( Eugene J. Volker and Clyde schultzl shepherd College Shepherdstown. West Virginia 25443
The Microbial Conversion
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Most commercial vitamin C i s produced synthetically from glucose t ~ a s e do n a series of reacGons originally described by H e i c h s t e i ~(~I ) . O n e s t e p of t h e reaction sequence is t h e microhial oxidation of 1)-st~rhitol111to 1.-sorb(fi;e !111. . . . emr~lovina . . t h e b a c t e r i u m ~ A t o b a c t e sr u b & d a ~ . ~
of D-Sorbit01 to L-S0rb0se An interdisciplinary experiment illustrating an industrial process yield if second crops are taken into consideration. One recrystallization is usually sufficient to obtain crystals for a mixed melting point determination with commercial material. L-sorbose may he further characterized by its specific rotation, ir spectrum, and by the melting paint of its wazone. (Since toxic phenylhydrazine is required for making the osazone, this part should either be done in a hood or be omitted.) The experiment cnn he interrupted at srwral pointri by storing the reactiun vrssrl in n refrigerator. This perm~tsconiiderul,le flerihility in arranging work schedules. No experience in microbiology other than the ability to make sterile transfers is necessary for s u ~ c e s s . ~
Experimental Preparation of the lnoculurn
M a n v oreanic textbooks ( 2 )m a k e reference to this industrially L p & t a n t use of microbes which provides an excellent illustration of the selectivity characteristic of enzyme-catalyzed reactions. T h e transformation c a n n o t be achieved i n comparable yield with conventional laboratory chemicals. T h e product of t h e reaction, sorbose, owes i t s i m p o r t a n t place in chemical manufacturing-many millions of pounds a r e m a d e each year-solely to its ;ole as &I intermediate in t h e synthesis of vitamin C. An euneriment.. based on the studies of the sorhitol-sorbose conveminn hv -.. .,Wells -~~ and coworkers (3) . has heen introduced in the o m i e chwmstry laboratory program of Shepherd College and has elicitrd a fwornhle rracrim f n m the strongly life-scienceorirnvd nudiencr. Potentially this experiment can be used in classes of chemistry or biology. The new experiment has the following advantages
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1) In contrast to other microbial reactions which have appeared recently in this Journal (4), the oxidation of sorbitol to sorbose can be carried out in fairly concentrated solution. and therefore amounts of the productin terms of grams rather than milligrams are obtained. 2) The stlrirng mawrial and the product (a hexitol and a ~irnplrsugar) as wll ns the solvent (water) and the micrcwrganirm used are non-tamir and inexp~nrivt.,provldmg a safc Inhoratory experience for a l a q e number of itudcnrs. 3) Srvemd interesting extensions of the experiment are pcm~ihle.such as the inwstignrwn of reartam yields and rarer as a functiun of substrate concentration, explorat& of the osmotic tolerance limit of the microomanism. and the conversion of sorbose to vitamin C. A recent, if sokewhat lengthy, procedure for this last purpose is available which is suitable for more advanced students (5). ~
Aeetobacter suboxydons (ATCC 621) is received from our supplierl as a freeze-dried sample in a glass ampule. The ampule contents are transferred according to the attached instructions into 5 ml of a sterile broth made up according to this formula: 0.5 g yeast extract, 0.3 g peptone, 2.5 g mannitol and 100 ml of distilled water. This stock culture is incubated at rwm temperature with occasional shaking and is subcultured once a month using sterile technique. Alternatively, the organism may he maintained on agar slants (same composition as the broth hut with the addition of 1.5 g agar). The best source of vigorously growing cells far a large class is a strongly turbid sample from a "pilot" sorbitol-sarbwe conversion run, as described below.
The Microbial Oxidation of DSorbitol to L-Sorbose
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Two and one half crams of D-sorbitol h ~ d r a t.e0.1 . ~ e of veast extract, and 23 ml old~$fllledwater are &tined i n a 12;)-mlErlwmeyer flask, giving a wlutkm which is apprmximarrly lob. (u. v, in sorbitol hydrate after inoculation.The flask is stoppered with a cotton plug and is sterilized in an autoclave for 20 min, then allowed to coal to room temperature. If an autoclave is not available, a large pressure cooker may be used in its place. One milliliter of the stock culture of A. suboxydans isadded to the iwine~ sterile t techniaue. contents of the Erlenmever flaskwith a ~ and the flask is mauntei on a recioroeal shaker.'jU1f the inoculum is
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must he taken into account when calculating the yield of the product. The course of the reaction can he foUowed by withdrawing aliquots for polarimetry with a sterile pipet and centrifuging for 30 min at 3300 rpm or for a shorter time at a higher speed. (The removal of bacterial cells before palarimetry can also be effected by vacuum filtration using a filter paper covered with a 5-mm layer of diatomaceous earth filter aid.) The approximate value of the expected rotation can be calculated from a consideration of the volume, the nominal concentration of the
In our lahoratorv we find it convenient to work with 25-ml samnles of -~ 10%sorhitol solkon. hut there are no nrohlems in "&e un';the 'Undergraduate research participant in chemistry. - - -~~ ZFor a recent review of this and related reactions see Kulhhnek sample volume. Because of the significant difference in specific rotation between D-sorbitol ( [ a ]= ~ -2.0') ~ ~ and L-sorhwe ( [ a ] ~ ~(8). ~ 3For a practical guide to microbiological technique for the organic = -43.Z0), the reaction can he followed hy palarimetry; however, in chemist see Fonken and Johnson (9). order to make optical measurements it is necessary to first remove T h e American Type Culture Collection, 12301 Parklawn Drive, the suspended cells of the microorganism by centrifugation or by Rockville, MD 20852. vacuum filtration. 5Better results were obtained with D-sorbitol hydrate (Nutritional Although the time needed for essentially complete conversion of Biochemicals Div., ICN) than with anhydrous D-sorbitol (Fisher sorbitol to sorbose varies with the nature of the inoculum and the Scientific Co.) as the stating material. efficiency of oxygenation of the highly aerobic A. suborydans, a T h e shaker used wes manufactured bv theEberbach Cormration. 48-hour period may be regarded as typical if the experimental proAnn Arbor. Mich ; i t wasoperated at IMcycle* min. hlnst reciprornl cedure given here is followed. or rota,). shakers would he rqunlly suitable. The product crystallizes easily from water-ethanol in up to 8Wb ~
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Volume 55, Number 10, October 1978 1 673
sample, and the specific rotation of the optically active starting material and product; the small amount of yeast extract makes a negligihle contribution. When conversion to sorbme is wsentially complete, an aliouot of the reaction mixture described here shows a rotation of nhnnt -7.R0. .~ wine a 2-dm wlarimeter t u h t 7 In everv lot of flasks a t ~~"~~ leost one is rhrckt.d by polnrimetry hefore wurkup is initialed to be cermin that the runversion has yon. Ioc~~mplrtion. Allholl~hn 18-hr reaction time is usually needed if the "pilot flask" method is followed, no harm is done hy Leaving samples on the shaker for longer periods. A titrimetric method, hased on the fad that in the course of the sarbitol-sorbose conversion a reducing sugar is formed from anonreducing hexitol, can be used as an alternative to polarimetry for following the reaction (6). Workup is initiated by centrifuging or fdtration, as described above. Next, the reaction mixture is evaporated under reduced pressure with a rotary evaporator to about 5 ml of a syrup and 25 ml of ordinary ethanol is added. Overnight standing in a refrigerator usually hrings ahout massive crystal formation, but the process can he speeded up by "seeding" the solution. The product crystals are vacuum filteredwith a small Hirsch funnel and washed with ethanol. The weieht. .. . mn. . .mixed mo with authentic I.-gorhow, the sperifir rotation, and the 8r s p e c t r u m in a KHrdisk are determmed. If dpsirnl, tht.ow,one (mp 15fiD.nfterrecryslalliwtion) ~~
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7This is the average experimental value of the rotation measured with the polmimeter when the product solution derived from a nominally 10%sorbitol hydrate sample is examined.
674 / J m i of Chemical Education
may be prepared (7). Yields of 1.2-1.6 g of sorbose, mp 162-4' (5371% hased on sorbitol monohydrate) are common for the first crop of crystals. If a second crop of somewhat lower melting crystals is isolated from the mother liquor, the combined yield is significantly increased and can reach 80%. Sorbwe mav be recnistallized from water-ethanol; a little charcoal helps to remove colored impurities. Pure L-sorhose melts at 165'.
Acknowledgment The authors wish to thank Mr. Michael Gasper1 for his contributions to the development of this experiment and Dr. James A. Moore of the Department of Chemistry of the University of Delaware for his valuable comments on the manuscript. Literature Cited 11) Reiehtein,T.. and Glilssner, A..Holu. Chim Aero. 17.311 (19341. (2) Among others see Brown, R. F., '"Organic Chemistry," Wadsworth Publishing Co., Belrnont, Cd., 1975,~.629; Geissman, T A., "Principle801 O'ganic Chemistry,'. W. H. F m m n and Co.. Sen Frsnciseo, 1977, p. 307: Gutsche. C. D., and Pasto, D. J., "Fundsmenfalr of Ormnie Chemistri." Pmntiee-Hall Inc., E n g l e w d Cliffs. N.J., 1975, p 1026. (3) Wells, P. A., S t u b h , J. J., Lwkwiwd, L. B., and h , E . T., l n d Eng. Chom., 29,1385 (19371. (4) Kumler, P. L., and W o n g . P. J.. J. CHEM. EDUC.. 52, 475 (1975); Volke.. E. J.. J. CHEM. EDUC. 54.65 119771. (5) Rumpl, P., and Marlier. S.. Bull. Soe Chim France. 187 (19591. i s s h d r e r , ~~. . , ~ d ~ F.,J.B~OI. ~ l t them., ~ ~ 45,365 ~ ~ w, ~~ i .. (7) *dams, R.,Joh"so". J. R., and Wilrn~,Ch. F., Jr.,"Labnra*ri Exprimenlain omit Chemistry,"6th ed., Maonillan Co.. London, 1970, p. 268. 181 KuUIBnek. M.."FmentationPmeeuesEmolwed in VitaminCSvnttesh." in Adwnees in ~ p p l i s d ~ i r r o b i o l o g 12,11(1969). y. (9) Fonken G., and Johnson. R., "Chemical Oxidatiane wivith Miowrganiema,'. Mareel Dekker Inc,New York. 1972.p. 243f.
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