JPS6122951B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to choline oxidase (hereinafter abbreviated as COD) and betaine aldehyde dehydrogenase (hereinafter abbreviated as COD).
This paper relates to a method for purifying COD and BADH (abbreviated as BADH), and more specifically, to purify COD and BADH, which are simultaneously produced in the bacterial cells of Penicillium genus bacteria, by hydro-orbit chromatography using an alkyl-sepharose or aminoalkyl-sepharose column. The present invention relates to a method for purifying both of them individually or at the same time using the graphite method. Generally, COD oxidizes choline in the presence of _O2 ,
It is an enzyme that catalyzes the reaction that produces betaine aldehyde and hydrogen peroxide, while BADH is an enzyme that oxidizes (dehydrogenates) betaine aldehyde in the presence of NAD (P) and catalyzes the reaction that produces betaine. Both of these enzymes are also known as useful enzymes used in the measurement of cholinesterase activity and the quantification of lecithin in the field of clinical testing. Conventionally, COD has been diagnosed in JP-A No. 54-35284 in bacteria belonging to the genus Cylindrocarpon, Fusarium, and Gibberella, and in JP-A No. 53-52687 in bacteria belonging to the genus Aspergillus, and BADH has been reported in Pseudomonas aeruginosa A- 16 (Pseudomonas
aeruginosa A-16) [Agric.Biol.Chem.40 volumes,
1743-1749 (1976)], the productivity of each is known. The present inventors screened a wide range of microorganisms in search of a more effective method for producing COD and BADH, and as a result, many strains belonging to the genus Penicillium were found.
We learned that it can simultaneously produce COD and BADH and has a high enzyme production ability. In separating and purifying COD and BADH from this culture, it is necessary to purify COD and BADH individually or simultaneously, depending on the purpose of use. For example, there are two methods for measuring cholinesterase activity: oxidizing choline produced from choline ester by cholinesterase with COD and quantifying hydrogen peroxide or betaine aldehyde produced at the time. In the method for measuring cholinesterase activity using the method of quantifying hydrogen peroxide, BADH is not required, and only COD is required, whereas in the method for measuring cholinesterase activity using the method for quantifying betaine aldehyde, the enzyme used is COD and
This is because both BADH and BADH are required. Therefore, the present inventors searched for an industrial purification method suitable for this purpose, and as a result of intensive study, the general formula CH ₃
-(CH ₂ ) _o - Water-insoluble carrier conjugate (where n is 1
It has been discovered that it is extremely effective to use adsorption and elution methods for NH ₂ -(CH ₂ ) _n -water-insoluble carrier bound substances (where m indicates 2 to 8) or NH 2 -(CH 2 ) n - . That is, the present invention was completed based on this knowledge, and a liquid containing COD and BADH is converted into a liquid containing the general formula CH ₃ −
(CH ₂ )n-water-insoluble carrier conjugate (where n is 1 to
5 is shown. ) or NH ₂ -(CH ₂ )m-water-insoluble carrier conjugate (where m is 2 to 8) to adsorb COD and BADH, and then separate and elute them singly or simultaneously using an eluent. This is a method for purifying COD and BADH. The liquid containing COD and BADH in the present invention is
Any bacteria can be used as long as it can simultaneously produce COD and BADH.
Penicillum ochlochlorone No. 79, which the present inventors newly isolated from the soil
ochro-chloron No.79) can be used effectively. This strain was sent to FERM-P
It has been deposited as 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 most are 10-150μ x 2-3μ, but 500μ
Some reach μ. Smooth or slightly rough surface. It branches asymmetrically and forms a conidial structure consisting of a basal branch, a basal basidium, and a phialide. The basal division technique and basal basidia are spread out at a wide angle. The basal basidium is 10-20μ x 2
~3μ, Fiallide is 7~11μ in totsukuri shape
×2μ. Conidia are formed from phialides and are chained, but not columnar. Sub-salvation form~
It is oval in shape with one end pointed. The surface is slightly rough
It is 28~3.2μ×2.0~27μ. 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 conidiation structure belongs to the phiaro-type conidial system, the tip of the conidiophore is penicillary, and the conidia are chained together, so it is classified in the genus Penicillium. Searching according to the AManual of Penillia, the conidiation 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. Furthermore, the conidia are grayish-yellow-green in color, the conidial chains are tangled and not columnar, and the colony is not rope-shaped.
Belongs to the Penicillium janthinellum series.
In addition, the underside of the vegetative hyphae and colonies are not strongly colored orange-red or reddish-purple, the conidiophores are smooth or slightly rough, the conidia are slightly rough. Depending on the quality of growth on extract agar medium, the strain may be Penicillium ochro-
It was identified as belonging to chloron. Raper, KBand C. Thom: A Manual
of Penicillia, Hafner Publishing Co.,
NewYork and London (1968) Examples of medium components suitable for culturing the bacteria used in the present invention include 1% choline hydrochloride, 0.2% dipotassium phosphate, 0.1% common salt, 0.05% magnesium sulfate,
The yeast extract is 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 to 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. and grinding them, and other methods. Any method using physical force such as sound waves, water pressure, or freezing and thawing may be used. Water or an appropriate buffer is added to the crushed bacterial cells obtained by these methods, and the suspension is centrifuged or the liquid is used as a crude enzyme solution. The solution containing COD and BADH used in the present invention may be such a crude enzyme solution or a partially purified enzyme solution that has been purified to a certain degree. In this case, purification methods include salting out, gel filtration, ion exchange chromatography, etc. alone or in combination. The adsorbent used in the present invention has the general formula
CH ₃ −(CH ₂ ) _o −NH ₂ (where n indicates 1 to 5)
Alkylamine represented by or general formula NH ₂ −
It can be obtained by binding an α,ω-diaminoalkane represented by (CH ₂ ) _n -NH ₂ (where m is 2 to 8) to a water-insoluble carrier. Further, specific examples of the alkylamine or α,ω-diaminoalkane used herein include ethylamine, propylamine, butylamine, pentylamine, hexylamine, ethylenediamine, 1.
3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,
1,8-diaminooctane is mentioned. In addition, the water-insoluble carrier is preferably an insoluble carrier that is inert, sparse, insoluble, and hydrophilic, such as agarose (registered trademark Cephalose, manufactured by Pharmacia Fine Chemicals), dextran (registered trademark), Trademark security index:
Pharmacia (manufactured by Fine Chemicals), cellulose powder, polyacrylamide gel (registered trademark Biogel, manufactured by Bio-Rad), and the like are used. In the binding reaction, first the water-insoluble carrier is bromocyanine,
Activated with sodium periodate, epichlorohydrin, etc., and then treated with alkylamine or α, ω-
This is accomplished by adding a diaminoalkane-added buffer. The obtained _CH3- ( _CH2 ) _o -water-insoluble carrier-conjugate or _NH2- ( _CH2 ) _n -water-insoluble carrier-conjugate was brought into contact with a solution containing COD and BADH at pH 6 to 9, and both enzymes were combined. Let it absorb. At this point, only COD and BADH are adsorbed, and miscellaneous proteins flow down without being adsorbed. Therefore, the carrier-bound product with both enzymes adsorbed is prepared at pH 6 to 9 in a solution containing an electrolyte such as salt or hexyl-enzyme at an appropriate concentration. In the case of Sepharose, etc., highly pure COD or BADH or a mixture of COD and BADH can be easily obtained by treating with a solution containing an appropriate hydrophobic reagent. Thus, Penicillium ochro−chloron
No.79FERM-P COD obtained from No.5303 and
We will discuss the physical and chemical properties of BADH. () Properties of COD (1) Action: Oxidizes choline and produces betaine aldehyde and hydrogen peroxide. (2) Optimum pH: The optimum pH for this enzyme is around 8. (Showed in Figure 2) (3) Stability: PH7 to 1 hour treatment at 25℃
Stable within the pH range of 9. (It is shown in Figure 3.) (4) Km: Km when choline is used as a substrate is 4×
10 ^-4 M. (5) Activity measurement method: 0.1M phosphate buffer (PH
8) Mix 2.8ml, 0.1ml of 0.2M choline hydrochloride solution, and 0.1ml of enzyme solution in a quartz cell, and heat at 25℃.
Measure the increase in absorbance at 500 nm. The amount of enzyme that can oxidize 1 μmol of choline per minute under these conditions is defined as 1 unit. () Properties of BADH (1) Action: Oxidizes betaine aldehyde in the presence of electron acceptors (NAD, etc.) to produce betaine. (2) Optimal PH: The optimal PH of this enzyme is between 8 and 9. (See Figure 4.) (3) Stability: Stable at PH6 to PH8 when left at 25°C for 1 hour. (As shown in Figure 5) (4) Activity measurement method: 0.1M phosphate buffer (PH8) 0.7
ml, NAD solution (10mg/ml) 0.05ml, 0.1M betaine aldehyde 0.025ml, enzyme solution 0.025ml 1
ml in a quartz cell and heated at 340 nm at 25°C.
Measure the increase in absorbance. The amount of enzyme that can oxidize 1 μmol of betaine aldehyde per minute under these conditions is defined as 1 unit. Next, test examples and examples of the present invention will be shown. Test Example 1 Using each strain of Penicillium, choline hydrochloride 1%, ammonium nitrate 0.3%, dipotassium phosphate 0.2%, common salt 0.1%, magnesium sulfate 0.05
COD of the extract obtained by culturing in a medium containing 0.3% yeast extract, grinding the cultured cells with alumina, and further extracting with 10mM phosphate buffer (PH8) containing Triton X-100. and BADH activity was investigated. The results are shown in Table 1.

【table】

[Table] As is clear from Table 1, strains belonging to the genus Penicillium simultaneously produce large quantities of both COD and BADH. Example ₂ CH3- equilibrated with 20mM phosphate buffer (PH7)
(CH ₂ ) _o -Sepharose and NH ₂ -(CH ₂ ) _n -Sepharose columns, respectively, obtained in Example 1.
Pour Penicillium ochro-chloron No. 79 crude enzyme solution, and after washing, wash the column with 20mM phosphate buffer (PH7.0) containing 0.1M NaCl, and then wash the column with 20mM phosphate buffer (PH7.0) containing 0.5M NaCl. (PH7.0) to separate and collect COD and BADH.
The results are shown in Tables 2 and 3.

【table】

[Table] As evident from Tables 2 and 3, COD
and BADH are completely adsorbed on these adsorption carriers. And in the case of _CH3- ( _CH2 ) _o -cepharose (ie, alkylcephalose), when eluted with a sodium chloride gradient, BADH is eluted first, followed by COD. That is, in Table 2, when n = 5 (hexyl-cepharose), BADH is eluted with 0.5M common salt, but COD is not eluted, and a more highly concentrated salt solution or an appropriate hydrophobic reagent is used. It is clear that by using a buffer containing (for example an alcohol such as propanol) COD can be seriously eluted. On the other hand, in the case of _NH2- ( _CH2 ) _n -cephalose (i.e., aminoalkyl-cephalose), when eluted with a salt gradient, COD is eluted first, contrary to the case of alkylcephalose.
BADH is then eluted. As an example
Penicillium ochro−chloron No.79 FERM−P
FIG. 1 shows the elution pattern of aminohexyl-sepharose when crude enzyme solution No. 5303 was used as a sample. As described above, it can be seen that COD and BADH can be eluted individually or simultaneously regardless of whether the carrier is alkyl-cephalol or aminoalkyl-cephalose. Example 1 Penicillium ochro-chloron No.79 FERM-P
No.5303 with choline hydrochloride 1%, dipotassium phosphate
0.2%, sodium chloride 0.1%, magnesium sulfate 0.05%, and yeast extract 0.3% aerobically cultured at 30° C. for 2 days to obtain cultured bacterial cells. 20 of these bacteria
After suspending in mM phosphate buffer (PH7) and crushing with glass beads in a Dyno Mill, Triton In addition, a precipitate was obtained by centrifugation. This precipitate was extracted with 20mM phosphate buffer (PH7), and the resulting supernatant was desalted using a Sephadex G-25 column equilibrated with the same buffer to obtain a crude enzyme solution. The obtained crude enzyme solution was added to 20mM phosphate buffer (PH7).
The mixture was applied to an aminohexyl-Sepharose column (100 ml) equilibrated with . COD and BADH were completely adsorbed and the column was washed with 50mM NaCl.
When 700 ml of 20 mM phosphate buffer (PH7) was passed through the tube, 960 units of COD were eluted. Furthermore, when 500ml of 20mM phosphate buffer (PH7) containing 180mM sodium chloride was poured, 105 units of BADH were eluted, and COD
We were able to separate and purify BADH. The specific activities of COD and BADH in this case were about 30 times and about 35 times that of the extract, respectively. 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. A crude enzyme solution was obtained by processing according to the same procedure. 20ml of this crude enzyme solution
Pour it into a butyl-Sepharose column (250 ml) equilibrated with M phosphate buffer (PH7), wash it, and then
20mM phosphate buffer (PH7) containing M NaCl 1
When the solution was run, an eluate containing 890 units of COD and 180 units of BADH was obtained. The specific activities of COD and BADH in this case were both about 13 times that of the extract. Example 3 Penicillium ochro-chloron No.79.FERM-P
No. 5303 was cultured 5 times in accordance with Example 1, and a recombinant enzyme solution was obtained in the same manner as in Example 1. This crude enzyme solution was equilibrated with 20mM phosphate buffer (PH7) on an aminooctyl-Sepharose column (100mM).
ml) and column with 20mM NaCl containing 100mM NaCl.
After washing with M phosphate buffer (PH7), 500 ml of 20mM phosphate buffer (PH7) containing 500mM sodium chloride was flowed, and an eluate containing 1000 units of COD and 120 units of BADH was obtained. COD in this case and
The specific activities of BADH were approximately 12 times higher in each case.

[Brief explanation of the drawing]

Figure 1 shows the separation and purification diagram of COD and BADH from the crude enzyme solution using aminohexyl sepharose, Figure 2 shows the PH activity curve of COD, and Figure 3 shows the PH stability of COD. Figure 4 shows the activity curve of BADH, and Figure 5 shows the PH stability curve of BADH. However, in Figure 1, A is COD and B is BADH.
In Figures 2 to 5, ○ is acetate buffer, ● is phosphate buffer, △ is Tris-HCl buffer,
▲ indicates the case where a sodium carbonate buffer solution was used.

Claims (1)

[Claims] 1 A solution containing choline oxidase and betaine aldehyde dehydrogenase is converted into a compound with the general formula _CH3- ( _CH2 ) _o -water-insoluble carrier conjugate (where n represents 1 to 5) or _NH2- ( _CH2 ) _n - A method for separating and purifying choline oxidase and/or betaine aldehyde dehydrogenase, which comprises adsorbing the enzymes onto a water-insoluble carrier conjugate (where m represents 2 to 8) and then eluting both enzymes simultaneously or separately. .