JPH03266977A - Incubation of beta-tyrosinase-containing microbe - Google Patents

Abstract

PURPOSE:To obtain in high yield microbe containing a high concentration of beta-tyrosinase by incubation in a medium with the glucose concentration falling between 0.3 and 0.01% (W/V). CONSTITUTION:beta-tyrosinase-contg. microbe belonging to Escherichia sp. such as Escherichia coli TD-001 (FERM P-10838) is aerobically incubated, while stirring under aeration, in a medium containing (A) a nitrogen source such as ammonium sulfate, (B) an inorganic salt such as potassium hydrogenphosphate, (C) a growth promotor such as thiamine, and (D) L- or DL-tyrosine. During this process, glucose is either continuously or intermittently added to the medium so that the glucose concentration in the medium be maintained within the range 0.3%-0.01% (W/V) throughout the incubation. Thence, after incubation, the culture fluid is centrifuged to recover wet microbe, Thereby, the objective microbe containing a high concentration of beta-tyrosinase can be obtained in high yield, thus producing said microbe in high efficiency.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to culturing a β-tyrosinase-containing bacterium belonging to the genus Escherichia by adjusting the glucose concentration in the medium to increase the concentration of β-tyrosinase. The present invention relates to a culture method for obtaining high yields of bacterial cells.

[Prior Art] L-tyrosine is an important amino acid as a raw material for the synthesis of thyroid function enhancers and the like, and it is expected that a cheaper manufacturing method will be established.

Conventionally, L-tyrosine has been produced by extraction from soy protein hydrolyzate or fermentation, but in recent years there have been problems such as high purification costs and waste disposal methods.
A method for producing tyrosine by an enzymatic method using β-tyrosinase-containing bacterial cells is attracting attention.

However, there are almost no reports on methods for preparing bacterial cells containing high concentrations of β-tyrosinase in high yields.

[Problems to be Solved by the Invention] An object of the present invention is to provide an efficient culture method for obtaining a high yield of bacterial cells containing β-tyrosinase at a high concentration.

[Means for Solving the Problems] In the present invention, when culturing a β-tyrosinase-containing bacterium belonging to the genus Escherichia, a medium containing L- or D-L tyrosine is provided with a glucose concentration of 0.000 during the culture period.
This is a method for culturing β-tyrosinase-containing bacteria, which is characterized by culturing while adding glucose so that the concentration is maintained within the range of 3% to 0.01% FW/V).

The present invention will be explained in more detail below.

The β-tyrosinase-containing bacteria belonging to the genus Escherichia used in the present invention are not particularly limited, but include, for example, Escherichia coli (Escherichia coli).
coli) TD-001 (FE RMP-108
38) is preferably used.

The β-tyrosinase-containing bacteria belonging to the genus Escherichia described above can be cultured in a medium containing the following nutrients such as a carbon source, a nitrogen source, an inorganic salt, and a growth promoting substance.

Nitrogen sources to be included in the medium include, for example, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium sulfate, and ammonium phosphate; nitrates such as potassium nitrate, sodium nitrate, and ammonium nitrate; organic nitrogen such as glutamic acid, glutamine, aspartic acid, and asparagine;
Ammonia etc. can be used alone or in combination, and examples of inorganic salts include potassium hydrogen phosphate,
Potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, and the like can be used alone or in combination. Substances that promote the growth of microorganisms include vitamins such as diamine and biotin; amino acids such as methionine and cysteine; or yeast extracts containing all or part of these, polypeptone, meat extracts, cornstap liquor, casamino acids, etc. is used.

The amount of DL-dyrosine added to the medium is 0.01
-2% (W/V), Sarani Favorite L<Ha005~05
%FW/V).

Glucose is added to the medium continuously or intermittently so that the concentration of glucose in the medium during the culture period is maintained within the range of 0.3% to 0.01% FW/Vl.

Medium temperature is 10-45°C, preferably 25-40°C
The pH of the medium is 3 to 10, preferably 5 to 10.
It is in the range of 9. If there is a change in pH during culture,
It is desirable to maintain the temperature constant within the above range using ammonia, caustic soda, caustic potash, etc. Culture time is usually 5~
It will be held for 48 hours.

Furthermore, it is desirable that the culture be carried out under aerobic conditions, with aeration and agitation so that dissolved oxygen during the culture does not become a rate-limiting factor.

"Effects of the Invention" According to the culture method of the present invention, a high yield of bacterial cells containing a high concentration of β-tyrosinase can be obtained, and therefore β-tyrosinase can be efficiently produced.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

In the Examples below, β-tyrosinase activity was measured by the following method.

Cells centrifuged (4,000 rpm, 15 minutes, 4°C) from culture solution 20- were added to 50 mM phosphate buffer (p
After suspending in 20-H8,0) centrifugation again (4,000
rpm, 15 minutes, 4°C) and collect the bacterial cells. After suspending the collected bacteria in the above-mentioned buffer solution 2-, the microorganisms were subjected to ultrasonic disruption treatment (Sonifier 200 manufactured by Branson L), and the treated material was centrifuged (
(12,00 Orpm, 40 minutes, 4°C) The soybean supernatant liquid was used as the test enzyme solution.

For the reaction, 0.1- of the above enzyme solution was mixed with 0.9- of the following reaction solution.
After adding to the water, reacting at 30°C for 15 minutes,
The sample was immersed in C boiling water for 1 minute, and the treated solution was further diluted 10 times with distilled water, and the diluted solution was passed through a filtration membrane (2,2P) to separate the enzyme protein. The amount of tyrosine in the permeate was measured by high performance liquid chromatography (Shimadzu LC-5A).
It was quantified.

<Reaction solution composition> Contains 10 g of phenol, 40 g of L-serine, 10 g of NH4CQ, 2 g of EDTA, and 4 g of Na2SOs in 1f2 of 5'OmM phosphate buffer (pH 8,0).

In addition, the mass of kumbaku in the test enzyme solution can be measured using Lowry
etc. (Journal of Biologica
lChemistry, vol, 193. p, 26
5. (19511).

The glucose concentration in the medium was measured using Glucose C Test Wako [manufactured by Wako Pure Chemical Industries, Ltd.].

The bacterial cell yield was measured as the amount of wet bacterial cells collected after centrifugation (8.00 rpm, 20 minutes, 4°C) from 50 cells of the culture solution.

Example 100 of the culture medium shown in Table 1 was dispensed into a 500i Erlenmeyer flask and sterilized at 120°C for 15 minutes.
After aseptically adding 50% (W/Vl glucose solution (sterilized at 120°C for 15 minutes) for 1 i, Escherichia coli TD-001, (FERM
P-10838) was inoculated and cultured with shaking at 37°C for 1 day.

1.000 of a medium prepared in the same manner as 20 of the culture solution
MFF was inoculated and heated at 37°C with a rotational speed of 1. OOOrpm, air flow rate 1vvm, pH 7.2 (
(adjusted with 28% ammonia water) for 8 hours. In addition, while measuring the glucose concentration in the culture solution over time using an aqueous solution (sterilized at 120°C for 15 minutes) of 50% FW/V), the concentration of glucose in each experimental group shown in Table 2 was measured. It was added continuously or intermittently in an aseptic manner so as not to exceed the upper limit, and the addition was stopped when the total amount added in each experimental group reached 20 g.

Table 1 L-tyrosine KH2PO4 K 2 HP O4 (NH,) 2SO.

M g SO4・7H20 FeSO4・7H20 Casamino acids (DIFCOI Yeast extract Add distilled water 2,0g 1,6g 5,5g 3.0g 0,1g 0mg 3,0g 3,0g 1,000- (pH7,2 ) After the completion of the culture, 20 μl of each culture solution was centrifuged, and β-tyrosinase activity was measured using the method described above.
Bacterial cells cultured only by adding 0 g/12 were used.

Table 2 × Relative value with the activity of comparative example (20 g/f2 added at the beginning of culture) as 1,0

Claims (1)

[Claims] When culturing β-tyrosinase-containing bacteria belonging to the genus Escherichia, a medium containing L- or DL-tyrosine is used with a glucose concentration of 0.3% to 0.0% during the culture period.
1. A method for culturing β-tyrosinase-containing bacteria, the method comprising culturing with the addition of glucose so that the ratio is maintained within a range of 0.01% (W/V).