrations do not affect the growth of the fermenting organism, but improves productivity.

Additional nitrogen component, such as urea, can be added in the form of a liquid, spray, powder or granules.

Suitable microbial strain for industrial fermentation method can be any type strain wild-type, producing a valuable connection, interest, provided that the wild-type strain has good growth characteristics.

In addition, suitable microbial strain for industrial fermentation method can be a strain, which receive and/or enhance the fact that the parent strain of interest is subjected to classical mutagenic treatment sludge and transformation of recombinant DNA, also p and the condition, that the resulting mutant or transformed microbial strain has good growth characteristics. Thus the characteristics of the growth of the parent strain will depend, will be l and the resulting mutant or transformed strains have improved or similar growth characteristics compared to the characteristics of the parent strain.

The fermentation method using this fermentation medium is improved in relation to one or more parameters selected from the group consisting of concentration of product (product/volume), product yield (formed by the product/the consumed carbon source) and the formation of the product (formed by the product of volume and time), or other additional style parameters and their combinations.

As will be borne in mind by a competent expert in the field of technology, the optimal concentration of inorganic salts aid of the coal will vary from clone to clone, although the end result in all cases is to obtain higher titers in less time in the acid.

The term "fermentation medium" or "fermentation medium" refers to the environment in which carry out the fermentation, which includes fermentation substrates and other raw materials used fermenting microorganisms for the formation of specific drugs is i.i.d. product.

Fermentation medium in the present invention must contain suitable carbon substrates. Suitable substrates may include, but are not limited to, monosaccharides such as glucose and fructose, polysaccharides, such as starch or cellulose or mixtures thereof and other ingredients, corn syrup, molasses sugar beet, glycerin and barley malt. In addition, the carbon substrate may also be a one-substrates, such as carbon dioxide or methanol, which is shown metabolic conversion into key biochemical intermediates. Therefore stipulate that the source of carbon used in the present invention can cover a wide range of carbon-containing substrates and will only be limited by the choice of the organism. In addition to an appropriate carbon source of the medium, aeration must contain suitable minerals, salts, buffers and other components, known to the competent specialists in the field of technology that is suitable for crop growth and stimulation of expression of the desired protein or the final product.

"Nitrogen components are substrates, raw materials or components, which are a source of assimilated nitrogen in the fermentation medium.

Suitable nitrogen sources may include, but without the OTF is the limit listed, soybean flour, cottonseed flour, gelatin, yeast extract, casein, casein hydrolysates, corn syrup and inorganic salts of ammonium, nitrates and nitrites.

According to a significant aspect of the invention, the preferred additive in a fermentation medium are amides of carbonic acid, such as urea. They will include compounds containing N-CO-N, or related groups. The present invention provides for the use of urea derivatives, such as demethylation, determation, N-acetyl-N-phenylacetone, isopropylpyrimidine, N-phenylacetone and the like, or combinations thereof.

Used the term "effective amount" is a quantity of urea or its derivatives, the introduction of which, according to the invention, in a fermentation medium leads to the formation of a significant quantity/output the formed product, in addition, for short periods of time without affecting the growth of yeast cells.

The term "fermenting organism" refers to any microorganism suitable for use in a desired fermentation process. According to another aspect of the invention, the preferred microorganisms are strains of bacteria, actinomyces and/or species of fungi, preferably for use as a body-producer include, but without limiting the texts listed, E. coli, Streptomyces sp, Actinoplanes sp, Aspergillus sp, Rhizopus sp, Penicillium sp, etc.

The term "recombinant"as used in this context to describe a protein or polypeptide means a polypeptide produced by expression of recombinant floor nucleotide. The term "recombinant"as used in this context in the cells means cells that can be or have been, used as recipients for recombinant vectors or other transferred DNA, and include the progeny of the original cell, which was transliterowany. It should be borne in mind that the offspring of a single parent cell may not be completely identical in morphology or in genomic complement or the total DNA of the original parent body due to random or directed mutation.

The term 'polypeptide', 'protein', 'peptide' refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term also does not include or exclude postexplosion modifications of the polypeptide, although chemical or postexplosion modifications of these polypeptides can be included or excluded as specific embodiments. In one embodiment, the molecule is a polypeptide or its sister the e analogues or derivatives thereof. Preferably, when the polypeptide is a cyclic peptide. According to another preferred variant implementation, the polypeptide isone acyclic peptide. In another preferred embodiment, the recombinant protein is produced by fermentation using a fermentation medium corresponding to the present invention.

One of the embodiments of the present invention relates to the production of granulocyte colony-stimulating factor. Granulocyte colony-stimulating factor is a pharmaceutically active protein, which regulates proliferation, differentiation, and functional activation of neutrophilic granulocytes (see Metcalf, Blood 67: 257 (1986); Yan, et al. Blood 84(3): 795-799 (1994); Bensinger, et al. Blood 81(11): 3158-3163 (1993); Roberts, et al., Expt'l Hematology 22: 1156-1163 (1994); It is, et al. Blood 81(7): 1960-1967 (1993)). G-CSF means the natural or recombinant protein, preferably a human, as obtained from traditional sources, such as tissue, protein synthesis, cell culture with natural or recombinant cells. Include any protein having the activity of G-CSF, for example, mutiny or otherwise modified proteins.

"Secondary metabolite" is a compound derived from primary metabolites, which produces the body, is not the tsya primary metabolite and is not required for growth of the microorganism in standardsapproved. Connection of secondary metabolites can be converted into useful compounds by subsequent chemical conversion or subsequent biotransformation. In this case ensuring high data availability intermediate compounds would, in addition to high-end products useful compounds, which in itself can be regarded in this context as a secondary metabolite.

In one aspect of the invention, the Protocol fermentation may include two phases: periodic and periodic injection (optional).

Brief description of drawings

Figure 1: Comparison of the profile of biomass predecessor-IN-105 with the addition of/without the addition of urea.

Figure 2: Comparison of concentration profiles of the predecessor product IN-105 with the addition of/without the addition of urea.

Figure 3: Comparison of the profile of the biomass precursor of insulin with the addition of/without the addition of urea.

Figure 4: Comparison of concentration profiles of the product of the precursor of insulin with the addition of/without the addition of urea.

Figure 5: Comparison of the profile of biomass predecessor glargine; with the addition of/without the addition of urea.

Fig.6: Comparison of concentration profiles product predecessor glargine; with the addition of/without the addition of urea.

Fig.7: Comparison of the profile of biomass predecessor basis with the addition of/without added what I urea.

Fig: Comparison of concentration profiles product predecessor basis with the addition of/without the addition of urea.

Fig.9: Comparison of concentration profiles of the product of the enzyme lipase with the addition of/without the addition of urea.

Figure 10: Profiles of biomass, obtained at various concentrations of urea during fermentation predecessor-IN-105.

11: concentration Profiles of product obtained at different concentrations of urea during fermentation predecessor-IN-105.

Fig: Profiles of biomass, obtained at various concentrations of urea during fermentation predecessor of insulin.

Fig: concentration Profiles of product obtained at different concentrations of urea during fermentation predecessor of insulin.

Fig: residual concentration of urea, and the maximum concentration of the product to obtain IN-105.

Fig: Profile of residual concentration of urea and the maximum concentration of the product to obtain the precursor of insulin.

Fig: Comparison of the profile of biomass predecessor-IN-105 when the level of feeding methanol to about 20 g/l/h.

Fig: Comparison of concentration profiles of the predecessor product IN-105 when the level of feeding methanol to about 20 g/l/h.

Fig: Learning connections, other than urea, testing other related compounds in terms of osdate on the productivity of the fermentation of Pichia.

Fig: Effect of urea on the residual concentration of phosphate ions in the broth. The effect of urea on the metabolism of phosphate strain.

Fig. The profile of growth culture for the education of pravastatin.

Fig. The titer of pravastatin adding urea.

Fig. The percentage conversion compactin in pravastatin adding urea.

Fig. Comparison of the density of the medium cell culture (SPC) for the formation of G-CSF in E. coli.

Fig. The effect of urea on the specific productivity of G-CSF.

Fig. Comparison of the SEC for the formation of streptokinase in E. coli.

Fig. The effect of urea on the specific productivity of streptokinase.

Fig. The effect of urea on the production of lipase using Rhizomucor sp.

The implementation of the invention

The present invention further define the following examples. It should be borne in mind that these examples, although they show preferred embodiments of the invention, lead only to illustrate. From the above discussion and these examples competent specialist in the field of technology can establish the main features of this invention and, without leaving its nature and scope, can make various changes and modifications of the invention to adapt it to various usage and conditions. These examples should not be istokov the VAT as limiting the scope of the invention. The following examples represent preferred embodiments of the present invention. Example 1:

Two load fermenter provided the use of Pichia pastoris for expression of the precursor IN 105. In a single load (experiment # 1) half the concentration of the control environment mistaken for the original environment with the exception of glycerol. After the boot stage injected methanol with speed feed approximately 8 g/l/h. The fermentation is continued for about 8 days. The maximum concentration of the product reaches 3.0 g/l within 7 days and stabilized. In another load (experiment # 2) use a medium of the same composition, and optionally in the fermenter add 0.1 M urea. After the boot stage are fed with methanol with 4% wt./about. the urea. The fermentation is continued for 8 days. The maximum concentration of the product reaches 3,5 within 7 days. A significant difference in terms of profiles of cell growth is not supervised. Profiles of biomass and product concentrations obtained from the above experiments, presented in figure 1 and 2, respectively.

Example 2:

The expression of the precursor of insulin urea explore using fermentation of Pichia pastoris for determining the level of expression of the product. In this study, using the composition of the control environments and methanol with 4% urea is injected at a higher level of 20 g/l/h. In this study, the maximum product concentration reaches 4,21 g/l for 137 relatively 4.26 deaths g/l for 182 hours, when urea is not added to the fermenter. No difference in the profile of cell growth is not observed, indicating that the addition of urea in the fermentor increases the level of expression of the product without affecting the profile of growth and reduce the fermentation time. Profiles of biomass and product concentrations obtained from the above experiments, presented in figure 3 and 4, respectively.

Example 3:

Two loading of the fermenter is carried out with the use of Pichia pastoris for expression of the precursor of glargine;. In a single load (experiment # 1) half the concentration of the control environment mistaken for the original environment with the exception of glycerol. After the boot stage injected methanol with the speed of feeding 8 g/l/h. The fermentation continued for 10 days. The maximum concentration of the product reaches of 1.03 g/l for 10 days and stabilized. In another load (experiment #2) use a medium of the same composition and in addition to the initial fermentation medium add 0.1 M urea. After the boot stage are fed with methanol with 4% urea. The fermentation continued for 9 days. The maximum concentration of the product reaches 1.75v for 9 days. Significant difference in relation to profile the growth of cells not see. Profiles of biomass and product concentrations obtained from the above experiments, presented in Figure 5 and 6, respectively.

Example 4:

Two loading of the fermenter is carried out with the use of Pichia pastoris for expression of the precursor of the basis. In a single load (experiment #1) half the concentration of the control environment mistaken for the original environment with the exception of glycerol when fed with glycerol. After the stage of feeding glycerol injected methanol with speed feed 11 g/l/h. The fermentation is continued for 6 days. The maximum concentration of the product reaches 0.74 g/l for 6 days and stabilized. In another load (experiment #2) use a medium of the same composition, and optionally add it to the fermenter 0.1 M urea. After the boot stage are fed with methanol with 4% urea. The fermentation is continued for 5 days. The maximum concentration of the product reaches 0,78 within 5 days. A significant difference in terms of profiles of cell growth is not supervised. Profiles of biomass and product concentrations obtained from the above experiments, presented in Fig.7 and 8, respectively.

Example 5:

Two loading of the fermenter is carried out with the use of Pichia pastoris for expression of the enzyme lipase. In a single load (experiment #1) half the concentration of the control environment p is inimum for the original environment with the exception of glycerol. After the boot stage injected methanol with speed feed approximately 6 g/l/h. The fermentation is continued for about 8 days. The maximum concentration of the product is approximately 1650×106 units of lipase within 7 days and stabilized. In another load (experiment #2) use a medium of the same composition, and optionally in the fermenter add 0.1 M urea. After the boot stage are fed with methanol with 4% wt./about. the urea. The fermentation is continued for 8 days. The maximum amount of product reaches approximately 2500×106 units of lipase within 7 days and then stabilized. A significant difference in terms of profiles of cell growth is not supervised. The General profile of the product obtained in the above experiments, presented in Fig.9.

Example 6:

Investigate the effect of concentration of urea in the downloads with methanol, used to obtain the predecessor IN 105, 1, 2, 3, and 5% urea in methanol at boot time in the recharge of methanol. The other parameters remain the same as in example 1. The maximum concentration of the product reaches of 3.0, 3.2, and of 2.5 to 3.3, 3.5, and 2.8 g/l for 7 days in downloads with 0, 1, 2, 3, 4 and 5% urea, respectively, in methanol. Increased productivity observed when using 4% urea in methanol during the phase of induction with methanol. Profiles of biomass and product concentrations is illustrated in Figure 10 and 11, respectively.

Example 7:

In another study with the level of feeding methanol and 20 g/l/h. The concentration of urea vary to determine the most effective concentration of urea, providing the maximum concentration of the precursor of insulin. The maximum concentration of the product reaches 4.26 deaths, 4,03, 3,39, 4,16, 4,21 and 5.31g/l at 182, 166, 140, 164, 137 and 170 hours, respectively, in the downloads where recharge of methanol is carried out with the concentration of urea 0, 1, 2, 3, 4 and 5%, respectively. Profiles of biomass and product concentrations to illustrate Fig and 13, respectively. The study shows that the addition of urea increases the level of production of the precursor of insulin, the expression level is highest when feeding methanol carried out with 5% urea.

Example 8:

Explore the experimental load, in which during the phase of induction with methanol support the residual concentration of urea at different levels of 0.1, 0.3, 0.5mm, of 0.7, 1.2 and 1.5 M in the cell-free supernatant, and study the effect on productivity. Take all the load parameters and the composition of the medium, similar to that shown in example 1. Residual urea is supported by a separate feeding urea. The results show that the maximum product reach, when the urea level support in the area of 1 M in the cell-free supernatant in EMA fermentation for download. The maximum concentration of the product of 4.46 g/l is reached, when the support level of residual urea approximately 0.5 M In this experiment, the total contribution of urea equivalent 0, 0,2, 0,5, 0,9, 1,2, 1,8, 2,3 and 2.9 M in a final volume of broth in the study, where the support level of residual urea 0, 0,1, 0,3, 0,5, 0,7, 1,0, 1,2 and 1.5 M, respectively. The results of the present Fig.

Example 9:

They also study the fermentation of the precursor of insulin. In this study, recharge of methanol is carried out at the level of 20 g/l/h. In the fermentation of the precursor of insulin is shown that the concentration of this product maximum support when the residual concentration of 0.7 M. In this study, the total contribution of urea equivalent 0,0, 1,6, 2,7, 3,5, 7,0, 8,8, 11,7 and 13.3 M final volume of broth in the study, where the support level of residual urea 0, 0,1, 0,3, 0,5, 0,7, 1,0, 1,2 and 1.5 M, respectively. The results show that culture can consume significant amounts of urea, as present Fig.

Example 10:

The other party IN 105 spend with standard control environments and with the addition of about 20 g/l/h. methanol with 4% urea. In this study reach product concentration 3,71 g/l for 113 hours relative to output 3,76 g/l for 183 hours, when urea is not added to the fermenter. The results of the research is ment to illustrate Fig and 17.

Example 11:

In the next group of experiments urea replace various other compounds to show their effects on the productivity of the fermentation. Thus, in a separate download test thiourea, diimide, carbodiimide, thiocarbamide at a concentration of 1%. Show that all of them increase the productivity relative to the control, as shown in Fig.

Example 12:

In experiment show that feeding urea during fermentation increases the consumption of phosphate yeast, resulting in the depletion of phosphate in the environment earlier in the boot, where the contribution of urea is omitted. Thus, accelerated phosphate and increased productivity (g/l/h.) are the result of a metabolic shift is achieved by introducing urea in standard or modified protocols fermentation of Pichia order to obtain peptides and proteins.

Example 13:

Prepare the environment for the growth of containing soy flour 5.0 g, dextrose monohydrate 20,0 g soy peptone 5.0 g, CaCO31.0 g, K2HPO40.1 g in 1000 ml of water. the pH of the seed medium down to 6.8±0.1 s using NaOH solution. Sterilized environment for inoculum inoculant the bottle suspension of spores of the culture of Streptomyces sp (BICC 6826) and incubated at 28±1°C for 48 hours under aerobic conditions. Then the grown seed transfer in Fe is mentation Wednesday, contains soy flour 37,5 g, dextrose monohydrate 22,5 g, cottonseed flour 3.75 g, corn syrup and 7.5, NaCl 7.5 and antifoam SAG 0.5 g in 1000 ml of water (pH down to 7.0±0,1). After 48 hours incubation add sterile solution compactin together with small amounts of dextrose. Every 24 hours is collected from the flasks of several similar flasks to test the bioconversion compactin in pravastatin. The procedure is repeated every 24 hours until the total recharge compaction will not be 3.0 g/L.

Before beginning experiments using modified fermentation medium in a fermentation medium was added different levels of urea, to set the level of toxicity of urea to the body. Concentration 0,0, 0,5, 1,0, 1,5, 2,0, 2,5, 3,0, 3,5 and 4.0 g/l added to the flask containing fermentation medium, then incubated as described above. After 48 hours incubation monitorium growth in each of the flasks. Data represent Fig.

Concentrations greater than 3.0 g/l of urea, show the inhibition of culture growth. In depth study shows similar results. As a consequence, the concentration below 3.0 g/l is taken to check the effect of urea on the bioconversion.

As described previously, a similar experience conducting environment for the formation of a product containing urea as additional components the NTA. The concentration of urea in the medium for education product support level 0,0, 0,5, 1,0, 1,5, 2,0, 2,5 and 3.0 g/l and add it to the time of inoculation.

After 48 hours incubation contribute sterile solution compactin together with water dextrose. The bioconversion assessed after 24 hours by collecting one of the many flasks, which are carried out in similar conditions. This procedure is repeated every 24 hours, while in General do not contribute to the recharge of compactin 3 g/L. the Bioconversion appreciate relative to the total number compactin consumed for the formation of pravastatin. The results of the experiment relative to the titer and percent conversion shown in Fig and 22, respectively.

The flask with the concentration of urea 1.5 g/l show the maximum level of conversion compactin in pravastatin 85,4% compared with the control flask with a conversion rate of 59.5%.

Example 14:

The expression of granulocyte colony-stimulating factor urea studied using fermentation of E. coli to determine the expression level of the product. The environment used in the study consists of preplant environment, planting environment and environment for product education. Preplant environment consists of soy peptone 10.0 g NaCl 10.0 g, yeast extract 10.0 g in 1000 ml of water. Sowing environment consists of 1.2 g of ammonium sulfate, 2.4 g of magnesium sulfate, 10 g of al is Zhevago extract, 11 g DMH, 5 g K2HPO440 ml of trace elements in 1000 ml of water. Environment for product education consists of dextrose monohydrate 11 g of ammonium sulfate 2.4 g, magnesium sulfate 4.8 g, yeast extract 20 g, 10 g K2HPO4, trace elements 40 ml in 1000 ml of water. pH down to 7.0 with ammonia. After completion of the loading phase, the biomass in the fermenter increase permanent makeup dextrose and yeast extract. Then induce cell mass and lead the process the next 8 hours to obtain a product of interest.

Spend three download fermentors to establish the effect of urea in a fermentation process using E. coli. First boot (experiment #1) is a control boot without adding any urea. Your environment described above. Download induce at about 180 g/l SEC. The fermentation is continued until 8 o'clock. after induction. Reach a peak of 6.6 g/l specific activity 0,028 g/SEC. The second load (experiment #2), in the fermenter add 1 g/l of urea in addition to the above environments. To download make the feed containing the same amount of urea (1 g/l). Download induce at about 180 g/l SPK and continue fermentation for 8 hours after induction and receive 7,4 g/l product with a specific activity 0,033 g/SEC. A third load (experiment #3) about the W ill result is similar to experiment #2, but with the addition of 2 g/l urea. Made the final product is to 6.58 g/l with a specific activity to 0.032 g/SEC.

The results show that not observe any significant difference in the profile of cell growth, indicating that the addition of urea increases the education level of the product without affecting the profile of growth. In General receive approximately 18% increase in specific productivity. The SEC profiles obtained by conducting the above experiments, presented at Fig, and profile specific productivity - Fig.

Example 15:

A similar experiment is carried out using E. coli as expressing system for receiving streptokinase. Use the same environment as mentioned in example 13, and the same test concentration of urea. As described in example 14, have the same three boot with a urea concentration of 0 g/l 1 g/l and 2 g/l Control boot without urea (experiment #1) gives the final productivity 7,06 g/l after 8 hours of induction with specific productivity was 0.026 g/SEC. The maximum titer of reach boot with 1 g/l urea (experiment #2)with the productivity of 10.5 g/l and specific productivity 0,041 g/SEC. Unexpectedly shown that loading with 2 g/l urea (experiment #3) gives only 6,56 g/l product specification the specific productivity of 0.022 g/SEC. For this product increased concentrations of urea greater than 1 g/l resulted in a sharp drop in specific productivity and titer.

As in example 2, there are no significant changes in the values of the SEC three studies that clearly shows that productivity improvement is the increased level of education of the product and is not associated with the biomass. In General receive approximately 57% increase in specific productivity. The SEC profiles obtained by conducting the above experiments, presented at Fig. Profile title presented on Fig.

Example 16:

Rhizomucor sp (BICC 362), which is known to produce the enzyme lipase, also show higher productivity when adding urea to the environment. The lipase generated from this culture, can be widely used for bioconversion reactions, for example, esterification and hydrolysis. For this process, take two download fermenter (volume 10 l), one without the addition of urea and the other with the addition of 0.5 g/l of urea. Conduct a study with a higher concentration of urea (1 g/l), which results in lower productivity and a very high level of consumption of caustic soda to maintain the pH. Environment for growth for Rhizomucor sp (BICC 362) consists of Maida to 41.4 g of sucrose, 10 g peptone of 3.06 g, ammonium sulphate 2 g, yeast extract 2 g, potassium phosphate 0,85 g, calcium chloride, magnesium sulfate and sodium chloride, 1 g each. Complete medium was adjusted to 1 l with water. Grown seed (10%) transfer to a medium for the formation of a product consisting of dextrose 12.5 g, soy peptone 37,5 g soy flour 25, phosphate potassium 2,5, of 0.625 magnesium sulfate, soybean oil, 12.5 g in 1000 ml of water. the pH of the medium down to 6.0 and then support at 6.0 during boot by adding caustic soda.

The addition of urea (0.5 g/l) shows the increase of the educational level of lipase in comparison with the control load. A higher concentration of urea shows a lower productivity. Data represent Fig.

It should be borne in mind that this invention is not limited to the described specific methods, protocols, cell lines, species or genera components and environments, as they may vary. In addition, it should be borne in mind that the terminology used in this context, serves only for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the attached claims. The above description is for the purpose of training an ordinary specialist in the field of technology, as almost COI is lesofat the present invention, and it is not intended to detail all those obvious modifications and variations which will be obvious to a competent specialist when reading the description.

1. Fermentation medium for the production of recombinant proteins selected from the group comprising insulin, a precursor of insulin and insulin analogs, using induced by methanol fungi species Pichia pastoris, supported characterized by the concentration of urea or its derivatives in the range from about 0.3 M to about 1 M and containing per liter of water basic salts in the following amounts:

orthophosphoric acid (85%)from 2.67 to 133,5 ml
calcium sulfatefrom 0,093 to 4.65 g
potassium sulfatefrom 1,82 to 91 g
magnesium sulfate-7H2Ofrom 1.49 to 74.5 g
the potassium hydroxidefrom 0,413 to 20,65 g
glycerinfrom 4 to 200 g

and per liter of water trace elements in the following amounts:
the copper sulfate-5H2O from 0.6 to 30 g
the sodium iodidefrom 0.008 to 0.4 g
manganese sulfate-H2Ofrom 0.3 to 15 g
molybdate sodium-H2Ofrom 0.02 to 1 g
boric acidfrom 0.002 to 0.1 g
the cobalt chloridefrom 0.05 to 2.5 g
zinc chloridefrom 2 to 100 g
ferrous sulfate ferrous-7H2Ofrom 6.5 to 325 g
Biotinfrom 0.02 to 1 g
sulfuric acidfrom 0.5 to 25 ml

2. Environment according to claim 1, in which derivatives of urea selected from the group comprising dimethyloxetane, dieselmachine, N-acetylphenylalanine, isopropylidenediphenol, prilocaine or combinations thereof.

3. Environment according to claim 1, in which urea or its derivatives are used in liquid form, in the form of a spray, powder or granules.

4. Environment according to claim 1, in which the insulin is an insulin IN-105.

5. Environment according to claim 1, in which the insulin is an insulin glargine.

6. Environment according to claim 1, in which recom is inantly protein is the basis.

7. A method of obtaining a recombinant protein selected from the group comprising insulin, a precursor of insulin and insulin analogs, including the stage of reproduction induced by methanol fungi species Pichia pastoris and the stage of expression of recombinant protein using a fermentation medium according to claim 1, in which the contribution of methanol with a speed of from about 6 g/l/h to about 20 g/l/h

8. The method according to claim 7, in which derivatives of urea selected from the group comprising dimethyloxetane, dieselmachine, N-acetylphenylalanine, isopropylidenediphenol, prilocaine or combinations thereof.

9. The method according to claim 7, in which the produced insulin is the insulin IN-105.

10. The method according to claim 7, in which the produced insulin is an insulin glargine.

11. The method according to claim 7, in which the resulting recombinant protein is the basis.



 

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9 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of cultivating Bacillus brevis strain 101 for producing gramicidin S. Submerged cultivation of a culture is carried out on a synthetic culture medium. The medium contains yeast autolysate and casein hydrolysate in concentration of 0.1 g/l and 0.2 g/l on amine nitrogen, glycerine in concentration of 20 ml/l, edible 40% lactic acid 2.0-4.0 ml/l, ammonium phosphate-chloride 0-3.4 g/l, di-substituted ammonium phosphate 0.85-4.5 g/l, mono-substituted potassium phosphate 0-1.0 g/l, magnesium sulphate heptahydrate 0.9 g/l, sodium citrate 1.0 g/l. Content of amine nitrogen in the initial medium is equal to 1.3-1.6 g/l. When concentration of amine nitrogen falls to less than 1.4 g/l, a concentrated culture solution is added to the medium until achieving concentration of 1.75 g/l. The concentrated culture solution contains glycerine, edible 40% lactic acid, di-substituted ammonium phosphate and chloride and magnesium sulphate with ratio of concentration of glycerin, lactic acid, nitrogen, phosphorus and magnesium equal to 1:(0.008-0.032):(0.027-0.036):(0-0.008):(0.002-0.008). During growth, the rate of stirring is increased from 200 to 500 rpm. pH is kept at 6.5-6.8 by adding potassium and sodium hydroxide. The process is stopped 2-6 after the onset of a stationary phase.

EFFECT: method enables reproducible production of a large amount of gramicidin S.

10 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.

EFFECT: invention enables increase of vitamin K2 content in the product.

11 cl, 5 tbl

FIELD: chemistry.

SUBSTANCE: invention discloses acylamidase enzyme AA37 from Rhodococcus erythropolis 37 All-Russian collection of industrial microorganisms Ac-1793, with a sequence given in the description. A nucleotide sequence which codes this enzyme is defined. The invention describes a method for synthesis of N-substituted acrylamides in aqueous medium from acrylamide and amines in the presence of an acylamidase biocatalyst in isolated state or in a composition with E.coli cells.

EFFECT: invention enables to obtain N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines in an aqueous medium.

3 cl, 14 ex

FIELD: medicine.

SUBSTANCE: method for producing an Escherichia coli cell fraction showing protease inhibiting activity provides bacteria cells preservation in the presence of buffered 80-90% glycerine, processing by 3% triton X-100 for removing a cell membrane. Produced cytoplasm proteins are extracted by the increasing salt concentration, namely 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanole. It is followed by affine sepharose 4B chromatography with immobilised trypsin and estimation of protease inhibiting activity in eluates.

EFFECT: invention can be used in analysis of molecular-genetic mechanisms of procaryote cell structure formation and roles of protein components in their organisation, and also genome remodelling nature that is necessary for disclosing of regulation pathways of mechanisms of macro-and microorganism action, and also search of new drug targets and development of ecologically safe new drugs.

3 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: method of bacterial cell immobilisation provides introduction of bacterial cells Erwinia rhapontici B-9292 in the concentration 10 % in a poly-N-vinylpyrrolidone solution of molecular weight 500 kDa in the concentration 1 mg/ml in an acetate buffer pH=6.0. The received reaction mixture is kept for 3 hours at temperature 22-25°C, and then the immobilised bacterial cells are centrifuged. The isomaltulose yield is 92-95%.

EFFECT: method allows to intensifying the saccharose transformation process and providing higher isomaltulose yield.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: Brevibacillus laterosporus bacteria strain, Russian National Collection of Industrial Microorganisms No. B-10531 is produced by multistage selection from natural Brevibacillus laterosporus 16-336 strain. An algicide activity of the strain makes 10.5-52.5 % of residual optical density in 24-h incubation. A diameter of a zone of growth retardation of plant pathogenic fungi makes 3-14 mm. A diameter of a zone of growth retardation of pathogenic bacteria makes 5-12 mm.

EFFECT: strain produces a wide spectrum of biologically active compounds for control of microscopic algae of various taxonomic types, plant pathogenic fungi and pathogenic bacteria.

5 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to preparing physiologically active compounds, and concerns a bacteriorhodopsin producer strain. The Halobacterium salinarum ST 2 strain is produced by multistage selection of a Halobacterium salinarum halophilic bacterial strain, Russian National Collection of Industrial Microorganisms No. B-9025, without chemical pretreatment and is deposited in Russian National Collection of Industrial Microorganisms No. B-10425.

EFFECT: Halobacterium salinarum strain Russian National Collection of Industrial Microorganisms No B-10425 exceeds both a prototype, and a parent strain in efficiency.

2 ex, 2 tbl

FIELD: food industry.

SUBSTANCE: pasteurised food product containing a proline-specific protease has water activity equal to at least 0.85. Used as the enzyme is protease extracted from Aspergillus or belonging to the S28 serine proteases family. The optimal activity of the said protease is at a pH value from 1 to 7, preferably - at a pH value from 2 to 6. Additionally proposed is a food product containing less than 1 wt % of protein or peptides. The said food products are produced by way of addition of a proline-specific protease to them.

EFFECT: such products consumption ensures gluten peptides splitting and is recommended to patients suffering from gluten intolerance.

17 cl, 5 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining a yeast extract involves treating Saccharomyces cerevisiae yeast cells with purified phospholipase A and separating the yeast extract from treated yeast cells. Yeast cells undergo proteolysis during treatment with phospholipase. Treatment with phospholipase is carried out at pH 2-10, and the amount of phospholipase ranges from 0.001 to 1 mg enzymatic protein/g dry substance. Yeast extract output is equal to 68.8%, protein output is equal to 67.4% and degree of hydrolysis is equal to 71.8%.

EFFECT: method enables to obtain an end product with high output, protein content and degree of hydrolysis.

6 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method for preparing isotope-marked secondary metabolic products of fungi provides immobilised fungi growing on an inert carrier with an artificial fluid culture medium added. All carbon, nitrogen and/or sulphur atoms in the specified medium are substituted by stable isotopes chosen from the group including 13C, 15N, 33S and 34S. Also, an isotope-marked secondary metabolic product made of fungi is presented.

EFFECT: high-purity end product preparation.

13 cl, 2 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Method of obtaining lipids from microorganisms includes processing cells of microorganisms, grown in fermentative medium, for release of intra cellular lipids, dissolving part of present proteins. Fermentative medium is divided into layers: heavy aqueous and light, containing lipids, separation being carried out in medium, which contains less than 5% of non-polar organic solvent, without allowing non-polar organic solvent to extract lipids. Then heavy layer is separated from light layer and lipids are obtained from light layer.

EFFECT: creation of efficient and economical method of obtaining lipids from microorganisms.

45 cl, 6 tbl, 1 ex

FIELD: wood chemical industry.

SUBSTANCE: method involves treatment of balsamic poplar vegetative part with water to isolate essential oil and a solid residue. Treatment with water is carried out by hydrodistillation followed by extraction of solid residue with 94-96% ethyl alcohol to obtain alcoholic extract and oil cake. Wax is isolated from alcoholic extract and evaporated to obtain lipid concentrate, and oil cake is wetted with vat liquid prepared in hydrodistillation and subjected for biodestruction with fungi of genus Trichoderma. Method provides preparing the complex of biologically active substances from balsamic poplar vegetative part.

EFFECT: improved preparing and processing methods.

2 cl, 2 ex

FIELD: biotechnology, microbiology, pharmacology.

SUBSTANCE: invention relates to preparing a medicinal preparation using microorganism-producers. Lecanicillium sp. G16 strain is isolated from a soil sample from Teberdinsky reservation and deposited in Collection of microorganism cultures GNTSA at number 347A. Invention provides preparing a complex preparation possessing high hypolipidemic and antitumor activity and prolonged effect in low concentrations.

EFFECT: valuable medicinal properties of strain and preparation.

3 tbl, 1 ex

FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.

SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella polycephala micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object and stored there during 45 days and used in space feeding.

EFFECT: prolonged shelf life and improved quality of citrus fruits.

FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.

SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella parvispora micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object, stored there during 45 days and used in space feeding.

EFFECT: prolonged shelf life and improved quality of citrus fruits.

FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.

SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella pulchella micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object, stored there during 45 days and used in space feeding.

EFFECT: prolonged shelf life and improved quality of citrus fruits.

FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.

SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella gamsii micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object and stored there during 45 days and used in space feeding.

EFFECT: prolonged shelf life and improved quality of citrus fruits.

FIELD: biotechnologies.

SUBSTANCE: method includes conservation of cells in presence of buffered 80-90% glycerine with the following removal of cell shells with 3% triton X-100, extraction with growing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% b-mercaptoethanol, extraction of positively charged proteins from above fractions with the help of ion-exchange chromatography with amberlite IRC-50 in the continuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% in 0.1 M potassium-phosphate buffer pH 6.8 and detection of inhibiting activity in them relative to tripsin.

EFFECT: invention may be used to analyse molecular-genetic mechanisms of procaryote cell structure generation and role of protein components in their organisation, and also features of genome remodelling.

9 dwg, 1 ex

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