JPS6137909B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing betaine aldehyde dehydrogenase-producing bacteria belonging to the genus Penicillium and obtaining betaine aldehyde dehydrogenase from a culture such as the resulting cultured bacterial cells. Betaine aldehyde dehydrogenase is known as an enzyme that oxidizes or dehydrogenates betaine aldehyde in the presence of NAD (P) and catalyzes the reaction to produce betaine. Its uses include measuring cholinesterase activity and quantifying lecithin. It can be offered. Conventional betaine aldehyde-producing bacteria include Pseudomonas aeruginosa A-16 (Pseudomonas aeruginosa A-16).
aeruginosa A-16) [Agricultural and Biological Chemistry
and Biological Chemistry), Vol. 40, pp. 1743-1749 (1976)], and there was nothing noteworthy in industrial production. The present inventors screened a wide variety of microorganisms in search of an industrially inexpensive production method for betaine aldehyde dehydrogenase, and as a result found that many strains belonging to the genus Penicillium have a high ability to produce betaine aldehyde dehydrogenase. The invention has been completed. The present invention is a method for producing betaine aldehyde dehydrogenase, which is characterized by culturing betaine aldehyde dehydrogenase-producing bacteria belonging to the genus Penicillium and collecting betaine aldehyde dehydrogenase from the culture. The bacteria used in the present invention are betaine aldehyde dehydrogenase-producing bacteria belonging to the genus Penicillium, and are exemplified below. Penicillium ochlochlorone No.79
(Penicillium ochro−chloron No.79) Penicillium carnescens IFO7955)
(Penicillium carnescens IFO7955) Penicillium jancinerum IFO7905
(Penicillium janthinellum IFO7905) Penicillium janthinellum IFO8070
(Penicillium janthinellum IFO8070) Penicillium citrinum IFO6026
(Penicillium citrinum IFO6026) Of these, Penicillium ochro−chloron No.79
is a newly isolated bacterial strain from soil and is being sent to the Microtech Institute.
It has been deposited as FERM-P No. 5303. Next, Penicillium ochro−chloron No.79,
The mycological properties of FERM-P No. 5303 are shown. That is, the morphological properties of this strain on malt extract agar and Czapek agar are shown in parentheses, the growth state in parentheses, and the physiological properties in parentheses. () Morphological properties Conidiophores arise from aerial hyphae or basal hyphae and are mostly 10-150μ x 2-3μ, but can reach 500μ. Smooth or slightly rough surface.
It branches asymmetrically and forms a conidial structure consisting of a basal branch, basal basidium (metre), and phialide. The basal branches and basal basidia are widely spread. The basal basidium is 10-20μ x 2-3μ,
The phialide is shaped like 7 to 11μ x 2μ. Conidia are formed from phialides and are chained, but not columnar. Subspherical to oval with one end pointed. The surface is slightly rough and 2.8 to 3.2
μ×2.0 to 2.7μ. Ascospores and sclerotia are not formed. () Growth status Growth in each medium is the result of culturing at 23°C for 10 to 12 days. Tuapetsk agar medium Growth is rather slow, colony diameter is 30-40
It is about mm. Woolly. Aerial mycelium is white to yellowish white. Conidia are poorly formed and are grayish-yellow-green in color. The underside is light yellowish brown. There are no water-soluble dyes. Malt extract agar medium: Grows well and colonies are 60-70mm in diameter. Woolly. Aerial hyphae are almost white in color. Conidia are well formed and are grayish yellow-green in color. The underside is pale yellow-brown to dark yellow. There are no water-soluble dyes. () Physiological properties Optimum growth conditions Temperature is around 30℃, pH is 4-6. Growth range Slight growth at 7℃. It grows well at 37℃, but weakly at 40℃, and does not grow at 45℃. Grows between PH2.5 and PH9. The conidial structure belongs to the phiaro-type conidial system,
The tip of the conidiophore is penicillary, and the conidia are linked together, so it is classified as a member of the genus Penicillium. A
A search according to the Manual of Penicillia reveals that the conidiation-forming structure is irregularly branched, the basal branches and basal basidia are spread out at wide angles, and the phialides are shaped like an asymmetrical fungus. It is a group. In addition, the color of the conidia is grayish-yellow-green, the conidial chains are tangled and not columnar, and the colony is not rope-shaped.
Belongs to the janthinellum series. Moreover, the vegetative hyphae and the underside of the colonies are not strongly colored orange-red or reddish-purple, the conidiophores are smooth or slightly rough, the conidia are slightly rough, and the surface of the conidia is slightly rough. Depending on the quality of growth on malt extract agar medium, the strain may be Penicillium.
It was identified as belonging to ochro-chloron.
Raper, KBand C. Thom: A Manual of
Penicillia, Hafner Publishing Co., New York
and London (1968) In the present invention, a normal nutrient medium to which choline or a choline-containing substance is added is used for culturing betaine aldehyde dehydrogenase-producing bacteria. Choline is used as either a carbon source or a nitrogen source, but glucose, starch, etc. may be added as a carbon source, and ammonium nitrate, ammonium chloride, ammonium phosphate, meat extract, yeast extract, etc. may be added as a nitrogen source. As the inorganic salt, dipotassium phosphate, magnesium sulfate, common salt, etc. are used. Examples of medium components suitable for the present invention include 1% choline hydrochloride, 0.3% ammonium nitrate, 0.2% dipotassium phosphate, 0.1% common salt, and magnesium sulfate.
0.05%, yeast extract 0.3%, and the culture conditions are preferably aerobic culture at a temperature of 20 to 40°C for 1 to 4 days. The pH of the medium is preferably 5-7. The bacterial cells obtained by culture are washed and crushed to prepare a crude enzyme solution. Methods for crushing the bacterial cells include mixing the bacterial cells with glass beads, activated alumina, etc. Any method using physical force such as sound waves, water pressure, or freezing and thawing may be used. After adding water or an appropriate buffer to the crushed bacterial cells obtained by these methods to make a suspension, the supernatant or liquid obtained by centrifugation or filtration is used as a crude enzyme solution. . The crude enzyme solution thus obtained contains various enzymes or contaminant proteins in addition to betaine aldehyde dehydrogenase. To remove these impurities, salting out, gel filtration, ion exchange chromatography, electrophoresis, etc. are performed alone or in combination. Next, Penicillium obtained in the present invention
The properties of betaine aldehyde dehydrogenase obtained from ochro-chloron No. 79 will be described. Note that betaine aldehyde dehydrogenases obtained from other bacterial strains also exhibit similar properties. (1) Action: Oxidizes betaine aldehyde in the presence of electron acceptors (such as NAD) to produce betaine. (2) Optimum reaction pH: The optimum reaction pH of this enzyme is between 8 and 9 (as shown in Figure 1). (3) Stability: PH6 to 1 hour at 25℃
It is stable at pH 8 (shown in Figure 2). (4) Activity measurement method: 0.1M phosphate buffer (PH8) 0.7
ml, 0.05 ml of NAD solution (10 mg/ml), 0.025 ml of 0.1 M betaine aldehyde, and 0.025 ml of enzyme solution are mixed in a 1 ml quartz cell, and the increase in absorbance at 340 nm is measured at 25°C. Under these conditions, the amount of enzyme that can oxidize 1 μmol of betaine aldehyde per minute is 1
Unit. Next, test examples and examples of the present invention will be shown. Test Example 1 Each strain shown in Table 1 was treated with 1% choline hydrochloride, 0.3% ammonium nitrate, 0.2% dipotassium phosphate, and salt.
0.1%, magnesium nitrate 0.05%, yeast extract 0.3
% for 2 days with aeration and agitation, the cultured cells were ground with alumina, and further treated with Triton X-
The betaine aldehyde dehydrogenase activity per 100 ml of the extract obtained by extraction with 100 mM phosphate buffer (PH8.0) was examined, and the results are shown in Table 1.

【table】

[Table] Example 1 Penicillium ochro−chloron No.79, FERM−
P No.5303 with choline hydrochloride 1%, dipotassium phosphate 0.2%, salt 0.1%, magnesium sulfate 0.05%
30°C in medium 5 consisting of 0.3% yeast extract, 2
The cells were cultured aerobically for days to obtain cultured bacterial cells. The cells were suspended in 10mM phosphate buffer (PH8) and
After crushing with glass beads in a mill, Triton X-100 with a final concentration of 0.2% was added to the suspension of the crushed bacterial cells, and 800 ml of an extract was obtained by centrifugation. The extract contained 0.200 units/ml of betaine aldehyde dehydrogenase. Example 2 Penicillium citrinum IFO6026 was cultured in medium 10 consisting of 1% choline hydrochloride, 0.3% ammonium nitrate, 0.2% dipotassium phosphate, 0.1% table salt, and 0.3% yeast extract, and the resulting bacterial cells were cultured in Example 1. Extract 1.5 was obtained by processing in the same manner. The extract contained 0.150 units/ml of betaine aldehyde dehydrogenase. Example 3 Penicillium ochro−chloron No.79, FERM−
P No. 5303 was cultured for 5 times according to Example 1, and an extract was obtained in the same manner as in Example 1. Ammonium sulfate was added to this extract, and the precipitate obtained between 0.2 and 0.6 saturation was dissolved in 10 mM phosphate buffer (PH8), and the betaine aldehyde dehydrogenase-containing solution 100
Got ml. This solution contained 1.52 units/ml of betaine aldehyde dehydrogenase. The specific activity was able to be about 4 times that of the extract.

[Brief explanation of the drawing]

FIG. 1 is a PH activity curve of betaine aldehyde dehydrogenase obtained by the production method of the present invention,
Figure 2 also shows the PH stability. ○ indicates acetate buffer, ● indicates phosphate buffer, △ indicates Tris-HCl buffer, and ▲ indicates sodium carbonate buffer.

Claims (1)

[Claims] 1. A method for producing betaine aldehyde dehydrogenase, which comprises culturing a betaine aldehyde dehydrogenase-producing bacterium belonging to the genus Penicillium and collecting betaine aldehyde dehydrogenase from the culture.