RU2485181C2 - Method of ion-exchange isolation of lysine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: biomass is isolated by microfiltration. The native solution is clarified. The clarified solution is desalted by passing through the ion-exchange system of 10 identical columns with a height to diameter ratio of 0.8, which are connected in the sequence: anion exchanger in OH-form - cation exchanger in H⁺-form, at a rate of 2-2.5 of solution volume per volume of the system. The desalted solution is passed through the column with the anion exchanger in OH-form. The resulting solution of lysine-base is passed through the column with the cation exchanger. Elution of lysine with cation exchanger 3-5N is carried out, preferably 4N, with alkali liquor. The gaseous hydrogen chloride is bubbled to the resulting concentrated lysine eluate, causing simultaneously neutralisation and crystallisation of lysine.

EFFECT: method enables to eliminate the need of evaporation in the technological, which results in power consumption economy.

2 cl, 1 ex

Description

The invention relates to the field of microbiological industry and relates to the production of crystalline λ-lysine. Lysine is used as a feed additive to improve the digestibility of cereal proteins, which lack 30-35% lysine for complete protein synthesis in poultry and pigs. Currently, more than 900 thousand tons of crystalline lysine for fodder purposes and 15 thousand tons of highly purified lysine for food and medical purposes are produced in the world.

For the first time, Japanese firms Ajinomoto Ko and Kiova Hakko began to produce crystalline lysine in the 60s of the last century. The technology included enzymatic lysine biosynthesis, separation of biomass, acidification of the native solution with sulfuric acid, sorption of lysine as a doubly charged ion on a fixed bed of sulfostyrene cation exchange resin, elution of lysine with ammonia, removal of excess ammonia by vacuum evaporation, acidification of lysine with concentrated HCl, crystallization of lysine, centrifugation.

The main disadvantage of this method was the use of large quantities of cation exchange resin, water and heat energy during evaporation of the eluate. These disadvantages are eliminated in the lysine isolation process proposed by the Belgian engineering company CHemviron Carbon (www.chemvironcarbon.com). This method is implemented at the factories of the main producers of lysine Ajinomoto Co., Kiova Hakko (Japan), ADM (USA) and C J (South Korea). The method involves a continuously operating ion-exchange system (ISEP) of 30 ion-exchange columns connected in series in the form of a vicious circle in which the culture fluid, water and eluent move towards the moving ion exchanger.

The ion-exchange columns are mechanically moved clockwise in a circle in the form of a carousel and thereby fall under the supply and drain of the processed fluids.

In the ion exchange installation, there are 4 exposure zones:

- washing zone;

- adsorption zone;

- rinse area;

- elution zone of the product.

All other stages of the production process: separation of biomass, acidification of the native solution before sorption, ammonia elution - clarification of the eluate, vacuum evaporation of the eluate, crystallization of lysine, centrifugation of crystals and drying do not differ from the previously used method.

Despite the fact that the ISEP system reduces the amount of cation exchanger by 17 times compared with a fixed resin layer. This method has disadvantages. First of all, this is the lack of selectivity of the exchange and chromatography of substances, due to which lysine, cations and pigments go along one front, and the entire pigment enters the eluate. A large amount of activated carbon is used to remove the pigment. But even with this, the lysine solution is evaporated only to a concentration of 50%.

In this case, larger crystals can be obtained than at a lysine concentration of 65%, even in a highly colored solution, however, only half of the lysine crystallizes, and the lysine remaining in the mother liquor is returned to the process. Thus, a compact ion-exchange unit does not make lysine cleaner and cheaper.

Another technological concept is reflected in USSR copyright certificates No. 677501, No. 1112779 and RF Patent No. 1578196. It consists in the fact that a native solution acidified to pH 1.5 containing lysine L ²⁺ is passed through columns with cateonite in the Na ⁺ form, lysine is eluted from the half of the columns with alkali and the eluate is passed through the second half of the columns. The amount of lysine in the columns doubles according to the equation L ²⁺ + L ± = 2L ⁺

- L ²⁺ - sorb.

- L ± - e.

- 2L ⁺ - sorb.

In this process, pigmented substances pass along with the drains and are not adsorbed on the resin.

The difference between alkaline and ammonia elution is that the alkali elutes lysine equivalently and the lysine eluate has a pH corresponding to the isoelectric point of lysine, i.e. 9.7. At this pH, which does not change during the entire elution process, the pigment does not desorb and the eluate remains clean.

A significant excess of the eluent is necessary for the desorption of lysine by ammonia, the pH of the eluate reaches 12-12.5, and the pigment is desorbed with lysine. The process of recharging lysine on the column allows you to get twice as concentrated lysine eluate, and therefore reduce the amount of evaporated water. In the present invention, alkaline elution and a fixed layer of cation exchange resin are used, therefore, the closest prototype is the method described in RF Patent No. 1578196. The method includes the following operations. After fermentation, the culture fluid is filtered on a microfiltration unit on ceramic membranes with a pore size of 100 kDa. The filtrate was acidified to pH 1.5 with nitric acid and passed through six columns with Ku-2 × 8 cation exchange resin in the H ⁺ form until they were completely saturated. An alkali solution dosed so that its amount in g-eq is passed through three columns. corresponds to 95% of the exchange capacity of the resin in the column. The eluate from each column with a pH of 10.2 is passed through a lysine saturated column. In this process of recharging lysine on the resin, the pH of the filtrate from the columns is 5-7. Lysine is absent in the filtrate. Columns with lysine are eluted with an alkali solution, the amount of which in g-eq. is 105% of the exchange capacity of the resin in the column. The eluate from the three columns is combined and a certain amount of ammonia adsorbed on the resin together with lysine is evaporated under vacuum. Then, lysine is neutralized with concentrated HCl, clarified on IA-4 microporous oinosorbent, the lysine solution is evaporated to a concentration of 65%, crystallized at a temperature of 15-18 ° C, centrifuged on a filter centrifuge at 5000 rpm and dried on a dryer in a suspended layer. The mother liquor from crystallization is returned to the process at the clarification stage. The biomass concentrate is dried on a spray dryer. The final product is a white powder with a lysine content of monochlorohydrate of 98.5-98.7%. The product can be used in food conditions as additives in bread, pasta and other products. The yield of the product is 85-88%.

The disadvantage of this method is the large amount of cateonite, the need for evaporation of a large amount of water and multi-stage. The aim of the present invention is to improve ion-exchange technology and address these disadvantages. This is achieved by the fact that after microfiltration of the culture fluid, the filtrate is desalted using a special ion-exchange column consisting of ten layers of ion exchanger, which can rotate relative to each other. In one position, the cateonite layer alternates with the anion exchange layer. By passing the solution at a rate of 2 volumes per total column volume, all inorganic salts are removed from the lysine solution. During regeneration, layers of cateonite and anion exchange resin enclosed in cylindrical containers rotate 90 ° C relative to each other and move to another position. In this case, the cateonite layer is under the cateonite layer, and the anionite layer under the anionite layer, i.e. two columns are practically formed. Layers of cation exchanger regenerate 2N HNO ₃ , and layers of anion exchanger - alkali solution. After washing with water, the layers are again moved to their original state.

или Cl^-). Таким образом получают цвиттер-ион лизина Л±. Пропуская обесцвеченный раствор цвиттер-иона через колонну с катионитом Ку-2×8 в Н⁺-форме, смолу полностью переводят в лизиновую форму. По сравнению с сорбцией цвиттер-иона лизина, идущей по уравнению: Л±+Н⁺R→ЛН⁺R, сорбция на катионите двухзарядного лизина из подкисленного нативного раствора требует в 2,7 раза больше смолы. Другое преимущество данного способа состоит в том, что пропуская через колонну, насыщенную лизином, 4н раствор щелочи, подогретый до 50°С, получают элюат лизина с концентрацией до 70% лизина основания. Кристаллизацию осуществляют, подавая в раствор газообразный HCl или концентрированный раствор HCl. После центрифугирования и сушки кристаллов получают белый негигроскопический порошок с содержанием основного вещества более 98,5%.The desalted solution is passed through a clarification resin column IA-4 in the Cl ^- form. The clarified solution is passed through a column with EDE-10p anion exchange resin, on which the anion bound to lysine is adsorbed (

or Cl ^- ). Thus, the zwitterionic lysine ion L ± is obtained. By passing a bleached solution of the zwitterion through a column of Ku-2 × 8 cation exchanger in the H ⁺ form, the resin is completely converted to the lysine form. Compared with the sorption of the lysine zwitterionic ion according to the equation: L ± + H ⁺ R → LH ⁺ R, sorption on doubly charged lysine cation exchange resin from an acidified native solution requires 2.7 times more resin. Another advantage of this method is that passing through a column saturated with lysine, a 4N alkali solution, heated to 50 ° C, obtain a lysine eluate with a concentration of up to 70% of lysine base. Crystallization is carried out by supplying gaseous HCl or concentrated HCl solution to the solution. After centrifugation and drying of the crystals, a white non-hygroscopic powder is obtained with a basic substance content of more than 98.5%.

Examples of carrying out the invention

Example 1

400 l of culture fluid with a concentration of lysine 100 g / l and biomass 45 g / l, obtained by biosynthesis of a Brevibacterium culture on a medium containing glucose syrup, ammonium sulfate, potassium hydrogen phosphate, vitamins and trace elements, are microfiltered in a plant with a polymer membrane with pores of 100 kDa. The biomass concentrate is diluted with water (100 L) and microfiltration is continued to a biomass concentration of 25%. Get 80 l of concentrate containing 18 kg of dry biomass and 2 kg of lysine. The concentrate is dried on a spray dryer with a capacity of 10 l / h by evaporated moisture. Get 20 kg of protein containing 10% lysine. The product is a complete feed protein supplement. 420 l of permeate is passed through a desalination ion-exchange system consisting of ten two-liter columns with a diameter of 15 cm, a height of 12 cm, loaded with EDE - 10p anion exchange resin in the H ^- form and Ku-2 × 20 cation exchanger in the H ⁺ form. The columns are connected in series: Anionite-Cationite. The speed of the solution is 2-2.5 volumes per volume of the system, i.e. 40-45 l / hour. The solution is displaced from the columns with water at the same speed. Get 440 l of a solution with a pH of 6.8 light brown, not containing inorganic salts. The desalted solution is passed through a column loaded with 50 l of IA-4 brightening microporous resin at a rate of 40 l / h. The solution is displaced from the column with water at the same speed. Lysine sulfate is converted into a zwitterionic ion by passing a lysine solution through a column with EDE - 10p resin in an N ^- form with a diameter of 25 cm, a height of 200 cm and a volume of 100 l. The speed of the solution in the column is 75 l / h. The solution is displaced from the column with water at the same speed. Get 530 l of a colorless clear solution with a pH of 9.8. Sorption of the lysine zwitterion on Ku-2 × 8 cation exchanger in the H ⁺ form occurs by the chemisorption mechanism and is released with energy. Due to this, lysine fills all exchange spaces on the resin, i.e. the resin capacity for lysine is actually equal to theoretical, 5.1 mEq / g. Lysine is eluted from cateonite using a 4N NaOH solution heated to 50 ° C. The eluent speed is 30 l / h. In the elution process, the solution is fed from above so as not to erode the elution front. A viscous, light yellow solution of the base lysine comes out from the bottom of the column. In total, after displacing the alkali from the column with deionized water, 55 liters of eluate are collected.

Hydrogen chloride gas was obtained in a 100 L glass reactor, gradually pouring concentrated H ₂ SO ₄ into 30 L of concentrated HCl. Lysine is crystallized in a 100 liter glass reactor. The device is equipped with a bubbler connected by a polyethylene hose to a source of hydrogen chloride. When bubbling HCl with stirring, crystallization of lysine monochlorohydrate begins. A suspension of white crystals becomes viscous, and the pH of the mixture reaches 5.2. The concentration of lysine in suspension is 64%. The suspension is centrifuged on nylon tissue at 5000 rpm. 41.7 kg of crude crystals of lysine with a moisture content of 18% are dried on a dryer in a suspended layer at an inlet air temperature of 110 ° C. 16 l of the mother liquor containing 7 kg of lysine is returned to the process at the stage of passing the solution through anion exchange resin. The yield of dry crystals of lysine monochlorohydrate is 34.2 kg, or 86%. The quality of the powder is fully consistent with the certificate for food lysine.

The advantage of this process over existing industries is that it uses little water and there are no evaporation processes that require high energy costs.

Claims (2)

1. The method of ion-exchange isolation of lysine from the culture fluid, including the separation of biomass by microfiltration, clarification of the native solution, sorption of lysine on a polystyrene cation exchange resin, elution of lysine with an alkali solution, crystallization of lysine, centrifugation of crystals and drying, characterized in that the solution is desalted before sorption on the cation exchange resin, passing through an ion exchange system of 10 identical columns with a height to diameter ratio of 0.8, connected in the sequence: anion exchange resin in OH ^- form - cation exchange resin in H ⁺ form, with a rate of 2-2.5 volumes of solution per volume of the system; the desalted solution is passed through a column with anion exchange resin in the OH ^- form, the resulting solution of the lysine base is passed through a column with cation exchange resin and gaseous hydrogen chloride is bubbled into the concentrated lysine eluate, while simultaneously neutralizing and crystallizing lysine. 2. The method according to claim 1, characterized in that for the elution of lysine from cation exchange resin use 3-5n, preferably 4n, an alkali solution.