IL24955A - Process for the production of diphtheria toxin - Google Patents

Description

PATENT ATTORNEYS 0 EJ REINHOLD COH MICHAEL COHN I Π D Ί N ISRAEL SHACHTER 1 D c AND DESIGNS ORDINANCE SPECIFICATION PR00E8S FOR THE P 0OU0TI0N OF Dl A THE STATE OF PRIME MINISTER S A LABORATORIES AN ISRAELI OF EMERY AN ISRAELI OF IRVAT YOVEL I EVEN LEA AN ISRAELI BENYAMIN METUOELA STREET JERUSALEM AND ANY I AN I6RAELI OITIZEN OF BRAS L STREE do hereby declare the nature this invention and in manner the is to he particularly described ascertained in b the This invention relates to the production of diphtheria toxin referred to as for toy submerged cultures of the bacillus Corynebacterium production of this toxin is as a rule by growing the bacillus in a standardized liquid nutrient medium as principal both and containing carbon sources proportions which have been found to be optimal with a view to achieving the highest possible yield of toxin for a given volume of nutrient The carbon source conventionally used for purpose is a at a concentration of not more than grams per 100 ml of carbo source consists as a of enzymatically degraded animal protein at an approximate concentration of to mg of a total nitrogen per milliliter It is known that the production of toxin comes to a standstill at a when the medium still contains appreciable amounts of the carbon source while the carbon source may have been used up more or less Experiments tending to increase the final toxin titer of the fermented medium by means of higher concentrations of the carbon source have and it is even found that a substantial increase of an initial content a maltose content of up to or in the absence of maltose an even smaller increase of a much smaller initial glucose content produces considerable depression of the final toxin by as much as Of yields in relation to the composition of the nutrient can only he evaluated as the average of a fair number of experimen for even under seemingly equal conditions the toxin production is apt to vary within rather wide limits from hatch to hatch for no known or apparent This cautionary statement holds good for toxin production method as well as for the process according to the and all indications concerning the latter made in the present specification are to be understood as being made on a number experiments sufficiently large to permit typical titer values to be and the specific experiments reported herein as examples of the performance of the invention have been selected so as to approach such typical averages as closely as It has now been in accordance with this that a substantial increase in the toxin can be achieved if to an otherwise conventional nutrient medium a supplementary amount of carbo source of carbon source is added in the course the toxin preferably at a time when the of toxin fermation at its optimal in a typical submerged culture is about to 34 hours after the inoculation of the but it is apt to vary for different strains of the bacterium and with variations in the initial composition of the nutrient The carbo source to be be the aa that contained initially in the nutrient Even more striking results have been achieved with some compounds known to be ormed in the course of the enzymatic degradation of lactic acid or its acetic acid or its or For a supplementary amount about of maltose added at the optimal time to a fermenting medium whose original content is increases the toxin titer by 25 to and even up 1Q0 while similar increases in th toxin titer ca obtained very much smaller supplementary amounts of any one of the degradation products mentioned At any both before the inoculation and during the growth and producing the pH of the medium should be maintained at a level not belo The increase in the toxin titer achieved by a supplementary amount of carbon source added at the appropriate time to the fermenting medium is even more surprising in view of the faot as a fermented standard medium contains a considerable proportion of unutilised organically bound nitrogen after the toxi production has come to an In terms of amino nitrogen the utilizatio is hardly ever higher and frequently The following amino acids have been found to increase the toxin titer i an especially efficacious manner when added as a supplement to the fermenting medium at the appropriate glutamic The process according to the invention can be carried out as a bato process by means of containers or in a Continuous performance of the process is also in which the addition of supplementary amounts of carbon source will be repeated from time to The invention will be described below with reference several In all of the following technique The bacillus was the Park Williams 8 strain of 2000 obtained om the National Institute of Public The medium contained a papain digest of beef muscle and composed as described by et with the exception maltose content was The was sterilized by filtration through and steamed for 20 The toxin produced was estimated by method with a purified antitoxin calibrated against standard antitoxin for flocculation of the The growth was evaluated by the optical density of washed and diluted determined in a B at 590 e maltose utilization was estimated in the supematants of method of Somogyi content of the was estimated by the method of Pope Stevens while the method of Ma Zuamaga was used for the estimation of the total nitrogen in the The protein in the supernatants was precipitated by trichloroacetic acid and the precipitate was washed times in trichloroacetic The protein nitrogen in the precipitate was estimated either by the method of et or by the method of Parr Randall The production of toxin was carried out in liter each containing 100 of Each flask with 5 of an and mounted on a reciprocating shaker and shaken for 48 hours an incubato Samples of flasks were removed at tested microscopically for purity of the pooled and submitted to the tests described 1 20 litres of culture medium are prepared as follows s of muscle of ver good quality is ground and tap water added to 20 Floating fat is carefully removed by filter The temperature is adjusted to and the fat again collecting on the surface is removed once As soon as no fat globules can be the is adjusted to by the addition of The temperature is then raised and the mass is stirred by means of a electric stirrer 200 The pE measured and to The meat is then digested at in seven stages of 30 minutes each immediately following upon one ion stage 8 gr of papain is ground and added to the few minutes the pH is checked and adjusted to the addition of The is the checked and adjusted every 10 stag Θ gr of papain are to the mass at the end of the first digestion and the pH is maintained at At th end of this stage the pH is adjusted to Again gr papain are added to the mass and the latter is stirred for 30 minutes while the pH is being kept at 4th digestion stage Addition of another 8 gr of the being kept digestion stage Addition of another 8 gr of papain at At the end of this digestion stage the temperature raised to digestion stage Addition of another 8 gr of papain at and at Addition of another 8 gr of papain at a temperature of and at At the end of this last stage the pH is lowered to by the addition of concentrated HO 70 The mixture is heated to boiling and boiled for 10 Then glacial acetic acid is added to lower the to 100 The mixture is then left to t n Ove n ht clear filtrate is made up with water to about 19 then the pH is raised to the addition of of strength and the temperature is raised to this liquid an aqueous suspension of 70 g of yeast is The temperature is maintained at one hour the pH is regularly adjusted The mixture is then filtered through filter To the filtrate there is added ml of a aqueous solution of lactate and ml of The sodium lactate solution is as follows 40 g of pure solid sodium is dissolved in 47 g of distilled the solution is cooled and then slowly added to 100 g of lactic acid of solution is composed as follows Thiamin g g g Nicotinic acid g in water with the addition of a few drops of concentrated hydrochloric Pimelic acid ml 10 5 ml EXAMPLE 2 Example Each flask is inoculated with ml of an shaken mounted on reciprocating shaker and shaken for about 48 in an incubator at Samples of 2 to 7 flasks were removed at tested microscopically for purity of the and their toxin content was determined by the In general one run consisted of 54 To five groups of g o g respectively of sodium lactate and g and respectively of lactic acid as 100 were added at the time of inoculation whereas a sixth group was left as control without The results obtained after 48 of shaking were the Addition Toxin titer None 340 g of maltose 260 g of lactate 320 g 300 g lactic acid 320 g no toxin 3 ive groups of prepared similarly as in Example given at zero g and g of acetic acid aft g and g of while a sixth grou was left as control without The results after 48 of shaking were the following Addition Toxin titer None 250 g of acetic g of glucose 200 g tt 200 g 120 Five of prepared similarly as in Example were inoculated and the incubator for 24 then four of them were given additions of g of sodium g of lactic as pure and g of while the fifth group was left as control without The bottles were placed on the After a total time of 48 hours the results were the Hone 280 g of maltose of sodium lactate 320 of acid 300 g o glycerophosphate 320 5 5 groups of prepared as described in Example were shaken in the incubator for 25 then they were given additions respectively g of g of g of of acetic while the fifth group was left as a control without After a total of 48 hours the results were the followin Addition Hone 90 of maltose 180 g of sodium lactate 140 g of glucose 220 g of acetic acid 220 run carried out as described in After 24 hours of shaking the ollowing additions were given g of g of g of acetic g of sodium g of sodium A sixth was left as a control without After a total shaking time of 48 hours the results were following Addition Toxin None 180 g of maltose 260 g of glucose 290 g of acetic 220 g of sodium acetate 240 g of sodium acetate 270 7 run was carried out described in Example a shaking time of 24 hours two groups of flasks received additions 1 mg of methionine and 1 mg of methionine plus 20 mg of cystine and g of glutamic per A third group was left without addition a After a total of 48 hours of shaking the results were the following Addition Toxin None 220 1 mg of methionine 260 1 of methionine 20 mg of cystine 320 g of glutamic 8 The run carried out as described in Example with the addition of a furthe grou of flasks which received g of maltose per After a total of hours of shaking the results were the followin Addition Toxin 140 1 of methionine 160 1 mg of methionine 20 mg of 185 g of glutamic g of maltose 245 EXAMPLE 9 Two A and containing each 3 litres of the nutrient medium according to Example were inoculated with 100 ml of a 24 hours shaken culture of The fermentors were stirred by means of impellers working at 1400 and aerated at the rate of 475 ml of The fermentors were run in parallel for 24 when the toxin content determined in each of Fermentor contained 140 and fermentor B 100 Starting the 26th hour a solution of maltose was added drop by drop to fermentor B up to a total addition of 45 g of equal 15 mg of maltose per ml of culture After 47 hours the stopped in both fermentors and toxin content was determined 180 and contained 240 10 The run was begun similarly as in Example At the end of 24 hours the toxin titer in fermentor was 60 I that of fermentor B Starting from the 26th hour a solution of acetic acid was added drop by drop to feraiento A up to a total quantity of 60 ml to ml per ml culture After 52 hours the fermentation was stopped in both end toxin content was fermentor contained 190 and fermentor 3 contained 130 The above Examples allow following conclusions to be All xamples show that no apparent reasons the toxin tite of control cultures ary addition of or carbon vary within Bach run to be considered by itself and no between the results of different runs are Examples 2 and 3 show the depressing effect of the addition of supplementary of carbon sources at aero Examples 4 to 6 sho the ter increasing effect of the addition of carbon sources to fermenting shaken flask cultures after 24 or 25 Example 7 shows a similar the supplementary addition of containing carbon Example 8 the for and containing carbon Examples 9 and 10 show a similar due to the supplementary addition of carbon in the case of fermentor insufficientOCRQuality

Claims (1)

HAVING particularly described and ascertained the nature of our said invention and in what manner is be declare what we claim is for the production of diphtheria toxin by submerged cultures of Corynebaoterlum in a nutrient medium containing containing carbo wherein a supplementary amount of ee sources added to the nutrient medium in the course of preferably a height process according to Claim wherein the nutrient medium contains Initially maltose as a carbon preferably in an amount of g process according to Claim wher i the nutrient medium contains initially meat as a carbo process according to Claim 2 o wherei maltose or an compound Known to be formed by the decomposition o such as lactic or acetic acid or their or are used the supplementary carbo Λ process according to any of Claims 1 to wherein cystine glutamic acid are used as the supplementary carbon Λ process according to any of

1. Claim 1 to wherein the pH of the nutrient medium both before the inoculation and during growth and periods is maintained at a value not A process for the production of diphtheria toxin by submerged cultures of Corynebacterium in a nutrient medium containing and according to any of claims 1 to conducted containing carbon substantially as described herein with reference to the Diphtheria when produced by the processes according to any of Claims 1 to Dated this 10th da of January For the Applicants insufficientOCRQuality