KR0169215B1 - Method for increasing fastness of nitrile hydratase on acrylamide - Google Patents

Abstract

The present invention provides a method for improving the resistance to acrylamide of a Rhodococcus strain having nitrile hydratase activity for converting acrylonitrile to acrylamide, which is a method of Rhodococcus rhodochrorls M33 (Russia). Deposited as VKM Ac-1515D in strain center IBFM) is characterized in that the strain is cultured in a medium containing an iron ion compound as a medium for the growth of the composition. The present invention also provides a method for producing acrylamide using the strain obtained by the above method.

Description

How to increase the resistance of nitrile hydratase to acrylamide

The present invention relates to a method of increasing the resistance of nitrile hydratase of Rhodococcus microorganisms to acrylamide, and more particularly, by incubating Rhodococcus rhodochrous M33 in a medium containing iron ions A method of increasing the resistance of nitrile hydratase to amides.

Acrylamide is a white crystal with specific gravity of 1.122 (30 ° C), melting point of 84.5 ° C, boiling point of 87 ° C (2mmHg) and 125 ° C (25mmHg), and soluble in water, alcohol and acetone, but insoluble in benzene and heptane. Acrylamide is used as a strength enhancer, wastewater treatment agent, flocculant, thickening agent, coating material, and is used in various industries such as wastewater treatment, oil recovery and civil engineering.

Acrylamide is manufactured in a chemical process using copper as a catalyst and a biological process in which acrylonitrile is hydrated using nitrile hydratase. Among the biological processes, high temperature, high pressure, or inert processes are not required. Its application is gradually expanded due to its simple and high stability of operation and operation. Furthermore, the biological process has the advantage that the efficiency of conversion from acrylonitrile to acrylamide is very high, such as 99.9%, so that a process for separating unreacted acrylonitrile after the reaction is not required.

Microorganisms used in the biological production process of acrylamide have nitrile hydratase activity that hydrates acrylonitrile and converts it into acrylamide, for example, Bacillus, Micrococcus and Brevibacterium. US Patent 4,001,081, Korean Patent Publication No. 91-7850, Nocardia (US Patent 4,48,968), Pseudomonas (US Patent 4,880,739), Rhodococcus (US Patent 5, 334519) is used.

In order to commercialize the acrylamide-containing reaction solution obtained by a biological method without further treatment, the concentration of acrylamide in the reaction solution must be at least 40%, and to achieve this purpose, nitrile hydratase is effectively resistant to acrylamide. Should be indicated.

The present inventors were developing a process for converting to acrylamide at around 20-25 ° C using Rhodococcus Rhodocross M33 (VKM Ac-1515D deposited in Russian strain center IBFM, Korean Patent Publication No. 95-18447). It has been observed that the inhibition of nitrile hydratase by acrylamide can be reduced when culturing M33 in medium with iron ions. When cultured in a medium containing iron ions, the microbial cell concentration did not decrease, the nitrile hydratase activity was slightly decreased, but the resistance to acrylamide was increased and 40% acrylamide could be prepared quickly. Accordingly, the present invention has been completed.

That is, an object of the present invention is a medium of the composition containing iron ions as a medium for the growth of Rhodococcus Rhodocross M33 (VKM Ac-1515D deposited in the Russian strain center IBFM, available for sale upon application) strain. It is to provide a method for improving the resistance to acrylamide of nitrile hydratase of Rhodococcus Rhodocrose M33 characterized by culturing the strain.

Another object of the present invention is to provide a method for producing a high concentration of acrylamide from acrylonitrile using a strain having improved resistance to acrylamide by the above method.

The above and other objects and features of the present invention will become apparent to those skilled in the art by the following detailed description.

Hereinafter, the present invention will be described in more detail.

When iron ions are added to the medium of the microorganism for preparing acrylamide, the genus Brevibacterium (US Pat. No. 4,001,081), Pseudomonas genus (US Pat. No. 4,880,739), Astrobacter genus (Ramakrishna Desai, Biotechnol. Lett, 15) , 267, 1993), and in the case of the J1 microorganism belonging to the genus Rhodococcus, it is reported that the addition of iron ions to the growth medium greatly reduces the activity of the nitrile hydratase (US Pat. No. 5,334,519).

In contrast to the effect of inhibiting the nitrile hydratase activity of iron ions reported for certain strains of the genus Rhodococcus, for the Rhodococcus Rhodocross M33, the nitrile hydratase for acrylamide was cultured by culturing in a medium containing iron ions. It is based on finding increased tolerance.

The nitrile hydratase of M33 incubated in a medium containing iron ions is not inhibited by acrylamide as a product, so that the reaction time can be shortened. As a result, it is possible to increase the concentration of acrylamide in the reaction solution to 40%, thereby allowing the reaction to proceed quickly and eliminating the process of separating and removing acrylonitrile remaining after the reaction is terminated. .

The medium that can be used for culturing Rhodococcus Rhodocrose M33 in the present invention is not limited to the type thereof, and any medium can be used as long as it is used for the culture of Rhodococcus strains. Selection of such a medium and selection of conditions for culturing the strain of the present invention using the medium can be performed by those skilled in the art without difficulty.

According to the present invention, examples of the iron ion compound added to the medium capable of growing Rhodococcus Rhodocross M33 include iron sulfate, iron chloride, ferric sulfate, and the like. Mg / l, preferably 1.5-4.0 mg / l. If the iron ion concentration is lower than the lower limit, there is a disadvantage in that the incubation time is longer, and if the iron ion concentration is exceeded, a proportional increase in proportion to the increase of the amount is not observed.

According to the present invention, a process for producing acrylamide by an enzymatic method using Rhodococcus Rhodocross M33 having improved resistance to acrylamide can be carried out under ordinary conditions, and such conditions can be easily selected by those skilled in the art. . However, in general, it can be carried out by incubating for 30-100 hours at a temperature of about 25-35 ℃. At this time, the nitrile hydratase activity tends to decrease over time as the incubation time increases, and when iron sulfate is added as an iron ion compound, the concentration of iron sulfate is about 5.0-15 mg / L, especially about 6.0-8.5 mg. / l, reaction time of about 30-40 hours, optimum conditions are formed in terms of enzyme activity and resistance to acrylamide.

In the present invention, in order to evaluate the improvement of resistance of nitrile hydratase to acrylamide according to the addition of iron ions, culturing M33 by adding iron ions to the strain culture medium by concentration, the presence of nitrile hydratase activity and acrylamide Nitrile hydratase resistance and cell concentration were measured.

The resistance of nitrile hydratase to acrylamide is determined by whether M33 cultivated in a medium to which iron ions have been added is determined by whether the activity of the nitrile hydratase is changed by the presence of acrylamide, and acrylamide at a specific concentration. It is expressed by the activity of nitrile hydratase in the presence of.

That is, 2% acrylonitrile and 50% acrylamide were added to 1.6 ml of 10 mM phosphate buffer (pH 7.6), and 1.0 mg of the cultured cells were dispersed and added to 0.4 ml of phosphate buffer solution at a dry weight at 20 ° C. Stir for 10 minutes. 30 µl of concentrated hydrochloric acid was added to stop the reaction, and acrylonitrile and acrylamide were analyzed by gas chromatography. The conditions of gas chromatography were as follows: Using a column in which 10% Carbowax 20M was added to chromosolve W (80-100 mesh), and the quantitative conditions were a detector and an injection port temp. Temperature 210 ° C., column temperature 160 ° C., and flow rate FID helium gas 30 ml / min (Lee et at., Enzyme Microb, Technol., 15, 980, 1993).

The nitrile hydratase activity of M33 was dissolved in 2% acrylonitrile in 1 ml of 10 mM phosphate buffer (pH 7.6), and 0.04 mg of cultured cells were added to 1 ml of phosphate buffer in dry weight, followed by 10 minutes at 20 ° C. The reaction was stopped by addition of 30 μl of concentrated hydrochloric acid and the reaction was determined by analyzing the acrylonitrile and acrylamide by gas chromatography.

In addition, when acrylonitrile was injected, the production time of acrylamide produced by M33 cells according to iron ion concentration was compared.

In the above, Rhodococcus Rhodocrose M33 cultivated in a medium in which iron ions were added in an amount of 1.5-5.5 mg / l, in particular 1.5-4.0 mg / l, was not obtained when acrylonitrile concentration was added at 2% level. In comparison, a reaction solution having an acrylamide concentration of 40% was prepared in a short time.

Hereinafter, the present invention will be described in detail through the following non-limiting examples.

Example 1

A medium consisting of glucose 20.0 g / l, ammonium chloride 5.0 g / l, potassium monophosphate 0.5 g / 1, potassium diphosphate 0.5 g / 1, magnesium sulfate 0.5 g / 1, cobalt chloride 0.015 g / 1 (weakly referred to as medium) Rhodococcus Rhodocross M33 obtained by culturing in a medium containing 5 mg / 1 of iron sulfate was inoculated into a medium containing 0.125, 2.5, 5.0, 7.0, 10.0, or 15.0 mg / L of iron sulfate, and cultured at 30 ° C. The cells were cultured until the cell concentration reached 7.7 g / 1.

The culture was stopped and the effect of adding iron sulfate was evaluated by measuring the incubation time, the activity of nitrile hydratase, and the resistance of nitrile hydratase.

The results are shown in Table 1.

Above, the nitrile hydratase activity and the resistance to acrylamide when the cell concentration reached around 7.7 g / L were maintained or increased with the addition of iron sulfate.

In addition, the addition of iron sulfate 5.0mg / ℓ, nitrile hydratase activity reached 215 U / mg cells when the incubation time was increased to 40 hours, it is confirmed that the addition of iron sulfate can reduce the incubation time.

Example 2

The same procedure as in Example 1 was carried out, except that iron chloride having the concentration shown in Table 2 was added to the medium instead of iron sulfate.

Example 3

The same procedure as in Example 1 was carried out except for the addition of ferric sulfate in the amount shown in Table 3 instead of iron sulfate. The results are shown in Table 3.

From the results in Tables 2 and 3 above, when iron chloride or ferric sulfate was added in addition to iron sulfate, the activity of nitrile hydratase and the resistance to acrylamide when the cell concentration reached 7.7 g / L were determined. It can be seen that it is maintained or increased with the addition of.

Example 4

Each of the M33 cells by iron sulfate concentration obtained in Example 1 was recovered by centrifugation, washed with distilled water and dried. 0.35 g of dried M33 cells were transferred to a 1.5 liter reactor and dispersed by adding 800 ml of distilled water. The reactor was maintained at 20-25 ° C. and a total of 370 g was injected into the reactor to maintain acrylonitrile at 2% concentration. Then, after reacting for the time shown in Table 4, the amount of remaining acrylonitrile and produced acrylamide in the reaction termination solution was measured. The results are shown in Table 4.

Rhodococcus cultivated by adding iron sulfate to 5-15mg / ℓ (1.5-5.5mg / ℓ in terms of iron ions), especially about 7mg / ℓ (about 2.5 mg / ℓ in terms of iron ions) Cross M33 shortened the production time when producing acrylamide from acrylonitrile, and the concentration of the final acrylamide was maintained at 40% or more, and commercialized immediately.

Claims (6)

Rhodococcus rhodochrous M33 (deposited as VKM Ac-1515D in the Russian strain center IBFM) as a medium for the growth of Rhodococcus rhodochrous Rhodo characterized by culturing the strain in a medium containing an iron ion compound A method of improving the resistance to nitrile hydratase of coccus rhodoprose M33 to acrylamide. The method of claim 1, wherein the iron ion compound is iron sulfate, iron hydrochloride, or ferric sulfate. The method according to claim 1 or 2, wherein the iron ion compound is contained in an amount of 1.5-4.0 mg / l in terms of iron ions. Following steps; (a) Rhodococcus rhodochrous M33 (deposited as VKM Ac-1515D in the Russian strain center IBFM) as a culture medium for the growth of the above cultures in a medium containing an iron ion compound nitrile hydra Obtaining Rhodococcus Rhodocrose M33 with improved resistance to other agents of acrylamide; And (b) culturing the strain obtained in step (a) in a medium containing acrylonitrile and iron ion compound to obtain acrylamide. The method of claim 4, wherein the iron ion compound is iron sulfate, iron hydrochloride, or ferric sulfate. 6. The method according to claim 4 or 5, wherein the iron ion compound is contained in an amount of 1.5-4.0 mg / L in terms of iron ions.