THE OXIDATION OF LACTOSE AND MALTOSE TO BIONIC ACIDS BY PSEUDOMONAS*

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1 THE OXIDATION OF LACTOSE AND MALTOSE TO BIONIC ACIDS BY PSEUDOMONAS* BY FRANK H. STODOLA AND LEWIS B. LOCKWOOD (From the Fermentation Division, Northern Regional Research Laboratory,t Peoria, Illinois) (Received for publication, August 8, 1947) Studies in this Laboratory on the biochemical activities of the genus Pseudomonas have led to the isolation of gluconic and 2-ketogluconic acids (l), ar-ketoglutaric acid (2), and the pentonic acids (3) as oxidation products of various monosaccharides. The analogous production of bionic acids from reducing disaccharides by oxidation of the free aldehyde group to a carboxyl group seemed to be a logical extension of these studies. The difficulty in isolating and characterizing the very water-soluble bionic acids has undoubtedly retarded investigation of them. Gluconic acid is readily obtained in the pure state by crystallization of its calcium salt; no such simple procedure is available in the case of the bionic acids. The only known crystalline metallic salt of a bionic acid, calcium lactobionate, precipitates in such a gelatinous form as to be useless for purification. The bionic acids were first prepared by Fischer and Meyer (4) as early as 1889, but only in recent years have they received extensive study. A number of attempts (5-7) have been made t.o increase the low yields obtained by brollrine water oxidation; these culminated in the electrolytic method of Isbell and Frush (8) at the National Bureau of Standards. In addition, Isbell prepared for the first time the crystalline lactobionic &lactone (9) and studied the preparation and properties of the crystalline double salt of calcium bromide and calcium lactobionate (10). The biochemical product ion of bionic acids, on the other hand, has not been referred to in the literature. The oxidizing action of Pseudomonas on reducing disaccharides has, however, been studied by Lembke (11) who made preliminary manometric measurements of oxygen uptake by lactose and maltose. It was found that lactose was oxidized to about the same extent as glucose by Bacterium pyocyaneum, B. fiuorescens, and B. syncyaneum, while B. putidum was without action on lactose. These four species showed little or no oxygen uptake with maltose. No attempt * This paper was presented at the Atlantic City meeting of the American Chemical Society, April, t One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Department of Agriculture. 213

2 214 OXIDATION OF LACTOSE AND MALTOSE appears to have been made by Lembke to isolate or identify oxidation products. Investigation of the formation of bionic acids by microorganisms has no doubt been discouraged by the wide-spread ability of microorganisms to cleave enzymatically the glucosidic linkage of the reducing disaccharides. In fact, the question of whether or not any microorganisms can metabolize reducing disaccharides without prior hydrolysis has been a subject of controversy. It was, therefore, one of the objectives of our work to seek a definite answer to this question, at least for some members of the genus Pseudomonas, by attempting the isolation of the bionic acids. In our oxidation studies we examined fifteen species of Pseudomonas and found that P. graveolens was capable of oxidizing lactose to calcium lactobionate, in rotating drums, with a yield of 75 per cent in 165 hours. Some of the species (P. putida, P. mucidolens, P. myxogenes, P. aeruginosa (Vendrell strain), P. pavonacea, P. putrifaciens, P. Jluorescens, P. chlororaphis, and P. syncyanea) were also able to oxidize lactose to an acid in considerable amount. The fermentation time, however, was so long that this group of microorganisms was not studied further nor was the nature of the oxidation products determined. The remaining species (P. ovalis, P. mildenbergii, P. synxantha, Chromobacterium iodinum, and P. saccharophila) were almost without action on lactose. With maltose, Pseudomonas graveolens again proved to be the best of the eighteen species tested for bionic acid production. A 77 per cent yield of calcium maltobionate was obtained in 50 hours in a rotating drum. P. fragi also oxidized maltose at a good rate, but the high calcium values suggested oxidation beyond hhe bionic acid stage. The remaining species (P. putida, P. mucidolens, P. myxogenes, P. mildenbergii, P. aeruginosa (Vendrell strain), P. pavonacea, P. jluorescens, P. boreopolis, P. syncyanea, P. ovalis, P. schuylkilliensis, P. synxantha, Chromobacterium iodinum, P. putrefaciens, P. saccharophila, and P. chlororaphis) oxidized maltose too slowly to be of interest for the preparation of the bionic acids. The first nine species of this group were also found to hydrolyze maltose. The crude calcium lactobionate was obtained by concentration of the filtered culture liquor to dryness. The salt was a white, non-hygroscopic, amorphous powder showing analytical figures in fair agreement with those for calcium lactobionate. This crude calcium salt from the fermentation could be readily purified by means of the difficultly soluble basic calcium salt. The identity of the calcium lactobionate obtained by fermentation was established by hydrolysis to n-galactose and calcium n-gluconate. The galactose, which was recovered in 85 per cent yield, was characterized by its melting point, rotation, x-ray diffraction pattern, and by conversion to

3 F. H. STODOLA AND L. B. LOCKWOOD 215 the characteristic o-tolyl hydrazone. The calcium gluconate, isolated in 86 per cent yield, was characterized by its rotat ion and x-ray diffraction pattern. Further confirmation of the identity of the lactobionic acid was supplied by conversion of the crude calcium salt into the crystalline double salt of calcium bromide and calcium lactobionate. This double salt gave an x-ray diffraction pattern identical with that of a synthetic sample kindly supplied by Dr. H. S. Isbell of the National Bureau of St,andards. Crude and purified calcium maltobionate samples were obtained in the manner already described for calcium lactobionate. The identit y of the natural maltobionic acid was est,ablished by comparison of its brucine salt with the corresponding salt of synthetic maltobionic acid prepared by the electrolytic method. These preliminary studies indicate that the biochemical production of bionic acids in quantity, in good yield, and in high purit,y can easily be accomplished by using the proper strain of Pseudomonas. It has been established, thereby, that at. least some microorganisms can metabolize disaccharides without prior hydrolysis. Materials and Methods Preliminary Fermentation Studies-To find species of sufficient interest to warrant larger scale studies, fermentations were carried out with 100 ml. of solution. The culture solution was contained in Pyrex tubes incubated at 30 and fitted with finely porous stones through which air flowed at the rate of 100 ml. per minute. The inoculum for each of t,he 100 ml. cultures was grown for 24 hours in 8 ml. of a liver extract medium which initially contained 0.2 per cent of glucose. The fermentation medium contained, in addition to approximately 100 gm. of disaccharide, 0.6 gm. of KHzPOd, 0.25 gm. of MgSOJ.- 7Hz0, and 5 ml. of corn steep liquor per liter. 3 drops of soy bean oil were added to each culture as an antifoam agent. At the time of inoculation, 1 ml. of sterile 20 per cent urea solution and 2.5 gm. of CaC03 (sterilized dry) were added to each culture. Drum Runs-The rotating drums were those described by Herrick, Hellbach, and May (12). For the 3 liters of fermentation liquor, a 100 ml. culture such as was used for the preliminary studies served as inoculum. Nutrients were supplied to the drum cultures in the same concentrations as to the test-tube cultures. The air flow through the 3 liter cultures was 1200 ml. per minute, the temperature was 25, and the rate of rotation was 9.5 R.P.M. Calcium was determined on filtered culture liquors by permanganate titration after oxalate precipitation. Maltose and lactose were determined

4 216 OXIDATION OF LACTOSE AND MALTOSE by the method of Shaffer and Hartmann (13) by use of specially prepared curves for the copper-sugar ratios. Culture numbers appearing in the experimental part are those of the Culture Collection, Fermentation Division, Northern Regional Re,search Laboratory, Peoria, Illinois. EXPERIMENTAL Studies on Lactose Preliminary Fermentations-100 ml. cultures containing 9.60 gm. of lactose were fermented with fifteen different species of Pseudomonas, and samples were removed periodically for sugar determinations. After 6 days of incubation, the P. graveolens 14 fermentation was harvested. There was no residual lactose. The other cultures were harvested after 13 days, at which time all gave positive tests for reducing substances with the Shaffer-Hartmann reagent and negative tests for monosaccharides with Barfoed s reagent. Nine of the species (P. putida 13, P. aeruginosa (Vendrell strain) 23, P. mucidolens 16, P. myxogenes 19, P. pavonacea 24, P. putrifaciens 77, P. jtuorescens 334, P. chlororaphis 560, and P. syncyanea 652) showed some dissolved calcium, in some cases a considerable amount. The remaining five strains (P. ovalis 8, P. mildenbergii 21, P. synxantha 79, Chromobacterium iodinum 141, and P. saccharophila 628) consumed almost no lactose. Drum Ru.n on Pseudomonas graveolens 14-3 liters of culture solution, which contained initially 9.30 per cent lactose (anhydrous), were inoculated with a 100 ml. culture of P. graveolens 14. After 165 hours the culture contained no residual sugar. Lyophiiization of 100 ml. of the filtered culture liquor gave a white powder which, after drying to constant weight at 100 (3.5 hours at 1 mm.), weighed 7.94 gm. [CX] z5 = (c 5.1; HzO). The analytical values approach those of calcium lactobionate. Analysis-C2rH4?0&a (754.6). Calculated. C 38.2, H 5.61, Ca 5.31 Found. 38.4, 5.95, 5.10 Based on the calcium analysis, 96 per cent of this crude product was calcium lactobionat,e, which corresponds to a 75 per cent yield for the fermentation. Hydrolysis of 79.6 mg. of the anhydrous salt gave 38.8 mg. of galactose (calculated for pure calcium lactobionate, 38.0 mg.) as estimated by the Sha.ffer-Hartmann method. The hydrolysis conditions (0.2 N HCl for 30 minutes at 15 pounds autoclave pressure) were established with calcium lactobionate prepared by the electrolytic method of Isbell and Frush (8). The method gave values accurate to f3 per cent. Hydrolysis of Na,tural Calcium Lactobionate to Galactose and Calcium Gluconate-The lactobionic acid obtained by means of an ion exchange

5 F. H. STODOLA AND L. B. LOCKWOOD 217 resin from 3.02 gm. of the crude salt was heated on the steam bath for 4 hours with 1 N HCl. The cooled hydrolysis mixture was freed of chloride ion with Ag,C03 and the excess of silver removed with H$. A slight excess of CaC03 was added and the filtered solution concentrated to 10 ml. Addition of 23 ml. of hot methanol precipitated a gum which was washed three times with 15 ml. portions of hot methanol. The gum was dried in vacua to 1.64 gm. of white powder, from which 1.42 gm. of calcium gluconate were obtained by crystallization from aqueous ethanol. The product showed the correct x-ray pattern and rotation (+9.3 ; c 2.9; HzO) for that salt. Based on 96 per cent purity of the crude calcium salt, the isolation of 1.42 gm. of calcium gluconate represented an 86 per cent recovery for the hydrolysis. The combined methanol extracts from the precipitated gum were concentrated to a syrup which crystallized after standing for several days. Filtration gave 1.17 gm. of white crystals which were shown by their melting point of , their melting point on admixture with pure n-galactose of melting point 164, their x-ray diffraction pattern, and their rotation ([a] i5 = +79 ) to be somewhat impure n-galactose. The n-galactose was characterized by conversion in 76 per cent yield to the o-tolyl hydrazone, the identity of which was established by a mixed melting point test with an authentic sample. Based on 96 per cent purity of the crude calcium salt, the isolation of 1.17 gm. of galactose represented an 85 per cent recovery of galactose. Isolation of Calcium Bromide Double Salt-The crude calcium salt from the fermentation (782 mg.) was dissolved in 0.51 ml. of water containing 200 mg. of CaBrz. By seeding with a few crystals of the double salt, obtained through the courtesy of Dr. Isbell, a heavy deposit of fine crystals was obtained. Addition of 50 per cent alcohol and filtration yielded 230 mg. of white crystals showing an x-ray pattern identical with that of the synthetic product of Isbell. The natural product showed a calcium content of 7.27 per cent; the calculated value for Ca(ClzHzlO&.CaBrP.- 6H20 is 7.54 per cent. Isolation of Pure Calcium Lactobionate from Crude Fermentation Product-For purification, 22.5 gm. of crude fermentation product were converted to the insoluble basic calcium salt (14). After treatment with carbon dioxide, removal of the calcium carbonate, and lyophilization, 14.7 gm. of a white powder were obtained. It gave good carbon and hydrogen figures for calcium lactobionate, but the calcium value was 0.4 per cent high, owing to inorganic salts. To remove these, the salt was dissolved in 45 ml. of water and 25 ml. of absolute alcohol added dropwise until the solution became cloudy. The amorphous precipitate that slowly settled out was filtered off and the filtrate treated with more alcohol to precipitate a

6 218 OXIDATIOK OF LACTOSE AND UALTOSE gum. Trituration with alcohol converted this gum to a granular powder (12.7 gm.). This salt, however, was found by carbon, hydrogen, and ethoxyl determinations to retain a molecule of alcohol even after 2 hours drying at 98 in vucuo. This alcohol was removed by dissolving the salt in water and lyophilizing. On analysis the results corresponded with those for calcium lactobionate after the compound was dried for 2 hours at 100 in vacua. [a] E5 = (c 5.2; HzO). Analysis-CuHlzOt&a (7M.6). Calculated. C 38.2, H 5.61, Ca 5.31 Found. I 38.1, 5.58, 5.38 Our synthetic calcium lactobionate showed [CX] F = f23.8 (c 5.9; HzO). We have been unable to find any report in the literature on the rotation of this salt. Xtudies on Maltose Preliminary Fermentations-In the same manner already described for lactose, eighteen strains of Pseudomonas were grown in 100 ml. of culture liquor. After 4 days the culture of P. graveolens 14 had completely oxidized the maltose and was harvested. On the 12th day, the P. fragi culture showed no more reducing value and was worked up. The remaining cultures were harvested on the 13th day, although there was residual reducing material present in all cases. Of this group, nine strains (P. putida 13, P. mucidobens 16, P. myxogenes 19, P. mildenbergii 21, P. aeruginosa (Vendrell strain) 23, P. pavoncacea 24, P. jluorescens 334, P. boreopolis 550, and P. syncyanea 652) gave good positive tests for monosaccharides with Barfoed s reagent, indicating hydrolysis of the maltose. Either glucose or 2-ketogluconic acid gives positive reactions in this test. The remaining seven strains (P. ovalis 8, P. schuylkilliensis 9, P. putrifaciens 77, P. synxantha 79, Chromobacterium iodinum 141, P. chlororaphis 560, and P. saccharophila G28) failed to give positive Barfoed s tests at time of harvest. There was considerable soluble calcium in the cultures of P. ovalis 8, P. schuylkilliensis 9, P. mucidolens 16, P. myxogenes 19, Chromobacterium iodinum 141, P. boreopolis 550, and P. syncyanea 652. As a result of this preliminary work, Pseudomonas graveolens 14 and P. fragi 25 were selected for further study in the rotating drums. Drum Run on Pseudomonas graveolens I.&In the manner already described for lactose, P. graveolens 14 was grown on 3 liters of culture solution containing 8.90 per cent of maltose. After 50 hours the culture solution contained less than 0.3 per cent of maltose and was harvested. Lyophilization of 100 ml. of the filtered culture liquor gave a white powder which, after drying to constant weight at 100 (3.5 hours at 1 mm.), weighed

7 F. H. STODOLA AND L. B. LOCKWOOD gm. [CX] z5 = +105 (c 6.0; HzO) (Glattfeld and Hanke (15), f98.3 ). It contained 2.63 per cent of maltose. Analysis-ChH,~Ot,Ca (754.6). Calculated. C 38.2, H 5.61, Ca 5.31 Found. 38.0, 5.55, 4.83 Based on the calcium analysis, 91 per cent of this crude product was calcium maltobionate, which represents a 77 per cent yield for the fermentation. Hydrolysis of 89.7 mg. of the crude anhydrous calcium salt gave 41.0 mg. of glucose compared to a calculated value of 42.2 mg. for calcium maltobionate containing 2.63 per cent maltose. The hydrolysis conditions (0.2 N HCl for 30 minutes at 15 pounds autoclave pressure) were established with pure barium maltobionate ([a] E5 = ; c 6.1; H,O) prepared by the electrolytic method. The accuracy of the method was f3 per cent. Brucine Salt of Natural Maltobionic Acid-The identity of the natural maltobionie acid was established by comparison of its brucine salt with the corresponding salt of synthetic maltobionic acid. The synthetic brucine salt was prepared as follows: 2 gm. of barium maltobionate were freed of barium with an ion exchange resin and the lyophilized residue warmed on a steam bath with 10 ml. of water and 1.53 gm. of brucine until a clear solution resulted. After filtration and lyophilization, the remaining 2.72 gm. of white powder were dissolved in 2.7 ml. of water, and absolute alcohol (15 ml.) was added almost to the point of cloudiness. Compact bars came down slowly and were filtered off after 6 days. Yield, 960 mg. Dried in air, the product melted at On analysis it proved to be a pentahydrate, as did a number of other samples prefiared in the same way. Analysis-C&~H,801~N,*5HzO (842.8). Calculated. C 49.9, H 6.94, N 3.32 Found. 50.0, 7.23, 3.42 (Dumas) Drying at 56 to constant weight gave the anhydrous product of melting point (evolution of gas) described by Glattfeld and Hanke (15). Analysis-C3sHtsOlQd (752.8). Calculated. C 55.8, H 6.43, N 3.72 Found. 55.7, 6.18, 3.73 (Dumas) The brucine salt of the natural mdtobionic acid was prepared in the manner just described for the synthetic salt. 1 gm. of the crude calcium salt gave 510 mg. of crystalline brucine salt. Recrystallization of 460 mg. of this product gave 240 mg. of pure pentahydrate of melting point Analysis-C&H~sOl~N~.5H~O (842.8). Calculated. C 49.9, H 6.94, N 3.32 Found. 50.2, 6.90, 3.41 (Dumas)

8 220 OXIDATION OF LACTOSE AND MALTOSE This material gave the same x-ray pattern as the synthetic pentahydrate. On drying to constant weight, the anhydrous product of melting point (evolution of gas) was obtained. Exposure of the dried sample to air overnight resulted in the formation of the pentahydrate (m.p ). Isolation of Calcium Maltobionate from Crude Fermentation Product- The crude fermentation product was converted to the insoluble basic calcium salt. After treatment with carbon dioxide, removal of the calcium carbonate, and lyophilization, a product was obtained which gave a high value for calcium. For further purification the salt was precipitated from water by absolute alcohol. Since the product so obtained retained alcohol very tenaciously, as in the case of calcium lactobionate, it was dissolved in water and the solution lyophilized. The analytical figures on this product after drying at 100 for 2 hours in vacua were as follows: Analysis-C&H420&a (754.6). Calculated. C 38.2, H 5.61, Ca 5.31 Found. 38.1, 5.61, 5.58 The rotation was [a]e5 = +103 (c 6.1; HzO). Drum Run on Pseudomonas jragi 25-3 liters of solution which contained 9.2 per cent maltose were oxidized by P. jragi 25. After 94 hours only 0.2 per cent of maltose remained and the culture was harvested. Glucose determinations on the culture liquor after hydrolysis showed 3.24 per cent glucose, which is equivalent to 6.2 per cent maltobionic acid. This amount of acid would require a calcium content of 0.33 per cent for the culture liquor; the value 0.47 per cent found indicates the presence of lower molecular weight acids. SUMMARY A strain of Pseudomonas graveolens has been found capable of oxidizing lactose and maltose to the corresponding bionic acids. These acids were isolated as the calcium salts in yields of about 75 per cent, and with a purity of the crude fermentation product exceeding 90 per cent. This provides a clear cut example of the oxidation of reducing disaccharides by a microorganism without prior hydrolysis. BIBLIOGRAPHY 1. Lockwood, L. B., Tabenkin, B., and Ward, G. E., J. Bact., 42,51 (1940). 2. Lockwood, L. B., and Stodola, F. H., J. Biol. Chem., 164,81 (1946). 3. Lockwood, L. B., and Nelson, G. E. N., J. Bact., 52,581 (1946). 4. Fischer, E., and Meyer, J., Ber. &em. Ges. 22, 361, 1941 (1889). 5. Stoll, A., and Kussmaul, W., U. S. patent 1648,368, Nov. 8, Goebel, W. F., J. Biol. Chem., 72, 809 (1927). 7. Hudson, C. S., and Isbell, H. S., BUY. Standards J. Res., 3,57 (1929).

9 F. H. STODOLA AND L. B. LOCKWOOD Isbell, H. S., and Frush, H. L., BUT. Standards J. Res., 6,1145 (1931). 9. Isbell, H. S., Bur. Standards J. Res., 11, 713 (1933). 10. Isbell, H. S., BUT. Standards J. Res., 17,331 (1936). 11. Lembke, A., Vorratsp$ege u. Lebensmittelforsch., 6, 265 (1942); Chem. Abstr., 38, 5872 (1944). 12. Herrick, H. T., Hellbach, R., and May, 0. E., Ind. and Eng. Chem., 27,681 (1936). 13. Shaffer, P. A., and Hartmann, A. F., J. Biol. Chem., 45,365 ( ). 14. Isbell, H. S., U. S. patent 1,980,996, Nov. 20, Glattfeld, J. W. E., and Hanke, M. T., J. Am. Chem. Sot., 40,991 (1918).

10 THE OXIDATION OF LACTOSE AND MALTOSE TO BIONIC ACIDS BY PSEUDOMONAS Frank H. Stodola and Lewis B. Lockwood J. Biol. Chem. 1947, 171: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1

assimilated, there being no indication of acid accumulation.

assimilated, there being no indication of acid accumulation. THE OXIDATION OF PENTOSES BY PSEUDOMONAS LEWIS B. LOCKWOOD AND GEORGE E. N. NELSON Fermentation Division, Northern Regional Research Laboratory,' Peoria, Illinois Received for publication August 17, 1946