JP4841162B2 - Microbial culture method - Google Patents

Description

  The present invention relates to a method for culturing microbial cells, and more specifically, in a fed-batch culture method for microbial cells having nitrilase activity, which is performed without using an inducer such as a nitrile compound or cyclic amide. The present invention relates to a microbial culture method for obtaining cells having nitrilase activity at a high concentration.

  Examples of microbial cells having nitrilase-producing ability include, Microorganisms belonging to the genus, Acinetobacter genus and the like are known.

  However, since the nitrilase activity in these microorganisms is generally low, in order to increase the nitrilase activity, the nitrilase-inducing substance is added to the medium and cultured. As a method for enhancing the nitrilase activity by adding a nitrilase inducer to a medium, for example, a method of culturing a microorganism by adding a nitrile compound such as acetonitrile, isobutyronitrile, benzonitrile, ethylene cyanohydrin ( For example, Patent Documents 1 to 4), a method of culturing a microorganism belonging to the genus Rhodococcus by adding a lactam compound such as ε-caprolactam (for example, refer to Patent Document 5), and the like.

  Further, as another microorganism culture method for enhancing nitrilase activity, a method of continuous culture under a carbon restriction using a carbon source containing a nitrile or a nitrile precursor (for example, Patent Document 6) is disclosed.

Further, as another method for obtaining a microorganism having high nitrilase activity at a high concentration, after the logarithmic growth phase is completed, a method in which only a carbon source is added to the culture solution (see, for example, Patent Document 7), or the growth of the microorganism. A method of exchanging the culture solution with a hypotonic solution after completion (for example, see Patent Document 8) is described.
Japanese Examined Patent Publication No. 63-2596 Japanese Patent Laid-Open No. 4-40898 JP-A-8-173152 JP 2003-274933 A Japanese Patent No. 3009421 International Publication No. 97-6248 JP-A-11-341979 JP 2001-292762 A

  However, among the methods of culturing microbial cells having high nitrilase activity known so far, in the method of adding a nitrile compound, the nitrile compound is not only a carbon source but also toxic to the microbial cell. It is difficult to add a large amount from the beginning of the culture. In addition, some nitrile compounds are highly volatile and flammable, and some of them emit a significant odor and are difficult to use in industrial culture.

  In addition, since lactam compounds and ethylene cyanohydrin are kept at a constant concentration in the system, the nitrilase-inducing effect is stable, but it is difficult to assimilate and thus remains in the culture medium after culturing. There is a problem of culture waste liquid treatment.

In addition, as described in Patent Document 7, after the logarithmic growth phase is completed, the method of adding only the carbon source to the culture solution has a problem that the once-expressed nitrilase activity decreases in the later stage of the culture, As a solution, a method of exchanging the culture solution with a hypotonic solution after the growth of the microorganism described in Patent Document 8 has been developed. In this method, a filtration membrane for exchanging the inside of the culture tank with a hypotonic solution, etc. However, there is a problem that the culture equipment itself becomes large-scale.
Under such circumstances, the present invention provides a culture method capable of obtaining a high concentration of microorganisms having high nitrilase activity with a simple apparatus without using a nitrilase inducer such as a nitrile compound or a lactam compound. It is for the purpose.

  As a result of intensive studies to solve these problems, the present inventors have added high vegetable nitrilase activity without adding an inducer such as a nitrile compound by adding vegetable oil and controlling the medium composition. A method for culturing microbial cells at a high concentration has been found and the present invention has been completed.

That is, the present invention is a microorganism culture having the nitrilase activity shown in the following (1) to (7).
(1) A fed-batch culture method of microbial cells having nitrilase activity, using Acinetobacter strains as microorganisms, without adding a nitrile compound or a lactam compound, and wherein the use of soybean oil as a nutrient source Microorganism culture method.
(2) culture the microbial cultivation method according to (1) the soybean oil steady concentration in the nutrient solution is not more than 10 g / L or more 250 g / L.
(3) The microorganism culturing method according to any one of (1) to (2) , wherein a yeast extract of more than 0 and 20 g / L or less is used as a nutrient source.
(4) The microorganism culture method according to any one of (1) to (3), wherein transition metal ions of more than 0 and 1000 ppm or less are present in the culture solution.
(5) The microorganism culture method according to (4), wherein the transition metal ion is an iron ion. ( 6 ) Acinetobacter sp. Strain having nitrilase activity is Acinetobacter sp. AK226 or Acinetobacter SP. The microorganism culturing method according to any one of (1) to (5), wherein the microorganism culturing method is AK227.
( 7 ) Acinetobacter strains having nitrilase activity are Acinetobacter sp. ( 6 ) The microorganism culturing method according to ( 6 ), which is AK226.

  By using the present invention, it is possible to obtain microorganisms having high nitrilase activity at a high concentration with a simple apparatus without adding inducers such as nitrile compounds and lactam compounds to microorganisms having nitrilase activity.

Hereinafter, the present invention will be specifically described.
Examples of the microbial cell having nitrilase activity in the present invention include, for example, Caseobacter, Pseudomonas, Alkaligenes, Corynebacterium, Nocardia, Rhodococcus, Bacillus, Bacteria, Micrococcus, Arthrobacter , Gordona genus, Acinetobacter genus and the like.

  Among these microbial cells having nitrilase activity, Gram-negative bacteria include, for example, Acinetobacter genus, Alkaligenes genus, etc., but any microorganism may be used as long as it has nitrilase-producing ability. Absent.

  Specifically, Acinetobacter SP. AK226 (FERM BP-08590) or Acinetobacter SP. AK227 (FERM BP-08591) may be mentioned. These strains are described in Patent Document 1. The microorganism in the present invention may be a microorganism into which a nitrilase gene that has been naturally or artificially improved is incorporated by a genetic engineering technique.

  The fed-batch culture method in the present invention is a culture method in which a medium is continuously or intermittently fed without extracting a culture solution, and is a method similar to a method generally called fed-batch culture. The method may be performed, for example, by having an excess of nutrients other than the carbon source, and gradually introducing the carbon source as the microorganism grows and continuing to introduce the carbon source until other nutrients are consumed. Can do.

The dry weight of microorganisms in the suspension is measured by the following method.
[Method for measuring dry weight of microorganisms]
First, an appropriate amount of a microorganism suspension having an appropriate concentration is taken, cooled to −80 ° C. and frozen, and then completely dried using a freeze dryer and the weight is measured. From this dry weight, the microorganism concentration in the suspension weighed first is calculated. Dilute the microbial suspension at a known concentration to a suitable concentration, measure the turbidity with a turbidimeter, create a calibration curve for the turbidimeter, and obtain the factor. From this factor and the turbidity indication value of the turbidimeter, the microbial dry weight of an arbitrary microbial suspension is calculated.

The specific activity in the present invention is expressed by the weight of ammonium acrylate produced by 1 g of dry cells per hour when acrylonitrile is used as a substrate.
The specific activity of microbial cells having nitrilase activity is determined by the following method.
[Method for measuring nitrilase specific activity]
A suspension of microbial cells having nitrilase activity is washed with 30 mM aqueous ammonium acrylate (pH = 7) and suspended in the same aqueous solution, and then 5 mL of the suspension is kept at 30 ° C. Acrylonitrile (100 μL) was quickly added to the suspension maintained at 30 ° C., and the reaction was started at the time when the addition was started and shaken at 30 ° C. for 20 minutes. The concentration of ammonium acrylate in the reaction solution or supernatant after the reaction was quantified by neutralization titration after trapping ammonium ions as hexamethylenetetramine by formalin treatment. At this time, a sample to which acrylonitrile was not added was prepared at the same time, and the same treatment as that for the sample to which acrylonitrile was added was performed. The titration value was set to a blank value (ammonium acrylate concentration before reaction), and the value subtracted from the titration value The ammonium acrylate concentration was used.

  In the present invention, any carbon oil may be used as the carbon source. For example, olive oil, corn oil, cottonseed oil, soybean oil and the like can be used, but preferably soybean oil is used as the carbon source. To do. When soybean oil is used as a carbon source in the present invention, the soybean oil steady concentration in the culture solution is 10 g / L or more and 250 g / L or less, preferably 10 g / L or more and 150 g / L or less. If the steady concentration of soybean oil in the culture solution is less than 10 g / L, the carbon source concentration in the system becomes too low, and the culture time may become long. Moreover, when the soybean oil steady concentration in a culture solution exceeds 250 g / L, the growth rate of microorganisms falls extremely similarly to the case where the carbon source concentration in a system becomes too high and a carbon source concentration becomes too low. The culture time may become longer.

The steady soybean oil concentration in the present invention refers to the soybean oil concentration in the culture solution from the start of microbial cell culture until the end of soybean oil feed into the system.
The soybean oil concentration in the present invention is determined, for example, by the following method.
[Measurement method of soybean oil concentration]
An appropriate amount of a lipase solution is added to 100 μL of a sample solution diluted with a surfactant, and the fatty acid is decomposed by reacting at 37 ° C. for 10 minutes. To 200 μL of the solution, an extract composed of copper sulfate and chloroform / n-heptane was added, shaken for 10 minutes, centrifuged, and 1 ml of the supernatant was added with a coloring solution (chloroform 40 ml / n-heptane 60 ml / vasocuproin 0.02 g). / 3 (2) -t-butyl-4-hydroxyanisole 2 g) 1 ml is added and measured at OD 480 nm. Prior to sample measurement, soybean oil having a known concentration is measured in the same manner, a calibration curve is prepared, and the soybean oil concentration in the sample is calculated using the calibration curve.

  As the nitrogen source in the present invention, any one generally used for microbial culture may be used, for example, amines, amino acids, nitrates, ammonia, ammonium salts of various inorganic acids and organic acids, and other nitrogen-containing compounds. , Peptone, tryptone, polypeptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal, soybean meal hydrolyzate, etc., preferably yeast extract.

  When using yeast extract as a nitrogen source, the concentration used is in the range of more than 0 to 20 g / L, preferably more than 0 to 15 g / L, more preferably 0.1 g / L to 10 g. / L or less. Even if the yeast extract is used in excess of 20 g / L, a dramatic effect cannot be expected.

  Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, iron sulfate, and calcium carbonate.

  Examples of transition metal ions used in the present invention include copper ions, cobalt ions, iron ions, zinc ions, manganese ions, nickel ions, and the like, and at least one of these transition metals can be used. However, it is particularly preferable to use iron ions among them. The use concentration of the transition metal is more than 0 and 1000 ppm or less, preferably 20 ppm or more and 300 ppm or less. Even if the transition metal is used in excess of 1000 ppm, a particularly dramatic effect cannot be expected.

In culturing microbial cells in the present invention, the pH of the culture solution is 5 or more and 10 or less, preferably 6 or more and 8.5 or less. The culture temperature is 20 to 40 ° C., preferably 25 to 35 ° C.
Hereinafter, although an example explains concretely, the present invention is not limited at all by these examples.

Example 1
Acetic acid 1% by weight, yeast extract 0.3% by weight, sodium glutamate 0.5% by weight, ammonium sulfate 0.5% by weight, sodium chloride 0.1% by weight, potassium dihydrogen phosphate 0.15% by weight, hydrogen phosphate Dipotassium 0.2 wt%, magnesium sulfate heptahydrate 0.2 wt%, manganese chloride 0.02 wt%, calcium chloride 0.01 wt%, soybean oil 2 wt%, yeast extract 0.05 wt%, 200 mL of a liquid medium containing 0.5% by weight of sodium glutamate and 0.8% by weight of acetic acid is placed in an Erlenmeyer flask, adjusted with sodium hydroxide to a pH of 7, sterilized at 121 ° C. for 20 minutes, and then added to Acinetobacter. SP. AK226 was inoculated and shake-cultured at 30 ° C. 18 hours after the start of the culture, acetic acid and ammonium sulfate were added, and the mixture was further cultured at 30 ° C. for 24 hours (preculture).
On the other hand, sodium chloride 0.1% by weight, potassium dihydrogen phosphate 0.15% by weight, dipotassium hydrogen phosphate 0.2% by weight, magnesium sulfate heptahydrate 0.2% by weight, ferrous sulfate heptahydrate 0.003% by weight of Japanese product, 0.5% by weight of ammonium sulfate, 0.02% by weight of manganese chloride, 0.002% by weight of copper sulfate pentahydrate, 0.002% by weight of zinc sulfate heptahydrate, yeast extract 0 3 L of a liquid medium containing 0.05% by weight, 0.5% by weight sodium glutamate, and 2% by weight soybean oil was placed in a jar fermenter, adjusted to pH 7 with sodium hydroxide, and sterilized at 121 ° C. for 20 minutes. Thereafter, the jar fermenter was inoculated with the pre-cultured solution. Soybean oil was fed from 8 hours after the start of the culture. The pH was controlled to 7 with phosphoric acid and aqueous ammonia. After the end of the culture, the final concentration of the dry cell of the microorganism was measured. As a result, the specific activity when using acrylonitrile as a substrate was 110 g-ammonium acrylate / g-dry cell / hour.

Example 2
The same operation was performed except that acetic acid was used instead of phosphoric acid used for pH adjustment in Example 1. When the final concentration of the dried cells of the microorganism was measured after the end of the culture, the specific activity was 55 g / L and 127 g-ammonium acrylate / g-dried cells / hour when acrylonitrile was used as the substrate.

Comparative Example 1
Sodium chloride 0.1 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate heptahydrate 0.05 wt%, ferrous sulfate heptahydrate 0.005 wt%, ammonium sulfate 0.1 wt% %, Potassium nitrate 0.1% by weight, manganese sulfate pentahydrate 0.005% by weight of the culture solution 250mL is placed in an Erlenmeyer flask and adjusted with potassium hydroxide so that the pH is 7, and sterilized at 121 ° C for 20 minutes After that, 2.5% by weight of acetonitrile was added. Acinetobacter SP. AK226 was inoculated and shake-cultured at 30 ° C. After the completion of the culture, the final concentration of the dry cell of the microorganism was measured. As a result, the specific activity when using acrylonitrile as a substrate was 110 g-ammonium acrylate / g-dry cell / hour.

Comparative Example 2
A culture solution 3L having the same composition as the medium described in Comparative Example 1 was placed in a jar fermenter, sterilized at 121 ° C. for 20 minutes, then inoculated with the culture solution described in Comparative Example 1, and aerated and stirred at 30 ° C. Acetic acid was fed 2 hours after the start of the culture. The pH was controlled with phosphoric acid and aqueous ammonia so that the pH became 7. After the end of the culture, the final concentration of the dried microorganism was measured. As a result, the specific activity was 74 g-ammonium acrylate / g-dried bacteria / hour when acrylonitrile was used as the substrate.

Comparative Example 3
In Comparative Example 2, the same operation was performed except that ammonium acetate was used instead of acetic acid fed during aeration and agitation culture. After the end of the culture, the final concentration of the dried microorganism was measured. As a result, the specific activity when using acrylonitrile as a substrate was 62 g-ammonium acrylate / g-dried bacteria / hour.

  By using the present invention, a microorganism having high nitrilase activity can be obtained at a high concentration without adding an inducer such as a nitrile compound or a lactam compound with a simple apparatus.

Claims (7)

A fed-batch culture method of microbial cells having nitrilase activity, using Acinetobacter strains as microorganisms, without adding a nitrile compound or a lactam compound, a microorganism culture, characterized by the use of soybean oil as a nutrient source Method. Microbial culture method according to claim 1, wherein the soybean oil steady concentration in the culture solution is not more than 10 g / L or more 250 g / L. The method for cultivating microorganisms according to any one of claims 1 to 2 , wherein a yeast extract of more than 0 and 20 g / L or less is used as a nutrient source. The microorganism culture method according to any one of claims 1 to 3 , wherein a transition metal ion of more than 0 and 1000 ppm or less is present in the culture solution. The microorganism culture method according to claim 4, wherein the transition metal ion is an iron ion. Acinetobacter strains having nitrilase activity are Acinetobacter sp. AK226 or Acinetobacter SP. It is AK227, The microorganism culture method as described in any one of Claims 1-5 characterized by the above-mentioned. Acinetobacter strains having nitrilase activity are Acinetobacter sp. It is AK226, The microorganisms cultivation method of Claim 6 characterized by the above-mentioned.