GB1585909A - Method of producing -glycerophosphate oxidase - Google Patents

Description

(54) A METHOD OF PRODUCING -GLYCEROPHOSPHATE OXIDASE (71) We, EASTMAN KODAK COMPANY, a Company organized under the Laws of the State of New Jersey, United States of America, of 343 State Street, Rochester, New York 14650, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The invention relates to a method of producing a-glycerophosphate oxidase. a-Glycerophosphate oxidase and useful techniques for its preparation and extraction have been described by Koditschek, L. K. and Umbreit, W. W., "a Glycerophosphate Oxidase in Streptococcusfaecium F24", Journal of Bacteriology, Vol. 98, No. 3, P. 106368 (1969) and by Jacobs, N. J. and Van Demark, P. J., "The Purification and Properties of the a-Glycerophosphate Oxidizing Enzyme of Streptococcusfaecalis, 10CI," Archives of Biochemistry and Biophysics, Vol. 88, p.

250-55(1960). The enzyme is useful for the oxidation of a-glycerophosphate in the presence of oxygen to produce dihydroxyacetone phosphate and hydrogen peroxide.

Koditschek and Umbreit, supra, describe the properties of a-glycerophosphate oxidase in Streptococcusfaecium F24 using for their study various assay techniques including the monometric assay. Cultures of Streptococcusfaecium were maintained on stabs of AC agar consisting of 1% tryptone, 1% yeast extract, 0.5% K2HPO4, 0.1% glucose and 1.5% agar (sometimes referred to as AC medium below). The cells were separated from this medium and enzyme preparations were extracted for assay.

Jacobs and Van Demark, supra, who also used manometric assay techniques in their study, describe the properties of a-glycerophosphate oxidase in Streptococcus faecalis lOCi. They grew the Streptococcusfaecalis in an AC medium as described above but having 0.2% glucose. They report that the enzyme from Streptococcus faecalis is highly specific for L-a-glycerophosphate as a substrate with no demonstrable activity on p-glycerophosphate, glycerol, dihydroxyacetone phosphate, or 1,2-propanediol phosphate.

According to the present invention, high yields of a-glycerophosphate oxidase are obtained by growing a member of the family Lactobacillaceae, preferably Streptococcusfaecalis, in a medium comprising a pyruvate as a carbon source, and an inducer for a-glycerophosphate oxidase and extracting the a-glycerophosphate oxidase.

In addition to the above ingredients, it is preferred that the medium also contain glucose and additionally may beneficially comprise yeast extract, a hydrolysed protein, e.g. tryptone, and K2HPO4. In the preferred embodiment of the invention the medium containing a mixture of glucose and a pyruvate as carbon sources has a synergistic effect on the production of a-glycerophosphate oxidase.

In addition, inorganic salts and vitamins may be added to the medium to increase significantly the yield of a-glycerophosphate oxidase.

Among the species within the family Lactobacillaceae which may be employed in the practice of this invention are: from the genus Streptococcus: S. faecalis, S. cremoris, S. faecium, S. salivarius; from the genus Lactobacillus: L. plantarum, L. casei, L. delbrueckii, L. fermentum, L. pentoaceticus, L. lactis, L. buchneri, L.

Ieichmannii; from the genus Leuconostoc: L. mesenteroides; and from the genus Pediococcus: P. cerevisiae.

Of the species, S. faecalis is preferred and, most preferred, is S. faecalis ATCC 12755.

Streptococci are commonly grown in a medium called "STP Medium" herein, which is described by Wood, A.J. and Gunsalus, I.C., "The Production of Active Resting Cells of Streptococci", Journal of Bacteriology, Vol.44, p. 333--41 (1942).

Details are given below. When growing Streptococcusfaecalis ATCC 12755 in the STP Medium, which is described below, 6080 U per litre of a-glycerophosphate oxidase is produced.

The microorganism mentioned in the preceding paragraph and elsewhere in this specification is that which was originally submitted to the American Type Culture Collection as Streptococcus faecalis ATCC 12755 and which was listed in their catalog under that name up to the Eleventh Edition in 1974. This same strain was renamed Streptococcus faecium ATCC 12755 in the Twelfth Edition of the catalogue.

The inducer for a-glycerophosphate oxidase is preferably glycerol. The addition of glycerol as the inducer has been found to increase the yield of aglycerophosphate oxidase by as much as 3 times that obtained without it. Other useful cr-glycerophosphate oxidase inducers are glycerol analogues which include, for example, 3-methoxy-l,2-propanediol, 1,3-propanediol, 1,2-propanediol, 2,3butanediol, 1,2,4-butanetriol, monoacetin, l-monopropionin, l-monobutyrin, monostearin, monoolein and trilaurin.

A useful amount of inducer has generally been found to be in the range of from 1.0 to 10 grams per litre of medium. Preferably from 2.0 to 5.0 grams per litre of inducer is used.

The STP Medium as described by Wood and Gunsalus contains glucose (1.0 g/litre), yeast extract (10 g/litre), tryptone (10 g/litre), and K2HPO4 (5 g/litre). Useful ranges for these ingredients in the present method have been found to be: glucose, from 0.1 to 3.0 litre; yeast extract, from 1.0 to 20 g/litre, preferably 2.0 to 10 litre; tryptone, from 5 to 20 g/litre; and K2HPO4, at least 3.0 litre, preferably from 3.0 to 5.0 g/litre.

Other protein hydrolysates which may be employed in the growth medium in place of tryptone specified above include, for example, Soy Peptone Type T, Edamin (Type T), Ferm Amine Types I, II, III and IV, N-Z Amine Types AT, BT, ET and YTT, all available from Sheffield Chemical Div. of Kraftco Corp.; Union, New Jersey; Casein Hydrolysate; Anatone, Microbiotone, and Pharmatone, available from Cudahy Laboratories, Omaha, Nebraska; Pharmamedia available from Traders Protin Div. Traders Oil Mill Co., Fort Worth, Texas and ammonium sulphate. It will be appreciated, however, that when one of the above protein hydrolysates is substituted for tryptone in the growth medium, the optimum amount of such protein hydrolysate may be somewhat more or less than the optimum amount of tryptone. A particularly useful substitute for tryptone is the Soy Peptone Type T which has increased yields of a-glycerophosphate oxidase to more than 5 times that obtained when using tryptone.

Other useful carbon sources which can be substituted into the growth medium in place of glucose include, for example, citric acid, lactic acid, sodium acetate, sodium succinate, aspartic acid, glutamic acid, corn syrup, molasses, fructose, lactose, maltose, and sucrose.

In a most preferred embodiment of the invention, 0.5 to 3.0 litre of glucose and 0.5 to 2.0 litre of sodium pyruvate are added to the growth medium. The combination of glucose and sodium pyruvate in the growth medium appears to have a synergistic effect on the yield of a-glycerophosphate oxidase. When using this combination of carbon sources the yield of enzyme has been increased up to 8 times that obtained when using 1 gm/litre of glucose as the carbon source.

It has also been found advantageous to add inorganic salts and vitamins to the growth medium in an amount effective to increase the production of enzyme. The exact amount of such salts and vitamins to add depends upon the types of salts and vitamins. This quantity is generally small and can easily be determined by those having skill in the art by routine experimentation.

When growing microorganisms such as Streptococcus faecalis to produce high yields of enzyme in a large scale fermentor, foaming is often encountered. In order to control such foaming, the use of a foam control agent is advisable. One such foam control agent found useful in the practice of this invention is Polyglycol P-2000, available from Dow Chemical Co. (Midland, Michigan). Up to 0.5 g/litre of this antifoam may be used in the . growth medium without inhibiting the production of enzyme. Generally, however, about 0.1 litre has been found sufficient to control foaming. Other foam control agents can also be used; the main criterion for selection and use being the lack of inhibition of enzyme synthesis at a concentraion level that will control the foam.

The microorganisms may be grown over a reasonable range of temperatures to produce a-glycerophosphate oxidase. Good results can be obtained in a temperature range of 2542"C. Best results have been achieved at a temperature of 30"C. After the cells have been grown, the a-glycerophosphate oxidase can be extracted by means of conventional techniques.

In the following examples which are presented so that the practice of the invention may be better understood, the following definitions apply: 1. Culture Streptococcusfaecalis ATCC 12755 was used for all the experiments described in Examples I11.

2. Media a) Culture maintenance medium. Micro Assay Culture agar (Difco Laboratories, Detroit, Michigan) with 0.2% glycerol was used for the culture maintenance. b) Fermentation media g/litre i. STP Medium (Wood and Gunsalus, 1942) Glucose 1.0 Yeast extract 10.0 Tryptone 10.0 K2HPO4 5.0 Distilled water to 1 litre ii. Modified STP Medium -- 1 g/litre Glucose 1.0 Tryptone 10.0 Yeast extract 10.0 K2HPO4 5.0 Glycerol 2.0 Distilled water to 1 litre iii. Modified STP Medium - 2 g/litre Sodium pyruvate 2.0 Yeast extract 2.0 Tryptone . 10.0 K2HPO4 5.0 Glycerol 2.0 Distilled water to 1 litre iv. Modified STP Medium -3 g/litre Sodium pyruvate 2.0 Yeast extract 2.0 Tryptone 10.0 K2HPO4 5.0 Glycerol 2.0 Salt solution C 5.0 Vitamin solutiion 1.0 Distilled water to I litre v. Modified STP Medium - 4 g/litre Sodium pyruvate 2.0 Yeast extract 2.0 Tryptone 10.0 K2HPO4 5.0 Glycerol 2.0 Salt solution PYS (See below) 20 ml Vitamin solution 1.0 ml Distilled water to 1 litre vi. Modified STP Medium -5 g/litre Glucose 2.0 Sodium Pyruvate 2.0 Yeast extract 2.0 Tryptone 10.0 K2HPO4 5.0 Glycerol 2.0 Salt solution PYS 20.0 ml Vitamin solution 1.0 ml Distilled water to 1 litre vii. Modified STP Medium - 6 litre Sodium Pyruvate 2.0 Yeast extract 2.0 Tryptone 10.0 K2HPO4 5.0 Glycerol 2.0 Salt solution PYS 5 ml Vitamin solution 1 ml Distilled water to I litre viii. Modified STP Medium-7 g/litre Sodium Pyruvate 2.0 Yeast extract 2.0 Tryptone 10.0 K2HPO4 5.0 Glycerol 2.0 Salt solution PYS 2.5 ml Vitamin solution 1 ml Tap water to 1 litre c) Salt solutions: i. Salt Solution A Part 1 and Part 2 were added in equal volumes to give salt solution A. g/litre of Part 1 0.1N HCl MgSO4#7H2O 100.0 FeSO4.7H2O 10.0 MnSO4,H2O 1.0 NaMoO4.2H2O 0.5 0.1NHCl to 1 litre Part 2 CaCl2 10.0 Distilled water to 1 lit ii. Salt Solution C g/litre of 0.1N HCl MgSO4.7H2O 25.0 CaCl2#2H2O 0.1 FeSO4.7H2O 2.8 MnSO4.H2O 1.7 ZnSO4.7H2O 0.06 NaCI 0.6 iii. Salt Solution PYS g/litre Na3C6H5O7#2H2O [Sodium citrates 5.0 MnCl2#4H2O 3.0 ZnCl 2.0 FeCl3#6H2O 2.0 MgCl2#6H2O 50.0 CuCl2.2M2O 0.2 CaCl2.2M2O 0.75 CoCl2#2H2O 0.2 NaMoO4#2H2O 0.1 Na2B4O7#10H2O 0.1 Distilled water to 1 litre d) Vitamin Solution mg/litre Thiamine#HCl 200 p-Aminobenzoic acid 200 Pyridoxin MCI 200 Riboflavin 200 D-Pantothenic acid (calcium salt) 200 Folic acid 2.0 Biotin 2.0 Riboflavin goes into solution on warming. The vitamin solution was filter-sterilized and was added to the medium after sterilization. e) In all large scale fermentations, the media also contained 0.01% polyglycol P-2000 as an antifoam agent.

3. Maintenance of Culture: The culture was maintained on the stabs of Micro assay culture agar with 0.2% glycerol; it was transferred to a fresh stab at least once every week. After 48 hours of incubation at 300 C, the stabs were stored at 40C. Another process used for the preservation of the organism was the storage in liquid nitrogen. For this purpose, the culture was grown in STP medium for 20 hours. The cells were then separated and resuspended in sterile 10% aqueous glycerol with Allen's salt solution (Allen, M.B., Archives of Mikrobiology, Vol. 32, p. 270-277 (1959). A small volume, 0.5-2.0 ml, of this suspension was added to a sterile glass ampoule which was then sealed and stored in liquid nitrogen.

4. Small Scale Fermentation: a) Preparation of inoculum -- The culture grown on Micro assay culture agar for 48 hours was transferred to 50 ml of STP medium or Modified STP medium-2 in a 250 ml Erlenmeyer flask. The flask was shaken at 300C and 200 RPM. After 20 hours of incubation, the contents were centrifuged in a Sorvall refrigerated centrifuge (Model RC IIB, DuPont Instruments Co., Newtown, Connecticut) at 12,000 x g for 15 minutes at 40C. The supernate was discarded, and the cells were resuspended in the same volume of sterile water as the original. This suspension was used as inoculum. b) Production of the enzyme -- Twenty-five ml of fermentation medium in a 250 ml Erlenmeyer flask was inoculated with 1.5 ml of inoculum prepared as described in (4.a). The flasks were shaken at 200 RPM at 300 C. After shaking the flasks for 20 hours, 2.5 ml of sample were withdrawn from each flask. The samples were centrifuged at 19,000 x g for 15 minutes in a Sorvall refrigerated centrifuge. The cells were resuspended in 2.5 ml of deionised water. This suspension was diluted 10 fold. The diluted suspension was used for the determination of dry cell weight and for the assay of a-glycerophosphate (a-GP) oxidase.

5. Large Scale Fermentation: a) Preparation of inoculum The inoculum for 150-litre fermentor was prepared by three different methods.

1) Six 2.8-liter Fernbach flasks containing 500 ml of Modified STP Medium 2 were inoculated with Streptococcusfaecalis ATCC 12755, grown for 48 hours on stabs of Micro assay culture agar with 0.2% glycerol. One stab was used to inoculate each Fernbach flask. The flasks were shaken at 125 RPM and at 30 C. After incubation for 20 hours, the contents of the flasks were aseptically centrifuged at 6000 x g in a refrigerated centrifuge (Model RC IIB, DuPont Instrument Co., Newtown, Connecticut)-for 15 min. The supernatant liquid was discarded, and the cell pellet was resuspended in sterile distilled water. A total volume of 500 ml of distilled water was used to resuspend the cells from 6 flasks. This cell suspension was used to inoculate a 150-litre fermentor.

2) The procedure was the same as above except that the contents of the flasks were not centrifuged. The whole fermentation broth from 6 flasks was used to inoculate a 150-liter fermentor.

3) This method of inoculum preparation was used to prepare inoculum in most of the experiments reported herein. A 250 ml Erlenmeyer flask containing 50 ml of Modified STP Medium-2 was inoculated with Streptococcusfaecalis ATCC 12755 grown for 48 hours on a stab of Micro assay culture agar with added 0.2% glycerol. The flask was shaken at 200 RPM and at 300C. After shaking for 12 hours, the contents of the flask were used to inoculate a 14-litre fermentor. Prior to the inoculation, this fermentor was charged with 10 litres of desired fermentation medium and was sterilized for 1 hour. The medium was cooled to 300C and inoculated as described above. The medium was agitated with 3 flat-bladed turbine impellers at 1300 RPM. A ring sparger was used for aeration. The air flow rate was 0.2 VVM (volumes of air per volume of media per minute) or 2 litres per minute. This gave a mass transfer coefficient, K, a, of 8 min-'.

The temperature was maintained at 300C. After 12 hours, the whole broth was used to inoculate a 150-litre fermentor. The purity of the culture was monitored throughout the development of inoculum.

Microscopic examination and plating technique were used for this purpose. b) Production of a-GP oxidase A- ~150-litre ~ fermentor was charged with 100 litres of appropriate fermentation medium and was sterilized for one hour. The medium was cooled to 300C. It was inoculated by aseptically transferring the contents of the 14-litre fermentor prepared as described in method 3 above. The medium was agitated with 3 flat-bladed turbine impellers at 250 RPM and aerated with a ring sparger. The rate of aeration was 0.18 VVM or 20 litres of aidmin. Under these conditions, the value of K,.a was 1.15 min-'. The temperature was kept at 300 C. Samples were aseptically withdrawin every hour with an automatic sampler (New Brunswick Scientific Co., Inc., New Brunswick, New Jersey), and the cell growth as well as a-GP oxidase production was measured. Dissolved oxygen concentration was monitored with a membrane electrode (IL 530, Industrial oxygen measuring system, Sensorlabs, Division of Instrumentation Laboratories, Inc., Lexington, Massachusetts). The pH was measured with an Ingold electrode (Type 764-31B, Dr. W. Ingold Ltd., Zurich, Switzerland). The fermentor was cooled to 100C with cold water when the enzyme level reached the desired value and the cells were harvested in a refrigerated continuous centrifuge (Cepa centrifuge Model Z81G, Carl Padberg GMBH, West Germany).

Average fermentation time was 9-10 hours.

6. Determination of Dry Cell Weight: A calibration curve correlating dry cell weight to absorbance at 660 nm was prepared. The absorbance was measured on a Spectronic (trade mark) 20 Spectrophotometer (Bausch and Lomb, Rochester, New York), and the dry cell weight was computed from the calibration curve.

7. Assay of a-GIycerophosphate Oxidase Activity: The a-GP oxidase activity was determined by measuring the peroxidase catalyzed oxidation of a leuco dye by H202 released during the oxidation of a glycerophosphate by a-GP oxidase. One unit of enzyme activity (U) was defined as that amount of the enzyme which converts I Mmole of substrate into product per minute at 370C and a pH 7.0. a) Reagents i) KP buffer: 0.1 M potassium phosphate buffer, pH 7.0. ii) Substrate solution: Dissolve 17.288 g of a-glycerophosphate in 100 ml of KP buffer. iii) Dye solution: Dissolve 1 g of 3,3'-dimethoxybenzidine dihydrochloride (o-dianisidine) in 100 ml of deionized water. iv) Detergent solution: Dissolve 1 g of Triton (trade mark) X--100 (a polyethoxy ethylene surfactant commercially available from Rhom & Hass, Phila., Pa) in 10 ml. of KP buffer. v) Buffer solution: Dissolve 3.3 mg of horseradish peroxidase type II in 50 ml of KP buffer. Add 1.1 ml of dye solution and 6.6 ml of detergent solution. Make the volume to 100 ml with KP buffer. b) Procedure Six ml of buffer solution and 1 ml of substrate solution in a test tube were equilibrated at 370C for 15 minutes in a waterbath shaker (New Brunswick Scientific, New Brunswick, New Jersey). The samples were diluted to contain 5-25 mU of a-GP oxidase per ml. One ml of approximately diluted sample was added to the equilibrated tubes. A blank was prepared with I ml of deionized water in place of the sample. The tubes were shaken at 370C in a water bath shaker. The colour development was measured every 5 minutes with a Spectronic 20 Spectrometer at 430 nm for 1 hour. The extinction coefficient was determined for the dye and was used to convert the optical density into concentration of the substrate utilized.

8. Determination of Mass Transfer Coefficient (K,.a) The mass transfer coefficent was measured by monitoring the dissolved oxygen concentration and the outlet oxygen concentration. The dissolved oxygen concentration was determined with a membrane electrode as described in paragraph 5.(b) (Production of a-GP oxidase) above. The outlet oxygen concentration was measured with a paramagnetic analyzer (Type CA150, Servomex Controls Ltd., Crowborough, Sussex, Eng). The following relationship was used to calculate K'a.

N = K,-a (C* - C,) wherein: N = Rate of mass transfer K, = Mass transfer coefficient a = Interfa ial area C*.- Saturation concentration of dissolved oxygen CL = Instantaneous dissolved oxygen concentration Examples 1--5 demonstrate the use of small scale techniques as described above.

Example 1.

Inductive Effect of Glycerol The addition of glycerol had a dramatic effect on the production of the enzyme. The maximum production was obtained in the medium containing 2 g of glycerol per litre (Table 1). Further increase in glycerol concentration did not increase the yield of the enzyme. The growth of the culture was not affected to a great extent; at most, a 20% increase in the dry cell weight was noted, whereas there was a more than 3-fold increase in the enzyme yield.

TABLE 1 Effect of Glycerol Concentration of Production of a-GP Oxidase Glycerol Dry Cell Weight litre litre U/litre % of Control 0.0 (Control) 0.34 62 100 2.0 0.41 205 330 5.0 0.38 203 328 10.0 0.34 178 287 The culture was grown in STP Medium supplemented with glycerol at concentrations shown in the table.

Example 2.

Effect of Carbon Sources a. Effect of glucose - As shown in Table 2, the growth of the culture was strongly dependent on the concentration of glucose. A three-fold increase in the dry cell weight was obtained when 3 g of glucose per litre was added to the medium as compared to that obtained in medium without glucose. The concentration of glucose also had a significant effect upon the production of a-GP oxidase; omission of glucose from the medium caused a 38% reduction in production. The production of the enzyme increased with increasing glucose concentration up to 1.0 g per litre.

Further increase in the concentration of glucose caused repression of cr-GP oxidase synthesis.

TABLE 2 Effect of Glucose Concentration a-GP Oxidase Production of Glucose Dry Cell Weight g/litre g/litre U/litre % of Control 0 0.22 52 62 0.5 0.29 62 76 1.0 (Control) 0.32 82 100 2.0 0.57 30 36 3.0 0.61 39 47 The culture was grown in STP Medium as described hereinabove. The concentration of glucose was varied as indicated in the table. b. Effect of sodium pyruvate -- The replacement of glucose with sodium pyruvate considerably improved the growth and the production ofcr-GP oxidase as shown Table 3. However, high concentrations of pyruvate ( > 3 litre) reduced the production of the enzyme. The organism was grown on Modified STP Medium I as described hereinabove except that the glucose in the medium was replaced with sodium pyruvate at concentrations shown in the table.

TABLE 3 Effect of Sodium Pyruvate Production of α-GP Oxidase Concentration of Dry Cell Carbon Source Weight Carbon Source g/litre g/litre U/litre % of Control None 0.0 0.19 176 88 Glucose (Control) 1.0 0.33 199 100 Sodium pyruvate 0.5 0.36 352 176 Sodium pyruvate 1.0 0.46 386 193 Sodium pyruvate 2.0 0.60 484 242 Sodium pyruvate 5.0 0.55 314 157 c. Combined effect of glucose and sodium pyruvate - It was observed that the combined addition of 2.0 g/litre of glucose and 2.0 g/litre of pyruvic acid sodium salt increased the growth of the cluture and, surprisingly, greatly increased the production of enzyme at the same time as shown in Table 4. This modification, called Modified STP Medium 5, represents a preferred medium for the present invention. The growth medium employed was Modified STP medium 4 with variations in the concentrations of sodium pyruvate and glucose indicated in the Table.

TABLE 4 Effect of Supplementation with Glucose Concentration of α-GP Oxidase Production Concentration of Pyruvic Acid Dry Cell Glucose Sodium Salt Weight g/litre g/litre g/litre U/litre % of Control 0 0 0.33 237 30 0 0.5 0.48 314 40 0 1.0 0.63 384 48 0 2.0 (Control) 0.58 794 100 0.5 0 0.51 282 36 0.5 0.5 0.68 412 52 0.5 1.0 0.80 580 73 0.5 2.0 0.74 10 52 133 1.0 0 0.49 194 24 1.0 0.5 0.81 500 63 1.0 1.0 0.81 540 68 1.0 2.0 0.79 1069 135 2.0 0 0.62 418 53 2.0 0.5 0.95 490 62 2.0 1.0 0.99 651 82 2.0 2.0 1.00 1539 194 3.0 0 0.72 27 3 3.0 0.5 1.05 315 40 3.0 1.0 1.13 369 46 3.0 2.0 1.06 998 123 Example 3.

Effect of Vitamins Vitamins stimulated the enzyme production by as much as 30%, though there was no effect on the growth of the culture (Table 5). It was also shown that the increase in the enzyme production was not proportional to the increase in the concentration of vitamins.

TABLE 5 Effect of Vitamins Volume of Vitamin a-GP Oxidase Production Solution Added Dry Cell Weight ml/litre g/litre U/litre % of Control 0.0 (Control) 0.64 766 100 1.0 0.63 808 106 2.0 0.66 939 123 3.0 0.63 882 116 5.0 0.62 908 119 10.0 0.65 1011 133 The culture was grown in Modified STP Medium-3. The concentration of vitamin solution was changed as shown in the table. For the composition of the vitamin solution, refer to paragraph 2. d) Vitamin Solution above.

Example 4.

Effect of Inorganic Salts The stimulation of enzyme production by the addition of trace elements indicated that trace elements may be the other limiting nutrient(s) in our fermentation medium. Trace elements are those which the culture requires in very small, trace quantities, of the order of parts per million, or less. The culture required a number of elements in trace amounts, and as more of these were supplied, the better the enzyme yields became. Tables 6A, 6B and 6C illustrate that salt solution PYS, which contains more trace elements than salt solution C, was better than salt solution C in improving a-GP oxidase production. Salt solution C, in turn, was better than salt solution A for the same reason.

TABLE 6A Effect of Various Mixtures of Trace Elements: Effect of Salt Solution C a-GP Oxidase Production Volume Added of Dry Cell ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Salt Solution C Weight ml/litre g/litre U/litre % of Control 0.0 (Control) 0.49 439 100 0.5 0.62 630 143 1.D 0.59 598 136 2.5 0.60 669 152 5.0 0.61 729 165 10.0 0.64 747 170 The culture was grown on modified STP Medium-2, supplemented with salt solution C in concentrations as described in the table.

TABLE 6B Effect of Various Mixtures of Trace Elements: Effect of Salt Solution A a-GP Oxidase Production Volume Dry Cell ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Mixture of Trace Added Weight Elements Added ml/litre g/litre U/litre % of Control None (Control) - 0,57 541 10 0 Salt Solution C 5.0 0.57 1037 192 Salt Solution A 2.0 * 0.61 837 155 * Optimum concentration of salt solution A.

The medium used in this experiment was Modified STP Medium-2 supplemented as shown.

TABLE 6C Effect of Various Mixtures of Trace Elements: Effect of Salt Solutions PYS a-GP Oxidase Production Dry Cell ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Mixture of Trace Volume Added Weight Elements Added ml/litre g/litre U/litre % of Control None (Control) - 0.62 582 100 Salt Solution C 5.0 0.63 811 140 Salt Solution PYS 5.0 0.69 923 158 Salt Solution PYS 10.0 0.70 1000 171 Salt Solution PYS 20.0 0.67 978 168 Salt Solution PYS 50.0 0.75 1130 194 Modified STP Medium-3 was used in this experiment supplemented as shown.

Example 5.

Effect of Vitamins and Trace Elements As described above, the enzyme production increased when the medium was supplemented with either the vitamin mixture or various mixtures of trace elements. A synergistic effect on the enzyme production was observed upon the addition of both the vitamin solution and salt solution C (Table 7). There was no significant effect on the growth of the culture.

TABLE 7 Effect of Vitamins and Trace Elements a-GP Oxidase Production Dry Cell Weight Additions g/litre U/litre % of Control None (Control 0.60 530 100 5.0 ml salt solution C per litre 0.65 577 109 1.0 ml vitamin solution per litre 0.59 582 110 5.0 ml salt solution C and 0.63 811 153 1.0 ml vitamin solution per litre The culture was grown on Modified STP Medium -2. Additions to the medium were as shown in the table.

The following examples demonstrate the use of a 150 litre fermentor employed in the large scale fermentation techniques described above.

Example 6.

Effect of Size of Inoculum Three procedures for inoculum preparation, described above, were tested.

The size of inoculum (6-10 litres) did not affect the growth of the culture or the production of the enzyme. However, the increase in the size of inoculum did reduce the time required to reach the maximum growth and the enzyme production by 30%. Results, shown in Table 8, indicate that whole broth was a satisfactory inoculum compared to centrifuged and resuspended cells.

TABLE 8 Effect of Size of Inoculum Growth a-GP Oxidase Dry Cell Time Required Average Time Required Weight to Reach Yield to Reach Size of Inoculum g/litre Maximum Hrs. U/litre Maximum Hrs.

6litres centrifuged 0.61 10.5 837 11.3 6-litres uncentrifuged 0.65 9.25 787 9.25 10-litres 0.62 7.75 8.44 7.75 (14-litres fermentor) Modified STP Medium-3 was used in these experiments.

Example 7.

Effect of Glycerol Increasing glycerol concentrations above 2 litre did not appear to improve the enzyme yield per litre in flasks or in the large fermentor. However, increasing glycerol concentrations above 2 litre did affect the growth of cells in the large fermentor. In contrast to the flask studies, in which growth was not affected, the growth of the culture in the fermentor was increased by 48%, as shown in Table 9.

The optimum concentration of glycerol for enzyme synthesis was approximately 2 g/litre.

TABLE 9 Effect of Glycerol Concentration of Glycerol Dry Cell Weight a-GP Oxidase g/litre g/litre U/litre 2.0 (Control) 0.63 1054 3.0 0.93 1039 Modified STP Medium-6 was used in this 150-litre fermentor experiment. The concentration of glycerol was varied as indicated. The inoculum was prepared in a 14-litre fermentor.

Example 8.

Effect of Glycerol Analogues We have shown that a-GP oxidase is an inducible enzyme, and that it is induced by glycerol. Since glycerol is a precursor of the substrate, aglycero phosphate, as well as a readily utilizable carbon source, its concentration must reduce during the fermentation. Therefore, it would be useful to find an inducer whose concentration did not decrease during the fermentation, i.e., a gratuitous inducer. For this purpose, we tested a number of glycerol analogues and monoglycerides at a concentation of 2 g/litre. The following experiments were carried out in shake flasks.

Induction of a-GP oxidase was obtained with all of the glycerol analogues tested (Table 10). The enzyme level was reduced to 38% of the control when glycerol was eliminated from the medium. This level was increased to 50% of the control when the glycerol analogues, with the exception of ethylene glycol, were added. Ethylene glycol strongly repressed the enzyme synthesis.

TABLE 10 Effect of Glycerol Analogues Dry Cell , a-GP Oxidase Concentration Weight Production Glycerol Analogues a M g/l % of Control None - 0.31 38 Glycerol (Control) 0.002 0.41 100 Ethylene glycol 0.032 0.30 17 3-methoxy 1,2,propanediol 0.019 0.30 49 1,3-propanediol 0.026 0.30 42 1,2-propanediol 0.026 0.30 47 2,3-butanediol 0.022 0.30 52 1,2,4-butanetriol 0.019 0.31 49 monoacetin 0.015 0.40 98 1-monopropionin 0.13 0.38 70 1-monobutyrin 0.012 0.37 92 monostearin 0.006 0.34 34 monoolein 0.006 0.28 41 Trilaurin 0.003 0.32 46 The culture was grown in Modified STP Medium1 with 2 g yeast extract per litre.

Glycerol was substituted with the above.

Example 9.

Effect of Temperature A reduction in fermentation temperature from 30"C to 25"C reduced the enzyme production by 22% and only slightly increased the growth. The temperature down-shift, however, had a dramatic effect on the time required to reach the maximum growth and the enzyme production; it took twice as long at 25"C as it did at 300C (Table 11).

TABLE 11 Effect of Temperature Growth a-GP Oxidase Dry Cell Time Required Average Time Required Temperature Weight to Reach Yield to Reach OC g/litre Maximum Hrs. U/litre Maximum Hrs.

30 (Control) 0.63 6.5 1054 6.5 25 0.71 12.0 822 13.0 The medium used was Modified STP Medium6. The size of inoculum was 10 litres.

Example 10.

Effect of Change of Nitrogen Source We have evaluated alternate nitrogen sources to replace tryptone. Trypticase peptone was found to be a good substitute. The a-GP oxidase production in the medium with trypticase peptone was comparable to that in the medium with tryptone, although it did not support the growth of S. faecalis as well. Results are shown in Table 12.

TABLE 12 Effect of Change of Manufacturer of Nitrogen Source Nitrogen Source Dry Cell Weight a-GP Oxidase Generic Name Trade Name g/litre U/litre Trypsin digest Tryptone* 0.63 755 casein Pancreatic digest Trypticase 0.54 788 of casein peptone** - * Product of Difco Laboratories, Detroit, Michigan **Product of BBL, Div. of Becton Dickinson Co., Cockeysville, Maryland Modified STP Medium-7 was used in these studies. Tryptone was replaced with the same concentration of trypticase peptone. The inoculum was grown in 14-litre fermentor.

Example 11.

Kinetics of a-GP Oxidase Production The enzyme production was shown clearly to be growth associated. The figure of the accompanying drawings shows the growth of the culture at 300C in Modified STP Medium-4, the production of a-GP oxidase, dissolved oxygen concentration and pH during the fermentation. Growth reached a maximum in 6 hours; enzyme production reached a maximum in 9 hours. Enzyme concentration remains stable for about 2 hours after reaching maximum. The preferred fermentation time is 6 to 12 hours.

Example 12.

Use of varying microorganisms The following bacilli, all members of the family Lactobaccillaceae were cultured in Modified STP Medium-7. The a-GP oxidase produced per litre of medium for cultures grown at 300 and 37"C is recorded in Table 13 below.

Claims (16)

TABLE 13 a-GP oxidase U/litre Culture 30 37 Streptococcus Faecalis, ATCC 12775 270 Streptococcus Faecalis. ATCC 8043 157 28 Streptococcus Faecalis, ATCC 19433 143 Streptococcus Cremoris, NRRL B634 41 110 Lactobacillus Casei, ATCC 7469 116 107 Lactobacillus Delbruckii, NRRL B445 141 54 Lactobacillus Fermentum, NRRL 9338 107 105 Pediococcus Cerevisiae, ATCC 8042 33 41 Pediococcus Cerevisiae, ATCC 8081 83 24 WHAT WE CLAIM IS:

1. A method of producing a-glycerophosphate oxidase by growing a member of the family Lactobacillaceae in a medium comprising a pyruvate and an inducer for a-glycerophosphate oxidase and then extracting the a-glycerophosphate oxidase.

2. A method as claimed in claim 1 wherein the member of the family Laetobacillaceae is Streptococcus faecalis, Streptococcus cremoris, Streptococcus salivarius, Streptococcus faecium, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus pentoaceticus, Lactobacillus lactis, Lactobacillus buchneri, Lactobacillus leichmannii, Leuconostoc mesenteroides or Pediococcus cerevisiae.

3. A method as claimed in claim 1 in which the bacillus Streptococcusfaecalis is grown.

4. A method as claimed in claim 3 wherein the Streptococcusfaecalis is ATCC 12755.

5. A method as claimed in any of claims 1--4 wherein the medium contains glucose.

6. A method as claimed in any of claims 1--5 wherein the inducer is glycerol, 3-methoxy- 1 ,2-propanediol, 1,3-propanediol, 1,2-propanediol, 2,3-butanediol, 1,2,4-butanetriol, monoacetin, I-monopropionin, I-monobutyrin, monostearin, monoolein or trilaurin.

7. A method as claimed in claim 6 wherein the inducer is present in an amount of from 1.0 to 10 grams per litre of medium.

8. A method as claimed in any of claims 1--7 wherein the medium contains from 0.5 to 3.0 grams of sodium pyruvate per litre of medium.

9. A method as claimed in claim 8 wherein the medium contains from 0.5 to 2.0 grams of sodium pyruvate and from 0.5 to 3.0 grams of glucose per litre of medium.

10. A method as claimed in any of claims 1--9 wherein the medium contains inorganic salts and citamins in an amount effective to increase the production of crglycerophosphate oxidase.

11. A method as claimed in any of claims 1--10 wherein the medium contains from 1.0 to 20 grams of yeast extract per litre of medium.

12. A method as claimed in any of claims I11 wherein the medium contains from 5 to 20 grams of a protein hydrolysate per litre of medium.

13. A method as claimed in any of claims 1--12 wherein the medium contains from 3 to 5 grams of K2MPO4 per litre of medium.

14. A method as claimed in any of claims 1--13 in which the medium is held at 30"C during cell growth.

15. A method according to claim 1 substantially as described herein and with reference to the Examnles.

16. α-Glycerophosphate prepared by the method of any of claims 1-15.