JPS58216688A - Production of alpha-glycerophosphate- oxidase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL FIELD The present invention relates to a process for producing α-glycerophosphate oxidase.

Prior art α-glycerophosphate oxidase and useful
Its preparation and extraction technique is described by Kodltseh@k.

Neat mexidase, Journal op Pacteriology, Vol. 98, No. 3, pp. 1063-1068 (1969
Jacobs, N, J- and Van De
Mark, P. J., “Purification and properties of α-glycerophosphate oxidase from Stregutococcus faecalis”
, Archives op Biochemistry and
Biophysics, Vol. 88, pp. 250-255. This enzyme is useful for oxidizing alpha-glycerophosphate in the presence of oxygen to produce dihydroxyacetone phosphate and hydrogen peroxide.

Kodi tsehek and [Jmbreit] studied the ΔL and α-7'(f')J-1i''24 using a variety of potency testing techniques, including manometry assays.
Explains the properties of glycerophosphate oxidase. Streno Cocus 7 Culture of ichium 1% trigton, 1% yeast extract, 0.5 thium 2HP04.0
．． St.
ab). These cells were separated from the medium and the enzyme products were extracted for assay.

Jacobs and Van Demark, supra, have also described the properties of the α-glycerophosphate oxidase of Stregutococcus faecalis 10C1 in their work using a manometry assay. They used the above-mentioned AC medium (however, the enzyme from glucose received 1s, ￥
For L-α-glycerophosphate, j is the commercially acceptable % isomerism, β-glycerophosphate, glycerol, dihydroxyacetone phosphate 4
, 2-7'O)f has no appreciable activity towards diol phosphate.

Jaeab, N., J. and Van Demark, P.
*J, Journal of Bacteriology, 79
vol., pp. 532-538 (1960). Comparison of glycerol oxidation mechanisms in aerobically and anaerobically grown Stregutococcus faecalis. Taxonomy using aerobically and anaerobically grown cells from Stregtococcus faecalis. Glycerol substitute in research ii!
11 are discussed. This Nostreno cocus faecalis is grown in the AC medium described above.

Gunsalus, 1. C, and Umb r s i
``Oxidation of glycerol by leg cocus fucus'', Journal of Bacteriology, Vol. 49, 347-357, ``T, W, W,'' and A. We have reported the results of an assay for the oxidation of glycerol by a suspension of active ray 1 foam from Mugi Yue.

By adding pyruvate to the assay, they converted pyruvate into H2O. Because it acts as a scavenger, the glycerol oxidation level is 1! tM, but the activity of α-glycerophosphate oxidase has not been described.

Gunsalus, 1. C.'s "Strenotococus 7"
Bulk glycerol fermentation products by Echaris (Journal of Bacteriology, Vol. 54, 239-2)
44 Negative (1947)) reported on the growth of L'legtococus 7 echaris in a medium containing glycerol and yeast extract up to part 1. Gunsalus has shown that glycerol causes a slight growth of Stregutococus faecalis (aerobic growth), but almost no growth (anaerobic growth) is observed.

Clarldge, (H, A, and) (endlin,
D. One of the glycerol produced by Stregutococus faecalis (Journal of Bacteriophore)
No. 84, pp. 1181-1186 (1962) reports a study of glycerol utilization by Strezotococus faecalis using the general Warburg test. They grew Legtococus faecalis in a medium containing 1/i/l glucose supplemented with 1 part glycerol (10 g/l) in order to obtain cells that rapidly oxidize glycerol in their assay. It's all clear that it will. α-Glycerosulfate oxidase activity is not described.

Several of the aforementioned publications generally report studies on the oxidation of glycerol. The first two of these documents, namely “Kodlt@ehek and Umbr
eit” and “J a c ob! + and VB
n l) Only emark' indicates the presence of α-glycerophosphate mexidase in their assay. However, none of the literature indicates that α-glycerophos-7 ethoxidase can be produced in unexpected commercial yields by growing bacteria in combination with (1) pyruvate, and preferably (2) glucose. Not yet. Other documents do not even mention the presence of α-glycerophosphate oxidase in the microbial strain.

The purpose of the present invention is to produce α-glycerophosphate mexidase in high yield by growing microorganisms in a medium-quality manner.

The method for producing α-glycerophos 7ate oxidase according to the present invention comprises Strezotococcus ≦W=yi2. By growing members of the genus Lactobacillus and Leuconostoc
The entire production of -glycerophosphate oxidase is characterized in that the -glycerophosphate oxidase is grown in a medium containing pyruvate, and then α-glycerophosphate oxidase is extracted.

Specific explanation of Ni composition and effectiveness of the invention In addition to the above-mentioned components, it is also preferable that the medium contains glucose, and further contains yeast extract, trinoton, and 2HPO4.
In a further preferred embodiment of the invention, inorganic salts and vitamins can be added to the medium to significantly increase the yield of α-glycerophos7ate oxidase.

Specific species of microorganisms that can be used in carrying out the present invention include Z]±! -shi-zf) Cocus of the genus Δ
Streptococcus faeca
lii) s leg cocus ri1 morris (Stre
ptoeoecus cremoris), Streptococcus sa1 I des (Streptococcus
gallvarlu+s), stregtococasus five-engineering f
') Soil (Streptcoecus faecium)
; Lactopasillus grantherum of the genus Lactoba yyx (
Laetobacillus plantarum),
Lactobacillus-/7-1=Δ(Laetobacillus
lus easel), Lactobaelllus delbru@cki
S, λri1ヱ is planned 5 Z 7 − j 711− (
Lactohaeillus f ermen t
um), Lactopacillus benthositeka Z (La
etobacillus psntoaeetieus
), Lactobacillus lactis
lus 1actXi 59) LaetobaClllus buchn@r, Lactobacillus
usleiehmannii);
onostoemesenteroides) and Vediococcus seritypee (Pe
dloeocausc @rswisia
, and Gunsalus, 1. "Manufacture of active resting cells of St. flathead" by C. (Journal of Bacteriology, Vol. 44, pp. 333-341 (1942))
rs'rp medium, as described in . It grows normally in medium. As described below, in the STP medium, Tregutococcus faecalis ATCC 12755
When grown, 60-80 U/A of α-glycerophosphate oxidase is produced.

Stregtococcus soecalis AT as described herein
The microorganism named CC12755 was first developed by ATCC (A
merican Type Culture C
It was deposited in the ATCC catalog (1974, 11th edition) under that name as Stregtococcus faecalis ATCC 12755 (!).
The name has been changed to 2755.

According to the invention, said microorganism, in particular Strenotococcus
Faecalis pyruvate as a total carbon source and preference 1
Alternatively, α-glycerophosphate oxidase can be recovered in high yield by increasing the concentration in the modified STP medium by adding glucose.

8 named by WOOd and Gunsalus
TPts quality ij: , glucose-1,01/l,
Yeast-T4-su-10g/l, Torinoton 10
F! /l and 2) IPO4-5g/l'Ac included. A useful range of these ingredients is about 0.1 to 3.
011/it1 yeast extract about 1.0-20 p/l, preferably about 2.0-10.17/l.

about 5 to 201 tons/l and at least about 3.0 g/l of 2HP04, preferably from about 3.0 to about 5.01
/l was confirmed.

Other protein melts that can be used in place of Trinoton in the growth medium V include, for example, 5heffl.
eld Chemical Dlv, of Kraf
Soy Pegton Type T (Soy Psp) commercially available from Tao Corp, (Union, New Chassis)
ton@Type T), Erlamin
) Type T1 Farm Amine (Fe+rm Am1
n@) Type I, II, III and IV
, N-Z amine (Amin@) type AT, BT
, ET and YTT ; Cudahy Laborat
(i) Commercially available casein hydrolysates
ate), Anatone, Mlerobiotone and Pharmatone: Traders Pr
otln DLv, Trad@rsOLl Mill
Pharmamedlg, commercially available from Co., Ltd. (Dexus, Fort Worth), as well as ammonium sulfate.

However, if one of the protein lysates described above is used in place of tryptone in the growth medium, the optimum of the protein lysate may vary somewhat from that of tryptone. A particularly useful alternative to tryptone is Soy Pezoton TyfT, which increases α-glycerophosphatoxidase status by more than five times compared to when using tryptone.

Other useful carbon sources that can be used in place of glucose in the growth medium include, for example, citric acid, lactic acid,
Sodium acetate, sodium succinate, ame/4' lachic acid, glutamic acid, corn syrup, corn syrup, molasses, fructose, lactose, maltose, and sea rose.

In the most preferred embodiment of the invention, about 0.5 to about 3.0 g/13 of glucose and about 0.5 to about 2.0 g/13 of sodium pyruvate are present in the growth medium. Add Ojl/13 k16i. The combination of glucose and pyruvate sodium in the growth medium appears to have a complementary effect on the production of alpha-glycerophosphate oxidase. When using this combination of carbon sources, the enzyme yield increased by a factor of as much as 8 times compared to when glucose 19/11 was used as the carbon source.

It has also been found to be advantageous to add effective amounts of inorganic salts and vitamins to the growth medium.

As used herein, the term "effective amount" refers to a sufficient amount of inorganic salts and vitamins added to the growth medium to increase the yield of the enzyme. The addition needle for such inorganic salts and vitamins varies depending on the types of salts and vitamins. This dish generally has a small number of nests and can be very easily ground by those skilled in the art through routine testing.

When growing microorganisms such as Stregutococcus noecap in large-scale fermentation groups to obtain enzymes,
Foaming often occurs. To control this foaming,
In particular, when producing enzymes in large patches, it is preferable to use a foam control agent.

One example of a foam control agent useful in the practice of the present invention is Polyglyaol JP-2000, available from Dak Chemical Company (Midland, Mich.). It can be used in amounts up to 0.59/l in the medium.However,
A typical volume of about 0.1 M/l is sufficient to control foam. Other foam control agents (defoamers) can also be used. The criteria for its selection and use is that it does not inhibit enzyme synthesis at a level of control that controls foaming.

The microorganisms used in the present invention can be grown in a reasonable temperature range to produce α-glycerophosphate oxidase. Good results can be obtained in a temperature range of 25-42°C. Best results are achieved at a temperature of about 30°C. After growing the cells, glycerophos 7ate oxidase can be extracted according to conventional methods.

The following conditions were used in the following example, illustrating the practice M' of the present invention.

(2) Strezotococcus faecalis ATCC 12755 was used in Examples 1 to II under dark culture.

2. Medium a) Culture Preservation Medium For culture preservation, Micro As5ayCult was added in 0.2% glycerol.
ure agar) (Difco Laborator
ies, Detroit, Michigan) f: used.

h) Fermentation medium (1) STP @ material (Wood & Hi I/11Gu
nsalus, 1942) Glucose ■0 Yeast extract 10.0 Trizotone
10.0 to 2HPO45.0 Distilled water Amount below 1e for the whole company (11) Store 100% L'E Gu, 1/ass 1,0 Trizoton 10.0 Yeast extract
10.0 to 2HPO45.0 Glycerol 2.0 Distilled water
Amount to make the total amount 11 (fil) Improved STP I
Sodium pyruvate - 2l/l 2.
0 Yeast Extract 2.0 Trigton 1040 to 2HPO45・O Glycerol 2.0 Distilled Water
Shun (IV) to make total 1t11 Improved STP l-% Quality-31713 Pyruvine Vinegar Sodium 2.0 Yeast Extract
2.0 trizoton
10.0 to 2HPO45.0 Glycerol 2.0 Salt Solution C5.0 Vitamin Solution 1.0 Distilled Water
Adjust the total amount to 11 Lid (V) Dorongyoku ■ 21 Sodium Sodium Kebyrubate 2.0 Yeast Extract
2.0 Trizotone 10.0 to 2HPO45.0 Glycerol 2.0 NordSolution PY8 20m/vitamin solution g
1. Oml distilled water Volume to 11 (vl) Improved STP medium - 5g/l Glucose 2,0 Sodium pyruvate 2.0 Yeast extract
2.0 tryptone 10,0
K2HPO45.0 Glycerol 2.0 Salt Solution pyS20.0m Vitamin Solution
LOme distilled water Amount to make total 11 & l [) Improved STP 8% -611/1 Sodium pyrubin 2.0 Yeast extract
2,0 trizoton
10.0 to 2HPO45.0 Glycerol 2.0 Salt Solution, PY8 5 me Vitamin Solution
1 inasu e distilled water total, t 11
plate (nll improved STP medium-7yZl pyruvate natnatri 9m 2.0 yeast extract
2.0 tryptone
10.0 to 2HPO45.0 Glycerol 2.0 Salt Solution PYS 2.5 m Vitamin Solution
1-Tap water Amount to bring the total volume to 14 C) Salt solution/ (1) Salt solution/A Par with Part 1 below) 2ff: Mix equal volumes and add salt solution A to 1. Made by H.

Nozo-to 1 False 5≦!
L engineering: engineering yJ, tzo IJ3= and (,2-Mg504-7
H20100,0 FeS04'7H2010,0 Mn504#H201,O NaMo 04 ・2H200,5 0,I NHCL Amount to make the total amount 11) and -t 2
g/jlCa CL 2
10-0 Distilled water Amount to bring the total volume to 11 (II) Salt Sorieci, N CJ
/Height 1Mg5O4-7H2025,0 CaC12''2H200,1 FeS04'7H202,8 MnSO4'H201,7 ZnS04'7H200,06 NaCA
O,6011n 7xh)') --> 7PY
S f//l! [°2", 鑵-J-Fchitsu... 00 ,75 CoC12"2H200,2 N!12M0O4' 2H200,l NaB4O7@l0H200,1 Distilled water Quantity to set the panoramic view to 11 - Insect LL Om
Night 5/L Thiamine/HCA
200p-aminobenzoic acid
200 Pyridoxine-H, CL 2
00 Riboflavin 200 D-pantothenic acid (calcium salt) 200 Folic acid 2.0 Biotin
2.0g Flavin is dissolved in the solution by heating. This vitamin solution was sterile filtered and added to the medium after sterilization.

3. Preservation of culture Cultures were preserved on microassay culture agar punctures containing 0.2% glycerol. Cultures were transferred onto fresh punctures at least once a week. After incubation at 30°C for 48 hours, the punctures were stored at 4°C. Another method for preserving living organisms is storage in liquid nitrogen. For this purpose, the culture is STP
Grow in medium for 0 hours. The cells were then dissociated and treated with Allon's salt solution solution (A11e).
n+M*BT Archives of Microbiology, Vol. 32, p. 270-, p. 277, 1959).

A small amount of this suspension was added to a sterile glass ampoule, which was then sealed and stored in a liquid chamber.

Culture Ji2 grown for 48 hours in microassay culture Agar
50 Wf, 50 ml in an Erlenmeigy flask,
Transferred into STP medium or modified STP medium-2. The flask was shaken at 30°C and 200 rpm. After 20 hours of incubation, the contents were transferred to a Sorvall refrigerated centrifuge (RCIIB model, DuPont Instruments Inc.,
Newtown, Connecticut) at 4°C, 12,0
Centrifuged at 00Xg for 15 minutes. The shoe fluid was discarded and the cells were resuspended in the same volume of sterile water. This suspension was used as an inoculum.

b) Preparation of the enzyme 25 ml of fermentation medium in a 250 ml five-cell flask was inoculated with 1.5 ml of the inoculum prepared as described in section 1 (4, a). 2 at 30℃ in total flask
It was pounded at 0.00 rpm. After shaking the flasks for 20 hours, a 2.5 ml sample was taken from each flask. This sample was placed in a Sawpal refrigerating centrifuge for 19 minutes.
．． Centrifuged at 00 oxg for 15 minutes.

This whole cell was resuspended in 2.5 liters of deionized water.

This suspension was diluted 10 times. This diluted suspension was used for determination of dry cell weight and α-glycerophosphate (α-G
P) Used for oxidase assay.

5. Large-scale fermentation a) Preparation of inoculum 1501 Inoculum for the fermenter was prepared in the following three ways.

(1) 500d (7) Improved STP *Quality-2efi
tr6 2.81 Fernpach flasks with 0.2
% glycerol and grown for 48 hours by puncturing microassay cultured Agar! The upper three sides were inoculated with 7x J. lily ATCC 12755'fc. 1 puncture each 7
Erumpa 1. It was used for inoculation of half flasks. 3 flasks
Shake at 125 rpm at 0°C. After 20 hours of warming, the contents of the flask were transferred to a refrigerated centrifuge (RCIIB model, DuPont Instruments, Newtown, CT).
Aseptically centrifuge at 600 oxg for 15 minutes. The supernatant was discarded and the entire cell pellet was resuspended in sterile distilled water. Total urine and 500 ml of distilled water were used to resuspend the cells from 6 flasks. This cell suspension was
It was used to inoculate the Oe fermenter.

(2) This preparation method is the same as above except that the contents of the flask were not centrifuged. The entire fermented broth from 6 flasks was used in the 1501 fermenter fermenter.

(3) This inoculum preparation method was used to prepare most of the inoculants for the experiments described herein. A 50 ml 250 me Erlenmeigie flask containing modified STP IN quality-2v, 1 was inoculated with Stref0 Tococus faecalis ATCC 12755 grown for 48 hours by puncture of microassay culture agar supplemented with 0.2% glycerol. The flask was shaken at 30°C and 200 rpm. After shaking for 12 hours, the flask contents were used in a 141 fermenter. Before inoculation, the fermenter was charged with 10/1 of the desired fermentation medium and sterilized for 1 hour. The medium was cooled to 30°C and inoculated as described above. 1300 r using this medium fc 3 blade turbine type impeller.
Stirred at pm.

A ring sparger was used for ventilation. Air current is 0.2WM (volume per unit volume of medium per minute)
, Mochi” 21! /min. This is 8 in the shape of a bar.
Mass transfer coefficient KL-af of m1n: given.

After 121 td, the whole broth was used to ferment the 1501 fermenter. Culture purity was monitored throughout the development of the inoculum. Microscopic examination and plating techniques were used for this purpose.

b) Production of α-GP oxidase A 15 ol fermenter was charged with 100 A of suitable fermentation medium,
Sterilized for 1 hour. The medium was cooled to 30'C. This was inoculated by aseptically transferring the contents of a 14A fermenter prepared as described in method (3) above. This medium is passed through a turbine-type impeller with three thousand blades for 25 or
The mixture was stirred at m and aerated using a ring-shaped gas jar. Air flow rate was 0.18 WM or 201 air/min. The value of KL'a under such conditions is 1.15 m1n-1
I was disappointed. The temperature was maintained at 30°C. Samples were aseptically removed hourly using an automatic sunglasser (211 Planswick Scientific, New Brunswick, New Chassis) to measure cell growth and α-GP mexiguse production. Dissolved oxygen concentration is measured by membrane electrode (I
L530, industrial oxygen measurement system, Sensor Labs,
The Division of Instrumentation was supervised by Radley's Corporation, Lexington, Massachusetts). Also, Ingold electrode (type 764-31B,
PII was also measured by Dr., W., Ingold & Co., Kürich, Switzerland). When the oxygen level reached the desired value, the fermenter was cooled to 10°C with cold water and the cells were collected in a refrigerated continuous centrifuge (Model 281G, Karl Padauberg GMBH-, West Germany). .

Average fermentation time was 9-10 hours.

6. Measurement of dry cell weight A calibration curve showing the relationship between dry cell weight and absorbance at 660 nm was prepared. Sp@etronie 20Spect
The absorbance was measured using a rophotometIIIr (Palash & Rohm, Rotinister, Ny-York), and the amount of dry cell nails was calculated from the calibration curve.

Assay of Activity α-GP oxyguse activity was determined by measuring peroxidase-catalyzed oxidation of the leuco dye by H2O2 released upon oxidation of α-glycerophosphate by α-GPffl oxidase. One unit of oxygen activity (U) is defined as the amount of oxygen that converts 1 μmole of substrate to product per minute at 37° C. and pH 7.0.

a) Reagent (+) KP Puffer 20. 1M potassium phosphate buffer, 17.0 (11) Substrate solution: Dissolve 17.288 g of α-glycerophosphate in 100 ml of KP buffer, 01
1) Dye solution: 100 ml O deionized, 3 to 47 ml water
,3'-dimethoxy-pentidine dihydrochloride (
Dissolve Triton X-100 (Roam and
(■) Buffer solution: Horseradish vermexidase type 13.3 mi in KP buffer 50 me.
, l and add dye solution 1.1+++/! and detergent solution 66- were added, and the total volume was diluted to 10% with KP buffer.
Set to 0-.

h) Method: 1 ml of the buffer solution in test 1 and the substrate solution was equilibrated for 15 minutes at 37°C in a water bath shaker (221 Prinswick Scientific, New Plantswick, New Chassis). . This sample was diluted to contain 5-25 mU of α-GP, 4-oxidase. 1 ml of the correctly diluted sample was added to the equilibrated tube. A blank was prepared with deionized water 1- in place of the sample. The tubes were shaken at 37°C in a water bath shaker. Color development was measured every 5 minutes for 1 hour at 4301 m using a Spectronic 20 spectrometer.

The extinction coefficient for the dye was determined and used to convert the optical density to the concentration of the gold-utilized substrate.

8. Determination of mass transfer coefficient (KL-IL) The mass transfer coefficient was determined by monitoring the dissolved oxygen concentration and outlet oxygen setting. Dissolved oxygen concentration is determined from 5. above. Term (b) (α-GP,
It was determined using the membrane electrode described in (Production of 4-oxidase).

The outlet acid accumulator is magnetic analysis fit (type CA150, v
-gyt Cusco/Trows Limited, Cloborrow, Sussex, UK).

The following relational expression was used for the calculation of K (, @a.

NA = KL ``a (C'' CL ) In the formula, NA two-matter transfer rate L = Bfa mass transfer coefficient a = interfacial area CL: saturated concentration of dissolved oxygen CL = instantaneous dissolved oxygen concentration Example 1-5 is the above-mentioned small Illustrate the use of scale techniques.

Example 1: Induction effect of glycerol Addition of glycerol has a revolutionary effect on enzyme production. The greatest fibrosis results were obtained in a medium containing 2 g/13 glycerol (see Table 1).

Furthermore, increasing the glycerol at 0 degrees did not increase the enzyme yield. Culture growth was not significantly affected. ν11, while the amount of dry cells M increased by about 20 degrees in each case, it was more than three times as large in the case of enzyme extractor J.

Table 1 0.0" 0.34 62 100
2.0 0.41 205 3305.
0 0.38 203 32810.0
0.34 178 287a: Control cultures were STP supplemented with glycerol at the indicated times.
It was grown in a medium.

As shown in Table 2, the growth of the culture was completely dependent on the concentration of glucose. Compared to the recorded cells obtained in glucose-free medium, the amount of dry cells increased threefold when 3 Vl of glucose was added. Glucose has a powerful effect on the production of α-GP and 3-gysidase! In the absence of glucose in the medium, a production drop of 38 g was observed. The amount of enzyme produced increased as the glucose concentration increased up to 1.01/it of glucose a. As the glucose concentration further increased, the amount of α-GP4 oxidase produced decreased.

0 0.22 52 620.5
0.29 62 761.0aO
, 3282100 2,00,5730' 36 30 0.61 39 47a = Control Cultures were carried out in STP medium as described above. The amount of glucose was varied as indicated.

b) Effect of Sodium Pyruvate As shown in Table 3, the growth and production of α-GP oxidase was significantly improved by replacing glucose with sodium pyruvate.

However, high concentrations of pyruvate above 3 g/13 reduce enzyme production. Improved STP *Quality-2゜3.
4, the carbon source was replaced with glucose at an optimum a degree of 2.0 g/13. Organisms were grown in modified STP medium-1 as described above, except that the glucose in the medium was replaced with IJ at the indicated concentration of pyruvate.
No blank space below for table 3 0.0 0.19 176 88um// 1.0 0.46 3
86 193tr 2.0
0.60. 484 242p
5.0 0.55 314
157a: Control C) Combination effect of glucose and pyruvate As shown in Table 4, glucose 2.01/il and sodium pyruvate 2.0 g/1. When -f was co-used, the growth of the culture was increased and, surprisingly, it was noted that at the same time, the amount of enzyme produced was significantly increased. This improved STP
A modified medium called Medium-5 is the preferred medium of the present invention.

Table 4 0 0 0.33 237
300 0.5 0.48 314
400],,0 0.63
384 480 2.0' 0
．． 58 794 1000.5 0
0.51 282 360.5
0.5 0.68 412 520.
5 1.0 0.80 580
730.5 2.0 0.74 105
2 1331.0 0 0.49
194 241.0 0.5 0.
81 500 631.0 1.0
0.81 540 681.0
2.0 0.79 1069 1352.0
0 0.62 418 53
2.0 0.5 0.95 490
622.0 1.0 0.99
651 822.0 2.0 1
．． 00 1539 1943, (l O
O,72273 3,00,51,0531540 3,01,01,1336946 3,02,01,06998123 a: Control cultures were grown in modified 5TPiJ-4 at the above glucose and sodium pyruvate concentrations.

Example 3: Effect of vitamins Vitamins do not affect the growth of the culture, but they stimulate the production of enzymes by more than 30% (see Table 5).

The increase in enzyme production is not proportional to the increase in vitamin concentration.

Table 5 0.0' 0.64 766 1001.0 0.6
3 808 106 2.0 0.66 939 123 3.0 0.63 882 116 5.0 0.62 908 119 10.0 0.65 1011 133a: Control culture grown in improved STP medium*-3 Ta. The concentration of the vitamin solution was varied as shown in the table. Regarding the composition of the vitamin solution, refer to the above-mentioned r 2. (d) Male tamine solution”
See section.

Example 4: Effect of inorganic salts The promotion of enzyme production by the addition of trace elements showed that trace elements are the other limiting nutrients in our fermentation media. Trace elements are elements that a culture requires in small, trace amounts of PPM order or less. The culture requires trace amounts of many elements, and the more these are supplied, the better the enzyme yield. 6th A, 6
The results for B and 6Cfe are the salt solution C,l
:! Salt solution PYS, which contains more trace elements, is better than salt solution C in improving the production of α-GP,4-oxidase. For the same reason, this salt solution +) x
-', y"yA'""j ('HIV; 6. Below.

Table 6A 0.0aO, 49439100 0,50,62630143 1,00,59598136 2,50,60669152 5,00,61729165 10,00,64747170 a: Control culture was prepared using Salt Solution Chiron C at the concentration shown in the table above. were grown in improved STP i-2 supplemented with

Below all f1 No Table 6B” -0,57541100 Salt Soryu 5.0 0.57 1037
192-John C Salt Solution 2.0 0.61 8
37 1555-John A a: Control b: Optimal concentration of Salt Solution A The medium used in this culture experiment was a modified STP medium supplemented as shown in the table above.
It is 2.

Below, there is no table of 6C of the whole Cretaceous period' 0.62 582
100 Salt Soleil 5.0 0.63
811 140-John C Salt Solution 5.0 0.69 9
23 158-Siya YS IQ, 5 0.70 1000
171// 20.0 0.67
978 168/I 50.0
0.75 1130 194a: Control, these flasks contain modified STP media not named Salt Solination C.

Modified STP medium-3 was used in this experiment. Salt solution C was replaced with a degree salt solution Δ-Sion PYS shown in the table above.

Example 5: Effect of vitamins and trace elements As mentioned above, enzyme production is increased when the medium is supplemented with either a vitamin mixture or various mixtures of one trace element. When both this vitamin solution and Salt Soli-John C were added, mutual effects as shown in Table 7 were observed. There was no significant effect on culture growth.

eLa 0.60 530 100
a: Control cultures were grown in modified STP medium-2. To this medium were added the additives shown in the table above.

Examples 6-11 below show 1
An example using a No. 501 fermenter was shown.

Example 6: P! j: Effect of starter size The three methods of inoculum preparation described above were tested.

Inoculum size (6-10A) had no effect on enzyme production or culture growth, but increasing inoculum size decreased the time required to reach maximum growth and enzyme production by 30 microns. Diminished. The results shown in Table 8 showed that whole broth was a satisfactory inoculum compared to centrifuged and resuspended cells.

61 centrifugation 0.61 10.5 837
11.361 Non-centrifugal 0.65 9.25
787 9.25 Improved STP medium-3 was used in this trial.

Details of the method are as described above.

Example 7: Effect of Glycerol Increasing the glycerol concentration beyond 21,711 did not reduce the improving effect on enzyme yield per liter in flasks or large fermenters. However, for cell growth in large fermenters, the glycerol concentration is 21/
The effect of increasing beyond l appeared. For flasks/experiments where no effect on growth was observed,
As shown in the table, culture growth in the fermenter increased by 48%.

The optimal concentration of glycerol for enzymatic synthesis is approximately 21/l
Out.

Table 9 2.0 ao, 63 1054 3.0 0.93 1039 a: Control In this 1501 fermenter experiment improved STP medium-6'
f:, used. The glycerol concentration was changed as shown in the table above. The inoculum was prepared in a 147 fermenter.

Example 8: Effect of glycerol analogs We have shown that α-oxidase is an inducible enzyme and is induced by glycerol. .

Since glycerol is a precursor of the substrate, α-glycerophosphate, and is a readily available carbon source, its concentration must decrease during fermentation. Therefore, it would be useful to find an inducer whose concentration does not decrease during fermentation, ie a free inducer. For this reason we tested a number of glycerol congeners and monoglycerides.

The following experiments were conducted in shake flasks. Induction of α-GP oxidase was obtained for all glycerol analogs tested (see Table 10). When glycerol was removed from the medium, the amount of enzyme was reduced to 38% of the control. This bond showed that when all glycerol congeners were added, with the exception of ethylene glycol, 50% of the control
It increased dramatically. Ethylene glycol is used in the synthesis of enzymes (i-
strongly suppressed.

No Table 10 0.31 38g'
J Sel0-b O,0020,41100 Ethylene glycol 0.032 0.30
172-methoxy-1,20,0190,304
9-Propanediol 1,3-Glopanediol 0.026 0.30
42-=le 1.2-gropangio 0.026 0.30
47-l2,3-butanedio-0,0220,3052l12,4-butanetri 0.019 0.31
19-ol monoacetin 0.015 0.40
981-monoglobionin 0.13
0.38 701-Monobutyrin
0.012 0.37 92 Monostearin 0.006 0.34
34 monomelain 0.006 0
．． 28 41 Trilawarin 0
．． 003 0.32 46a: All glycerol congeners tested at 2.011713.

b = Control culture was grown in modified STP medium-1 supplemented with 2 g/It yeast extract. Glycerol was substituted as shown in the table above.

Example 9: Temperature effect Decreasing the fermentation temperature from 30°C to 25°C reduces enzyme production by 22°C.
% and increased growth only slightly. However, lowering the temperature had a surprising effect on the time required to reach maximum growth and enzyme production. That is,
It was twice the value at 30°C at 25°C (Table 11 Table 11 30' 0.63 6.5 1054
6゜525 0.71 12.0
822 13. Oa: The control medium used is modified STP medium-6, and the inoculum size is i.
I got it in oz.

Example 10: Effects of the Hento of Muroyu Gen We evaluated other Yamoto sources instead of tryptone. We have found that trigticase (trlptease) peptone is a good basis. The production of α-GP oxidase in media containing trigticase peptone was comparable to that in media containing tryptone. However, trigticase peptone did not support Strezotococcus growth as well. The results were as shown in Table 12.

Table 12 *Products from Difco Laboratories (Detroit, Michigan)*tBBL% Products from Dickinson Company (Kottkeisville, MD) The medium used in these studies was Modified STP Media-7. there were. Tryptone was replaced with the same concentration of trigticase peptone. The seeds were grown in a #141 pot.

Example 11: Kinetics of α-GP mexidase production The results showed that production of the enzyme clearly correlates with growth. FIG. 1 shows the growth of modified STP medium 4 during fermentation, α-GP, 4
Oxidase production, dissolved oxygen concentration and pH are shown. Growth reached maximum at 6 hours and enzyme production reached maximum at 9 hours. The enzyme concentration remains stable for approximately 2 hours after reaching a maximum.

The preferred fermentation time is 6 to 12 hours. ◎ Results when using other Lactopasirachae H microorganisms listed in Table 13 in place of Streptococcus faecalis ATCC 12755, α-glycerophosphate We conducted an experiment to produce ogisidase.

The medium used was modified STP *quality-7, and the cultures were grown at 30°C and 37°C. The results are shown in Table 13.

Continuing from page 1 of table 13 in the margin below (1> Inventor Charles Thomas Gutsdepiyu 89 High School Spring Circle, Rotchester, New York, United States of America

Claims (1)

[Claims] 1. In producing α-glycerophosphate oxidase by growing members of the genus Stregutococcus, Bedeococcus, YILL-ZZ-presenting and Leuconostoc, growing the members in a medium containing pyruvate; A method for producing α-glycerophosphate oxidase, which is characterized in that α-glycerophosphate oxidase is then extracted.