Fermentation medium and method of producing recombinant proteins

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to a fermentation medium and a method of producing recombinant proteins using said medium. A fermentation medium for producing recombinant proteins selected from a group including GM-CSF, streptokinase and lipase, using microorganisms selected from a group including: E. Coli, Streptomyces sp. and Rhizomucor sp., is characterised by keeping concentration of urea or derivatives thereof in the range of 0.5 g/l to 2 g/l. The medium contains, per litre of water, basic salts in the following amounts: orthophosphoric acid (85%) 2.67-133.5 ml, calcium sulphate 0.093-4.65 g, potassium sulphate 1.82-91 g, magnesium sulphate-7H2O 1.49-74.5 g, potassium hydroxide 0.413-20.65 g, glycerine 4-200 g. The medium contains, per litre of water, trace elements in the following amounts: copper sulphate-5H2O 0.6-30 g, sodium iodide 0.008-0.4 g, manganese sulphate-H2O 0.3-15 g, sodium molybdate-H2O 0.02-1 g, boric acid 0.002-0.1 g, cobalt chloride 0.05-2.5 g, zinc chloride 2-100 g, iron (II) sulphate-7H2O 6.5-325 g, biotin 0.02-1 g, sulphuric acid 0.5-25 ml.

EFFECT: invention increases the output of end products.

6 cl, 27 dwg, 3 tbl, 16 ex

 

The present invention relates to the use of amides of carbonic acids, such as urea or its derivatives, carbamates, carbodiimides and thiocarbamide, as n additions in a fermentation medium to obtain recombinant proteins, such as G-CSF, streptokinase and lipase, with the aim of achieving increased levels of bioconversion by fermentation of suitable expressing organisms, such as E. coli, Streptomyces sp., Asp.ergillus sp., Rhizopus sp., Penillium sp. and Rhizomucor sp. Significant aspects of the invention, in particular, relate to the experimental process of fermentation using the optimized parameters of the culture media, responsible for higher product yield within shorter periods of education.

The level of technology

Expressing system on the basis of yeast, such as Pichia pastoris, are commonly used for expression of recombinant proteins, see Cregg, J. M. et al., in Dev. Ind. Microbiol. 29:33-41; 1988. Expressing system R. pastoris uses induced by methanol promoter of alcoholiday (AO1), which controls the gene that encodes the expression of alcoholiday, the enzyme that catalyzes the first stage of the metabolism of methanol (see J. M. Cregg et al. in Bio/Technology 11:905-910; 1993). R. pastoris has the potential for high levels of expression, efficient secretion of extracellular protein posttranslational m�of deficate, such as glycosylation, and growth to high values of density of cells on minimal medium in bioreactor cultures.

Method periodic fermentation with fed using Pichia pastoris as described in "Pichia fermentation Process Guidelines" (Guide to the Pichia fermentation process) company Invitrogen Co. (San Diego, CA), hereinafter designated as the control. To obtain recombinant proteins in transformed Pichia pastoris is grown to the desired biomass, the high density of cells on glycerol as a carbon source. Phase of product formation is initiated by feeding methanol, which serves as the inducer and sole carbon source for the culture. During the formation of biomass and the phase of product formation ammonia, which serves as a source of nitrogen, is used to control the pH.

Despite the various benefits of yeast expressing systems, there is a need to optimize impacted by nutrients physico-chemical environment for efficient and maximum production of the protein in bioreactors. Much needed is the achievement of high specific productivity. It can be obtained by optimizing the initial environment, the strategy of make-up methanol and physico-chemical parameters of the process. There are publications in which ammonium sulfate,ammonium phosphate, to diammonium phosphate, potassium nitrate, urea, corn syrup, soy flour, cottonseed flour, molasses of sugar cane and beet, Peptones, flour hydrolysates, etc. are used as a source of nitrogen for the cultivation of bacteria, yeast and fungi. The use of different sources of carbon and nitrogen for growth of microorganisms constitutes prior art.

However, the optimal combination of specific sources of carbon and nitrogen for efficient production of recombinant proteins, peptides and enzymes are not disclosed in the literature, corresponding to the prior art. For example, application WO/2007/005646 discloses the production of ethanol actually culturing the recombinant yeast in a holistic environment for the growth that contain complex carbohydrates, as well as a number of cheaper nitrogen sources such as corn syrup, corn steep liquor, yeast autolysis and urea. In addition, this method does not use induced by methanol, the mechanism for growth or production unlike developed in the present invention of a method for producing recombinant proteins. Similarly, U.S. patent 4288554 describes the method of continuous fermentation just to grow non-GMO (Nagano-modified) form of Candida with the use of urea in combination with other sources of nitrogen and primary�th salt environment. In this case there is no suggestion or description of where urea may be used during the fermentation process (periodic, periodic with feeding, continuous) using induced with methanol GMO Pichia pastoris for efficient production of recombinant proteins and peptides, such as human insulin and its analogues, or enzymes like lipase.

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

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

For many years the culture of mushrooms used for production of valuable biomolecules, such as enzymes, and other useful molecules. Mushroom culture, such as Rhizopus, Aspergillus, Penicillium, etc., used in classical fermentation, designed to get a wide range of enzymes, such as lipase, amylase, dextranase, etc., which are used in food, textile, leather, and other similar industries. Actinomycetes, are known as the basic components for producing antibiotics, are widely used to produce several secondary metabolites that are useful to man. One of the key properties of fungi and actinomycetes is the property "bioconversion", such as hydroxylation, esterification, etc. the Main advantage is that bioconversion of specific in relation to the target, 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 stirring and supply of oxygen, or mo�the Internet settings, relating to the composition of the nutrient media, such as modification of the concentrations of sugars during fermentation, changes in treatment during the process or changes related to natural properties of the microorganism, etc.

Unexpectedly found that the fermentation broth, characterized by a controlled addition of some rich and readily soluble sources of nitrogen, such as urea, to yield increased productivity and/or increased level of bioconversion and, thus, reduced production time.

Disclosure of the invention

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

Another primary object of the present invention is to provide a method of producing recombinant products, their derivatives or analogues.

Another main objective of the present invention to provide a recombinant protein product.

Accordingly, the present invention relates to a fermentation medium, intended for the preparation of recombinant proteins, their derivatives and their analogs by fermentation using microorganisms, with the specified environment differs in that it contains an effective level of�radio amide of carbonic acid; a method for producing recombinant proteins, their derivatives or their analogues, which provides for the multiplication induced or pendulumeca microorganism expressing a protein in a fermentation environment, and this environment differs in that it contains an effective concentration of the amide of carbonic acid.

The present invention relates to the fermentation medium 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.

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

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

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

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

In yet another embodiment of the us�Mr sage of the invention increased consumption of phosphate.

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 the multiplication induced or pendulumeca expressing a protein of a microorganism in a fermentation medium, wherein the specified environment differs in that it contains an effective concentration of the amide of carbonic acid.

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

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

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

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

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

In yet another variant implementation�of Tulane present invention, the level of make-up methanol is 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.

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

The invention provides a composition of nutrients intended for use in the development of a fermentation broth, wherein the composition contains nitrogen components, such as amides of carbonic acid, for example the above-mentioned urea and related forms or derivatives, together with one or more other components of the fermentation medium, which are specially optimized for high yield of recombinant proteins for shorter time periods of education.

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

Additional nitrogen component, such as urea, can be added in the form of liquid, spray, �orosco or pellets.

The main problem of the invention consists in the fact that the productivity of the process of fermentation of the recombinant protein by using the microorganism is strongly influenced by the content of urea in the medium for cultivation. As a consequence, the product yield is significantly increased, especially with shorter periods of fermentation, adding nitrogen component, such as urea, in the medium for cultivation.

According to the preferred embodiment of the invention, the addition of urea in the fermentation medium increases the level of consumption of the key ingredient "phosphate", which, in turn, improves productivity. Discovered that the faster the consumption of phosphate, the shorter the cycle time of fermentation and, consequently, higher productivity. Thus for the first time shown the metabolism of urea, along with phosphate, which increases the level of expression of the protein or peptide without affecting the profile of growth and shortens the fermentation time.

According to another aspect of the invention, the urea provides a higher level of product selection at the end of fermentation at any pH.

Thus, the present invention gives higher outputs protein product, reduced the cycle time of production, improved utilization of nutrients introduced into the process, and � generally reduces capital costs and production costs.

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

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

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

The term "fermentation broth" or "fermentation medium" refers to the environment in which the fermentation is carried out, which includes the fermentation substrate and other raw materials used by the 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 kinship group. The present invention provides for the use of derivatives of urea, such as demethylation, dialymotion, N-acetyl-N-phenylacetone, isopropylidenebis, N-phenylacetone, etc., or combinations thereof.

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

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 are Pichia pastoris, Pichia sp., Saccharomyces sp.,Saccharomycescerevisiae, Kluyveromyces sp. or Hansenula polymorpha.

The invention may also be suitable for expression of any recombinant peptide using inducible by methanol species of fungi, but not limited recombinante expressed peptides, proteins, insulin, precursors of insulin, insulin derivatives or insulin analogues.

The term "recombinant," as used in this context to describe a protein or polypeptide means a polypeptide produced by expression of the recombinant polynucleotide. The term "recombinant," as used in this context in relation to cells means cells that can be or have been used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected by. It should be borne in mind that the progeny of a single parental cell may not be completely identical in morphology or in genomic complement or 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�th length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. The term also does not refer to postexplosion modifications of the polypeptide, or excludes them, although chemical or postexplosion modifications of these polypeptides can be included or excluded as specific embodiments. In one embodiment of the molecule is a polypeptide or its related analogs or derivatives thereof. Preferably, when the polypeptide is a cyclic peptide. According to another preferred embodiment of the polypeptide is a non-cyclic peptide. In yet another preferred embodiment of the polypeptide selected from the group consisting Bloomington: Indiana, 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 b-chain of 30 amino acids. The chains are connected to each other 2 by disulfide bridges. This definition includes not only the use of natural insulin, but also insulin derivatives and analogs. The connection of insulin may, for example, to provide a compound of the mammalian insulin, such as human insulin, or derivatives �for analogs of the compounds of insulin.

Insulin derivatives are derivatives of natural insulins, namely human insulin or animal insulins, which differ from the corresponding in other respects identical to natural insulin substitute at least one of the natural amino acid residue and/or the introduction of 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 can be at least about 60% homologous to the natural insulin. Insulin derivative can be even more homologous, for example at least about 75%, or at least about 90% homologous to natural insulin. Typically, insulin derivatives have slightly modified the action compared with human insulin.

Upon receipt of the insulin and insulin derivatives by genetic engineering of the precursor of insulin often Express "proinsulin", containing b-, C - and a-chains. This proinsulin can be turned into insulin or an insulin derivative by enzymatic or chemical removal of P-chains after appropriate and proper stacking and the formation of disulfide bridges. Proinsulin can be derived by IU�Isha least 60% homologous on b - and a-chains of natural proinsulin. However, binding of C-peptide can be selected as completely different from any known C-peptide. Proinsulin derivative can be even more homologous, for example at least about 75%, or at least about 90% homologous to natural proinsulin.

Recombinant insulin product is an IN-105.

The resulting drug product specifically refers to the molecule IN-105. IN-105 is a molecule of insulin, conjugated to 6-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 may be nanoconjugates by A1, B1 and B29, deconjugating on various combinations of A1, B1 and B29 or trichohyalin on various combinations of A1, B1 and B29.

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

The Fe Protocol�documentation may include three phases: loading, feed (optional), and the phase of 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*7H2O29,8
K2SO4By 36.4
CON4,13
Glycerin40
H3PO4(density of 1.7)The level of 22.95
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. Solution of trace elements and D-Biotin (pre-sterilized by filtration) is aseptically added to the medium, each with a speed of 4.35 ml/l of medium (density of the solution of trace elements is 1.05, and D-Biotin 1,0). The composition of trace elements solution:

Components (salt)Quantity (g/l)
Copper sulphate, CuSO4*5H2O6,0
Sodium iodide, Nal0,08
The manganese sulfate, MnSO4*H2O3,0
Molybdate of sodium, Na2MoO4*2H2O0,20
Boric acid, H3BO30,02
Cobalt chloride, CoCl2*6H2O0,50
Zinc chloride, ZnCl220,0
Ferrous sulfate, FeSO4*7H2O65,0
Sulfuric acid, H2SO45,0 ml

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

Preparation of solution of Biotin:

D-Biotin 0.2 g/l

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

Feeding yeast extract and soy peptone:

In addition, recharge of yeast extract and soy peptone also introduced during the fermentation. It should be prepared in the following way:

ComponentsConcentration (g/l)
Soy peptone100
Yeast extract50

The components are dissolved and with the help of drinking water get the required volume. The solution was then sterilized at 121°-123°C for 90 min. Density of feed soy peptone approximately 1,05.

Make-up methanol:

12,0 ml of trace elements solution, 12 ml solutions of D-Biotin and 40 g of urea was added per liter of methanol before the introduction of the feed.

Method of fermentation:

Method of fermentation involves the growth phase of the cells in loading, an optional phase feeding with glycerol loading and phase of induction with methanol.

Phase of cell growth in the download

Monitoring and control loading

The production parameters of the fermenter set the input and control as follows:

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

The induction phase of methanol (FIM)

Feeding methanol begin immediately after the end of the loading phase. Methanol sterilized (in download mode) by filtration 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 6,3±0,1 depending on protein expression in the environment (varies from product to product and from clone to 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 of yeast extract and soy peptone also begin in the fermenter at a speed of 0.4 g/l/HR source volume.

Monitoring and control FIM

Temperature:18-30°C (varies from product to product and from clone to clone)
pH:3,0-7,0
DO:>1% (used to control�I methanol concentration in the broth)
Time: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 is prepared by culturing freeze-dried glycerin source culture on minimal glycerol medium (EASC). The main fermentation medium obtained from Control Pichia process guidelines (Guide for the control of processes using Pichia), contain phosphoric acid, dehydrated calcium sulfate, potassium sulfate, magnesium sulfate is heptahydrate, potassium hydroxide, glycerine, minerals and D-Biotin. Nutrient medium for cultivation should also include known compounds in small or trace quantities, which are usually administered in a fermentation culture medium, such as water-soluble compounds of CA, Mg, Mn, Fe, K, Co, Cu, Zn, In, Mo, Br and I. May also be present other trace elements. Solution of trace elements according to the present invention, in particular, includes copper sulfate pentahydrate, sodium iodide, manganese sulfate monohydrate, sodium molybdate dehydrate, boric acid, cobalt chloride hexahydrate, zinc chloride, ferrous sulfate heptahydrate. Although the concentration of each�wow ingredient environment specifically optimized for each product, below the lead of the control environment.

Control environment

Fermentation basic salt medium

1 liter mix the following ingredients:

Phosphoric acid85% (26,7 ml)
Calcium sulphate0,93 g
Potassium sulphate18,2 g
Magnesium sulfate-7H2O14,9 g
Potassium hydroxide4.13 g
Glycerin40,0 g
Waterto 1 liter

Add to the fermenter with water to an appropriate volume and sterilized.

Trace elements RTM

Mix the following ingredients:

Copper sulfate 5H2O6.0 g
Sodium iodide0.08 g
Sulfate manganese-N2On3.0 g
Molybdate sodium-2H2O0.2 g
Boric acid�TA 0.02 g
Cobalt chloride0.5 g
Zinc chloride20.0 g
Ferrous sulfate-7H2O65,0 g
Biotin0.2 g
Sulfuric acid5,0 ml
Watera final volume of 1 liter

Filtered, sterilized and stored at room temperature.

When mixing these ingredients may appear muddy sediment. Environments can be filtered, sterilized and used.

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

According to another aspect of the invention, the formation of biomass during the growth phase in the loading takes place until such time as in the original environment is present glycerin. In addition, the formation of biomass is not an important factor, and it is carried out only in a few cases.

According to a further aspect of the invention, after reaching the desired biomass culture induce the constant feeding methanol and urea. During the feeding of the methanol is carried out also feeding yeast ex�a terrorist attack and solution of peptone.

According to another aspect of the speed of feeding of methanol up to 20 g/l/h. As understands any competent specialist, optimization speed-UPS to further enhance levels of production contemplated by this invention.

In addition, the invention will now be described in connection with some preferred variants of implementation in the following examples, to better understand and appreciate aspects of it. To limit the invention data specific variants of implementation does not provide. On the contrary, intend to cover all alternatives, modifications and equivalent solutions because they can be included in the scope of the invention as defined in the attached claims. Thus, the following examples which include preferred embodiments of implementation, will serve to illustrate the practical implementation of this invention, and it should be borne in mind that the details are shown only as an example and for the purpose of illustrative discussion of preferred embodiments of the present invention and are present for the presentation of the material, which is considered the most useful and easily understood description of the methods of fermentation, and of the principles and conceptual aspects of the invention.

The invention ol�includes nutrient composition, intended for use in the preparation of the fermentation medium, wherein the composition contains nitrogen components, such as carbamide, such as urea and related forms or derivatives, such as carbamates, carbodiimides, 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 recombinant protein products such as insulin glargine IN 105, Bloomington: Indiana, lipase and carboxypeptidase during the fermentation process (periodic, periodic with feeding, continuous) using induced with methanol GMO Pichia pastoris provided by the addition of urea without affecting the growth of yeast cells.

According to one aspect of the invention, the nitrogen component, which specifically affects the output and production time, represents the amide of carbonic acid, such as urea and its derivatives and the above related compounds.

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

The present invention demonstrates the use of amides of carbonic acid, such as urea or its derivatives, carbamates, carbodiimides and thiocarbamide, as n additions in a fermentation medium to obtain proteins with the aim of achieving increased levels of bioconversion using E. coli, Actinomycetes and fungal cultures. Significant aspects of the invention, in particular, relate to the experimental process of fermentation using the optimized parameters of the culture media, which is 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 organism expressing.

The invention provides for a nutritional composition intended for use in the preparation of the fermentation medium, wherein the composition contains nitrogen components, such as carbamide, such as urea and related forms or derivatives, such as 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 PR protein�products, such as G-CSF, streptokinase, HGH, etc., during the fermentation process (periodic, periodic with feeding, continuous) using induced with methanol GMO Pichia pastoris using inducible E. coli, carried out by the addition of urea without affecting the growth of yeast cells.

It is also possible to increase the level of bioconversion to obtain high yields of products, such as fermentation method pravastatin (periodic, periodic with feeding, continuous) using actinomycetes and/or fungal cultures

The present invention, furthermore, increases the production of enzymes, such as lipase, amylase, cellulase, periodic, or periodic with recharge processes using fungal cultures.

According to one aspect of the invention, the nitrogen component, which specifically affects the output and production time are amides of carbonic acid, such as urea or its derivatives and the above related compounds.

According to another aspect of the invention, the preferred microorganisms are strains of the family Enterobacteriaceae, preferably for use as body-producent include, but without limitation enumerated E. coli.

According to another preferred aspect of the invention the microorganism�anistemi are strains of actinomycetes and/or family 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 competent professionals 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 merely as illustrations. Various changes and modifications included within the essence and scope of the invention disclosed will become apparent to competent professionals in engineering from reading the following descriptions and from reading the other sections of the present description.

The invention provides for a nutritional composition intended for use in the preparation of the fermentation medium, wherein the composition contains nitrogen components, such as amides of carbonic acid, such as urea and related forms or derivatives of the 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 (�reamer, urea and related forms or derivatives), in specific concentrations does not affect the growth of the fermenting organism, but increases productivity.

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

A suitable microbial strain for industrial fermentation method may be any wild type strain producing a valuable compound of interest, provided that the wild-type strain has good growth characteristics.

In addition, a suitable microbial strain for industrial fermentation method may be a strain that receive and/or will improve the fact that the parent strain of interest, is subjected to a classical mutagenic treatment or transformation of recombinant DNA, also provided that the resulting mutant or transformed microbial strain has good growth characteristics. Thus, the characteristics of the growth of the parent strain will depend on whether the resulting mutant or transformed strains having an improved or similar growth characteristics compared to the characteristics of the parent strain.

The fermentation method with the use of this enzymatic environment the impact�instown in respect of one or more parameters, selected from the group consisting of the concentration of the product (product/volume), the product (formed product/consumed carbon source) and formation of product (formed product/volume and time), or other additional parameters and their combinations.

As will be borne in mind by a competent specialist in the field of technology, the optimal concentration of powerups amides of carbonic acid will vary from clone to clone, although the ultimate result in all cases is to obtain higher titers in less time.

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

The fermentation medium in the present invention must contain suitable carbon substrates. Suitable substrates may include, but without limitation listed, monosaccharides such as glucose and fructose, polysaccharides, such as starch or cellulose or mixtures thereof and other ingredients corn syrup, sugar beet molasses, glycerin and barley malt. Additionally, the carbon substrate may also be one-carbon substrates such as dioxide ug�of erode, or methanol, which is shown metabolic conversion into key biochemical intermediates. Therefore stipulate that the source of carbon used in the present invention may cover a wide range of carbon-containing substrates and will only be limited by the choice of organism. In addition to an appropriate carbon source, the fermentation medium must contain suitable minerals, salts, buffers and other components, known to those skilled in the art, suitable for the growth of crops and stimulation of expression of the desired protein or the final product.

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

Suitable nitrogen sources may include, but without limitation listed, soya flour, cottonseed flour, Peptones, yeast extract, casein, casein hydrolysates, corn syrup and inorganic salts of ammonium ion, nitrates and nitrites.

According to a significant aspect of the invention, the preferred additive in the fermentation medium are amides of carbonic acid, such as urea. They will include compounds containing N-CO-N or kinship group. The present invention provides for the use of derivatives of urea,�x as dimethyloxetane, dialymotion, N-acetyl-N-phenylacetone, isopropylidenebis, N-phenylacetone, etc., or combinations thereof.

Used the term "effective amount" is an amount of urea or its derivatives, the introduction which according to the invention in a fermentation environment leads to the formation of substantial amounts of/output of 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, actinomycetes and/or species of fungi, preferably for use as body-producent include, but without limitation 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 the recombinant polynucleotide. The term "recombinant," as used in this context in relation to cells means cells that can be or have been used as recipients for recombinant vectors or other transfer DNA, and include �otomoto the source cell, which was transfected by. It should be borne in mind that the progeny of a single parental cell may not be completely identical in morphology or in genomic complement or 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. The term also does not refer to 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 of the molecule is a polypeptide or its related analogs or derivatives thereof. Preferably, when the polypeptide is a cyclic peptide. According to another preferred embodiment of the polypeptide is a non-cyclic peptide. In yet another preferred embodiment of the recombinant protein is produced by fermentation using a fermentation environment corresponding to the present invention.

One of the embodiments of the present invention relates to the production of granules�zitarrosa colony-stimulating factor.

Granulocyte colony-stimulating factor is a pharmaceutically active protein that regulates proliferation, differentiation and functional activation of neutrophil granulocytes (see articles 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); Neben, et al. Blood 81(7): 1960-1967(1993)). G-CSF means the natural or recombinant protein, preferably human, as obtained from a traditional source, such as tissues, 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 an organism that is not a primary metabolite and is not required for growth of the microorganism under standard conditions. Connection of secondary metabolites can be converted into useful compounds by subsequent chemical conversion or subsequent biotransformation. In this case, ensuring high data availability intermediates would, in addition to increased production of the final beneficial compounds, which in itself can be regarded in this context as a secondary metabolite.

In one TSA�the invention, the fermentation Protocol may include two phases: periodic and periodic with makeup (optional).

Brief description of the drawings

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

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

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

Fig.4: Comparison of concentration profile product precursor of insulin with the addition of/without the addition of urea.

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

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

Fig.7: Comparison of the profile of biomass predecessor, Bloomington: Indiana with the addition of/without the addition of urea.

Fig.8: Comparison of concentration profile product predecessor, Bloomington: Indiana with the addition of/without the addition of urea.

Fig.9: comparison of the concentration profile of the product of the enzyme lipase with the addition of/without the addition of urea.

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

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

Fig.12: Profiles of biomass obtained at time�ary concentrations of urea during fermentation precursor of insulin.

Fig.13: the concentration Profiles of the products obtained at different concentrations of urea during fermentation precursor of insulin.

Fig.14: residual concentration of urea and the maximum concentration of the product to obtain IN-105.

Fig.15: Profile of residual urea concentration and maximum concentration of the product to obtain the precursor of insulin.

Fig.16: Comparison of the profile of biomass predecessor-IN-105 at the level of the supply of methanol to about 20 g/l/h.

Fig.17: comparison of the concentration profile of the predecessor product IN-105 at the level of the supply of methanol to about 20 g/l/h.

Fig.18: the Study of compounds other than urea, testing of other related compounds in terms of their impact on the productivity of Pichia fermentation.

Fig.19: Effect of urea on the residual concentration of phosphate ions in the broth. The effect of urea on the metabolism of phosphate by a strain.

Fig.20. Profile growth culture for the formation of pravastatin.

Fig.21. Caption pravastatin adding urea.

Fig.22. The percentage of turning compactin in pravastatin adding urea.

Fig.23. Comparison of the density of the medium cell culture (SPC) for the formation of G-CSF in E. coli.

Fig.24. The effect of urea on the specific productivity of G-CSF.

Fig.25. A comparison of the SEC for the formation of streptokinase in E. coli.

Fig.26. Effe�tons of urea on the specific productivity of streptokinase.

Fig.27. The effect of urea on the production of lipase using Rhizomucor sp.

The implementation of the invention

The present invention further specify in the following examples. It should be borne in mind that these examples, although they show preferred embodiments of the invention, lead is only for illustration purposes. From the above discussion and these examples competent specialist in the field of technology can establish the main features of this invention and the comfort of 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 construed as limiting the scope of the invention. The following examples represent preferred embodiments of the present invention.

Example 1.

Two download the fermenter is carried out with the use of Pichia pastoris for expression of the precursor IN-105. In one batch (experiment # 1) half the concentration of the control environment is taken for the original environment with the exception of glycerol. After the boot stage methanol injected at a speed of recharge approximately 8 g/l/h. The fermentation was continued for about 8 days. The maximum concentration of the product reaches 3.0 g/l within 7 days and stabilized. In another downld�the ZEC (experiment # 2) use a medium of the same composition and optionally in the fermenter add 0.1 M urea. After the boot stage is carried out feeding methanol with 4% wt./about. urea. The fermentation was continued for 8 days. The maximum concentration of the product reaches 3.5 g/l within 7 days. Significant difference in terms of profiles of cell growth is not observed. Profiles of biomass and concentration of the product obtained as a result of these experiments, shown in Fig.1 and 2, respectively.

Example 2.

The expression of the precursor of insulin urea examined using Pichia pastoris fermentation for determining the level of expression of the product. In this study using the composition of the control environment and methanol with 4% urea is injected at a higher level of 20 g/l/h. In this study, the maximum concentration of the product reaches 4,21 g/l for 137 relative to 4.26 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 fermenter increases the level of expression of the product without affecting the profile of the growth, and reduce the time of fermentation. Profiles of biomass and concentration of the product obtained as a result of these experiments, shown in Fig.3 and 4, respectively.

Example 3.

Two download the fermenter is carried out with the use of Pichia pastoris for expression of the precursor GLA�hyna. In one batch (experiment # 1) half the concentration of the control environment is taken for the original environment with the exception of glycerol. After the boot stage methanol injected at a speed of feeding 8 g/l/h. The fermentation was continued for 10 days.

The maximum concentration of the product reaches 1,03 g/l for 10 days and stabilized. In another download (experiment # 2) use a medium of the same composition and in addition to the initial fermentation medium is added 0.1 M urea. After the boot stage is carried out feeding methanol with 4% urea. The fermentation was continued for 9 days. The maximum concentration of the product reaches 1.75 V for 9 days. Significant difference in terms of profiles of cell growth is not observed. Profiles of biomass and concentration of the product obtained as a result of these experiments, shown in Fig.5 and 6, respectively.

Example 4.

Two download the fermenter is carried out with the use of Pichia pastoris for expression of the precursor of Bloomington: Indiana. In one batch (experiment # 1) half the concentration of the control environment is taken for the original environment with the exception of glycerol when fed with glycerol. After the step of feeding glycerol methanol injected at a speed of recharge 11 g/l/h. The fermentation was continued for 6 days. The maximum concentration of the product reaches 0.74 g/l for 6 days and stabilized. In another download (experiment # 2) use a medium of the same composition and further added to the fermenter 0.1 M urea. After the boot stage is carried out feeding methanol with 4% urea. The fermentation was continued for 5 days. The maximum concentration of the product reaches 0.78 in for 5 days. Significant difference in terms of profiles of cell growth is not observed. Profiles of biomass and concentration of the product obtained as a result of these experiments, shown in Fig.7 and 8, respectively.

Example 5.

Two download the fermenter is carried out with the use of Pichia pastoris for expression of the enzyme lipase. In one batch (experiment # 1) half the concentration of the control environment is taken for the original environment with the exception of glycerol. After the boot stage methanol injected at a speed of recharge of approximately 6 g/l/h. The fermentation was continued for about 8 days. The maximum product concentration reaches approximately 1650×106 units of lipase within 7 days and stabilized. In another download (experiment # 2) use a medium of the same composition and optionally in the fermenter add 0.1 M urea. After the boot stage is carried out feeding methanol with 4% wt./about. urea. The fermentation was continued for 8 days. The maximum amount of product reaches approximately 2500×106 units l�grooves for 7 days and then stabilizes. Significant difference in terms of profiles of cell growth is not observed. The General profile of the product obtained as a result of these experiments, shown in Fig.9.

Example 6.

Investigate the effect of concentration of urea in the downloads with the methanol used to obtain the predecessor-IN-105, with 1, 2, 3 and 5% urea in methanol during loading in a portion of methanol. The other parameters remain the same as in example 1. The maximum concentration of the product reaches 3,0, 3,2, from 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 concentration of the product illustrated in Fig.10 and 11, respectively.

Example 7.

In another study, the level of make-up methanol 20 g/l/h. The concentration of urea varies to determine the most effective concentration of urea for maximum concentration of the precursor of insulin. The maximum concentration of the product reaches to 4.26, 4,03, 3,39, 4,16, 4,21 and was 5.31 g/l at 182, 166, 140, 164, 137 and 170 hours, respectively, in downloads, where a portion of the methanol is carried out with the concentration of urea 0, 1, 2, 3, 4 and 5%, respectively. Profiles of biomass and concentration of the product illustrated in Fig.12 and 13, respectively. Investigated�e shows that the addition of urea increases the level of production of the precursor of insulin, the expression level is highest when feeding with methanol is carried out with 5% urea.

Example 8.

Explore experimental load, in which during the phase of induction with methanol maintain residual concentration of urea at different levels 0,1, 0,3, 0,5, 0,7, 1.2 and 1.5 M in the cell-free supernatant, and examine the effect on productivity. Take all downloads with parameters and 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 max product reach, when the urea level support in the area of 1 M in the cell-free supernatant during the fermentation for download. The maximum concentration of the product 4,46 g/l is achieved when the level of residual urea approximately 0.5 M. In this experiment, the total recharge of urea equivalent 0, 0,2, 0,5, 0,9, 1,2, 1,8, 2,3 and 2.9 M final volume of the broth in the study, which support the level of residual urea 0, 0,1, 0,3, 0,5, 0,7, 1,0, 1,2 and 1.5 M respectively. The results are presented in Fig.14.

Example 9.

Also carried out a study of the fermentation of the precursor of insulin. In this study, feeding with methanol is carried out at the level of 20 g/l/h. In fermentati� predecessor of insulin shown that the concentration of this product is maximum when support residual concentration of 0.7 M. In this study, the total recharge of urea equivalent 0,0, 1,6, 2,7, 3,5, 7,0, 8,8, 11,7 and 13.3 M final volume of the broth in the study, which support the 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 substantial amounts of urea, as represent in Fig.15.

Example 10.

Another batch of IN-105 is carried out with a standard control environments and with the addition of about 20 g/l/HR of methanol with 4% urea. In this study reach the concentration of the product of 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 study illustrated in Fig.16 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 test downloads 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.18.

Example 12.

In the experiment indicate that feeding urea during fermentation increases the consumption of phosphate by yeast, resulting resulting to the depletion of phosphate in the fry�e before, than download where feeding urea is not carried out. Thus, the accelerated consumption of phosphate and increased productivity (g/l/h) are the result of a metabolic shift, which is attained by the introduction of urea in standard or modified protocols Pichia fermentation for the purpose of obtaining peptides and proteins.

Example 13.

Prepare the environment for growth, containing soybean meal 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 using NaOH solution. The sterilized medium for inoculum vial is inoculated with spore suspensions of the culture of Streptomyces sp. (BICC 6826) and incubated at 28±1°C for 48 hours under aerobic conditions. Then the grown inoculum is transferred to a fermentation medium containing soy flour 37,5 g, dextrose monohydrate 22,5 g, cottonseed flour 3.75 grams, corn syrup of 7.5, 7.5 and NaCl antifoam SAG 0.5 g in 1000 ml 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 receives one of the flasks from several similar flasks to check the bioconversion compactin in pravastatin. The procedure was repeated every 24 hours until the total recharge of the compact will not be 3.0 g/L.

Before beginning experiments with IP�altanium modified fermentation broth in a fermentation medium is added different levels of urea, to set the level of toxicity of urea for the body. Concentration 0,0, 0,5, 1,0, 1,5, 2,0, 2,5, 3,0, 3,5 and 4.0 g/l is added to the flask containing the fermentation medium, and then incubated as described above. After 48 hours incubation monitoryou growth in each of the flasks. Data are in Fig.20.

Concentrations exceeding 3.0 g/l of urea, show the inhibition of culture growth. In replications of the study shows similar results. Consequently, concentrations below 3.0 g/l take to check the effect of urea on the bioconversion.

As described earlier, a similar experiment is performed with the environment to produce a product containing urea as an additional component. 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 during inoculation.

After 48 hours of incubation make a sterile solution compactin together with a portion of dextrose. Bioconversion evaluated after 24 hours by collecting one of the many flasks, which is carried out in similar conditions. This procedure was repeated every 24 hours, while in General do not contribute to the recharge of compactin 3 g/L. 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 pokazania Fig.21 and 22, respectively.

The flask with the concentration of urea 1.5 g/l as the maximum level of conversion compactin pravastatin in 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 level of expression of the product. The environment used in the study consists of seedbed environment and seed environment and for product formation. Seedbed environment consists of soybean peptone 10.0 g, NaCl 10.0 g, yeast extract 10.0 g in 1000 ml of water. The sowing medium consists of 1.2 g of ammonium sulfate, 2.4 g of magnesium sulfate, 10 g of yeast extract, 11 g DMH, 5 g of K2HPO4, 40 ml of trace elements in 1000 ml of water. Environment for education product consists of dextrose monohydrate 11 g of ammonium sulfate 2.4 g, magnesium sulphate 4.8 g, yeast extract 20 g, K2HPO410 g, 40 ml of trace elements in 1000 ml of water. pH down to 7.0 with ammonia. After completion of the loading phase, the biomass in the fermenter to increase the constant feeding 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 fermenters to establish the effect of urea in the fermentation process using E. coli. �irst download (experiment # 1) is a control boot without adding any urea. Used environment described above. Downloading induce at about 180 g/l SEC. The fermentation was continued until 8 h after induction. Peaking at 6.6 g/l with a specific activity 0,028 g/SEC. In the second download (experiment # 2), the fermenter is added 1 g/l of urea in addition to the above-described environments. To download make makeup that contains a similar amount of urea (1 g/l). Downloading induce at about 180 g/l of SEC, continue the fermentation for 8 hours after induction and receive 7.4 g/l of product with a specific activity of 0.033 g/SEC. Third download (experiment # 3) was carried out analogously to experiment #2, but with the addition of 2 g/l of urea. Made the end product is to 6.58 g/l with a specific activity 0,032 g/SEC.

As the results show, not see no significant difference in the profile of cell growth, indicating that the addition of urea increases the level of product formation without affecting growth profile. In General get about an 18% increase in specific productivity. The SEC profiles obtained as a result of the above experiments are shown in Fig.23, profile and specific productivity - Fig.24.

Example 15.

A similar experiment carried out using E. coli as expression system for receiving streptokinase. Used�form the same environment, as mentioned in example 13, and the same test concentration of urea. As described in example 14, is carried out three similar download with urea content 0 g/l 1 g/l and 2 g/l Control download without urea (experiment # 1) gives the ultimate productivity 7,06 g/l after 8 hours of induction in specific productivity was 0.026 g/SEC. The maximum titer reached to load the content of 1 g/l of urea (experiment #2) with a productivity of 10.5 g/l and specific productivity 0,041 g/SEC. Unexpectedly shown that loading with 2 g/l of urea (experiment #3) gives only 6,56 g/l product specific productivity 0,022 g/SEC. For this product increased concentrations of urea greater than 1 g/l results in a sharp drop in specific productivity and titer.

As in example 2, no significant changes in the values of SEC three studies that clearly shows that increasing productivity is the result of an increased level of education of the product and not due to changes in biomass. In General receive approximately 57% increase in specific productivity. The SEC profiles obtained as a result of the above experiments are shown in Fig.25. Profile titer is shown in Fig.26.

Example 16.

Rhizomucor sp. (BICC 362), which, as is well known, produces the enzyme lipase, the�also shows the increase of productivity when adding urea to the environment. Lipase generated using a given culture, can be widely used for bioconversion reactions, such as esterification and hydrolysis. The process will take two download the fermenter (volume 10 l), one without the addition of urea and the other with addition of 0.5 g/l of urea. Also carried out 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 41,4 g, sucrose 10 g, peptone 3,06 g, ammonium sulfate 2 g, yeast extract 2 g, potassium phosphate 0.85 g, calcium chloride, magnesium sulfate and sodium chloride, 1 g each. The complete medium was adjusted to 1 l with water. Grown inoculum (10 vol.%) carry on Wednesday to produce a product consisting of dextrose 12.5 g, soybean peptone 37,5 g, soy flour 25, potassium phosphate 2,5, magnesium sulphate of 0.625, soybean oil 12.5 g in 1000 ml of water. pH down to 6.0 and then support at 6.0 for download by adding caustic soda.

The addition of urea (0.5 g/l) indicates an increase in the level of education of lipase compared with control download. Higher concentration of urea shows a lower productivity. Data are in Fig.27.

It should be borne in mind that from this�the acquisition is not limited to the described specific ways, protocols, cell lines, species, or genera and media components, as they may vary. In addition, it should be borne in mind that the terminology used in this context, is used only for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims of the invention. The foregoing description is for the purpose of training an ordinary specialist in the field of technology, how to use 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 descriptions.

1. Fermentation medium for the production of recombinant proteins selected from the group comprising G-CSF, streptokinase and lipase, with the use of microorganisms selected from the group including: E. Coli, Streptomyces sp. and Rhizomucor sp., characterized by the concentration of urea or its derivatives, are supported in the range from about 0.5 g/l to about 2 g/l by a constant supply of urea, basic salts in the following amounts (per liter):

Phosphoric acid (85%)from 2,67 ml to 133,5 ml
Calcium sulphatefrom 0.093 g to 4.65 g
Potassium sulphatefrom 1,82 g 91 g
Magnesium sulfate - 7H2O1,49 g to 74.5 g
Potassium hydroxidefrom 0,413 g to 20,65 g
Glycerinfrom 4 g up to 200 g

and salts of trace elements in the following amounts (per liter):
Copper sulfate - 5H2Onfrom 0.6 g to 30 g
Sodium iodidefrom 0.008 g to 0.4 g
Sulfate manganese - H2Ofrom 0.3 g to 15 g
Molybdate sodium - 2H2Ofrom 0.02 g to 1 g
Boric acidfrom 0.002 g to 0.1 g
Cobalt chloridefrom 0.05 g to 2.5 g
Zinc chloridefrom 2 g to 100 g
Ferrous sulfate - 7H2Ofrom 6.5 g to 325 g
Bioti� from 0.02 g to 1 g
and
Sulfuric acidfrom 0.5 ml to 25 ml

2. Fermentation medium according to claim 1, characterized in that the recombinant proteins are characterized by maximum product titer greater than 0.5 g/L.

3. Fermentation medium according to claim 1, characterized in that the urea or its derivatives selected from the group comprising dimethylketene, dieselmachine, N-acetylphenylalanine, isopropylidenediphenol, prilocaine or combinations thereof, and wherein it is added in the form of liquid, spray, powder or granules.

4. A method of producing recombinant proteins selected from the group comprising G-CSF, streptokinase and lipase, through the use of microorganisms selected from the group comprising E. coli, Streptomyces sp and Rhizomucor sp., in a fermentation medium according to claim 1, characterized by a residual concentration of urea maintained in the range of from about 0.5 g/l to about 2 g/l by a constant supply of urea.

5. A method according to claim 4, wherein the indicated recombinant proteins are characterized by maximum product titer greater than 0.5 g/L.

6. A method according to claim 4, characterized in that said urea or its derivatives selected from the group comprising dimethylketene, dieselmachine, N-acetyl�nimodipine, isopropylidenediphenol, prilocaine or a combination of, the above-mentioned urea or its derivatives are added in the form of liquid, spray, powder or granules.



 

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4 cl, 10 dwg, 7 ex

FIELD: food industry.

SUBSTANCE: invention relates to a fermented natural product manufacture method. The method envisages production of the first enzyme extract in the main fermenter by way of fermentation of raw materials chosen from fruit, vegetables, leguminous crops, mushrooms, nuts, wheat, rice, herbs, roots, leaves, flowers, separately or in combination, in the presence of microorganisms in an amount of 106 - 1012 cells/ml, preferably, in an amount of 108 - 1010 cells/ml. At least one part of the first enzyme extract is retrieved; the said part is transferred into at least one additional accessory fermenter. The said part of the enzyme extract is fermented in the presence of microorganisms in an amount of 106 - 1012 cells/ml, preferably, in an amount of 108 - 1010 cells/ml to produce at least one particulate enzyme extract. At least one partial enzyme extract is transferred into the main fermenter and mixed with the remaining first enzyme extract. The microorganisms propagated mass is cultivated till concentration in the starter culture in the propagator is equal to 1012 - 1016 CFU/ml. One adds the said propagated mass of microorganisms to the combined enzyme extracts in an amount so that at least approximately to double the microorganisms quantity.

EFFECT: method allows to manufacture a product strengthening immunity and having very high antioxidant potential.

16 cl, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and deals with a method of obtaining a liquid fraction, which contains isolated highly molecular capsular polysaccharides of Streptococcus pneumoniae. The claimed method includes obtaining a fermentation culture of bacterial cells Streptococcus pneumoniae of serotypes 19A, 6A, 19F or 6B, producing the capsular polysaccharides, which include a phosphodiether bond between the repeated units, introduction of CO2 into the fermentation culture; lysis of bacterial cells with obtaining the liquid fraction, which contains the said polysaccharides, isolation of the capsular polysaccharides from the cell lysate with obtaining the liquid fraction of the isolated highly molecular capsular polysaccharides with a molecular weight of 480 kDa.

EFFECT: claimed inventions can be used in production of vaccines for immunisation against diseases, caused by Streptococcus pneumoniae.

14 cl, 3 dwg, 7 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is device for obtaining nanoparticles by reduction of metals from initial salts in presence of cultivated cells of microorganisms. Device includes control computer (1), connected with it electronic block of regulation and control (2) of all functional units and blocks of fermenter (3), pH-stabilising block (4) with pH sensor (5) and hoses for supply of titering solutions by pumps (6, 7), block (8) for regulation of redox-potential of culture mixture, provided with redox sensor (9), independently controlled pumps (10, 11) for introduction of initial solutions of metal salts, reducing agents and growth factors into fermenter (3), block (12) for regulation of dissolved oxygen level with sensor pO2 (13), pump (14) for supply of growth substrate, block (15) for measurement of optic culture density with application of optic fibre sensor (16), block (17) for measurement of spectral characteristics of culture mixture with application of optic fibre sensor (18), isolated with impermeable for cells membrane with pore size 100-250 nm, block (19) for thermoregulation of fermenter (3), equipped with temperature sensor (20), block (21) for regulation of culture mixture mixing, which brings into motion blade mixer (22), block (23) for regulation of culture mixture illumination in case of cultivating phototrophic microorganisms and control of spectral parameters of submersible diode lamp (24), block (25) for ultrafiltration of sampled culture mixture with sterilising membrane with pore size 100-250 nm with possibility of output of only nanoparticle suspension from fermenter, condenser of output moisture (26), preventing loss of culture mixture.

EFFECT: invention contributes to extension of arsenal of technological methods of obtaining nanoparticles of metals and makes it possible to achieve controllability of modes of nanoparticle formation.

2 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with an isolated polynucleotide, coding a polypeptide, involved in biosynthesis of pyripyropene A, a vector and a host cell, including such a polypeptide, and methods of obtaining pyripyropene A precursors, including the host cell cultivation. The claimed polynucleotide codes the polypeptide, possessing any one or more of the activities - polyketide synthase, prenyltransferase, hydroxylase, acetyltransferase or adenylate synthase.

EFFECT: claimed inventions make it possible to synthesise pyripyropene A, which is an insecticidal agent, and can be used in the formation of plant resistance to pest insects.

16 cl, 11 dwg, 1 tbl, 11 ex

FIELD: biotechnology.

SUBSTANCE: inventions relate to a strain of the fungus Penicillium verruculosum B10 EGII and a method of production of the fodder complex enzyme preparation. The presented strain is a producer of endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase. It is obtained on the basis of a strain Penicillium verruculosum RNCM F-3764D, by transforming with its plasmids pSTA10 and pPrCBHI-EGII. In the plasmid pPrCBHI-EGII the nucleotide sequence of the structural gene of endo-1.3/1.4-β-glucanase of Penicillium verruculosum is aligned with nucleotide sequences of the promoter region, the signal peptide and the terminator of gene cbhI of cellobiohydrolase I Penicillium verruculosum. The method of production of the preparation containing endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase comprises cultivation of the strain of fungus Penicillium verruculosum B10 EGII on the optimised nutrient medium in the fermenter and filtration, ultrafiltration and lyophilisation of the culture liquid.

EFFECT: inventions can be used to produce fodder complex enzyme preparation enriched with endoglucanase.

2 cl, 1 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemical engineering and techniques for producing veterinary, medical and pharmaceutical preparations. The method of producing a novel antiviral substance based on 2,5-dihydroxybenzoic acid and gelatine includes oxidising 2,5-dihydroxybenzoic acid with laccase enzyme to intermediate phyenoxy radicals and semiquinones, which are then copolymerised with gelatine, and separating the obtained copolymer from low-molecular weight components by dialysis; optimum concentrations of components of the reaction mixture are as follows: 2,5-dihydroxybenzoic acid - 15-80 mM, gelatine - 1-13 mg/ml reaction mixture, laccase - 0.5-10 units of activity/ml reaction mixture.

EFFECT: obtained copolymer has antiviral activity on herpesvirus, particularly Aujeszky's disease virus.

2 tbl, 1 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method includes cultivation of a fungus of Fusarium sambucinum type on nutrient medium containing a source of carbon 3-4%, nitrogen 0.2-0.3%, phosphorus 0.2-0.3% and microelements 0.07-0.08%, under sterile conditions at temperature of 26-30°C with mixing and aeration within 36-72 hours with subsequent separation of the cultural liquid from fungus biomass. Besides, the cultural fluid is centrifuged at room temperature for 15-20 min. at 800-1500 rpm, and the target product is made from supernatant by separation of peptides with molecular mass from 3,000-60,000 D. Also before centrifugation the cultural fluid is heated up to 110-115°C and pressure of 1.3-1.5 atm. with rate of 1.5-2 degrees/min. with the following exposure for 1.5-2.5 hours.

EFFECT: adjuvant prepared in accordance with the invention has higher potency that stimulates immunity, is harmless within vaccines and other products of immunological purpose, areactogenic, may be applied both independently and in combination with oil adjuvants or adjuvants-sorbents on the basis of aluminium hydroxide gel and other substances.

2 cl, 3 tbl, 9 ex

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

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: chemistry.

SUBSTANCE: invention refers to biotechnology. What is presented is nucleic acid coding protein possessing acetyl-CoA-carboxylase activity making up the deficiency of acetyl-CoA-carboxylase in yeast, wherein a nucleotide sequence is specified in nucleic acid, which contains a nucleotide sequence: (a) coding protein consisting of the amino acid sequence SEQ ID NO:2; (b) hybridised in the hard conditions with nucleic acid complementary to SEQ ID NO:1; (c) SEQ ID NO:1; and (d) hybridised in the hard conditions with nucleic acid consisting of the complementary nucleic sequence coding protein SEQ ID NO:2; wherein SEQ ID NO:1 and 2 are disclosed in the description. There are also described: acetyl-CoA-carboxylase (SEQ ID NO:2) increasing the host-specific arachidonic acid content; a recombinant vector containing the above nucleic acid; and a cell transformed by the above vector for producing the fatty acid composition rich in arachidonic acid. What is presented is a method for producing the fatty acid composition involving culturing the above cell and collecting the fatty acid composition from the transformed cell culture.

EFFECT: invention enables producing the fatty acid composition rich in arachidonic acid in the host cell.

11 cl, 8 dwg, 5 tbl, 8 ex

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