RU2539101C2 - Biocatalyst, method of its preparation and method of reetherification of vegetable oils using this biocatalyst - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: biocatalyst for reetherification of vegetable oils is offered which contains as enzymatic active substation the partially destructed cells or cell lysates of the recombinant producer strain rE. coli/lip, carrier consisting of silicon dioxide and of nanostructured carbon, and maltodextrin or high molecular weight polysaccharide as the water-retaining agent. A biocatalyst is prepared by mixing of cells or cell lysates of the recombinant producer strain rE. coli/lip with the carrier and water-retaining agent of carbon nature with the subsequent drying and fractionating. The method of reetherification of vegetable oils is performed both in a periodic mode in a mixing reactor, and in a continuous mode in a flowing reactor with the fixed layer of a biocatalyst at the temperature 60-80°C.

EFFECT: high enzymatic activity of biocatalysts during reetherification of vegetable oils.

5 cl, 2 tbl, 25 ex

Description

The invention is directed to the development of biocatalysts with thermostable lipase activity, intended for transesterification processes involving vegetable oils, as well as methods for their preparation. The invention can be used to obtain valuable products of the oil and fat industry, such as specialized fats - the most important ingredients of margarine products and spreads, as well as equivalents and substitutes for valuable cocoa butter and milk fat - the necessary components of confectionery and ice cream. The invention can also be used to obtain components of biodiesel obtained in the process of transesterification of vegetable oils with low molecular weight alcohols / esters.

Currently, for the preparation of biocatalysts with lipase activity, silicate carriers are used - silicon dioxide (SiO ₂ ), natural minerals (kieselguhr), which are distinguished by chemical and microbiological inertness, as well as high mechanical strength.

A known method of preparing a biocatalyst [Salis A .; Meloni D., Ligas S., Casula M.F., Monduzzi M., Solinas V., Dumitriu E. // Langmuir. 2005. V. 21. No. 12. P. 5511-5516] by physical adsorption of lipase from the microorganism Mucor javanicus, followed by crosslinking with glutaraldehyde on mesoporous silica. The biocatalyst exhibits almost the same activity in the hydrolysis of triglycerides, including triolein (an analogue of olive oil), equal to 80 Activity Units (EA) / g. The disadvantage of this method is the use of toxic crosslinking agents (dialdehyde), as well as the high cost of a mesoporous silicate carrier with a narrow pore size distribution synthesized by hydrolysis of tetraethyl ortho-silicate in the presence of surface-active copolymers (Pluronic®). Since the biocatalyst is obtained in the form of a fine powder, it cannot be used in a flow reactor with a fixed bed because of the rapid compaction of the layer and high hydrodynamic resistance.

A known method of preparing a biocatalyst by incorporating yeast autolysates into nanocarbon-silicate matrices [RU 24515466, СРР 19/00, 05.27.2012]. The biocatalyst is intended for the process of sucrose inversion and the production of an invert syrup containing glucose and fructose, and is characterized by high stationary activity. The disadvantage of this biocatalyst is the lack of enzymatic activity in the hydrolysis and transesterification reactions involving vegetable oils.

Closest to the proposed invention is a method of preparing a biocatalyst by incorporating a lipase from Pseudomonas cepacia into a silicate matrix obtained by hydrolysis of various silane derivatives in the presence of a lipase [Noureddini H., Gao X., Joshi S., Wagner P.R. // J. Amer. Oil Chem. Soc. 2002. 79 (1), 33-40]. This method is as follows. First, silane derivatives (tetramethoxysilane or butylmethoxy silane) and a catalyst (sodium fluoride) are added to the aqueous lipase solution. During the hydrolysis of silanes, a silica hydrogel is formed containing a lipase, which is then dried, and the silica xerogel is triturated. The powder is washed with distilled water with vigorous stirring in order to remove toxic impurities - unreacted silane, methanol, silanol, soluble oligomers. After washing, the wet paste is dried again, then triturated again and used as a biocatalyst in the batch process of hydrolysis of triglycerides, such as soybean oil. The activity of the biocatalyst is 20-80 EA / g at 40 ° C. The disadvantage of this method is the use of toxic and expensive reagents (silane derivatives) for the preparation of biocatalysts, the preparation of a biocatalyst in the form of a fine powder that swells rapidly under reaction conditions, which does not allow the use of such a biocatalyst in a fixed bed flow reactor. The disadvantage can also be considered the use of an enzyme previously isolated and purified, which increases the cost of the biocatalyst.

The invention solves the problem of preparing biocatalysts with high activity of thermostable lipase in the form of mechanically strong granules for processes carried out both in batch and continuous modes, including for the process of transesterification of vegetable oils.

The problem is solved by the composition of the biocatalyst for transesterification of vegetable oils based on lipase and a carrier - silicate matrix, in which it contains partially destroyed cells or cell lysates of the recombinant strain rE as an enzymatically active substance. coli / lip, producing thermostable lipase, as a carrier - nanocarbon-silicate matrices containing nanostructured carbon, as a water-retaining agent of a substance of carbohydrate nature, and has the following composition, in wt. % solids:

rE cell or cell lysates. coli / lip - 20-40,

nanostructured carbon (carbon nanotubes of CNTs) - 5-10,

water-retaining agent of carbohydrate nature, namely,

maltodextrin - 10-20 or high molecular weight polysaccharide - 1-2.

silicon dioxide - up to 100.

The composition of the biocatalyst includes a xerogel of silicon dioxide as a matrix for the inclusion (immobilization) of the enzymatically active component, as well as a structurant and a binder, which allows the biocatalyst to be obtained in the form of mechanically strong granules. Partially destroyed cells or cell lysates of a recombinant producer of thermostable lipase producer from Thermomyces lanuginosus are used as the enzymatic active component of the biocatalyst, without preliminary isolation of the enzyme from the producer strain and its further purification. Recombinant thermostable lipase producing strains (designated rE. Coli / lip) are constructed by genetic engineering by cloning a chemically synthesized lipase gene as part of an expression vector in cells of a specialized E. coli E. coli strain. Partially destroyed rE cells. coli / lip is obtained during the freeze-thaw process. Cell lysates rE. coli / lip is obtained by treating cell biomass with lysozyme, DNase, RNase A, and ultrasound enzymes.

The biocatalyst also contains nanostructured carbon, a water-retaining agent of carbohydrate nature - oligosaccharide (maltodextrin), polysaccharide - carboxymethyl cellulose (CMC), or gum arabic, or starch. Maltodextrin is an oligosaccharide with a number of monomer (glucose) units of 25-30. Cellulose and its derivatives, as well as starch, are polysaccharides whose molecule contains from 600 to 9000 glucose residues. Gum arabic (GUM) is also a polysaccharide with a complex arabinogalactan structure, it dissolves in water, forming solutions with a relatively low viscosity. CMC, GUM and starch are high molecular weight polysaccharides of plant origin.

Nanostructured carbon is a fine powder obtained by high temperature pyrolysis of ethylene on Co, Fe catalysts and containing carbon nanotubes (CNTs) with a diameter of 9-11 nm and 21-22 nm with a specific surface area of 280-320 and 100 140 m ² / g, respectively. Fine powder contains CNTs in the form of aggregates with a size of 100-150 microns.

The problem is also solved by the method of preparation of the biocatalyst, which includes the stage of mixing the cells or lysates of the recombinant producer strain rE. coli / lip with nanostructured carbon, a water-retaining agent and a silica hydrogel until uniform. Then the resulting mass is dried for 5-20 hours at a temperature not exceeding 40 ° C and fractionated.

As a result, biocatalyst granules with a size of 0.1–2 mm are obtained, which have a composition in wt.% Solids: cells / lysates rE. Coli / lip - 20-40, CNT - 5-10, maltodextrin - 10-20, polysaccharide (CMC, or GUM, or starch) - 1-2, SiO ₂ - up to 100.

For the preparation of biocatalysts, a silica hydrogel is used, obtained by sol-gel technology through the formation of coagel as a result of the interaction of sodium silicate or potassium silicate (in the form of liquid glass) with ammonium salts of sulfuric or nitric or carbonic acid, as described in [RU 2372403, C12N 11/14, 11/10/2009]. The silicon dioxide hydrogel contains 10-12 wt.% Dry matter in terms of SiO ₂ . When the hydrogel is dried, a silica xerogel is obtained with a specific surface area of 200-250 m ² / g. The silica hydrogel and xerogel are absolutely inert, have high chemical and microbiological stability, are stored for years in a tightly closed container at room temperature without losing their original properties.

The problem is solved by the method of preparation of a biocatalyst with thermostable lipase activity by including cells or lysates of the recombinant producer strain rE. coli / lip in silicate and nanocarbon silicate composite matrices. Xerogel of silicon dioxide is used as a builder and the main binder component. Maltodextrin or CMC, or GUM, or starch are used as a water-retaining agent. Nanostructured carbon is used as a component with pronounced hydrophobicity, effectively adsorbing both an enzymatically active substance (cells or lysates of rE. Coli / lip) and hydrophobic molecules of substrates of triglycerides of vegetable oils, which increases their concentration inside the biocatalyst, where the enzymatically active substance is immobilized. All components (SiO ₂ -hydrogel, cells or cell lysates, CNTs, a water-retaining agent) are thoroughly mixed to obtain a homogeneous pasty mass, then the resulting mixture is dried at a temperature not exceeding 40 ° C (in most cases, at room temperature and air humidity 20- 30%), crushed and fractionated to obtain solid, mechanically strong biocatalyst granules with a size of 0.1-2 mm.

The problem is also solved by the method of transesterification of vegetable oils using a biocatalyst prepared in the form of mechanically strong granules. The transesterification process is carried out at 60-80 ° C in both batch and continuous modes. Dry biocatalyst is loaded into reactors of various designs - mixing reactors and displacement reactors. During a continuous transesterification process, the biocatalyst is loaded into a column steel reactor and the reaction mixture is pumped through the fixed bed with a variable flow rate. Contact time (τ), equal to the ratio of the biocatalyst volume to the volumetric flow rate, varies from 30 to 90 minutes.

, %, где С_исх и С_кон - содержание твердых триглицеридов при 35°C в исходной смеси и продуктах реакции, соответственно.To determine the activity of the prepared biocatalysts, a hydrolysis reaction of 0.2 M emulsified tributyrin to butyric acid at 20 ° C is used. The activity of biocatalysts is expressed in units of activity per 1 g. One unit of activity (EA) corresponds to a reaction rate of micromol / min. The concentration of butyric acid is determined titrimetrically using 0.025 N. caustic soda solution. To determine the activity of biocatalysts in the transesterification reaction, a mixture of 2-3 weight is used as the initial reaction mixture. parts of vegetable oil (sunflower, or soy, or olive) and 1 weight. part of a fully hydrogenated oil (salomas). The activity of biocatalysts in the transesterification reaction is evaluated by analyzing the cooling curves of the final products, as well as the curves characterizing the content of solid triglycerides (TSH) in the reaction mixture at various temperatures (from 15 ° C to 35 ° C). During the transesterification process, the amount of TSH decreases, and the transesterification product is characterized by a lower melting point compared to the initial mixture of vegetable oil and salomas. The conversion is calculated by the formula:

,%, where С _out and С _con are the content of solid triglycerides at 35 ° C in the initial mixture and reaction products, respectively.

The invention is illustrated by the following examples and tables.

Example 1

The recombinant thermostable lipase producing strain was constructed by genetic engineering by cloning a chemically synthesized mature lipase gene from Thermomyces lanuginosus as part of the expression vector pJExpress401 in E. coli BL21 (DE3) cells (designated rE. Coli / lip). The coding region of the gene is optimized for expression in E. coli using the Gene Designer2 computer program from DNA2.0 (USA) and taking into account the frequencies of codon pairs and the position of the second amino acid residue from the N-terminus of the polypeptide chain. Strain rE. coli / lip provides gene expression induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) under the control of the T5 bacteriophage promoter.

The biocatalyst is prepared as follows. The cells of the recombinant rE producer strain are thoroughly mixed. coli / lip (1.5 g), partially destroyed during freezing-thawing, with silica hydrogel (5.0 g) until smooth. Then, the resulting mass is dried for 8 h at 25 ° C, mechanically crushed, and fractionated to a granule size of 0.1–2 mm. The biocatalyst has the following composition, in wt.% Solids: rE cells. coli / lip - 44, SiO ₂ - 56.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 380 EA / g (which is 5 times more than in the prototype). The biocatalyst transesterifies in a mixture of vegetable oil (olive or soybean) and salomas in a batch mode for 5 hours at 60 ° C. The conversion is 55%.

Example 2

Similar to pr. 1, only cell lysates of the recombinant producer strain rE are used. coli / lip. The biocatalyst has the following composition, in wt. % solids: rE lysates. coli / lip - 38, SiO ₂ - 62.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst does not show activity in the transesterification reaction.

Example 3

Similar to pr. 2, only in the composition of the biocatalyst add a water-retaining agent of carbohydrate nature - maltodectrine. The biocatalyst has the following composition, in wt. % solids: rE lysates. coli / lip - 40, maltodectrine - 5, SiO ₂ - 55.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 880 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 30%.

Example 4

Similar to pr. 3, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 39, maltodectrine - 11, SiO ₂ - 50.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 880 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 48%.

Example 5

Similar to pr. 3, only the biocatalyst has the following composition, in wt.% Solids: cell lysates rE. coli / lip - 38, maltodectrine - 23, SiO ₂ - 39.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 860 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 43%.

Example 6

Similar to pr. 3, only the biocatalyst has the following composition, in wt.% Solids: rE.coli / lip lysates - 36, maltodectrine - 34, SiO ₂ - 30.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 820 EA / g The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 25%.

Example 7

Similar to pr. 3, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 39, maltodectrine - 45, SiO ₂ - 16.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 840 EA / g The prepared biocatalyst practically does not show activity in the transesterification reaction at 70 ° C. The conversion is less than 5%.

Example 8

Similar to pr. 4, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 12, maltodectrine - 11, SiO ₂ - 77.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 250 EA / g The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 15%.

Example 9

Similar to pr. 4, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 20, maltodectrine - 11, SiO ₂ - 69.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 610 EA / g The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 35%.

Example 10

Similar to pr. 4, only the biocatalyst has the following composition, in wt.% Solids: cell lysates rE. coli / lip - 52, maltodectrine - 10, SiO ₂ - 39.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 900 EA / g The mechanical strength of the granules decreases significantly with an increase in the content of lysates above 40 wt. %, the biocatalyst granules swell and are destroyed in the reaction medium. The prepared biocatalyst transesterifies in a mixture of vegetable oil (olive, or soy, or sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 25%.

Example 11

Similar to pr. 3, only carboxymethyl cellulose (CMC) is added to the biocatalyst instead of maltodextrin. The biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 30, CMC - 0.5, SiO ₂ - 69.5.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 700 EA / g Biocatalyst granules retain high mechanical strength and do not swell for 5 reaction cycles lasting 24-48 hours each, i.e. for more than 120 hours.

The prepared biocatalyst transesterifies in a mixture of vegetable oil (sunflower) and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 30%.

Example 12

Similar to pr. 11, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 39, CMC - 1, SiO ₂ - 60.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 35%.

Example 13

Similar to pr. 11, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 40, CMC - 2, SiO ₂ - 58.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 34%.

Example 14

Similar to pr. 11, only the biocatalyst has the following composition, in mass. % in wt.% solids: rE lysates. coli / lip - 30, CMC - 5, SiO ₂ - 65.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 650 EA / g The mechanical strength of the granules in the reaction medium is reduced, the granules are destroyed during 5 reaction cycles lasting 24 hours each.

The prepared biocatalyst carries out the transesterification reaction in a batch mode for 5 hours at 70 ° C. The conversion is 30%.

Example 15

Similar to pr. 11, only the biocatalyst has the following composition, in mass. % in wt.% solids: rE lysates. coli / lip - 30, CMC - 10, SiO ₂ - 60.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 650 EA / g The mechanical strength of the granules in the reaction medium decreases, the granules are destroyed within 2 reaction cycles of 24 hours each.

The prepared biocatalyst carries out the transesterification reaction in a batch mode for 5 hours at 70 ° C. The conversion is 30%.

Example 16

Similar to pr. 3, only gum arabic (GUM) is added to the biocatalyst instead of maltodextrin. The biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 39, GUM - 1, SiO ₂ - 60.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 24%.

Example 17

Similar to pr. 16, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 40, GUM - 2, SiO ₂ - 58.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 28%.

Example 18

Similar to pr. 3, only instead of maltodextrin, a natural polysaccharide - starch is added to the biocatalyst. The biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 30, starch - 1, SiO ₂ - 69.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 670 EA / g Biocatalyst granules retain high mechanical strength and do not swell for 5 reaction cycles lasting 24-48 hours each, i.e. for more than 120 hours

The prepared biocatalyst carries out the transesterification reaction in a batch mode for 5 hours at 70 ° C. The conversion is 40%.

Example 19

Similar to pr. 18, only the biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 30, starch - 2, SiO ₂ - 68.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 670 EA / g Biocatalyst granules retain high mechanical strength and do not swell for 5 reaction cycles lasting 24-48 hours each, i.e. for more than 120 hours

The prepared biocatalyst carries out the transesterification reaction in a batch mode for 5 hours at 70 ° C. The conversion is 40%.

Examples 1-2 show that biocatalysts prepared on the basis of partially destroyed rE cells. coli / lip, have ~ 2 times lower hydrolytic activity than biocatalysts prepared on the basis of cell lysates. On the other hand, these biocatalysts are highly active in transesterification and they do not need to be injected with water-retaining agents, unlike biocatalysts prepared on the basis of rE cell lysates. coli / lip described in examples 2-19.

From examples 2-7 and table 1 it is seen that in the biocatalysts according to pr. 2-7, the content of maltodextrin is equal to 10-20 wt. %, is optimal and provides maximum conversion in the transesterification reaction.

From pr. 4, 8-10 shows that the optimal content of cell lysates rE. coli / lip in biocatalysts is 20-40%. These biocatalysts have high enzymatic activity and are stable under reaction conditions due to the high mechanical strength of the granules.

It can be seen from Examples 11-15 that with an increase in the content of a component of a polysaccharide nature, for example, carboxymethyl cellulose, the mechanical strength of the biocatalyst granules to the action of an aqueous reaction medium (for the hydrolysis of triglycerides) decreases due to a marked swelling of the polysaccharide, leading to the destruction of the silicate matrix. The optimal polysaride content in biocatalysts does not exceed 2 wt.%.

Example 20

Similar to pr. 5, only in addition to the composition of the biocatalyst is introduced nanostructured carbon in the form of a fine powder of carbon nanotubes (CNTs) with a diameter of 9-11 nm with a specific surface area of 280-320 m ² / g, forming aggregates 100-150 μm in size. The biocatalyst has the following composition, in wt.% Solids: rE lysates. coli / lip - 30, maltodectrine - 20, CNT - 5, SiO ₂ - 40.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 26%.

Example 21

Similar to pr. 20, only the biocatalyst has the following composition, in wt. % solids: rE lysates. coli / lip - 30, maltodectrine - 20, CNT - 10, SiO ₂ - 40.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 27%.

Example 22

Similar to pr. 20, only the biocatalyst has the following composition, in wt. % solids: rE lysates. coli / lip - 30, maltodectrine - 20, CNT - 20, SiO ₂ - 40.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 870 EA / g (which is 11 times more than in the prototype). The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C, however, the mechanical strength of the biocatalyst granules is low.

Example 23

Similar to Project 5, only nanostructured carbon is introduced into the biocatalyst in the form of a fine powder of carbon nanotubes with a diameter of 21-22 nm with a specific surface of 100-140 m ² / g, forming aggregates 100-150 microns in size. The biocatalyst has the following composition, in wt. % solids: rE lysates. coli / lip - 30, maltodectrine - 20, CNTs - 10, SiO ₂ - 2-40.

The initial activity of the biocatalyst in the hydrolysis of tributyrin is 1040 EA / g (13 times more than in the prototype), which is 1.2 times more than the biocatalysts according to Projects 5 and 21. The prepared biocatalyst transesterifies in a mixture of vegetable (sunflower) oil and salomas in a batch mode for 5 hours at 70 ° C. The conversion is 35%.

It can be seen from Projects 20–22 that the optimal content of nanostructured carbon in the biocatalyst is 5–10%. It can be seen from Project 23 that when nanostructured carbon is introduced into the biocatalyst, carbon nanotubes with a diameter of 21-22 nm with a specific surface of 100-140 m ² / g in the form of aggregates of 100-150 μm in size, an increase in the activity of the prepared biocatalyst as in the hydrolysis , and in the transesterification reaction.

Example 24. The method of transesterification in continuous mode.

A biocatalyst (3-5 g) with an optimal composition, prepared according to pr. 4, or pr. 9, or pr. 23, is loaded into a steel flow reactor. A reaction mixture consisting of vegetable (sunflower or soybean) oil and salomas with a variable flow rate is pumped through a fixed bed of the biocatalyst. Contact times range from 30 minutes to 90 minutes. Process temperature 70-80 ° C.

The test results are presented in table 2.

The table below shows that the optimal contact time is at least 60 minutes. The biocatalyst half-inactivation time, during which ½ of the initial activity is maintained, is 120 hours at 70 ° C.

Example 25. The method of transesterification in periodic mode.

A biocatalyst (0.5 g) with an optimal composition, prepared, for example, according to pr. 9, is poured into a reaction mixture (10 ml) of the following composition: 10 vol. % sunflower oil, 20 vol. % ethyl acetate (or methyl acetate), 70 vol. % hexane. The transesterification resulting in the formation of ethyl (or methyl) fatty acid esters (biodiesel) is carried out at 40 ° C and stirring at 150 rpm for 24-48 hours. The conversion is 36%.

From the above examples it can be seen that biocatalysts prepared on the basis of cell lysates of a recombinant producer strain of thermostable lipase rE. coli / lip, the composition of which is optimally selected, is on average an order of magnitude more active than the prototype. The method of preparation of biocatalysts is simple, relatively low cost and availability _{of a} builder (SiO ₂ -hydrogel), as well as moisture-retaining agents, does not require the use of toxic reagents and crosslinking agents. Biocatalysts are prepared in the form of mechanically strong granules, which allows the transesterification of vegetable oils to be carried out both in batch mode in mixing reactors and in continuous modes in flow reactors with a fixed bed.

Claims (5)

1. Biocatalyst for transesterification of vegetable oils based on an enzymatically active substance and a carrier containing a silicate matrix, characterized in that it contains partially destroyed cells or cell lysates of a recombinant strain E. coli / lip producing a thermostable lipase as an enzymatically active substance , as a carrier - a nanocarbon-silicate matrix containing nanostructured carbon, a water-retaining agent of carbohydrate nature, and has the following composition, in wt.% solids:
cells or cell lysates of r E. coli / lip - 20-40,
nanostructured carbon, namely, carbon nanotubes CNT - 5-10,
a water-retaining agent of carbohydrate nature, namely, maltodextrin - 10-20 or high molecular weight polysaccharide - 1-2,
silicon dioxide - up to 100. 2. A method of preparing a biocatalyst for transesterification of vegetable oils, which includes the stages of mixing, drying and fractionation, characterized in that the biocatalyst is prepared by mixing cells or cell lysates of a recombinant E. coli / lip lipase producer strain r with nanostructured silica hydrogel carrier carbon, and a water-retaining agent of carbohydrate nature, followed by drying and fractionation to a grain size of 0.1-2 mm, the resulting biocatalyst has the following composition, in wt.% dry substances: the cells or cell lysates r E. coli / lip - 20-40, carbon nanotubes CNT - 5-10, humectant carbohydrate nature, namely maltodextrin - 10-20, or high molecular weight polysaccharide - 1-2, silica - up one hundred. 3. The method according to claim 2, characterized in that the carbon nanotubes of CNTs have a diameter of 20-22 nm and a specific surface area of 100-140 m ² / g in an aggregated state. 4. The method according to claim 2, characterized in that maltodextrin or a high molecular weight polysaccharide is used as a water-retaining agent of carbohydrate nature, and their content in the biocatalyst is 10-20 wt.% And 1-2 wt.%, Respectively. 5. A method for transesterification of vegetable oils, which is carried out both in a batch mode in a mixing reactor and in a continuous mode in a flow reactor with a fixed bed of a biocatalyst, characterized in that the biocatalyst according to claim 1 is used and the process is carried out at a temperature of 60-80 ° C .