KR100407470B1 - A method for producing Rhodococcus rhodochrous by a fed-batch culture method and process for manufacturing amide compound thereby - Google Patents

Abstract

To the present invention is Rhodococcus also cross the culturing cross also inert to as maintaining the (specific activity) increase Lactococcus of the nitrile hydratase to produce a high concentration, while keeping as low as possible the concentrations of glucose, characterized by intermittently adding cobalt also as Lactococcus also relates to a culture method of the cross, and also cultured by the culture method Lactococcus also further comprises a process for producing an amide compound from a nitrile compound using a cross. Therefore, the present invention can mass-produce nitrile hydratase at low cost and reduce the production cost of industrially useful amide compounds.

Description

A fed-batch batch culture method of Rhodococcus Rhodocrose and a method for producing an amide compound from a nitrile compound using Rhodococcus Rhodocross cultured by the culture method for manufacturing amide compound thereby}

The invention Rhodococcus to as nitrile, which cross the produce to as Rhodococcus while culturing the cross at a high concentration hydratase of the inactive (specific activity) of highly maintained Rhodococcus also to as cultured by the culturing method and the culture method of the cross Lactococcus also to It relates to a method for preparing an amide compound from a nitrile compound using cross .

Amide compounds are useful materials that are widely used throughout the industry. The representative amide compound acrylamide is used as an intensifier, wastewater treatment agent, flocculant, thickener and coating agent, and is used in wastewater treatment, oil recovery and civil engineering. It is used in various industries.

As a conventional method for obtaining such an amide compound, there is an industrial production method for obtaining an amide compound by hydrating a corresponding nitrile compound using a catalyst in a reduced state of copper. However, such a manufacturing method requires a lot of efforts to remove by-products such as unreacted nitrile and to remove them, and also has a problem such that the conditions of the reaction process are high temperature and high pressure and thus have a lot of risks.

In recent years, in order to improve the problem of this industrial manufacturing method, the method of obtaining an amide compound using the nitrile hydratase which is an enzyme produced | generated by microorganisms as a catalyst has attracted the spotlight. Nitrile hydratase is an enzyme that promotes the conversion of nitrile compounds to amide compounds. The biological manufacturing process of the amide compound using nitrile hydratase has a high conversion rate from the nitrile compound to the amide compound (99.9%), does not require a process for separating unreacted nitrile, and the device is simple because of the mild reaction conditions. It has the advantage of high stability of operation operation.

Microorganisms that produce nitrile hydratase is diverse and Bacillus (Bacillus), bacterial Stadium in (Bacteridium), micro-nose Syracuse in (Micrococcus) and Brevibacterium genus (Brevibacterium) (US Patent 4,001,081 call; Korea Patent Publication No. 91-7850), the genus Corynebacterium (Corynebacterium) or in no-carboxylic Dia (Norcardia) (U.S. Pat. No. 4,248,968), genus Rhodococcus (Rhodococcus) (U.S. Pat. No. 5,334,519), Pseudomonas species (Pseudomonas) (U.S. Pat. 4,880,739) and the like are typically used.

On the other hand, conventionally, in order to produce a large amount of nitrile hydratase by culturing a high concentration of microorganisms, fed-batch culture was generally performed to increase the amount of carbon source. In addition, as a carbon source used in conventional fed-batch culture, glucose or glycerol is common.

However, when glycerol is used as a carbon source, there is an advantage that less acetic acid, a by-product of cell metabolism, is produced than glucose, but the growth rate of cells is low and the price is high. In addition, when glucose is excessively used as a carbon source, the cells can be cultured at a high concentration, but since glucose causes substrate inhibition of enzyme production, it is difficult to increase the productivity of the enzyme compared to the high concentration of cells.

That is, when fed-batch cultivation using glucose as a carbon source for the high yield of nitrile hydratase, there is a problem that the enzyme inactivation is lowered. Therefore, a large amount of microorganisms must be used to obtain the desired amount of amide, which causes a problem of increasing the load on the post-process for removing the injected microbial cells and purifying the amide compound. In addition, it takes a lot of time to cultivate a large amount of microorganisms, there is not much production amount of nitrile hydratase, and there are many disadvantages in terms of economics such as lowering the activity of nitrile hydratase when culturing a large amount of microorganisms.

Therefore, in order to economically produce amide compounds, it is important to mass-produce nitrile hydratase from microorganisms at low cost in a short time and to maintain high activity of the produced enzyme, and to increase the yield and activity of nitrile hydratase. Development of a culture method is necessary.

Accordingly, the present inventors have studied a method for culturing Rhodococcus rhodoprose producing nitrile hydratase at a high concentration while maintaining high inactivation of nitrile hydratase. As a result, the glucose concentration in the Rhodococcus Rhodocross culture medium was kept as low as possible, and cobalt was optionally added intermittently into the culture medium, thereby suppressing glucose substrate inhibition and inactivation of nitrile hitratase. It was confirmed that it was possible to hold | maintain and to cultivate Rhodococcus rhodoprose at high concentration, and came to complete this invention.

Thus, at the same time as an object of the present invention is cultured cross also Rhodococcus in a high concentration, mass production of hydratase nitrile to maintain the inactive higher of the nitrile hydratase, and nitrile produced by the method hydratase inactive high Rhodococcus It is to provide a method for preparing an amide compound from a nitrile compound using Rhodocross.

1 is a graph illustrating the relationship between the inactivation of nitrile hydratase and cell growth according to glucose concentration in fed-batch culture of Rhodococcus Rhodocross strain M33.

<Simple Symbol Explanation>

Each of A, B, and C of FIG. 2 is a graph illustrating a relationship between inactivation of nitrile hydratase and cell growth according to the number of additions of cobalt chloride in fed-batch culture of Rhodococcus Rhodocross strain M33.

Here, the arrow indicates the time point of adding 0.01 g / L cobalt chloride (CoCl ₂ · 6H ₂ O).

In order to achieve the above object, the present invention maintains the specific activity of the nitrile hydratase produced by Rhodococcus Rhodocrose by keeping the concentration of glucose as low as possible and selectively adding cobalt intermittently. In the meantime, Rhodococcus Rhodocross is cultured at a high concentration, and Rhodococcus Rhodocross is cultivated by the above method, and Rhodococcus Rhodocrose cultivated by the cultivation method is provided.

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

The inventors batch culture Rhodococcus Rhodocross strain M33 by varying the glucose concentration in the culture medium and measured the inactivation and final cell concentration of nitrile hydratase at the time when each glucose concentration was exhausted (see Example 1). As shown in Table 2, as the initial glucose input concentration increased, the concentration of final cells increased but the inactivation of nitrile hydratase rapidly decreased.

In other words, it could be confirmed that the substrate inhibition of glucose indirectly, and as the initial concentration of glucose increases, the final concentration of the cells increases, but the production of nitrile hydratase does not increase proportionally. have. In other words, in the case of Rhodococcus Rhodocrose, glucose acts to inhibit the nitrile hydratase expression system (catabolite repression).

Based on the batch culture results, fed-batch culture was performed while continuously supplying the substrate solution containing glucose so that the glucose concentration in the fermentation medium was kept as low as possible during the culture period. As a result, as the glucose concentration in the fermentation broth was kept as low as possible, it was confirmed that the inactivation of the nitrile hydratase was maintained high (see FIG. 1 and Example 2).

Therefore, the present invention is to cultivate the Rhodococcus rhodoprose while maintaining a low glucose concentration in the fermentation medium by a fed-batch continuous culture method.

In this case, when the total amount of glucose supplied is the same, the final cell concentration is the same whether the batch fed batch method or the batch culture method of the present invention is used. However, when the glucose concentration is kept low, as in the case of the present invention, the activity of the enzyme is increased, and the total Activity is also increased.

On the other hand, the glucose is supplied to the culture medium by an exponential feeding method or an upper pH adjusting method as a method for maintaining a low glucose concentration.

Substrate solution supply method is a batch culture in which nothing is added until all of the glucose in the medium is depleted at the beginning of the culture, and then exponentially fed or the upper pH adjustment method from the time when the glucose is depleted. As a result, it is preferable to supply a total of 20 to 60 g / l of glucose while keeping the concentration of glucose in the culture medium as low as possible. When more than 60 g / l of total glucose to be supplied is added, the viscosity of the culture solution is increased and the fluidity is reduced, which causes a problem in air aeration, so that the cell concentration does not increase significantly.

In addition, the reason for the choice of the substrate solution supply method with the fed-batch addition of the present invention after the batch addition of glucose and the depletion of glucose is that it is difficult to accurately control the supply amount because the consumption of glucose is very small at the beginning of the culture. .

Equation 1 is used to supply glucose by an exponential supply method, and samples are taken at intervals of several hours to measure microbial cell concentration and glucose concentration, and specific growth rate (μ) and cell yield (Y _x ) are measured from the measured values. _{/ s} ) to determine the new flow rate.

Where F is the feed rate of the substrate solution (l / hour), μ is the cell specific growth rate (1 / hour), X _o is the initial cell concentration (g / l), and S _o is the glucose concentration of the substrate solution (g / L), Y _{X / S} is the growth yield (g-cell dry weight / g-glucose).

In addition, the upper pH adjustment method, by adjusting the pH of the culture medium to a value of 7.25 to 7.35, the minimum pH of 7.20 to 7.15, the glucose concentration is kept as low as 0 ~ 5g / L as possible. In other words, when glucose is depleted and the pH value is rapidly increased, this signal is transmitted to the metering pump to supply the substrate solution, whereas when glucose is consumed and the pH is lowered below the lower limit, the KOH solution is supplied to the glucose concentration in the culture medium. Keep it as low as possible.

On the other hand, in the present invention, in order to increase the specific activity of the nitrile hydratase, it is preferable to supply 0.04 to 0.05 g / L of cobalt three to four times intermittently during the culture period.

To increase the specific activity of nitrile hydratase, the addition of cobalt is described in US Pat. Nos. 5334519 and 5089411. However, in this method, cobalt is added to a culture medium containing 20 g / l of glucose at a concentration of 0.005 to 0.015 g / l at the beginning of the culture. However, in the fed-batch culture of the present invention supplying a glucose amount of about 60 g / l as in the present invention, microorganism cells are produced at a high concentration, so in order to increase the inactivation of nitrile hydratase at least 0.04 to 0.05 g / l. It is preferable to divide and supply cobalt 3-4 times. However, cobalt is added as a cofactor for nitrile hydratase, and when the initial dose is excessive, the growth of microorganisms is suppressed (see Table 3). Therefore, as in the present invention, 0.02 to 0.03 g / L is added at the beginning of the culture, It is preferable to add little by little intermittently during the culture. In addition, the amount of cobalt administered intermittently is preferably 0.005 to 0.01 g / L.

Therefore, when the cobalt is dividedly supplied during the incubation period, as shown in FIG. 2, the inactivation of nitrile hydratase can be increased at the end of the culture without inhibiting the growth of microorganisms.

The cobalt compound in the form for adding cobalt to the culture medium is not particularly limited, but typically, a compound supplying Co ²⁺ or Co ³⁺ in an aqueous solution, preferably a compound supplying Co ²⁺ may be used. For example, cobalt chloride, cobalt sulfate, cobalt acetate, cobalt bromide, cobalt borate, and the like can be used.

The fed-batch culture (feeding 60 g / l total ) of the Rhodococcus rhodoprose strain producing nitrile hydratase according to the method of the present invention is compared with that of batch culture in a culture medium containing 20 g / l glucose. The concentration and total activity of the enzyme were increased by about 3 times, and the productivity of the enzyme was increased by 1.5-1.9 times, and even if a large amount of glucose was supplied, the inactivation of the enzyme was maintained as high as that of the conventional batch culture (Example 5 and 6). Therefore, the present invention can increase the amount of enzyme produced while maintaining a high concentration of cells.

On the other hand, the method for producing acrylamide from acrylonitrile using Rhodococcus Rhodocrose cultured at high concentration by fed-batch culture according to the present invention, refer to the method for producing acrylamide described in Korean Patent No. 169213.

In addition, the method for producing acrylamide from acrylonitrile using a microorganism cultured at high concentration by fed-batch culture according to the present invention proceeds using the strain culture as well as the method described in Korean Patent No. 169213 described above. Can be carried out using methods well known in the art of conventional bioreaction. For example, microorganism cells cultured at high concentration may be used after being fixed to a carrier by a conventional method.

Hereinafter, the present invention will be described in more detail with reference to examples and various examples.

These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

<Use strain>

Nitrile hydratase production strains used in the following examples were Rhodococcus rhodochrous M33 VKM Ac-1515D and Rhodococcus rhodochrous M8 VKPM S-926 strains. M33 is described in Korean Patent Publication No. 0131276, and M8 is described in Russian Patent No. 1731814.

Microbial cell concentration measurement

To measure the microbial cell concentration in the culture medium, take a small amount of the culture solution and dilute it with appropriate distilled water, and then measure the OD (at 540 nm) using a spectrophotometer (Cary UV-Visible spectrophotometer, Varian Co., Australia). Absorbance) was measured. The cell dry weight was obtained by converting the absorbance values. At this time, the absorbance OD 1 is 0.2 mg-dry cells / ml as the dry weight of cells.

<Glucose Concentration Measurement>

Glucose concentration in the culture medium was measured by a glucose analyzer (YSI model 2700 biochemistry analyzer, Yellow Spring Instrument Co., Inc., USA).

Measurement of inactivity of nitrile hydratase

The method for measuring inactivity of nitrile hydratase is as follows. 1 ml of a dry weight 0.04 mg microbial cell suspension was mixed with 1 ml of a 10 mM phosphate buffer solution at pH 7.6 and 2% (w / v) acrylonitrile, followed by stirring at 20 ° C. for 10 minutes. After the reaction, 30 µl of concentrated HCl was added to stop the reaction. The concentration of acrylamide formed by the reaction was analyzed by gas chromatography and used for the inactivation calculation of nitrile hydratase.

The activity of nitrile hydratase is expressed in the following units.

One unit is defined as the amount of enzyme required to produce acrylamide from acrylonitrile at a rate of 1 μmol / minute under the conditions described above.

Specific activity is expressed as amide-μmol / min / dry cell weight-mg or units / mg-dry cells.

Total activity is expressed as amide-μmol / min / culture-ml or units / ml.

Reference Example 1 Batch Culture of Rhodococcus Rhodocrose M33

Rhodococcus Rhodocrose M33 spawn stored in 15% glycerol was inoculated into an Erlenmeyer flask containing 500 ml of the culture medium having the composition of Table 1, and cultured at 30 ° C. for 48 hours (cultivation).

Furtherance Volume (g / ℓ) Potassium Phosphate (KH ₂ PO ₄ ) Dipotassium Phosphate (K ₂ HPO ₄ ) Cobalt Chloride (CoCl ₂ · 6H ₂ O) Ferrous Sulfate (FeSO ₄ · 7H ₂ O) EDTA-2NaGlucose Urea (urea) Magnesium Sulfate (MgSO ₄ 7H ₂ O) 0.50.40.020.0030.0062031.2

Then, 90 ml of the culture solution was obtained and inoculated into 3 L of the same culture medium as in Table 1 except that 0.01 g / L of sodium chloride and 7.5 g / L of urea were added thereto, followed by incubation in a 5 L culture tank (Korea incubator, Korea). The batch was incubated for about 57 hours while maintaining a temperature of 30 ° C., pH 7.2 (using 10% KOH), agitation speed 500 rpm, and air supply rate 0.4 vvm (volume / volume min.).

Glucose concentrations were frequently checked during the incubation period. The final cell concentration when glucose was completely depleted in culture medium was OD 35.5 (cell dry weight 7.1 g / l), enzyme inactivity was 120 units / mg-dry cells, and total activity was 852 units / ml.

REFERENCE EXAMPLE 2 Batch Culture of Rhodococcus Rhodocross M8

Except for using the Rhodococcus Rhodocrose M33 spawn instead of the Rhodococcus Rhodocross M8 seed, the shaking culture (species culture) 42 hours and the batch culture were carried out in the same manner as in Reference Example 1 for 48 hours.

When glucose in the culture medium was completely depleted, the final cell concentration was OD 37 (cell dry weight 7.4 g / l), the enzyme inactivity was 70 units / mg-dry cells, and total activity was 518 units / ml.

Example 1 Batch Culture by Glucose Initial Addition

Rhodococcus Rhodocrose M33 spawn stored in 15% glycerol was inoculated into an Erlenmeyer flask containing 500 ml of the culture medium having the composition of Table 1, and cultured at 30 ° C. for 48 hours (cultivation).

Then, 90 ml of the culture solution was obtained, except that 0.01 g / l sodium chloride, 7.5 g / l urea, and the initial glucose addition amount were 5, 10, 15, 20, 25, 30, 35, and 40 g / l, respectively. Was inoculated in 3 l of the same culture medium as in Table 1, and Rhodococcus Rhodocross M33 was cultured in a batch under the same culture conditions as in Reference Example 1. Final cell concentration and inactivation of nitrile hydratase were measured immediately after glucose was depleted in the culture medium while checking the glucose concentration in the middle of the culture period.

Initial amount of glucose added (g / ℓ) Cell concentration (OD _{540 nm} ) Nitrile hydratase inactive (units / mg-dry weight) Nitrile hydratase total activity (units / ml) 510152025303540 11.121.533.042.252.458.667.677.2 14113212010294867466 313567792860985100710001019

As shown in Table 2, as the initial amount of glucose increases, the final cell concentration and the total activity of nitrile hydratase increase, but the inactivation of nitrile hydratase decreases rapidly.

From the above experimental results, it was confirmed that the expression system of nitrile hydratase was inhibited by glucose as a carbon source, and the production of enzyme was suppressed.

Example 2 Fed-Breed Culture by Glucose Maintenance Concentration During Cultivation Period

Rhodococcus Rhodocross M33 spawn stored in 15% glycerol was inoculated into a Erlenmeyer flask containing 500 ml of the culture medium having the composition of Table 1, and incubated at 30 ° C. for 48 hours. Then, 90 ml of the culture solution was obtained and inoculated into 3 l of the same culture medium as in Table 1 except that 0.01 g / l of sodium chloride, 7.5 g / l of urea and 5 g / l of glucose were added. From the point of time when the substrate solution (glucose 165g, 40%) in the culture medium to maintain the glucose concentration of 0, 5, 10g / ℓ using a metering pump (MasterFlex, USA) by the above-mentioned index supply method While feeding, the culture was carried out at a temperature of 30 ° C. in a 5 L culture tank. The operating conditions of the culture medium were initially 0.4vvm of air injection speed and 500rpm of agitation speed. When the cell concentration reached OD 50, the air injection speed was 1.2vvm and the stirring speed was changed to 700rpm. Was maintained. In addition, the pH of the culture medium was maintained at 7.2 using a 10% KOH solution.

1 is a graph showing the growth pattern of the cells and the inactivation and total activity of the enzyme with the cultivation time shown by the fed-batch culture. As the glucose concentration in the culture medium is kept low, the specific growth rate of the cells decreases. It can be seen that the inactivation and total activity of the nitrile hydratase increases.

The specific growth rate was calculated by the equation (2).

Where μ is the specific growth rate, OD (t) is the cell concentration (OD) at t hours, and OD (t-1) is the cell concentration at t-1 hours.

Example 3 Batch Culture by Initial Cobalt Addition

Rhodococcus Rhodocrose M33 spawn stored in 15% glycerol was inoculated into an Erlenmeyer flask containing 500 ml of the culture medium having the composition of Table 1, and cultured at 30 ° C. for 48 hours (cultivation).

Then, 90 ml of the culture medium was obtained, except that 0.01 g / L sodium chloride, 7.5 g / L urea, and initial cobalt chloride (CoCl ₂ 6H ₂ O) concentrations were different from those shown in Table 3, respectively. Inoculated into 3 L of the same culture as the composition, and Rhodococcus Rhodocrose M33 was incubated for 49 hours under the same culture conditions as in Reference Example 1. Then, final cell concentration and inactivation of nitrile hydratase were measured. As a result, as the initial amount of cobalt chloride increased, the inactivation of nitrile hydratase increased, and the growth rate of the cells and the inactivation of nitrile hydratase decreased at concentrations of 0.03 g / l or more. The results are shown in Table 3.

Cobalt chloride (CoCl ₂ ㆍ 6H ₂ O) initial addition amount (g / ℓ) Cell concentration (OD _{540 nm} ) Nitrile hydratase inactive (units / mg-dry cells) Nitrile hydratase total activity (units / ml) Not added0.0050.0100.0150.0200.0250.0300.0350.0400.045 29.541.638.533.232.637.534.326.611.60.3 1230326065861211049075 712492463984236458305532084.5

As shown in Table 3, the cobalt concentration in the medium in the batch culture, it is preferable to add 0.02 ~ 0.03g / L in consideration of the enzyme inactivity, total activity and the concentration of the cells.

Example 4 Fed-Batch Culture with Additional Cobalt

Rhodococcus Rhodocrose M33 was fed-batch cultured in the same manner as in Example 2, and the feeding method of the substrate solution was carried out in a batch culture without adding any of the glucose until 5 g / L of glucose in the medium was depleted at the beginning of culture. From the time point, the glucose was supplied by the above-described upper pH adjustment method and the glucose concentration in the culture medium containing 0.02 g / L cobalt was kept as low as 0 to 0.5 g / L. In addition, during the incubation period, cobalt chloride was intermittently supplied to the culture medium by 0.01 g / L, and then divided into 1 to 3 times.

In culture medium A, cobalt chloride was added once at 0.01 g / l at a cell concentration of 2 g dry weight / l (OD 10). In the culture medium B, cobalt chloride was added twice at 0.01 g / l when the cell concentration was 2 g dry weight / l (OD 10) and 4 g dry weight / l (OD 20). In C culture medium, 0.01 g / l cobalt chloride was used when the cell concentration was 2 g-dry weight / l (OD 10), 4 g-dry weight / l (OD 20), and 14 g-dry weight / l (OD 70). Three times each was added.

Figure 2 is a graph showing the growth pattern of the cells according to the fed-batch culture and the inactivation pattern of the enzyme with the addition of cobalt chloride as incubation time, the inactivation of nitrile hydratase at the end of the culture as the number of addition of cobalt chloride increases Increasing results were confirmed.

[Examples 5 and 6] Fed-Batch Culture of Rhodococcus Rhodocross

During the incubation period of Rhodococcus Rhodocrose M8 and Rhodococcus Rhodocrose M33, cobalt chloride was intermittently 0.01 g / l while 60 g / l of glucose was continuously supplied so that the glucose concentration of the culture medium was kept at 0 to 0.5 g / l. 3 times each, that is, when the cell concentration was 2g-dry weight / l (OD 10), 6g-dry weight / l (OD 30), 14g-dry weight / l (OD 70), and was supplied. In the same manner, fed-batch culture was performed for 100 hours . The results are shown in Table 4 in comparison with Reference Example 1 and Reference Example 2.

Rhodococcus Rhodocross M8 Rhodococcus Rhodocross M33 Batch Culture (Reference Example 1) Fed-Batch Culture (Example 5) Batch Culture (Reference Example 2) Fed-Batch Culture (Example 6) Total Glucose Added (g / L) Final Cell Concentration * Cell Yield ** Nitrile Hydrate Inactivation *** Nitrile Hydrate Total Activity **** Nitrile Hydrate Productivity ***** 207.40.377051810.8 6025.20.4265163816.4 207.10.3512085214.9 6024.00.4120288028.5 * G-dry weight / ℓ ** g-dry weight / g-glucose *** units / mg-dry cells **** units / ml ***** units / ml / h

As a result of culturing Rhodococcus Rhodocrose, which produces nitrile hydratase by the fed-batch culture method, compared with conventional batch culture, the microbial cell concentration and the total activity of the enzyme were about three times, and the productivity of the enzyme was 1.5 to 1.9. There was a fold increase and the inactivation of the enzyme remained at a similar level.

Example 7 Conversion of Nitrile to Amide

In Example 6 fed-batch Rhodococcus Rhodocrose M33 was centrifuged, the supernatant was discarded, washed twice with distilled water, and then microbial cells were appropriately suspended in distilled water and used as a whole cell enzyme source.

800 ml of distilled water was added to a 1.5 L reactor while stirring, the temperature was adjusted to 20 to 25 ° C., and 0.6 g of the cell solution was added to the reactor by dry weight of Rhodococcus Rhodocrose M33 prepared above and resuspended. Next, acrylonitrile was added to the reaction mixture while controlling the injection rate so that the acrylonitrile concentration in the reaction solution did not exceed 0.2%. A total of 370 g of acrylonitrile was added to the reactor in total for 7 hours, and after 8 hours a 40% acrylamide solution was obtained. After the reaction, unreacted acrylonitrile was detected at 20 ppm or less, and by-product acrylic acid was detected at about 10 ppm.

As described above, the Rhodococcus rhodoprose was fed-batch-cultured to maintain the glucose concentration in the culture medium as low as possible, and cobalt was added intermittently in the middle of the culture period, thereby maintaining high cell culture and inactivation of nitrile hydratase. It is possible to do Therefore, by the method of the present invention, it is possible to mass produce nitrile hydratase at low cost and to reduce the production cost of industrially useful amide compounds.

Claims (8)

Also capable of producing nitrile hydratase Rhodococcus comprising: inoculated cross (Rhodococcus rhodochrous) to the medium; In the method of producing a Rhodococcus Rhodocrose comprising the step of culturing Rhodococcus Rhodocrose , The Rhodococcus Rhodocrose is M33 VKM Ac-1515D strain or M8 VKPM S-926 strain, The Rhodococcus also also prior to the step of culturing a cross Rhodococcus species also incubated batch-wise, and a cross, In the step of culturing the Rhodococcus Rhodocross, the method for producing Rhodococcus Rhodocross, characterized in that the supply of glucose continuously so that the glucose concentration is maintained at 0 ~ 5g / L in the culture medium. delete delete The method of claim 1, wherein the supply of glucose is provided by an exponential supply method, but samples are taken every several hours to measure the microbial cell concentration and glucose concentration, specific growth rate (μ), cell yield (Y _{x / s)} Rhodococcus Rhodocross manufacturing method characterized in that the feed rate is adjusted by determining a new flow rate using the following formula as a constant). [Equation 1] Where F is the feed rate of the substrate solution (l / hour), μ is the cell specific growth rate (1 / hour), X _o is the initial cell concentration (g / l), and S _o is the glucose concentration of the substrate solution (g / L), Y _{X / S} indicates growth yield (g-dry cells / g-glucose). The method of claim 1, wherein the feed system of glucose is supplied to the upper limit pH control method, the upper limit is 7.25 to pH 7.35, the lower pH is also method for producing a cross-Rhodococcus, characterized in that 7.20 to 7.15. delete The medium of claim 1, wherein 0.02 to 0.03 g / L of cobalt chloride (CoCl ₂ · 6H ₂ O) is contained in the initial culture medium, and cobalt chloride (CoCl ₂ · 6H ₂ O) is added to the culture medium during the culture period. Rhodococcus Rhodocross manufacturing method , characterized in that it comprises selectively adding two to three times by 0.01g / l intermittently. A method for producing an amide compound, wherein the nitrile compound is converted to an amide compound by using Rhodococcus rhodoprose cultivated by the production method according to claim 1.