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,Author: Margaret E. LongDivision: Biological Research

RDR, 1968, No. 33

No. of Pages: 16

September 13, 1968Notebook Pages: 147122-50,165151-200, 151301-50,154951-5000, 157351-400,158701-50, 163701-50,167951-8000, 172351-400,

170751-800, 161001-49,174301-50, 166751-800,166151-200, 157351-81,175651-68.) /,)0, 4a7-5dDated: February 28, 1966through October 13, 1967Previous Reports: None

ENZYMATIC CONVERSION OF D-GLUCOSE TO D-FRUCTOSE:IS OLATION AN D IDEN TIF IC ATION OF AN AC TIVE N EW B AC TER IAL S PEC IES

OBJECT:To screen natural sources for new microorgartiisms which actively catalyze

the conversion of D-glucose to D-fructose.

S U M M A R Y :Culture 2453, a hitherto undescribed species of Arthrobacter, producesan intracellular enzyme which catalyzes the conversion of D-glucose to D-fructose. Initial cell preparations converted 1 molar glucose solutions to

mixtures of 36% fructose and 64% glucose. Isomerizing capacity exceeded thatof various other organisms examined including several known species ofArthrobacter. Preliminary studies indicated specificity for D-xylose as theprinciple substrate in the cultivation medium for maximum enzyme synthesis.

EJ3 )C O NADN1LL

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T A B L E O F C O N T E N T S PageOBJECT. . . . . . . . . ................... 1SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . 3A . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 4B . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . 4

I. Isol ati on of Organisms . . . . . . . . . . . . . . . . 4II. Identification of Initial Working Srain ....... 6

a. Morpho 1 ogy . . . . . . . . . . . . . . . . . . . . 6b. Cultural Characteristics . . . . . . . . . . . . . 6c. Physiological Characteristics . . . . . . . . . . 7d. Temperature Relations . . . . . . . . . . . . . . 7e. Special Staining Reactions . . . . . . . . . . . . 7f. Source . . . . . . . . . . . . . . . . . . . . . . 7g. Comparison with Standard Type Organisms ..... 7

III. Preliminary Studies of Enzyme Production ....... 8a. Glucose Isomerizing Properties of SeveralSpecies of Arthrobacter . . . . . . . . . . . . . 8b. Carbohydrate Specificity for Enzyme Synthesis .. 9

1. EFFECT OF SUBSTRATE TRANSFER . . . . . . . . 92. EF F EC T OF C R UDE XY LOS E AND XYLOS E-CONTAINING MATERIALS . . . . . . . . . . . . 103. T H E E F F E C T O F P U R IF IE D B A G A S S E X Y L O S E . .. . 12

C . DSCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . 1 3D . CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . 1 4E . RECOMMENDAT I ONS . . . . . . . . . . . . . . . . . . . . . . 1 4

1 . Fuure Work . . . . . . . . . . . . . . . . . . . . . . 1 4I I . Patentabi l i ty . . . . . . . . . . . . . . . . . . . . . 15

BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . . . . . 1 6

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LIST OF TABLES

Table No. Title PageI Media Used for Primary Screening of

Glucose Isomerase Producing Organisms . . . 5II Characters Which Distinguish Culture 2453

as Distinct from Presently DescribedChromagenic Arthrobacter Species . . . . . . 8

III Conversion of Glucose to Fructose bySeveral Species of the Genus Arthrobacter . 9IV Effect of Substrate Transfer on Productionof Cellular Glucose Isomerase . . . . . . . 10V Synthesis of Cellular Isomerase in Culturesof RJR 2453 Containing Crude Xylose Preparedby Hydrolyses of Bagasse and Corn Hulls . . 11

V I Enzyme Synthesis in Cultures of RJR 2453Containing Mixed Carbon Sources ...... 12VII Comparison of Pfanstiehl Xylose withXylose from Eastern Chemical Company asThe Carbohydrate Source for CellularIsomerase Production by Culture 2453 . . . . 13

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A. INTRODUCTIONSyrups derived from corn starch are primarily rich in glucose and

deficient in fructose; thus, they are characteristically less sweet thansyrups derived from sucrose. The level of sweetness may be increased con-siderably by partial conversion of glucose to a mixture of glucose andfructose; however, chemical methods, such as the Lobry de Bryun-vanEkenstein transformation, which employ heat and an alkaline catalyst,frequently result in the formation of objectional by-products. Therefore,an investigation of enzymatic methods for the conversion of glucose tofructose was proposed.

Marshall and Kooi (4), in 1957, reported conversion of glucose tofructose by an isomerase preparation obtained from cells of Pseudomonash dro hila which were cultivated in a nutrient medium containing D-xylose.This and a similar preparation derived from an uncharacterized species ofBacillus was the basis of a process for the enzymatic formation of fructosein corn syrups and other glucose containing liquors for which a patent wasgranted to Marshall (5) in 1960. The patent not only claims the processbut also the species of Pseudomonas and all members of the genus Bacillusas sources for the isomerizing enzyme.

Since 1960, several Japanese investigators (7, 8, 9, 10, 11) havedemonstrated microbial enzymes which catalyze the glucose to fructose trans-formation. Active preparations were observed from such varied types ofmicroorganisms as Aerobacter cloacae, Lactobacillus brevis, Brevibacteriumpentosoaminoacidicum, Paracolo actrum aerogenoides,BaciTlus megaterium,and Streptomyces phaeochromogenes. Developmental studies for practical useof the enzyme, however, were conducted only with Aerobacter cloacae andStreptomyces phaechromogenes by Tsumura and Sato 10 and Ta ascacT(7),respectively.

In view of the evidence presented in those publications, applicationof an enzymatic system to the production of invert sugar from D-glucoseappeared feasible. A program was initiated whereby new and more activemicroorganisms might be discovered in order to design and develop a novelprocess for the efficient production of fructose in high D.E. corn syrups.

B . E X P E R I M E N T A LI. Isolation of OrganismsPotato pulp and other waste substances collected from the Idaho PotatoPlant, local bakeries, and corn milling plants served as the starting materialin the search for isomerase producing organisms. All test materials weresuspended in sterile basal salts solutions, and the suspensions were blendedat low speed for approximately 30 seconds in sterile Waring blendors. Onedrop was placed on the surface of agar plates and streaked with a wire loopin the conventional manner. Two plating media, based on a formula proposedby Tsumura and Sato (10), were used for screening and were prepared as shownin Table I.

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TABLE IMEDIA USED FOR PRIMARY SCREENINGOF GLUCOSE ISOMERASE PRODUCING ORGANISMS

Medium X-I Medium X-II(NH4)2HP04 6.0 g. (NH4)2HP04 6.0 g.KH2P04 0.2 g. KH2P04 0.2 g.MgSO47H2O 0.25 g. MgSO47H2O 0.25 g.Xylose 20.0 g. Yeast Extract 1.0 g.Agar 20.0 g. Tryptone 5.0 g.

Tap Water 1,000 ml. Xylose 20.0 g.p H 6.9 Agar 20.0 g.

Tap Water 1,000 ml.pH 6.9

Both simple and enriched mineral salts solutions were sterilized for15 minutes at 120 C. Xylose (Eastern Chemical Corporation, Pequannock,New Jersey) was prepared as a 50% solution in distilled water, sterilizedby filtration, and added asceptically to the media as needed to give a finalcarbohydrate concentration of 2%.

The plates were incubated at 20 C., 25 C., and 30 C. for 48 hoursafter which all colonies were picked to slants of corresponding medium tobe held in stock prior to enzyme analysis. Cultures for primary examinationwere transferred to fresh slants and incubated at 30 C. for 24 hours.Growth from each culture was washed from the slant with 1 ml. of liquidmedium to serve as a 1% inoculum for flask cultures.Flask cultures, consisting of 100 ml. of the appropriate medium in 500 ml.

Erlenmeyer flasks, were incubated at 30 C. on a horizontal rotary shakingmachine (250 rpm, 0.5 in. stroke). Growth was produced in two stages, a 5%transfer being made to fresh medium at the end of 24 hours. The pH, opticaldensity, and enzyme production were observed periodically from the secondgrowth stage, usually up to 72 hours incubation. The pH was determined byBeckman Potentiometer and the optical density by Lumitron Colorimeter usinga B650 mu filter. In the early studies, cellular isomerase was measured aspercent conversion of glucose to fructose using a standard solution of glucoseand a constant volume of culture according to a method devised by Shiffert (6).All organisms capable of converting 20% or more of the standard glucose solution

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to fructose were retained as active and potentially useful cultures. Later,following enzyme characterization, activity was recorded as microunits perml. of packed cells resuspended to a specific concentration in distilledH20 as described by James (3).

II. Identification of Initial Working StrainWeak glucose isomerizing properties could be demonstrated in approximately80% of all isolates; however, an optimum glucose conversion activity of 36%was obtained with the cells of one organism which was therefore selected asthe initial working strain and assigned the RJR stock culture number 2453.Since culture 2453 was isolated on X-II medium at an incubation temperature

of 300 C., it was purified by repeated platings under the same conditions.Taxonomic characteristics were studied for the most part according to theManual of Methods for the Pure Culture Study of Bacteria (1952). Theclassical description of the organism is as follows.

a._ Morpholo_9y_Rods, irregular in shape and size, generally 0.5 to 0.7 by 1.0 to3.0 microns, occurring in angular and palisade arrangement. Cells maybe straight, curved, or bent, frequently clubbed or swollen with rudi-mentary branching. Coccoid cells, 0.6 to 1.0 micron in diameter, andoccasionally large spheresup to 3.0 microns in diameter occur in oldercultures. On transfer to fresh medium, coccoid cells give rise to rodforms by germination leading to fragmentation. Cells are nonmotile.Early cells are gram negative, many with gram positive granules. Mostolder rods and coccoid forms are gram positive, but a few gram negativecells may be also found.b. Cultural Characteristics--------------

days.Gelatin Stab: Slow stratiform liquifaction, up to 50% after four

Agar Colonies; Spindle or circular up to 2.5 microns in diameter;convex to umbonate; entire, smooth, glistening, cream to pale yellow.Agar Slant: Growth filiform, flat, soft, butyrous, yellow.Nicotine Agar: Abundant pale yellow growth. No pigment producedin medium.Asparagine Agar: Growth scant, flat, filiform, colorless.Broth: Turbid, dispersed at 24 hours; sediment cream to pale yellow

after four days.Potato: Growth heavy, bright yellow, butyrous to mucoid.

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c._ Physiological CharacteristicsLitmus Milk: Slow clearing without coagulation; alkaline afterfive days.Indole: Not produced.Hydrogen Sulfide: Not produced.Sugar Media: Slight alkaline reaction; but no gas formed afterfive days from glucose, lactose, fructose, maltose, mannitol, sucrose;neutral reaction in xylose broth.Methyl Red: Negative.Acetylmethylcarbinol: NegativeStarch: Hydrolyzed.Nitrites not produced from nitrates.Urease: Positive.Utilizes nitrates and ammonium salts as nitrogen sources; citrate

utilized as sole source of carbon.Catalase: Strongly positive.Aerobicd._ Temperature RelationsOptimum near 30 C.; good growth between 20 C. and 30 C.; lightgrowth at 10 C. and between 37 C. and 40 C.; no growth at 45 C.e._ Special Staining Reactions_Cells are nonacid fast.True spores could not be demonstrated. Although cells containvarying amounts of material which retains Malachite Green, the materialis heat sensitive (80 C. for 10 minutes).f._ Source_Isolated from potato waste. Habitat probably soil.u._ Cqjparison with Standard_ Type 0rganisms_The basic cultural behavior patterns of culture 2453 were comparedby parallel studies with several standard strains of Corynebacterium,Brevibacterium, Bacillus, and Arthrobacter. The distinct cyclic mor-phology during growth was in agreement with members of the genusArthrobacter. Of the species hitherto presented in Bergey's Manual of

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Determinative Bacteriology, 7th ed., culture 2453 was most closelyrelated to the chromogenic, nonvitamin requiring strains A. aurescens(Clark 1961) Phillips 1953, A. ureafaciens (Krebs and Eggleston 1939Clark 1955, and A. ox ydans Sguros 1954 (yellow biotype). The morefastidious chromagenic species, A. citreus Sachs 1954, was alsoincluded in the comparative studies. The physiological propertiesdid not coincide completely with those of any of the described species,and culture 2453 may be clearly distinguished from the other chromagenicspecies by the characters listed in Table II. Consequently, culture 2453was assigned to the genus Arthrobacter with the designation Spec. Nov.

TABLE I ICHARAC TERS WHICH D ISTINGUISH CULTURE 2453

AS DISTINCT FROM PRESENTLY DESCRIBEDCHROMAGENIC ARTHROBACTER SPECIES

A. A. A. A. Cultureaurescens citreus oxydans ureafaciens 2453Starch + - + - +Gelatin + + + + +Urease - - + - +MotilityH2S + - - -Nitrate + + + +ReductionNicotine - - +HydrolysisCitrate + - + +Potato Brownish Scant Abundantyellow dull yellowcheesy yellow to gray

varyingwithstrain

+Yellowish Abundant

brown brightcheesy yellowsmooth tomucoid

III. Preliminary Studies of Enzyme Productiona. Glucose Isomerizing Properties of Several Species of ArthrobacterPure cultures of the eight species which were used for the comparativecharacterization of culture 2453 were cultivated in the X-II medium in the

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substrates. On the other hand, introduction of other carboncompounds to flask cultures actively growing in xylose medium hadno adverse effect on enzyme production. Total cell yields, however,were slightly lower from mixed substrates than from pure xylose;thus, no particular advantage was gained. Some typical reactionsof cultures to the pure substrate and to substrate transfer areillustrated in Table IV.

TABLE IVEFFECT OF SUBSTRATE TRANSFER ON PROD UCTION

OF CELLULAR GLUCOSE ISOMERASE

Carbon Source1 Optical Density2 % Activity3Xylose .782 21.0Glucose .658 1.5Glycerol .638 1.7Glucose + xylose .403 5.0Glycerol + xylose .387 5.0Xylose + glucose .420 22.0Xylose + glycerol .432 26.012.0% concentration of pure carbon compounds in X-II medium; 1.5% con-centration total carbon compounds in mixtures in X-II medium.2Cells harvested at 24 hours. Carbon source additions made at 16 hours.Cultures incubated at 30 C. on horizontal rotary shaking machine(250 rpm, 0.5 in. stroke).3Enzyme activity measured as % conversion standard glucose to fructoseby constant volume of culture, Shiffert method (6).

2. EFFECT OF CRUDE XY LOSE AND XY LOSE-CONTAINING MATERIALSThe replacement of xylose in the X-II medium by either xylitol,xylan, or pulverized corn hulls (30 mesh) failed to support goodgrowth of culture 2453. Poor cell yields from cultivation in xylanand xylan-rich material suggested that culture 2453 was unable toutilize those substances as carbon sources. Therefore, crude xylosepreparations, obtained by either acid or steam pressure hydrolysisof corn hulls and also bagasse, were examined as substrates. Althoughthe hydrolysates supported good growth, they failed to stimulatecellular enzyme synthesis equal to that of pure xylose controls asshown in Table V.

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T A B L E VSYNTHESIS OF CELLUL AR ISOMERASE IN CULTURESOF RJR 2453 CONTA INING CRUDE XYL OSE PREPAR EDBY HYDROLYSES OF BAGASSE AND CORN HULLS

Carbohydrate AssayHydrolysate* % Enzyme AssayNumber Origin Xylose Glucose Arabinose Galactose p units/ml.Control-EasternChemicalCompany 96 Trace 61 1RJR- Corn 56 7 26 228167934 HullsRJR Corn 51 8 25 6 292167935 HullsRJR Corn 51 5 26 6 277167936 HullsRJR Corn 55 8 26 7 1 67167938 HullsRJR Corn 41 10 23 5 71168221 HullsPF Bagasse 101 2 -- -- 345167939-1PF Bagasse 9 7 -2 -- -- 395167939-2PF Bagasse 96 -2 -- -- 3661 6 7 9 3 9 - 3PF Bagasse 1 0 5 Trace -- -- 3561 6 7 9 3 9 - 4RJR Bagasse 80 7 5 2 21 7167940RJ R Bagasse 90 3 2-3 1 1 6816 7 9 4 1RJR Bagasse 87 3 -3 1 1 79167942Two percent final xylose concentration in X-II medium; 1% EtOH and 10 ppmFe2SO4 added to standard formula; carbohydrate assay by thin-layer and gaschromatography, James (3); enzyme assay, James (3).*Hydrolysates prepared by Organic Division, R. J. Reynolds Research Department..,

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with no loss in isomerizing activity. Also, substitution with hydrolysatesof rice stalks and waste syrup for pure carbohydrates met with some success.The enzyme, however, was unstable and deteriorated rapidly upon exposure toair. Conversely, substrate transfer by two-stage cultivation of culture 2453failed to improve either cell populations or cellular isomerizing activityover growth in D-xylose medium, but the preparations were highly stable.Both lyophilized and acetone dried cells retained isomerase activity evenafter ten conversion reactions with different reaction mixtures in test tubeexperiments by Shiffert (6). Reportedly, Streptomyces phaeochromogenes isthe only organism capable of utilizing xylan or xylan-rich substances assubstrates for enzyme synthesis. Takasaki (7) obtained active enzyme pre-parations from cell-free extracts when the strain was grown in medium con-taining from 1 to 4% wheat bran, corn hulls, or corn cobs. On the otherhand, except for D-xylose, refined sugars were ineffective.

No mention of the genus Arthrobacter has been found in any of the literaturerelating to the isomerization of sugars by microbial enzymes. Thus, the genusas a whole and culture 2453 in particular assumed practical importance in thedesign of a new process for the interconversion of glucose to fructose.Although preliminary indictions of substrate specificity could be of someeconomic concern because of the relatively high cost of pure xylose, thelevel and stability of isomerase activity of culture 2453 was considered tobe promising. The organism was retained for developmental investigationswhereby the enzyme yields might be increased by specific cultural or strainselection methods.

D . C O N C L U S I O N S1. Culture 2453 is a hitherto undescribed species of Arthrobacter.2. Culture 2453 synthesizes an intracellular enzyme which catalyzesthe conversion of D-glucose to D-fructose.3. The isomerizing capacity of culture 2453 is superior to numerous

other organisms screened from natural sources and also to all other speciesof Arthrobacter tested.4. Maximum enzyme activity is obtained when cells are cultivated inmedium containing D-xylose as the principle source of carbon.5. The presence of other sugars, particularly glucose and arabinose,during the growth cycle tend to inhibit enzyme synthesis.6. Xylan and xylan-rich substances are not utilized for growth.Hydrolysates and unrefined xylose preparations are poor substrates for enzymesynthesis.

E. RECOMMENDAT IONSI. Future WorkCulture 2453 and all substrains will be retained in permanent storage.

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BIBLIOGRAPHY

1. Hochster, R. and Watson, R., Enz matic Isomerization of D-Xyloseto D-Xylulose. ARCH. BIOCHEM. BIOPHYS., 48, 120-129 (1954).2. Ishimura, M., Hirose, Y., and Katsuya, N., Glucose Isomerase fromBrevibacterium. Presented at the Meeting of Agr. Chem. Soc., Japan,Kanto Branch, September 1964.3. James, William B., Automated Analysis of Fructose. RDM, 1968, No. 2(January 5).4. Marshall, R. 0. and Kooi, E. R., Enzymatic Conversion of D-Glucoseto D-Fructose. SCIENCE, 125, 648-649 (1957).5. Marshall, R. 0., Enzymatic Process. U. S. Patent 2,950,228(August 23, 1960).6. Shiffert, J. D., The Conversion of D-Glucose to a Mixture of D-Glucose

and D-Fructose Using Glucose Isomerase. RDR, 1967, No. 2 January 18.7. Takasaki, Y., Studies on Sugar Isomerizing Enzyme Production andUtilization of Glucose Isomerase from Streptomyces Sp. AGR. BIOL.

CHEM., 30, No. 12, 1247-1253 1966 .8. Takasaki, Y. and Tanabe, 0., Isomerization of Sugars by Bacteria.

I. Identification and Cultural Conditions of Glucose-IsomerizinBacteria. JOUR. AGR. CHEM. SOC. (Japan) _ 0069. Takasaki, Y. and Tanabe, 0., Studies on Isomerization of Sugars by

Bacteria. Part IX. NAD Linked D-G ucose-Isomerizing and D-Mannose-Isomerizing Enzyme from Paracolobactrum aerogenoides. JOUR. AGR.CHEM. SOC. (Japan) 30, 220-225 1966 .

10. Tsumara, N. and Sato, T., Enzymatic Conversion of D-Glucose to D-Fructose: Part I. Identification of Active Bacterial Strain andConfirmation of D-Fructose Formation. Part II. Some PropertiesConcerning Fructose Accumulation Activity of Aerobacter cloacae,Strain KN-69. AGR. BIOL. CHEM. (Japan) 25, 1123-1134 (1965).

11. Yamanaka, K., Sugar Isomerases. Part I. Production of D-GlucoseIsomerase from Heterolactic Acid Bacteria. art II. Purificationand Properties of D-Glucose Isomerase from Lactobacillus brevis.AGR. BIOL. CHEM. Japan) 27, 265-278j-1-963 .