Oxidation of Isomaltose by Pseudomonas Taetrolens
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JOURNAL OF BACrERIOLOoY, Aug. 1969, p. 623 Vol. 99, No. 2 Copyright 0 1969 American Society for Microbiology Printed In U.S.A. Oxidation of Isomaltose by Pseudomonas taetrolens M. STERNBERG AND L. B. LOCKWOOD Miles Laboratories, Inc., Elkhart, Indiana 46514 Received for publication 16 May 1969 Pseudomonas taetrolens NRRL B14 oxidized isomaltose without hydrolysis of the 1-6 glycosidic linkage. The resulting isomaltobionic acid was identified by chro- matographic studies of acid hydrolysates and reconversion to isomaltose. Pseudomonas graveolens NRRL B14 was shown Dowex 50 in the H+ form and the acids were to oxidize maltose and lactose to the correspond- absorbed on a column of Dowex 2X-4 in the ace- ing maltobionic and lactobionic acids, leaving tate form. A column (1,000 ml) of wet resin was intact the 1-4 glycosidic linkage (5). It was of used for 300 ml of filtered fermentation liquor. interest to determine whether the same organism, The column was eluted with a gradient of 0.1 to now named P. taetrolens, would oxidize an a 1-6 1 N acetic acid. Fractions of 200 ml were collected disaccharide, namely isomaltose, to isomalto- and assayed by the phenol sulfuric acid method bionic acid. (1). A major peak emerged between fractions 26 Isomaltose was prepared by the treatment of and 31. After evaporation at 40 to 42 C to 300 ml, maltose with transglucosidase. The enzyme was the concentrate was extracted three times with an obtained from a fermentation with Aspergillus equal volume of ethyl acetate to remove the acetic niger (3). After incubation of a 10% solution of acid and then was lyophilized. maltose with transglucosidase for 48 hr, the main Isomaltobionic acid was identified by trans- reaction products were isomaltose and glucose. forming into the lactone and reducing the latter to The glucose was fermented with bakers' yeast; isomaltose as follows. An aqueous solution of after alcohol precipitation of the proteins and 29% isomaltobionic acid was mixed with an equal activated carbon treatment, the solution was con- volume of dioxane and evaporated to an oily centrated to a syrup containing mostly isomaltose liquid. This procedure was repeated two times as shown by paper chromatography. after which it was assumed that the free acid had A culture of P. taetrolens NRRL B14 main- been converted into the corresponding lactone. tained on nutrient agar was transferred into The oil was brought to 100 ml with water and nutrient broth and shaken at 25 C for 72 hr. This reduced with 5 g of sodium borohydride. The culture was used to inoculate 50 ml of nutrient reaction medium was kept at pH 3 to 4 by drop- broth. After 24 hr, 5 ml of this culture was used to wise addition of 1 N H2SO4 over a period of 60 inoculate the following medium in Erlenmeyer min. The solution was deionized by pouring it flasks: 10 g of isomaltose, 0.5 ml of corn steep through a mixed bed of Dowex 50 and Dowex 2 liquor, 3 g of CaC03, and 100 ml of water. ion-exchange resins in the H+ and OH- forms, The flasks were shaken for 6 days at 25 C. At the respectively. Isomaltose was identified as the only end of the fermentation, the reducing value ex- reducing sugar present by the same thin-layer pressed as glucose dropped to 0.48% but rose to chromatography method that was used for the 4.3 % after hydrolysis for 1 hr at 120 C with 0.45 identification of sugars in the fermentation liquor N H2S04 . The yield of isomaltobionic acid, based of P. tetrolens. on the reducing value after hydrolysis, was It has to be assumed, therefore, that P. graveo- 76.40%. The thin-layer chromatography of sugars lens can oxidize not only 1-4 disaccharides but on cellulose layers developed with n-butyl also an a 1-6 disaccharide to the corresponding alcohol-pyridine-water (6:3:1) revealed only bionic acids without hydrolyzing the glycosidic glucose after aniline phthalate spraying (2). A linkcage. spray for acids made with xylose-aniline (4) re- vealed one major spot at Rp 0.16 which yielded L1IERATURE CITED glucose upon elution and hydrolysis. The 1. Launder, H. F. 1962. p. 388. In R. L. Whistler and M. L. H2S04 Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 1. microbial cells were removed by boiling for 2 min Academic Press Inc., New York. and filtering with 1% activated powdered carbon 2. Partridge, S. M. 1948. Nature 164:443. (Pittsburgh Chemical Co., Pittsburgh, Pa.) and 3. Pazur, J. H., and T. Ando. 1961. Arch. Biochem. Biophys. 2% Celite 512 (Johns-Manville Co., New York, 93:43-49. 4. Smith, F., and D. Spriestersbach. 1954. Nature 174:466-467. N.Y.) 5. Stodola, F. H., and L. B. Lockwood. 1947. J. Biol. Chem The cations were removed by treatment with 171:213-221. 623.