RU2333962C2 - Method of isolation of polyhydroxibutyrate from dry biomass of microorganism - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology, namely, to isolation of polyhydroxibutyrate (PHB) from dry biomass of microorganism obtained by fermentative synthesis. The method involves extracting dry biomass of microorganism of genus Azotobacter with content of PHB of 50 to 75%, that of nitrogen of 1.2 to 3.5% and ash of 10 to 25% by chloroform at temperature 30-40°C and concentration of PHB in chloroform of 0.3 to 0.55 g/l, supplying PHB solution in chloroform to isopropyl alcohol evenly with rate of 1 to 3 l/hour with further separation and drying of target product.

EFFECT: obtaining a method of isolation of polyhydroxibutyrate for medicinal purpose with a preset molecular weight, output of 95-98.5% and purity of 99.5-99.95%, at process duration of 8-10.5 hours.

1 tbl, 19 ex

Description

The invention relates to the field of biotechnology, in particular the selection of polyhydroxybutyrate (hereinafter - PHB) from biomass obtained by enzymatic synthesis.

By biomass is meant the totality of all organic substances that are produced under certain conditions by cells of animals, plants or microorganisms, including genetically modified forms. PHB is a plastic, biodegradable and biocompatible polymer that can be used primarily in medicine, as well as in agriculture and industry.

Separating PHB from cellular material is a very difficult task.

The use of traditional methods of isolation, widely used in the extraction of vegetable oils from oilseeds, such as mechanical pressing, sedimentation, do not give the desired result.

The methods for isolating various substances used in chemical technology and described in the literature are reduced mainly to the extraction of the target product, in this case, PHB, from dry or from raw biomass.

If the extraction is carried out from dry biomass, then it is preceded by processing the raw biomass containing the target product - PHB, with an organic solvent, insoluble PHB (hereinafter - the non-solvent), in order to free from water, lipids, inorganic salts and other impurities; separating the precipitate — the cell mass containing PHB, by known methods — by filtration or centrifugation and drying it (EP No. 0015123, С12Р 7/62, publ. 12/22/1982; US Pat. No. 3036959, NKI 435-146, IPC С12Р 7 / 62, published on May 29, 1962; US Pat. No. 3044942, NKI 195-47, IPC class - not specified, published on July 17, 1962). In particular, in EP No. 0015123 it is proposed for drying to first pass through a raw biomass gas stream at a temperature of at least 100 ° C to evaporate the water, and then treat it with acetone or methanol. Acetone is also used in the methods for isolating PHB, protected by US patents No. 3036959 and No. 3044942.

The very extraction of PHB from dry biomass is carried out with a PHB solvent, the cell mass is separated by known methods - filtration or centrifugation, then the target product is precipitated with non-solvent PHB, its separation and drying.

In the case of direct extraction of PHB from crude biomass, the latter is treated with a PHB solvent, the organic phase containing PHB is separated by filtration or centrifugation; then, PHB is precipitated with a non-solvent, the PHB precipitate is separated by centrifugation, filtration or in a heated separator, and the target product is dried.

It should be noted that recently, in most cases, direct extraction involves drying of raw biomass, for example, by centrifugation in a disk separator (German application No. 4036067, С12Р 7/62, publ. 05/14/1992), passing gas at a temperature of 100- 500 ° C (US Pat. No. 4,234,907, NKI 560-185, IPC S07C 67/56, publ. 04/13/1982).

Partially halogenated hydrocarbons, such as 1,2-dichloroethane, chloroform (EP No. 0015123, EP No. 0015669, С12Р 7/62, publ. September 17, 1980; US Pat. No. 4,324,907) are used as the extracting solvent; 1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane (US Pat. No. 4,310,684, NKI 560-185, IPC S07C 69/675, publ. 12.01.1982), methylene chloride (German application No. 4036067 ) As a rule, the process is carried out at a temperature of more than 40 ° C. Moreover, among them there are such as, for example, chloroform, with which a relatively good yield is obtained, and such as, for example, 1,2-dichloroethane, with which pure PHB is obtained.

In the patent of Germany No. 19533459 (C08G 63/89, publ. 11/28/1996) and the application of Germany No. 19623778 (C08G 63/90, publ. 06/14/1996), methyl and ethyl lactates are used, but the extraction temperatures are high (for example, for ethyl lactate - 154 ° С). The application of Germany No. 19712702 (C08G 63/06, publ. 01.10.1998) describes the use of acetic acid, and the application GDR No. 229428 (C12P 7/62, publ. 06.11.1985) describes acetic anhydride. But in these solvents, already near 70 ° C, gelation and mixing with side components of biomass occurs. At the same time, part of the solvent is stored in PHB, giving the product a specific smell. The possible hydrolysis of PHB with a decrease in molecular weight is also not ruled out. In the patent of Germany No. 19955381 (C12P 7/62, publ. 02.22.2001) describes the extraction of 1-methyl-2-pyrrolidone at 80-120 ° C, and during the extraction is added a water-binding substance, preferably acetic anhydride. According to US patent No. 3036959 extraction is carried out using pyridine at a temperature of more than 40 ° C.

To precipitate PHB from the organic phase, water, methanol or a mixture thereof, acetone, petroleum ether, non-polar solvents are used.

Drying the target PHB, proposed in various patents, is carried out at a temperature of from 50 to 80 ° C.

Closest to the technical nature of the proposed is a method for the isolation of PHB from biomass, described in the application of Germany No. 4036067 (С12Р 7/62, publ. 05/14/1992). In accordance with this source, dry biomass is treated to extract PHB with chlorohydrocarbon - methylene chloride (a solvent immiscible with water and having a boiling point below 100 ° C) - when heated for 30 minutes. Cell material is then separated by centrifugation at a speed of 2200 rpm for 30 minutes. Then, the remaining organic phase containing PHB is injected into water heated above the boiling point of the solvent (~ 80 ° C). Injection is carried out at a speed of 300 l / h by the action of water vapor with a pressure of 3 bar using a double nozzle with a diameter of 4 mm for a PHB solution and an annular gap of about 2 mm for steam. PHB falls out in the form of flakes, part of the water evaporates. The suspension is stirred for 30 minutes at a temperature of 80 ° C. Then the target product is separated by centrifugation and dried at 80 ° C for 24 hours.

The yield of the product is 85%, the purity is 99%.

It should be noted that previously, in the preparation of dry biomass, part of the water from the raw biomass is removed by centrifugation in a disk separator.

The disadvantages of this method include the following: although water is a cheap and non-toxic reagent, however, the use of large quantities of it, in particular, at the stage of methylene chloride removal, leads, on the one hand, to the necessity of conducting the process at elevated temperature (80 ° C) and, with another, to the process of polymer hydrolysis, which entails a significant decrease in its molecular weight and prevents the production of a polymer with a predetermined molecular weight. A decrease in the molecular weight of the polymer is also facilitated by the need for prolonged drying (up to 24 hours) of PHB extracted from a solution from water at an elevated temperature (80 ° C). A small finished product yield of 85% is also the result of the use of large amounts of water for its purification, and the presence of even a small admixture of methylene chloride makes it impossible to use such PHB for medical purposes.

The technical task of the invention is to obtain PHB for medical purposes with a predetermined molecular weight and high purity of the polymer, as well as accelerating the process of isolating the target product.

The technical result, consisting in obtaining PHB with a molecular weight of 300-1200 kDa, with a yield of at least 90% and a purity of at least 99.2% with a reduction in the process time, is achieved by the fact that in the method for the isolation of PHB from dry biomass of a microorganism, including the extraction of PHB with hydrogen chloride and separating the cellular material, followed by precipitation of PHB from chlorohydrocarbon with stirring, separating and drying the target product, use the biomass of a microorganism of the genus Azotobacter with a PHB content of 50 to 75% as a dry biomass of the microorganism and from 1.2 to 3.5% and ash from 10 to 25%, chloroform is used as chlorofluorocarbon, extraction is carried out at a temperature of 30-40 ° C and the concentration of PHB in chloroform is from 0.3 to 0.55 g / l, and deposition is carried out by uniformly supplying a solution of PHB in chloroform to isopropyl alcohol, the feed rate being from 1 to 3 l / h.

Raw biomass with a solids content of 24-32%, obtained by the enzymatic method using a microorganism of the genus Azotobacter, is prepared by any known methods: by washing it with isopropyl alcohol, vacuum filtering isopropyl alcohol with lipid compounds dissolved in it, drying and crushing the washed biomass to a powder state .

The proposed technical solution is illustrated by the following examples (1-19). It should be noted that in examples 1-13 and 17-19 used dry biomass obtained by the enzymatic method using a strain of the microorganism Azotobacter chroococcum 7B; in example 14, using a strain of the microorganism Azotobacter chroococcum 12A; in example 15, using a strain of the microorganism Azotobacter chroococcum 35 and in example 16, using a strain of the microorganism Azotobacter vinelandii 21.

Example 1

A three-necked 3-liter flask equipped with a mechanical stirrer, reflux condenser and thermometer is charged with 16 g of dry biomass with a PHB content of 70%, nitrogen 1.66%, ash 13% and 1500 ml chloroform. The stirrer is turned on and the contents of the flask are vigorously stirred for 2 hours at 38 ° C, placing the flask in a heated water bath. In the flask, a suspension of dry biomass in chloroform is obtained.

The resulting suspension is filtered on a Buchner funnel through two layers of filter material (belting and nylon) under a vacuum of 15 mm Hg. rest and room temperature. A precipitate forms on the filter, which is a by-product of the purification of dry biomass - cellular material.

The content of PHB in the chloroform solution is 11.2 g, which corresponds to 0.5% of its concentration in the solution.

Then, in a three-necked flask with a capacity of 8 l, equipped with a mechanical stirrer, thermometer, reflux condenser, dropping funnel, 4500 ml of isopropyl alcohol are charged. The mixer is turned on and, with vigorous stirring, the contents of the flask are fed from a dropping funnel 1,500 ml of a 0.5% solution of PHB in chloroform at a rate of 1.3 l / h. In this case, PHB is released from the solution in the form of white flakes. Upon completion of the introduction of a solution of PHB in chloroform into isopropyl alcohol, the mixer is turned off and the resulting gel is left to mature for 1 hour at room temperature. Then the gel is filtered on a Buchner funnel through two layers of filter material (belting and nylon) under a vacuum of 15 mm Hg. rest The gel is cleaned from the filter material and again placed in a 1-liter three-necked flask equipped with a mechanical stirrer, thermometer and reflux condenser, where 350 ml of isopropyl alcohol are charged, the stirrer is turned on and additional washing with isopropyl alcohol is carried out for 15 minutes at room temperature. Then the mixer is turned off, and the precipitate is filtered off from isopropyl alcohol on a Buchner funnel through two layers of filter material (belting and kapron) at a vacuum of 15 mm Hg. rest and room temperature. The precipitate is dried in an oven at a temperature of 58-60 ° C for 2 hours. 11.03 g (98.5%) of PHB are obtained with a basic substance content of 99.95%, nitrogen 0.1%, and a molecular weight of 300 kDa.

Raw biomass with a solids content of 24-32% is preliminarily prepared by washing it with isopropyl alcohol, vacuum filtration of isopropyl alcohol with lipid compounds dissolved in it, drying and crushing the washed biomass to a powder state.

Information on examples 2-16 (in accordance with the invention), 17-18 (comparative - with parameters that go beyond the stated limits) are carried out according to the method described in example 1, example 19 - reproduction of the prototype, are shown in the table.

Comparison of the proposed method with the method of the prototype is complicated by the fact that the method of the prototype is carried out not in laboratory conditions, but on a cameral installation. In this regard, the prototype method is reproduced by us in the laboratory.

Example 19 (prototype).

In a three-necked flask with a capacity of 2 l, equipped with a water bath, a mechanical stirrer, reflux condenser and thermometer, 39 g of dry biomass are charged. 156 ml of methylene chloride are added to it and heated under reflux for 30 minutes. The resulting mixture was centrifuged to separate the organic solution of PHB from the aqueous phase of the cellular material of microorganisms.

The organic phase is injected at 80 ° C. into a two-liter five-necked flask equipped with a thermometer, mechanical stirrer, reflux condenser, double nozzle and containing 320 ml of water. The organic phase is injected at a rate of 50 l / h by the action of water vapor with a pressure of 0.5 bar using a double nozzle with a diameter of 1 mm for a solution and an annular gap of about 0.5 mm for steam. The constancy of temperature is maintained by means of a water bath. In this case, the PHB flakes fall out and part of the water evaporates. At the end of the injection, the suspension is stirred for 30 minutes at 80 ° C. Finally, the mass is again centrifuged in the same centrifuge and the PHB flakes are dried in a shelf dryer at 80 ° C for 12 hours. In this case, 9.5 g of PHB (yield 85%) with a purity of 99.0% and a methylene chloride content of less than 1 ppm are obtained. The duration of the process is 14 hours.

To prepare dry biomass, a portion of the water is previously removed from the raw biomass by centrifugation in a disk separator.

The presented examples confirm the achievement of the following technical result:

- the obtained PHB has properties that can be used in medicine in the form of films and filaments, for example, as a suture biodegradable material that does not require removal over time;

- obtained PHB with a predetermined molecular weight of 300-1200 kDa;

- the output of PHB is 95.0-98.5% against 85% of the prototype;

- the purity of PHB 99.5-99.95% against 99.0% of the prototype;

- the duration of the process was reduced from 14 hours to 8-10.5 hours compared with the prototype.

In addition, the proposed technology for the isolation of PHB is low-waste, because most solvents return to the process and are in the cycle.

Table The characteristics of the feedstock, process parameters and technical result in comparison with the prototype Example No. The content of substances and the results of the analysis of dry biomass, wt.% The temperature of extraction, ° C Concentration PHB in chloroform,% PHB solution feed rate, l / h Will continue. process, h The yield of the target PHB,% The content of PHB in the final product,% Molek. mass of PHB, kDa PHB nitrogen ash one 2 3 four 5 6 7 8 9 10 eleven In accordance with the invention one 70 1,66 13 38 0.5 1.3 8.0 98.5 99.95 300 2 fifty 3.4 24 38 0.37 1,1 9.0 96.0 99.5 370 3 75 1.3 eleven 38 0.53 1,2 10.0 97.0 99.6 680 four 70 1,2 fifteen 35 0.45 1,5 9.5 98.0 99.85 500 5 56 3,5 twenty 37 0.42 1.8 9.0 96.8 99.7 700 6 72 1.48 10 35 0.52 2.0 9.0 97.0 99.5 430 7 54 3,1 25 40 0.38 1,1 10.0 97.5 99.7 450 8 52 3.3 23 thirty 0.35 1,1 10.5 96.0 99.6 560 9 65 2.12 16 40 0.47 2.0 8.5 96.5 99.9 380 10 fifty 3,5 25 38 0.3 3.0 8.0 95.0 99.7 1100 eleven 75 1,2 10.0 38 0.55 1,1 10.0 97.0 99.5 600 12 67 2.0 14.8 40 0.48 1,0 10.5 95.5 99.65 1000 13 60 2.6 19 38 0.43 3.0 8.5 97.0 99.8 1200 fourteen 71 3.2 19 37 0.46 1,1 9.5 97.5 99.6 500 fifteen 55 2,5 twenty 40 0.4 2.1 9.9 96.0 99.8 1000 16 68 1,5 12 36 0.5 1,5 10.0 97.8 99.9 350 For comparison 17 49 3.6 25.6 29th 0.28 0.9 11.0 90.0 99,2 1200 eighteen 76 1,1 9,4 41 0.57 3,1 12.0 93.0 99.5 1000 Prototype 19 72 1.3 10 80 - fifty fourteen 85 99.0 600

Moreover, the technical result obtained is not obvious. It would seem more logical for the deposition of PHB from a solution, since the reactor contains a solution of PHB in chloroform, and a precipitant, isopropyl alcohol, is fed there. However, it was found that with this procedure, the time of deposition of the polymer from the solution increases, the purity of the product decreases; with the reverse order of introduction of the reagents, i.e. with the uniform addition of a solution of PHB in chloroform to isopropyl alcohol at a certain speed, it is possible to obtain a product of improved quality while accelerating the process.

Claims (1)

A method of isolating polyhydroxybutyrate from the dry biomass of a microorganism, including the extraction of polyhydroxybutyrate with chlorohydrocarbon and separating the cellular material, followed by precipitation of polyhydroxybutyrate from chlorohydrocarbon with stirring, separation and drying of the target product, characterized in that the biomass of microorganism of 50 g of Azot is used as a dry biomass of the microorganism up to 75%, nitrogen from 1.2 to 3.5% and ash from 10 to 25%, chloroform, ext the action is carried out at a temperature of 30-40 ° C and a concentration of polyhydroxybutyrate in chloroform from 0.3 to 0.55 g / l, and the deposition is carried out by uniformly supplying a solution of polyhydroxybutyrate in chloroform in isopropyl alcohol, and the flow rate is from 1 to 3 l / hours