Technology of synthesis feed lysine

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Ministry of Education and Science of Ukraine

National Aviation University

Faculty of Environmental Safety, Engineering and Technology

Department of Biotechnology

TERM PAPER

(Explanatory note)

On discipline “General Biotechnology”

Theme: “Technology of synthesis feed lysine ”

Executor: student of 304 group of FESET

Chuba S.V.

Supervisor: Petjuch G. P.

Kyiv 2020



Міністерство Освіти і Науки України

Національний Авіаційний Університет

Факультет Екологічної Безпеки, Інженерії і Технологій

Кафедра Біотехнології

КУРСОВА РОБОТА

(пояснювальна записка)

З дисципліни «Загальна біотехнологія»

Тема: «Технологія одержання лізину»

Виконав студент 304 групи ФЕБІТ

Чуба Святослав В'ячеславович

Керівник: Петюх Г. П.

Київ 2020



National Aviation University

Faculty of Environmental Safety, Engineering and Technology

Department Biotechnology

Discipline General Biotechnology

Speciality Biotechnology

TASKS

of the student term paper of

Chuba S.V.

1. Theme of work. Feed L-LYSINE PRODUCTION 2. Deadline of completed student work_______________________

3. Content of explanatory note: 1. L-LYSINE PRODUCTION; 2. MICROORGANISMS AND STRAIN DEVELOPMENT ; 1.2. NUTRITIONAL ENVIRONMENT; 3 L-LYSINE FERMENTATION TECHNOLOGY; 2.1. L-LYSINE FERMENTATION RESULTS ; 1. Raw material and supplementary materials; 2. Preparation of the medium; 3 Obtaining of pure (axenic) and natural-pure yeast culture; 4 BIOCHEMISTRY AND REGULATION OF L-LYSINE FERMENTATION; 5. CURRENT AND FUTURE DEVELOPMENTS; 6. QUALITATIVE AND QUANTITATIVE ANALYSIS OF L-LYSINE; 7 AQUATIC L-LYSINE FOOD ADDITIVE.

4. Date of assignment

Student

Supervisor Petjuch G. P.

« » 2020 yr.



SUMMARY

carbon nitrogen oxygen lysin

Explanatory note for course work

“Technology of synthesis feed lysine”: 38 pp., 3 figures, 3 tables, 84 literature resources.

General technological scheme of L-LYSINE production, Raw material and other factors of growht, Preparation of the medium, Obtaining of pure and natural-pure microorganism, Technological processes of synthesis, BIOCHEMISTRY AND REGULATION OF L-LYSINE FERMENTATION

Object of investigation - microbiology agent of lysine synthesis.

Subject of investigation - the technological methods lysine production.

Aim of the work - Draw up a diagram of the technology for obtaining feed lysine, consider all aspects of biosynthesis, as well as the very method of cultivating producers

Methods of investigation - analysis of theoretical information.

Result of this work is the generalization of the informational data and its analysis concerning the production of yeast, from the raw material to storage of the finished product.



CONTENT

THE LIST OF ABBREVIATIONS

INTRODUCTION

1. L-LYSINE PRODUCTION

2. MICROORGANISMS AND STRAIN DEVELOPMENT

3. NUTRITIONAL ENVIRONMENT

3.1 Source of carbon

3.2 Source of nitrogen

3.3 Inorganic salts, microelements and growth factors

3.4 Effects of oxygen

3.5 Effect of temperature

3.6 Impact ph

3.7 Anti-foaming

3.8 Plasmid protection

4. L-LYSINE FERMENTATION TECHNOLOGY

5. L-LYSINE FERMENTATION RESULTS

6. BIOCHEMISTRY AND REGULATION OF L-LYSINE FERMENTATION

7. L-LYSINE ISOLATION PROCESSES

7.1 Separation of cells

7.2 Ion-exchange chromatography

7.3 Drying stage

7.4 Hard forage additive l-lysin

8. AQUATIC L-LYSINE FOOD ADDITIVE

9. CURRENT AND FUTURE DEVELOPMENTS

10. QUALITATIVE AND QUANTITATIVE ANALYSIS OF L-LYSINE

CONCLUSIONS

REFERENCES

APPENDIX 1

APPENDIX 2

THE LIST OF ABBREVIATIONS

ASВ - active synthesis biomass

SS - Solid substances

AA/aa - Amino acid/s

CW - Colling water

ST - Steam



1. NUTRITIONAL ENVIRONMENT

1.1 Source of carbon

Mutants of Corynebacterium and related microorganisms allow the production of inexpensive amino acids from cheap renewable carbon sources by direct fermentation. Various carbohydrates are used individually or as a mixture to produce L-lysine such as glucose, fructose, sucrose, molasses (sucrose, glucose, fructose, etc.), maltose, starch and starch hydrolysates, cellulose, cellulose hydrolyzate, organic acids such as acetic acid, propionic acid, benzoic acid, formic acid, malic acid, citric acid and fumaric acid, alcohols such as ethanol, propanol, inositol and glycerin, and of course hydrocarbons, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, as well as fatty acids such as palmitic acid, stearic acid and linoleic acid.16, 19, 20, 39, 42, 43, 60, 62,]

1.2 Source of nitrogen

Various nitrogen sources are used alone or as mixtures for the industrial and pilot production of L-lysine, including inorganic compounds such as gaseous and aqueous ammonia, ammonium salts of inorganic or organic acids such as ammonium sulfate, ammonium nitrate, ammonium. phosphate, ammonium chloride, ammonium acetate and ammonium carbonate. Alternatively, natural nitrogen-containing organic materials such as soybean hydrolyzate, soy protein hydrolyzate (total nitrogen about 7%), soybean meal, soybean meal hydrolyzate, corn extract, casein hydrolyzate, yeast extract, meat extract, malt extract, urea extract., peptones and amino acids can also be used [5, 19, 20, 43, 67, 68].

Interestingly, Inuzuka and Hamada (1976) reported an increase in the yield of L-lysine due to the enrichment of the L-lysine fermentation medium with a culture liquid (2-150 ml / L) of a microorganism producing L-leucine [12, 69]



1.3 Inorganic salts, microelements and growth factors

Additional components must be added to the fermentation medium at the beginning and / or periodically during the fermentation of L-lysine, such as inorganic salts of various metals such as magnesium (for example, magnesium sulfate), calcium, potassium, sodium, iron (for example, ferrous sulfate), manganese and zinc or traces of other metals. The source of phosphorus for L-lysine are phosphorus-containing salts of sodium and potassium [12,19,20,27]. Factors such as amino acids and vitamins (eg vitamin B1) and suitable precursors are also added to the culture in addition to the above substances, once or intermittently during culture [12, 20]. Table 1 illustrates the composition of a typical working environment for C. alkanoglutinousa [27,63].

The addition of certain amino acids such as arginine, aspartic acid, isoleucine or valine enhances the production of L-lysine, unlike leucine. In addition, the inhibition of bacterial growth caused by AEC (S-2-aminoethylcysteine) was reduced by adding 1 g / L of arginine to the culture medium.

Table 1 - Composition of L-Lysine Fermentation Medium of C. Alkanoglutinousa



1.4 Effects of oxygen

No significant information has been found in the patent literature on the effect of air saturation, and little is known about the actual effect of oxygen on L-lysine fermentation. Fermentation of L-lysine is an aerobic process [42,43,63] that requires a large amount of oxygen and is highly dependent on air saturation in the bioreactor. Under anaerobic conditions, lactic acid is produced as a by-product, which is reused after aerobic conditions have been created (unpublished data obtained by Anastassiadis and Stephanopoulos, Massachusetts Institute of Technology)

Aerobic conditions are maintained by aseptic addition to the culture of oxygen-containing gas mixtures, for example, atmospheric air or pure oxygen [9,12]. The cultivation of microorganisms producing L-lysine is carried out by shaking (250-300 rpm) or aeration (0.5-1.5 rpm) of the mixing bioreactors. In [14,16] laboratory fermentations are described operating at an aeration rate of atmospheric air of 2.1 rpm and a varying stirring speed during fermentation between 600 (at 0 h) and 900 rpm (at 19 h).

The enormous effect of air saturation (100% air saturation corresponds to saturation at an aeration rate of 1 rpm at 30 o C and a stirring speed of 600 rpm) on the continuous production of B. lactofermentum L-lysine was found in experiments to develop a chemostat process (unpublished data obtained by Anastassiadis and Stephanopoulos, MIT)

1.5 Effect of temperature

A wide range of optimum and operating temperatures is disclosed in the International Patent Bibliography for L-lysine fermentation to protect the entire usable range. In [43] it is stated that L-lysine is produced at a temperature of 24 to 37 C, preferably at 31-33 C. Operating temperatures in the range of 25 to 40 C are stated in [10] (preferably 30 to 40 o C) 63] describes the fermentation process, which states that the production of L-lysine by strains of fusion protoplasts occurs at temperatures from 20 to 40 C for 1-6 days. Alternatively, [67] claims an increase in the production of L-lysine using the thermophilic strain Corynebacteriumthermoaminogenes at temperatures from 25 to 50 C.

US Pat. No. 5,846,790 discloses an increase in temperature to 33-40 ° C, preferably above 34 ° C, at a certain intermediate stage in the fermentation of L-lysine and L-glutamic acid. Nocardia alkanoglutino usa grows at temperatures from 10 to 40 o C, producing L-lysine at 20-40 C, optimally at 27-37 C [64]. Incubation of E. coli during L-lysine fermentation is carried out with stirring with immersed air, shaking, or in stationary culture at temperatures between 30 ° C and 42 ° C, preferably at about 37 ° C.

1.6 Impact ph

pH is a very important factor that strongly influences microbial fermentation. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, urea, ammonia and ammonia gas, or inorganic acid compounds such as phosphoric or sulfuric acid and organic acids, are used to adjust the pH in L-lysine cultures in the range pH. 5 to 9 [7,9,11,12,20,43,63,67,70] discloses the production of L-lysine by mutant strains at a pH range of 5 to 8.5, [69] and [11] pH in the range from 5.8 to 8.5, preferably from 6.5 to 7.5. A suitable initial pH of the culture medium from 6 to 8 is described in [61], the production of L-lysine, described in [16, 67], occurs at pH 7.2 using NH3 or NH4OH and at a pH of 5.0 to 8.0 in [14]. And in US patent 4066501 - at pH from 7.2 to 8.0 by feeding acetic acid and ammonium acetate (30 C).

Nocardia alkanoglutinousa grows at a pH of 6 to 9 [27]. The fermentation of E. coli L-lysine is carried out at a pH of 5 to 8 using inorganic, acidic or alkaline gas or ammonia [61].,



1.7 Anti-foaming

Foaming that occurs during fermentation is controlled by the addition of antifoam agents such as polyglycol fatty acid esters [9,12,20] or silicone and polypropylene.

1.8 Plasmid protection

Suitable selective substances, for example Antibiotics are added to the medium to maintain the stability of the plasmids [12, 20].



2. L-LYSINE FERMENTATION TECHNOLOGY

Amino acids are commercially produced in batch or fed-batch processes. All nutrients are added at the beginning of fermentation in batch operations [16]. For shake flask experiments, the strains are first incubated on agar plates, for example. within 24 hours at 33 C With this culture, agar plates are inoculated with a preculture (eg, 10 ml of medium in a 100 ml baffled Erlenmeyer flask). MM medium is used as pre-culture medium. The preculture is incubated, for example, for 24 hours at 33 C and 240 rpm on a shaker. Subsequently, the main culture is inoculated using this pre-culture so that the initial optical density (eg OD at 660 nm) of the main culture reaches a certain level, eg OD 0.1 [12]. The production environment (for example, MM environment) is used for the main crop. The cultivation is carried out, for example, at 33 C and an air humidity of 80% [12], L-lysine and the carbon source are determined during and at the end of fermentation. In batch fermentation, the microorganism grows until one or more of the essential nutrients are depleted or until fermentation conditions become unfavorable (for example, product inhibition, oxygen restriction, pH drop in shake flasks, uncontrolled fermentation, etc.)...

In fed-batch fermentation or continuous fed-batch fermentation, one or more nutrients are continuously or intermittently added to the culture medium either from the beginning of fermentation, after the culture has reached a certain age, or when the nutrients are depleted.... The microorganism grows at a rate determined by the rate or timing of nutrient delivery. Typically a single nutrient, very often a carbon source (eg ethanol, glucose), is fed to the fermenter to overcome substrate inhibition and high osmotic pressure. In addition, continuous supply of complete medium simulates chemostatic conditions, gaining the combined benefits of both batch and chemostatic operation. An interesting option for extended fed-batch fermentation is re-batch or fed-batch batch or refill and ferment. A portion of the fermentation broth is removed at a specific time in the operation, while the feeding continues to expand the fermentation operation and increase high product concentrations, or the fermenter is filled with fresh medium at the end of the fermentation. Favorable fermentation conditions are maintained by controlling pH, temperature, oxygen concentration, supplying essential nutrients to the culture, or alternatively fed-batch mode (e.g. ramp or logarithmic mode) using sophisticated feeding strategies that significantly increase L-lysine production and In continuous fermentation or chemostat (Fig. 1), a continuous supply of complete medium is used, while the culture broth is continuously or semi-continuously withdrawn so that the working volume of the fermenter remains constant. Typically, one nutrient is the limiting factor for growth, such as nitrogen, phosphorus, amino acid, vitamin, or carbon source. In principle, continuous fermentation can continue indefinitely under steady state conditions. Continuous fermentation of L-lysine offers many advantages over batch fermentation (unpublished data obtained by Anastassiadis and Stephanopoulos, Massachusetts Institute of Technology, 1994). [11] describes a new method that maintains the total carbon source concentration at a low level of less than 5 g / L (for example, sugar concentration) in fed-batch cultures, continuous or continuous cultures with cell recirculation, by monitoring the increase in pH or dissolved oxygen (in depending on carbon consumption) and periodically adding media to the bioreactor at calculated feed rates using a computerized feed control device. Several goals have been achieved so far, including overcoming substrate inhibition, efficient carbon source utilization, easy product isolation and pollution prevention, combining the benefits of traditional continuous and batch fermentation at the same time. Oxygen consumption, carbon dioxide emissions, pH, by-product production and ammonia addition are additional parameters that were previously considered (predetermined proportionality factor) for ineffective control of the carbon source in the bioreactor [11]. In contrast to conventional fed-batch processes (simultaneous supply of carbon sources, trace elements and growth-limiting amino acids), [3] describes a new process that introduces a carbon source and limits the amount of amino acids through two or more feed streams. applies a continuous supply of the entire medium to a constant fermentation volume in order to achieve stable conditions. A single nutrient, such as a carbon source, nitrogen, sulfur, phosphorus, or alternatively oxygen, a vitamin, or an amino acid (applicable to auxotrophic microorganisms), will limit growth [16]. Continuous fermentation processes are described in [71] for the production of amino acids such as L-glutamic acid, while [11] describes a continuous process of cell recycling to produce L-lysine.



3. L-LYSINE FERMENTATION RESULTS

A small part of the work described in the patent literature has been devoted to the development and optimization of the fermentation process versus the genetic improvement of L-lysine-producing strains. According to the International Patent Bibliography, the development of the bacterial strain and process has resulted in a continuous increase in L-lysine titers and has led to the fermentation of L-lysine over the past 50 years [72-74]. Fermentation usually continues until the concentration of L-lysine or the desired product reaches its maximum. This goal is usually achieved within 10 to 160 hours for L-lysine production [12]. Table 2 compares the results of shake flask experiments between wild and mutant strain [12]. Large scale fermentation processes produce an impure broth containing 80-110 g / L L-lysine [75]. It has been reported that a fermentation time of 30 to 100 hours reaches 50-100 g / L of L-lysine produced by the mutant strains.

Table 2 - Comparison of Results Obtained by Wild and Mutant Strain



4. BIOCHEMISTRY AND REGULATION OF L-LYSINE FERMENTATION

Fig 1

The biosynthesis of lysine in various microorganisms proceeds in different ways. There are two fundamentally different pathways for lysine biosynthesis. One path starts from 2 - ketoglutaric acid, through 2 - aminoadipic acid to L-lysine (aminoadipic path).

In this way, lysine is formed in the cells of yeast, fungi, actinomycetes and some algae. The regulation of lysine biosynthesis by the aminoadipine pathway has been relatively little studied, but it is still known that it goes through the first enzyme, which catalyzes the conversion of 2-ketoglutaric acid into homolimonic acid, since an excess of lysine in the medium significantly inhibits the formation of the latter.

The second path (the diaminopimelic pathway) starts with aspartic acid and goes through 2,6 - diaminopimelic acid. This pathway takes place in bacterial cells. This pathway of lysine formation was studied in detail in E. Coli, and later it was proved that in the main producers of lysine, and in glutamate-synthesizing bacteria, biosynthesis proceeds along the diaminopimeline pathway. This scheme of lysine formation was deciphered using various mutants deficient in certain enzymes in the chain of conversions of aspartic acid to L - lysine. When comparing the regulation system of lysine biosynthesis by the diaminopimeline pathway in different bacteria, it can be seen that they are not the same.

Because of the importance of the economic aspect of L-lysine synthesis, biochemical pathways for L-lysine synthesis have been extensively researched and elucidated in order to increase lysine production and reduce production costs. During cellular absorption, glucose is phosphorylated to glucose-6-phosphate with the consumption of phosphoenolpyruvate (phosphotransferase system), while sucrose is converted to fructose and glucose-6-phosphate by the phosphotransferase system and an invertase reaction. Glucose is catalyzed by the Embden-Meyerhof-Pamas pathways (glycolysis) and pentose phosphate. During glucose catabolism, glucose-6-phosphate isomerase and glucose-6-phosphate dehydrogenase compete for the substrate glucose-6-phosphate, resulting in fructose-6-phosphate or 6-phosphogluconolactone. respectively [6]. The oxidative part of the pentose phosphate cycle converts glucose-6-phosphate to ribulose-5-phosphate, providing reductive equivalents in the form of NADPH. The continuing pentose phosphate cycle, pentose, hexose and triose phosphates mutually transform. 5-phosphoribosyl-1-pyrophosphate (pentose phosphate) is required for the biosynthesis of nucleotides and as a precursor of aromatic amino acids and L-histidine. NADPH acts as a reductive equivalent in numerous anabolic biosynthesis, while four NADPH molecules are consumed for the biosynthesis of one lysine molecule from oxaloacetic acid [6]. Thus, the flow of carbon to oxaloacetate (OAA) remains constant regardless of systemic disturbances [6, 77]. Pyruvate carboxylase (PEP carboxykinase), phosphoenolpyruvate carboxylase (phosphoenolpyruvate carboxylase; PEPC), and pyruvate carboxylase have been identified as anaplerotic enzymes that catalyze the reaction of adding 1 mol of carbon dioxide to pyruvate and phosphoenolpyruvate by acting as TCA and as TCA as TCA and the subsequent formation of lysine, threonine, isoleucine (they are formed from aspartic acid) during metabolism and in the production of amino acids formed from organic acids of the TCA cycle (for example, glutamic acid, glutamine, proline, arginine, citrulline, ornithine, etc.) [46]. The beginning of the pathway to lysine with L-aspartate, which is synthesized by transamination of oxaloacetate. C. glutamicum has the ability to convert the L-lysine intermediate piperidine 2,6-dicarboxylate to diaminopimelate in two different ways: by reactions involving succinylated intermediates or by a single reaction of diaminopimelate dehydrogenase [16]. In general, carbon flux into the pathway is regulated at two points: first, through feedback inhibition of aspartate kinase by levels of both L-threonine and L-lysine; and secondly, by controlling the level of dihydrodipicolinate synthase. Thus, increased production of L-lysine can be obtained in corynebacteria by deregulation, desensitizing feedback inhibition by L-lysine and L-threonine [16, 51] and by increasing the activity of these two enzymes. L-lysine-HCl concentrations between 6.3 g / L and 28.7 g / L L-lysine-HCl were produced in shake flasks after 5 days by mutant strains of N. alkanoglutinousa at 33 C, depending on the carbon source used and 52, 5 g / l. l after 96 h in a bioreactor at 33 C, pH 7.0 (ammonia water), 400 rpm and aeration rate of 0.5 rpm [27]. Manufacturing companies generally do not disclose L-lysine titers and yields due to today's fierce industrial competition. Approximately 170 g / L L-lysine is produced intermittently after 2 days using superior recombinant C. glutamicum strains (Research Center Julich, Germany, 2006). About 100 g / L L-lysine was continuously produced under optimized chemostat conditions with a short residence time by the B. lactofermentum (ATCC) strain without any genetic improvement, which highlights the still large hidden potential for the development of the process (unpublished data obtained by Anastassiadis and Stephanopoulos, 2004-2006, Massachusetts Institute of Technology, USA)

Besides the biochemical pathway for L-lysine synthesis, L-lysine export has been shown to be another important factor influencing the production of L-lysine in C. glutamicum. It has the following properties: (1) the transporter has a rather high Km value for lysine (20 mM); (2) the conveyor is the OH-symport system (the capture systems are the H + -antiport systems); and (3) the transporter is positively charged and the membrane potential stimulates secretion [16, 65].



5. L-LYSINE ISOLATION PROCESSES

Crystalline lysine is isolated from the CL after biomass separation.

For the production of feed products, lysine preparations with a basic substance content (lysine monochlorohydrate) of 70% and more are suitable. In this case, the crystals can be colored yellow and light brown. More stringent requirements are imposed on medicines for medical purposes; for parenteral nutrition, the content of the basic substance must be at least 99%.

In the normal course of the process, the share of side amino acids does not exceed 3% of the lysine content, the share of microbial cells is 1.5%. Self-discharging separators are used to separate biomass from CL, as well as filtration with a precoat layer either on a drum vacuum filter or on frame filter presses, followed by washing the sediment with water.

Solutions containing lysine, after acidification with hydrochloric acid (pH = 5.0 ч 5.2) and the introduction of a stabilizer (NaHSO3), are concentrated by evaporation in a vacuum to 45-50% DM. The resulting concentrate is subjected to crystallization, which is carried out at 5-12 ° C for 1-2 days. The sediment is separated from the mother liquor in flow-through industrial centrifuges and then dried in a spray dryer or in a fluidized bed. The finished product is usually brown in color and contains at least 70% of the main substance.

Another way of isolating lysine is by ion exchange. For this, the product solution is acidified with H2SO4 to pH = 1.6 ч 2.0, resulting in the formation of the amino acid dication. After chemisorption on a cation exchanger (KU-2x8) used in H + or form, impurities of neutral and acidic nature are separated. Amino acids are eluted from the cation exchanger with 0.5-5% ammonium hydroxide, the solution is evaporated, acidified with HCl to pH = 4.9-5.0, and the concentrate is crystallized at 5-12 ° C, yielding light yellow or light brown lysine monochlorohydrate crystals colors that, after drying, contain 90-95% of the basic substance and 1.0-12.5% of ash. To obtain a preparation of a higher purity, the purification scheme includes the stage of processing the solution with active carbon, recrystallization from 50% ethanol, etc.

Separation and purification of the product is a very important factor that greatly influences the efficiency of the fermentation process and production costs, which constantly requires improvement - elements of the recovery process of amino acids, especially L-lysine. L-lysine is extracted from the fermentation broth in a variety of ways. For many years, solid L-lysine-HCl has been produced after various stages such as fermentation, separation, purification, crystallization and drying. L-lysine from the obtained culture broth can be recovered by known conventional methods, such as using ion exchange resins or by direct crystallization of L-lysine from culture broths. After separating the cells by filtration or centrifugation, L-lysine can be isolated from the fermentation broth by an ion exchange step and then concentrated by evaporation and spray drying.

5.1 Separation of cells

The fraction of the L-lysine fermentation broth is obtained by any suitable separation methods such as ultrafiltration or centrifugation [4, 14]. To overcome the difficulties arising from the high viscosity of the culture broth, the filterability can be improved by adding a mineral acid (e.g. concentrated sulfuric acid) to bring the pH to 1-3, or by adding alkali to bring the pH to at least 9, and then heat the culture broth to ? 80 o C (100 o C) for at least 5 minutes (eg 20 minutes) [27].

5.2 Ion-exchange chromatography

Various methods of ion exchange chromatography are used to separate L-lysine. and other amino acids, either before or after removal of biomass by filtration or centrifugation. These include primary cation exchange resins based on the isoelectric point of the desired amino acid, passing a solution at pH 1.5-4, and elution of L-lysine from the resin with an eluent (e.g. ammonium hydroxide) at pH> 9.59, above the isoelectric point), and ion exchange chromatography using fixed bed or simulated moving bed resins adsorbing the desired amino acid to the resin. These purification methods are more economical than the costly crystallization step and also provide additional benefits and flexibility in the shape of the final product. It can be powder (spray dryer), coarse powder or fine-grained product (fluidized bed dryer), flakes (drum dryer) or intermediate granules (dryer-granulator) [8, 16].

Alternatively, calcium hydroxide is added to the culture broth to bring its pH to 11.0. The culture broth is then heated at 100 ° C for 30 minutes, aerated at a flow rate of 1 rpm for 2 hours. Concentrated sulfuric acid was added to adjust the pH to 5.0, followed by filtration. In particular, the filtrate is passed through an ion exchange resin (IR-120, NH +4 type) such as IR-120 or IRC-84 to adsorb L-lysine, which is washed with water and then eluted with ammonia solution. [27]. Eluate fractions containing L-lysine and impurities such as ammonia, potassium, calcium, sodium and magnesium ions and other amino acids are concentrated by evaporation. Ammonia is removed at the same time using multi-stage evaporators. They are then neutralized or acidified with concentrated hydrochloric acid and crystallized either with a batch crystallizer or with an evaporative crystallizer (crystallization process) to obtain L-lysine-HCl with a purity of more than 98%. Potassium (as K +) forms up to about 80% of the non-ammonium cationic impurities in the L-lysine liquid after the primary ion exchange operation. The wet crystals of L-lysine are separated from the mother liquor using a centrifuge or filter, and then dried in a conventional fluidized bed dryer that can be recycled, and some of the lysine is lost when the waste stream is purged. Impurities that are not separated from L-lysine in the primary (conventional) ion exchange resin stage are removed by passing the first solution through a secondary (also third) cation exchange resin (feed rate 12-30 m3 / h), which is located in the bed, works like fixed bed, as a countercurrent rotating fixed bed or as a countercurrent moving bed, which greatly reduces impurity levels, recovery losses, and capital and operating costs and thus does not require further purification by crystallization [8, 27].

Suitable primary and secondary cation exchange resins include the strongly acidic cation exchange resins DOWEX XUS 43518 and XUS 40406 and DOWEX C500ES (registered trademark of the Dow Chemical Company), which are typically sulfonated styrene-divinylbenzene copolymers with a resin capacity of 1.9 equivalents. /liter. The column is regenerated with 5-10% sulfuric or hydrochloric acid, and then washed with several volumes of demineralized water. US5684190 [8] provides a method for separating basic amino acids (e.g. L-lysine) from an aqueous solution (pH 4 by adding sulfuric acid) using cation exchange resin columns connected in series (DIAION SK-1B, Amberlite IR-120 and Duolite C-20, all are trademarks) involving sequential repeated steps of adsorption and elution (also cited in [16]). Cation exchange resins can be used in the form of ammonium, sodium, etc. [79]. According to [43], the supernatant is passed through a column of the "Amberlite IR-120" type after centrifuge. isolation of fermentation broth and removal of cells and other sediments. L-lysine, which is absorbed on the resin, is eluted with 3% ammonia water, the eluate is evaporated and HCl is added, so that on cooling, crystals of L-lysine-HCl2 H2O are obtained. In US 5268293 [14] purification is described. a procedure including bringing the filtrate to pH 5.0 with sulfuric acid, decolorizing with activated carbon, concentration, crystallization and then separation into crystals and a liquid fraction. L-Lysine is adsorbed onto the strongly acidic Duolite C-20N cation exchange resin by loading the eluent (10 ml) up the column at a volume velocity of 5. This upflow adsorption method removes microorganisms. centrifugation is not required, which reduces the loss of L-lysine. After removal of ammonia, the fraction enriched in L-lysine is mixed with the filtrate in an ultrafiltration step and subjected to pH adjustment, decolorization and concentration to obtain pure crystals (recovery 94% yield and more than 99% purity). After adsorption is complete, the resin column is rinsed sufficiently with water to remove microorganisms and particulate matter. The adsorbed lysine is eluted with 2NH4OH and the L-lysine fraction is collected, which is 0.6 times the volume of the resin (129 g / l L-lysine · HCl, 97% recovery from the culture liquid). Fractions with low L-lysine concentration are returned to the next cycle. Crystals obtained by this method are dried with hot air to obtain 99 g of L-lysine hydrochloride (extraction yield 94% and purity above 99.5%) [14]. The L-lysine-HCl solution is usually crystallized to obtain L-Lysine-HCl dihydrate crystals (L-lysine-HCl 2H 2 O), which are then dried to less than 1% moisture. This product still remains dusty and lumpy, resulting in loss of valuable material and sometimes incomplete composition. The use of ion exchange makes the process expensive. Extending the previous method, [4] describes a new method for the production of an L-lysine feed additive with a final L-lysine purity in the range of 35 to 80%.

5.3 Drying stage

The cleaning process also includes a drying step, which may include any suitable drying means such as a spray granulator, spray dryer, tray dryer, drum dryer, rotary dryer, and tunnel dryer [4, 67, 80]. Concentrated L-lysine solutions are prepared by heating fermentation broths under reduced pressure with steam at 130 ° C using a multipurpose concentrator or thin film evaporator. The microorganism is deactivated, while concentrated broths are stable for a long time, and L-lysine does not decompose during storage for six months at 20 C [68, 76]. Direct spray drying of L-lysine fermentation broth avoids additional purification steps associated with this. with the L-lysine-hydrochloride process, in particular using expensive ion exchange. However, it is difficult to achieve a stable concentration of L-lysine in the final dry product due to the significant change in the concentration of L-lysine in the fermentation broths. In addition, the dry product can be dusty and difficult to handle [4]. EP0923878B1 [67] describes a new process for the preparation of a pelleted feed additive L-lysine, which significantly dries up L-lysine-enriched broth, while the L-lysine content depends not only on the initial concentration of L-lysine in the fermentation broth [67].



5.4 Hard forage additive l-lysin

ETW0501996B [81] describes a process for producing an amino acid feed additive that still contains most of the fermentation broth solids. [68] describes a fermentation process for obtaining a stable long (6 months at 30 C) L-lysine sulfate (35 to 48% by weight of lysine) for animal nutrition with the addition of sulfuric acid or ammonium sulfate. Concentrated solutions for L-lysine fermentation have the same nutritional properties as purified lysine and biomass; other components of the medium or products derived from them do not have a harmful effect on animal health [68]. US Pat. No. 5,622,710 [82] describes a method for preparing a non-dusty biomass containing (or not) granular animal feed particles by spray drying a fermentation broth, which is converted into granules using expensive high shear mixing equipment. European application no. 91460051.5 describes a method for preparing a dust-free, free-flowing granular L-lysine product from a liquid solution or suspension using a spray granulation process. Care should be taken when using or properly disposing of the cell-rich L-Lysine broth, which is often viewed as a waste byproduct. Occasionally, for economic reasons, a non-granular L-lysine feed additive with a controlled amount of L-lysine purity is desirable. International publication number WO9523129 and application ser. US 08991145 (filed December 16, 1997) describes the preparation of non-stoichiometric L-lysine salts in granular form, whereby the amount of L-lysine in the final product can be controlled. The spray granulation stage can be replaced by alternative drying methods such as spray drying, drum drying, rotary drying, tray drying and tunnel drying [4]. In addition, stable granule animal feed additive is produced by granulation from fermentation broth. Biomass may be fully or partially present in granules or not at all [82]. US3089824 [83] Describes the use of a fluidized bed for the production of compressed tablets for medical use. Moreover, [12] offers feed additives of L-lysine, which do not contain only trace amounts or 90-100% of biomass and / or components from the resulting fermentation broth.



6. AQUATIC L-LYSINE FOOD ADDITIVE

The production of L-lysine by enzymatic synthesis is based on the preliminary chemical synthesis of DL-б-amino-е-caprolactam (ACL), which is then used for the selective enzymatic hydrolysis of L-ACL to L-lysine; D-LAC undergoes enzymatic racemization and is reused as a substrate for hydrolysis. The hydrolyzing enzyme is obtained from Cryptococcus laurendii cells, and the enzyme catalyzing racemization is obtained from Achromobacter obae cells.

When obtaining L-lysine, it is advisable to use the combined action of two enzymes on the substrate [7]. To do this, the required amount of yeast and bacterial cells exhibiting hydrolase and bacterial activity is introduced into an aqueous solution of DL-б-amine-е-caprolactam. The optimal conditions for the action of both enzymes are as follows: pH = 8.0 ч 8.5; temperature 30-50 ° С.

The yield of L-lysine in this process is 99.8% (molar) of the substrate.

Alternatively [84] describes various processes related to the production of an aqueous cell-free feed additive containing 300 to 600 g / L L-lysine to meet the different specific needs of individual customers. The L-lysine content depends not only on the L-lysine concentration in the bioreactor, and the cell-free amino acid solution can be further purified using chromatographic columns, for example, an ion exchange column. The above process can be applied to any amino acid that can be obtained by fermentation, such as L-lysine, threonine, and tryptophan [84].



7. CURRENT AND FUTURE DEVELOPMENTS

Qualitative and quantitative analysis of the L-lysine formed is also an important point in the international literature. After centrifugation and filtration of the fermentation broth, L-lysine is determined by various analytical methods. In US 4275157 [43], a colorimetric determination method is used. The concentration of L-lysine (as the monohydrochloride salt) accumulated in the culture broth is also analyzed by acidic ninhydrin or by high pressure liquid chromatography (HPLC, reverse phase HPLC) with precolumn derivatization with orthophthalic aldehyde and an amino group. acid separation, e.g. on a Hypersil AA column (Agilent). The amino acid composition in the culture liquid is analyzed using an amino acid autoanalyzer using ion exchange chromatography and post-column reaction with ninhydrin detection [6,7,9, 12, 14, 20]. In addition, L-lysine was measured by bioanalysis using a mutant Escherichia coli strain or using an amino acid analyzer, for example, from Eppendorf-BioTronik (Hamburg, Germany) [12, 27]. Glucose content is determined using a glucose analyzer [9] or can be analyzed using HPLC applications.



8. QUALITATIVE AND QUANTITATIVE ANALYSIS OF L-LYSINE

The production of L-lysine by fermentation methods using various mutant strains of corynebacteria and other microorganisms obtained by classical mutagenesis or recombinant technology remains the first. runner in biotech production of bulk chemicals. It is assumed that no other microbial process has been undertaken with such a massive effort to produce better mutant strains. Little work has been devoted to the development and optimization of the fermentation process, but there is still a lot of room for further improvements in terms of increasing productivity concentration and L-lysine yield. The results (about 100 g / L L-lysine) obtained in continuous cultures (chemostat) with a short residence time using a conventional ATCC strain producing L-lysine (Anastassiadis and Stephanopoulos, 1994-1996, Massachusetts Institute of Technology, USA), were confirmed these thoughts. In comparison, the legendary enzymatic production of citric acid reflects the development of fermentation technology and the development of processes that have taken place over the past 100 years of modern biotechnology.



CONCLUSION

As a result of the analysis of literature data, research on the technology of obtaining feed lysine was performed.

1. Data on the causes of such large volumes of lysine production, as well as its use in many industries and forms of the drug were summarized.

2. The characteristics of the main stages of the biotechnological process are clarified. Each technological operation affects the formation of the product, and the quality and properties of the future lysine concentrate depend on its proper conduct.

3. It is established that there is a certain technology for obtaining lysine and its control. (And all this is very well limited by licenses and patents) The process is controlled by using different methods of qualitative and quantitative analysis of the culture fluid during the oral fermentation process.

4. It is also established that the obtained drug is a complex of vitamins, microbial biomass and lysine, which increases its value as a feed additive.

5. Several variations of the technological process of lysine production and future prospects for the development of biosynthesis of this amino acid are considered.

It is concluded that the process of lysine synthesis requires extraordinary control to obtain the maximum yield of the required product.

It was also concluded that a sufficient level of lysine supply to livestock is a necessity, as well as the enrichment of the daily diet of people, especially those who are limited in the consumption of animal products rich in essential amino acids.

From personal observations I can connect the place of discovery of the first producers with a country that has a monopoly on licensing and patenting, which is quite a logical historical fact.



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APPENDIX 1

Component	Content
AA, mas. % of SS
Lysine	15-20
Glutamic a.	2,5-3,7
Val	1,2-4,8
Ala	1,3-3,1
Аsp	0,8-1,4
Leu	0,6-1,1
Pro	0,3-2,8
Gly	0,6-0,9
Arg	0,3-0,8
Tyr	0,4-0,7
Met	0,4-0,6
Iso	0,4-0,6
Phe	0,2-0,6
Try	0,5-0,6
Ser	0,4-0,6
Thr	0,3-0,6
His	0,2-0,3
Cys	0,2-0,3
Nitrogen compound, масс. % of mas SS
General N	5,2-7,9
Protein (Nґ6,25)	37,5-49,4
N:
Protein	1,9-3,6
б-amino	0,9-2,0
Amiac	0,3-1,4
Betaine	0,82-1,66
Betan	6,0-13
Vitamins mg/kg
В1	1,7-9,7
В2	84,2-160
В3	30-60
В4	10-20
В6	100-340
РР	8-10
Other organic compound. % of mas SS:
Reducing substances	4,6-12,7
Fats	1,3
Cellulose	0,3
Mineral compound % of mas. zoles
Zole mass. % of mass SS	19,0-28
Ca	5,2-12,5
K	28,6-33,6
Na	0,8
Mg	1,1-1,5
Fe	0,1-0,25
P	2,2-4,4
Si	10,9-11,5
microelements, мg/100 g
Zn	1821
Co	67,8
Kd	476,7
Mb	545,9
Mg	3071
Cu	280

Table 3 - Chemical compound of result fermentation



APPENDIX 2

Fig 2 - Scheme of producing lysine

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