RU2527050C2 - Method of biocatalytic conversion of dibenzothiophene - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of biocatalytic conversion of dibenzothiophene, which includes oxidation of initial compound with hydrogen peroxide in presence as biocatalyst of haemoglobin in mixture of buffer solution with acetonitrile, novelty of which consists in the fact that as buffer solution, taken is acetate buffer solution, molarity of which constitutes 40-60 mM, with pH equal to 5.0-5.5; process is carried out at room temperature with molar ratio of initial components: dibenzothiophene:hemoglobin:hydrogen peroxide as (1-2.5):1:(900-1100) and ratio of solution components in vol. %: acetate buffer solution:acetonitrile as (2.5-3.5):1. Technical result is as follows: novel method of dibenzothiophene conversion with reduced catalyst consumption, absence of process of protein denaturation and, as a result, presence of transparent solution with achievement of high selectivity of DBTO output and 100% SDT conversion has been elaborated.

EFFECT: invention can be used in petroleum refining industry in production of motor fuel.

3 cl, 1 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of bioorganic chemistry and can be used in the refining industry in the manufacture of motor fuel.

In connection with the increased use of motor fuel, the attention of researchers around the world is attracted by the problem of improving its quality. Sulfur compounds present in the fuel during combustion form sulfur oxides (SO _x ), which lead to air pollution, acid rain, damage to greenhouse gas emission control devices [Song, C. New design approaches to ultra-clean diesel fuels / C. Song, X. Ma // Appl. Catal. B: Environmental. - 2003. - V. 41. - P.207-238]. Over the past 20 years, researchers around the world have been actively exploring the possibility of using enzyme-based biocatalysts for oxidative desulfurization of hydrocarbon feedstocks.

The use of biocatalysts provides several advantages:

a) mild process conditions,

b) minimal environmental impact,

c) low energy costs,

d) ease of process control,

d) functioning in a wide range of pH, temperature and ionic strength.

However, the high price and low stability of biocatalysts inhibits their large-scale application.

The main marker for assessing the quality of motor fuel (according to the content of sulfur compounds) and monitoring the effectiveness of its processing (conversion) is dibenzothiophene (DBT). Ideally, DBT should not be present in motor fuel, i.e. the conversion process should be 100% efficient.

The oxidative desulfurization of DBT proceeds in two stages: in the first stage, dibenzothiophene sulfoxide (DBTO) is formed, and in the second, the corresponding sulfone (DBTO ₂ ).

To ensure the simplicity of analytical control over the quality of engine fuel processing, it is necessary that a high degree of DBT conversion is combined with a selective yield of practically only one product - DBTO. In addition, it is known that during the extraction of sulfoxides from fuel, the fraction of hydrocarbon impurities in the extracts is less than during the extraction of sulfones; therefore, the oxidation of sulfur compounds to sulfoxides, which will reduce fuel losses during desulfurization, is of the greatest practical interest [Sharipov A.Kh. , Nigmatullin V.R., Nigmatullin I.R., Zakirov R.V. Chemistry and technol. fuels and oils, 2006, No. 6, p. 45-51].

A known method of biocatalytic conversion of DBT, including the oxidation of the starting compound with hydrogen peroxide in the presence of peroxidase as a biocatalyst in a mixture of 200 mm phosphate buffer solution with a pH of 8.0 and acetonitrile in a molar ratio of the starting components of the reaction mixture: DBT: hydrogen peroxide as 0.267: 0.005, this ratio of solvent components is in vol.%: phosphate buffer solution: acetonitrile as 3: 1, with peroxidase activity of 0.06 IU / ml: the process is carried out at a temperature of 45 ° C for 60 min (da Silva Madeira, L., Santana Ferreira-Leitao, VS, da Silva Bon, Dibenzothiophene oxidation by horseradish peroxidasein organic media: Effect of the DBT: H ₂ O ₂ molar ratio and H ₂ O ₂ addition mode, EP Chemosphere. 2008. 71. 189-194) .

However, the maximum conversion of dibenzothiophene under these conditions was only 59%, and the yield of DBTO was only 12%.

A known method of biocatalytic conversion of DBT, including the oxidation of the starting compound with hydrogen peroxide in the presence of bovine hemoglobin as a biocatalyst in a mixture of 20 mM acetate-phosphate buffer solution at pH 5.2 and propanol at a molar ratio of the starting components: DBT: bovine hemoglobin: hydrogen peroxide as 23 : 2.5: 1000, while the ratio of solvent components is in vol.%: Acetate-phosphate buffer solution: propanol as 3: 1, the process is carried out at a temperature of 30 ° C and its duration is 1 hour. (Klyachko, N. L., Klibanov, A.M. Oxidation of Dibenzothiophene Catalyzed by Hemoglobin and Other Hemoproteins in Various Aqueous-Organic Media. 1992, Appl. Biochem. Biotechnol. 37, 53-68).

However, although the maximum conversion of DBT reaches 99% and the yield of DBTO is 89%, in selected conditions, in addition to bioconversion, hemoglobin is denatured, due to which it precipitates. Thus, the catalyst loses its catalytic activity and is removed from the reaction sphere, which leads to its halt and turbidity of the reaction mixture solution, which in turn further complicates the sample preparation of motor fuel for analytical control of the effectiveness of the biodesulfurization process.

A known method of biocatalytic conversion of DBT adopted for the prototype is the oxidation of the starting compound with hydrogen peroxide in the presence of bovine hemoglobin as a biocatalyst in a mixture of 60 mM phosphate buffer solution at pH 6.1 and acetonitrile in a molar ratio of the starting components: DBT: bovine hemoglobin: peroxide hydrogen as 3: 1.2: 3000, while the ratio of solvent components is in vol.%: phosphate buffer solution: acetonitrile as 5: 1, the process is carried out at room temperature, while the duration of its It is 4-5 hours, maximum DBT conversion reaches 74%, and 100% yield DBTO. (Ortiz-Leon, M., Velasco, L., Vazquez-Duhalt, R. Biocatalytic oxidation of polycyclic aromatic hydrocarbons by hemoglobin and hydrogen peroxide. Biochem. Biophys. Res. Commun. 1995. V. 215. P.968 - 973 .)

However, the duration of the process reduces its manufacturability, as well as the denaturation of hemoglobin, due to which it precipitates, which leads to the loss of catalyst activity and the reaction stops. In addition, the solution of the reaction mixture becomes cloudy, which further complicates the sample preparation of motor fuel for analytical control of the effectiveness of the biodesulfurization process.

The present invention solves the problem of creating the most technologically advanced process for conducting the maximum conversion of dibenzothiophene while achieving the maximum yield of DBTO.

The problem is achieved by the method of biocatalytic conversion of dibenzothiophene, including the oxidation of the starting compound with hydrogen peroxide in the presence of hemoglobin as a biocatalyst in a mixture of buffer solution with acetonitrile, the novelty of which is that acetate buffer solution with a molarity of 40-60 is taken as a buffer solution mm, and the pH is 5.0-5.5, and the process is carried out at room temperature with a molar ratio of the starting components: dibenzothiophene: hemoglobin: hydrogen peroxide to (1 - 2.5): 1: (900-1100) and the ratio of the components of the solution in vol.%: acetate buffer solution: acetonitrile as (2.5-3.5): 1.

The most stable results are obtained when carrying out the process at a temperature of 25-30 ° C with a molar ratio of the starting components: dibenzothiophene: hemoglobin: hydrogen peroxide as 2.3: 1: 1000, while the ratio of the components of the solution is in vol.%: Acetate buffer solution: acetonitrile as 3: 1.

The process, subject to the concentration parameters found by the authors of the present invention, allows the process to be carried out in the absence of protein denaturation and, as a consequence, the presence of a clear solution, which reduces the reaction time by 9 times. In addition, the consumption of the catalyst is reduced by 2.5 times, which significantly reduces the cost of the process, while achieving a high degree of selectivity of the DBTO yield up to 96%, with 100% DBT conversion.

Thus, the technical result of the invention consists in increasing the rate of DBT conversion, reducing the consumption of the catalyst, the absence of the protein denaturation process and, as a result, the presence of a clear solution while achieving a high selectivity of DBTO output and 100% DBT conversion. In addition, the present invention minimizes the sample preparation of motor fuel for analytical control of the effectiveness of the biodesulfurization process.

Short Description of Shapes

Figure 1 shows a chromatogram of bioconversion products of DBT under optimal process conditions (molar ratio of starting components: DBT: hemoglobin: hydrogen peroxide as 2.3: 1: 1000, ratio of solvent components in vol.%: Acetate phosphate buffer solution: acetonitrile as 3: one).

Table 1 shows the performance indicators of the inventive process for various ratios of the starting materials and the conditions for the bioconversion of DBT, where

* Optimal conditions for bioconversion of DBT

** Prototype, 60 mM phosphate buffered saline

The following examples confirm, but do not limit, the claimed method.

EXAMPLE 1. Biocatalytic oxidation of dibenzothiophene with hydrogen peroxide under optimal conditions.

To control the conversion of DBTI, a standard gradient elution method is used: eluent A is water, eluent B is acetonitrile (7: 3). Flow rate 0.5 ml / min. Data recording and chromatogram processing is performed using ChemStation software version A 10.02. A chromatograph with a diode array detector is used, since the reaction products are absorbed in the UV region (λ = 230 nm).

79 μl of acetonitrile (total (total content in the reaction mixture of 25 vol.%), I.e. ratio in vol.%: Acetate buffer solution, is added to 230 μl of 10 mM acetate buffer pH 5.2, the molarity of which is 50 mM. : acetonitrile is 3: 1.) 46 μl of a 2.55 mM dibenzothiophene solution are added dropwise (concentration in the reaction mixture is 230 μM dibenzothiophene); 45 μl of 0.112 mm hemoglobin solution (catalyst). 100 μl of a 50 mM hydrogen peroxide solution (oxidizing agent) is added dropwise with stirring. The total ratio of components in the mixture is in moles: DBT: hemoglobin: hydrogen peroxide as 2.3: 1: 1000, while the ratio of solvent components in vol.%: Acetate buffer solution: acetonitrile as 3: 1.

With the passage of the reaction, the solution remains completely transparent, there is no precipitate. At the time of completion of the addition of the oxidizing agent and mixing the solutions, a stopwatch is started and every minute, 5 μl of the solution is introduced into the chromatographic column using an autosampler.

The chromatogram obtained by the standard method using the gradient elution method at the ratio of the maximums of the initial and final DBT conversion products confirms (see Figure 1) that the process was completed in 5 minutes, while the DBT conversion is - 99 ± 1%. The content of DBTO is 94 ± 2%; DBTO ₂ -6 ± 2%.

To determine the dependence of the performance indicators of the proposed process for various ratios of the starting materials and the conditions for the bioconversion of DBT, additional studies were carried out, the results of which are shown in table 1.

As can be seen from the data presented in table 1, the process in the claimed parameters (examples 2-4) for the first time allows you to make the process of maximum DBT conversion the most technologically advanced, namely the simultaneous absence of protein denaturation and, as a result, the presence of a clear solution, shortening the reaction 9 times in comparison with the prototype. In addition, the catalyst consumption is reduced by 2.5 times, which significantly reduces the cost of the process, while achieving a high degree of selectivity of the DBTO output to 96%. Exceeding the above optimal ranges leads to insufficient selectivity for the output of the DBT - DBTO oxidation product (Example 1), a significant increase in conversion time (Example 5-6), protein denaturation and, as a consequence, loss of transparency of the solution (Example 6).

Table 1. METHOD FOR BIO-CATALYTIC CONVERSION OF DIBENZOTIOPHENE. No. The molar ratio of the components of the reaction mixture The environment for bioconversion, vol.% The ratio of solutions Bioconversion Conditions The degree of conversion (for DBT) and product yields, wt.% Solution transparency Process time DBT Hemoglobin H ₂ O ₂ Acetonitrile Acetate Buffer Solution (50 mM) pH T, ° C DBT DBTO DBTO ₂ t min one 0.75 0.8 800 one four 5.0 25 99 ± 1 76 ± 2 25 ± 2 + 5 2 one one 900 one 2,5 5.5 25 98 ± 2 93 ± 2 5 ± 2 + 5 3 2.3 * one 1000 one 3 5.2 25 99 ± 1 94 ± 2 6 ± 2 + 5 four 2.5 one 1100 one 3,5 5.0 25 98 ± 2 94 ± 2 7 ± 2 + 5 5 3.0 1.5 1200 one 3 6.0 thirty 68 ± 1 30 ± 2 2 ± 2 + 60 6 2.3 2.5 2000 2 6.0 45 42 ± 5 36 ± 4 1 ± 2 - 180 Prototype 3.0 ** 1.2 3000 one phosphate buffer solution (60 mM) 5 6.1 25 74 one hundred 0 - 240-300

Claims (2)

1. The method of biocatalytic conversion of dibenzothiophene, comprising oxidizing the starting compound with hydrogen peroxide in the presence of hemoglobin as a biocatalyst in a mixture of a buffer solution with acetonitrile, characterized in that an acetate buffer solution with a molarity of 40-60 mM and a pH of 5.0-5.5, and the process is carried out at room temperature with a molar ratio of the starting components: dibenzothiophene: hemoglobin: hydrogen peroxide as (1-2.5): 1: (900-1100) and the ratio of the components of the solution in vol.%: acetate buffer solution: acetonitrile as (2.5-3.5): 1. 2. The method according to claim 1, characterized in that the process is carried out at a temperature of 25-30 ° C with a molar ratio of the starting components: dibenzothiophene: hemoglobin: hydrogen peroxide as 2.3: 1: 1000, while the ratio of the components of the solution is in vol.% : acetate buffer solution: acetonitrile as 3: 1.