Microbial Levan Youn W. tase Unitad Stats Depertinoet of Agricltae “Agieluna esarch Service Soatars Regina! Research Conor "New Orleans, Louisa 70179 dein A Cw © Spmitow, & thton st Actvaton B: Hl 1, Goma Sian rope. rope _Gotpntonsnd opto of Dsl ply en hala aoe erence 1. Fndaon tan Vi ton tan 1 dat Gam aod Ps Etendee D. Oke appcatns 1 Introduction Industry uses lags quantities of natural polysaccharides, and new sources of polysaccharides are boing sought In recont years, atention hhas been diroced toward producing extracellular polyseccharides by ‘microbial fermentation. Examples are dextran and xanthan gum pro ‘duced by Leuconosioe mesenteroides and Xanthomonas campestris, respectively. Recently, the sugat industry has face intense comptition from high-fructose con syrup, which is used as a low-cost alternative ‘sweetener. This chapter investigates the possibility of producing micro- bial levan from sucrose for industrial applications evans are fructans, natural polymers of fructose, The two main ypes of fructans ore the levans, with mostly (2 ~» 6) linkages, and the Inulin, with B(2 > 4) linkages. Branched fructans with both types of ones riamscuercnenccr wines (49Dwm ‘you w 448 linkages also exis, awvan i common namie fora fructan in which mos fructose has (2 6) linkages. A more descriptive name wold be (2 —> Ghenfrucan, Lovans and inulins of low molecular weight (M,: usally ‘<5000} are abundant resorve carbohydrates in many plant tissues (Van smme and Derycko, 1983). Many microorganisms, wlen grown on ttcrae, produce exiraellularlevans of high molecuiat weight ‘Microbial lovan, ike dextran i offen an undesirable by-product of supa juice processing bacause it inereases the viscosity ofthe procoss- ing liquor (Avigad, 1968; Fuchs, 195). was ropored by Lippmann as ‘arly a8 1881, and the name rs propoaod forthe compound. Geoig-Smith (1902) showed that ‘when grown on Suerose. produced fructans, and the name “lovan” was introduced as analogous to “dextran.” The term lovulan now denotes partially de- sraded lovan fractions. Eaely reports about levan are confusing bocause ‘microbial nomenclature was unsystomatic and materials were inade- ‘quately described. Lovans have nover had extensive industrial use. ‘The ow existingroviow atcieson levan ether are obsolatorare of roviews af other micrabil polysaccharides (Avigad, 1968: Evans and Hibbert, 1946; Hohre, 1985: Pontis and Del Campillo, 1985). In this chapter, we present somo of our recont work on microbial reduction of Toven and review the earlier work of others 1 Occurrence ‘A variety of microorganisms produce extracellular polysaccharides as ‘capsule attached tothe cll wall orasa slime secreted into the growth ‘medium. These materials are formed as @ dofenso machanism or 35 @ food roservoir. Some soil microorganisms produce leven, espocially Bacillus sp. Oral bacteria such as Rothie dentocoriose, Streptococcus salivarius, and Odontomyees viscosus produce and accumulate Levan {in human dental plaque (Higuchi etal. 1970; Manly and Richardson, 1968; Newbrun, 1969). Several species of yeast (Fuchs et al., 1988: ‘Loewenburg and Roose, 1957) and fungi (Loewenbarg and Roose, 1957) ‘also preduce levan. Table I Iists some ofthe microorganisms that pro- ‘duce levan. ‘All species of bacili belonging to group 1 in Smith eta. (1952) (8. subiilis, B. megaterium, B. cereus, and B. purilus) produce levan from sucrose (Fuchs, 1959), Many strains of B,polymyaxa that belong group also produce polysaccharides—moslly hetropolysaccarides, which consist of o-glucase, o-mannose, galactose, o-ructose, glucuronic ‘acid, and pyruvate (McNeely and Kang. 1973: Glukhova et al. 1985: ‘Mitsuda eta, 1981; Madden otal, 1986; Fuku et el, 1985; Ninomiye ‘ecw ad Rose (57 ba (997 Warr an lo) ‘etree tne (9571 ‘erates Seinen ao Quel (1958) ‘ence vl is at (188) Fen Gain (G9shr Tas 199) ‘Athair wofacirs Tana ‘Azsabuctorchosseue Haun and Cold (153): Schatvk aera ‘aoc uc ompiqecins Mandan Pana 982) ‘ests meget rane and niet (040 Scr eer Hehe 961 ‘cts pose Nesrinel a 291) Ninoy and Kia (19705 in 089 Mari (982 Dedonder a6 oak (981 Hest t {0913 Mantle Pana (02 Park a enon (04 Yamamoto (105) oryetncteram evenforons Dat and tha 1962 Goyer tale Roa 1903) Clutonctteronydon” haa (081) Uecaraeoe mentees Cyan and Ray (578) Povetomme Fete (19: Er ds (19405. Crow ond ‘dst 987), ati dostoceroes var (08) Steplorecar sp (Cogan an Roby (1979 Shnamac (397) Steptaocorwiveriae Knell and Warchowal (sO) Puce (G9S6 ung oa 87 Root (8 [2981 Ly and Ped Lane (157 uch (958) Kenthomenas Zymamonas ah 0980, Dawes (1200 Lys ad vom ‘paisa ‘and Kizaki, 1970), The ratios of various sugars in the polysaccharides epend on the composition of the growth medium and the microbial strain uted. Polysaccharide yield increases with sucrose but decroasos ‘with glucose and arabinose: mannose hat the lowest polysacehva ef a, 1985), Hallas polymyae grown on sterase mopolysaceharides, eossating of O-100% fructoo. de gon the analytical method used (Murphy. 1952: lan. 1889). tevais ae monoeaiyodons, wheraas arccloscly rolated have dillerent structure aid properties. Mant levans eins, have a much lower molecular woight than bac: teria lovans (Pontis and Dol Campillo, 1985) Lovans are distributed throughout plants: leaves contain small amounts, but large quanitioe fare found in roots, bulbs, tubors, rhizomes, and someones iu mature Truit, whore they sorve as storage carbokydratos and ma resistance fo cold and drought (Meier and Reid, 1982, 1076}. The presence of evans (end fuctane in goneal) in plats has no apparent correlation with the prosance oF absence of starch. No co relation hae been found betwoon the prosenes of lovans{fructans) an the occurrence of C3 and C, photosyathet aye (Bendor and ‘Smith, 1973), The water-soaked appearance of plantsisofton caused by Tevans formod by infected bacteria (Gross and Rudolph, 1088) Lova may holp rolain moisture and aggregate sol at the plant rhizosphere (Webley otal, 1965), M. Biosynthesis A, Levansvonase ‘The biosynthesis of levan requires the extracllular enzyme leva sucrase (sucrose 6-fructosytransterase, EC2.4.1.10), which shows spoc- icity for sucrose. Most resoarch an the biosyathosis of levan has boon ‘conducted using the enzymes from B. subtilis, Aerobocter levanicur ‘and 8. salivarius. Theso enzymes have boon extensively purified at the mode of action expored (Bauer and Brisk, 1979; Eliseshvill, 1984; gold et ol. 1956; Fouet ota, 1984; LoBirun and Van Raponbusch, 1880; Mantsala and Puntala, 1982; Pascal and Dedonder, 1972; Reese and Avigad, 1908). Levansucrase of B. subtilis i inducible and exocel- lular, whereas that of A. levanicum is constitutive end endocallular (Dedonder, 1966). 1 is uncertain whether lovansucrase is one enzyme aor complex of multiple enzymes tha syothesize the main chain (26) ‘andthe branch ft» 1) linkages. Chains of levan, like dextran and starch, grow in sts by repeated \ransfor ofa hexosyl group fom a donor to growing acceptor molocule (estrin ef a, 1936; Hehe, 1935; Sato etal, 1984). ally MOR 3 ANC the 6 ttt ‘The action of levansucras is a slop-by-sop addition of single fee tofuranosyl units at the Ca hydroxyl af ructoe lene hal unit of a growing levan cliain (Fig. 1). The exact nature of this initiation stop ofthe polymer growth fom sucrose isnot clay under. stood. The levansucrase may catalyzs the reactions ofa teaily rovers ible primary step andl a subsequent irrversibile step as fallows a fran + semper vse $e e \whoro fru is fructose, enzis lovanauerase, and R represents a carbonyl of aldose, Possibly, the aldose part ofthe substrate molecule i replaced by an onzyme-linked group, end partial decomposition ofthis Tova precursor to aldose and kotosa furnishes the energy necessary for leva synthesis All levan-orming systems, ithe cellfec or whole-cell pro dduce fructose and a sores of oligosaccharides as products of tanstruc 2 in addition to glucose and the polymer isl, Tho yield of a vas molec rom sco aie by let176 youn w aN iz 40-40% of Frcsee 1968. i sucrose a “dnor substrate” with the Michie to 0.06 M. The optimal pl for Iovansuerase on Sucrose is 5.0-6.0.and the activity at pit 44nd 7.5 was half the rate at 16.0 (Dedonder, 1966; Kiss, 1968). The enzyme activity is stable for Several days at pl 5.0. The enzyme gradually loses its activity at 37°C find complete, almost immediate inactivation occurs at 100. Theaddi- ion of metals (eg, Fo", AP", Zn") increases the stability of the ‘onzyme in lation ta tomporatue, Lovansuerase propared trom B.sub- Uilis hae a sedimontation coeficiont of 27, difusion coefficient of {60 % 10°”, and a M, of ~40,000 (Dadonder, 1966). An enzyme feoze- riod oF kept at ~20°C shows no las of activity for long pee Bocauso hydrolytic breakdown of sucrose and palymertztion of fruc- tose occur simultanooutly, the oxact positon of oquilibrium in levan synthosis is difficult to define, The ratio of Ivan to sucrose at equilih- rium ie generally thought tobe >1, bocause a roversal of tho raction has not boon detected. A yield of lovan at high as 62% ofthe theoretical ‘maximum has boen reported (teh, 1055). Tho prosence of lovan isnot ‘zzential for lovensuersee formation, bul adding prolormed primer (lovan) to a lovan-aynthosizing system sccolarates the rate of polymer ization, incessos Rnal yield, and affects tha production of homogs ‘neous, high molocular weight lovan (Matloon et ol. 1955; Hestin, 1956). Howover, these effcts occur only under conditions of low ionic _trongth (Tanaka eta, 1990} The degre af polymerization of lovan is ‘genorlly regulated by fonicstrngth, and the enzymatic synthesis of {evan could occur in th absonce of an “adapter” (preformed prime). Docillus sublilis levansucrase synthesized lovan fr more effectively at lower then ambient temperatures (Tanaka tal, 1979) B. Srecincery ‘Tho spocifity of levansucrase depends not only on the vfructside but also on the aldose residue of the substrate (Hestrin etal, 1956). Levansucrase of A. levanicum acts on sucrose, raffinose, and invert ‘sugar a mixture of glucose and uctoss). Terminal fructose is generally belied to be necessary for the enzyme activity. However, some sub- strates with terminal fructose are not sultable for levan production. The fenzyme docs nol utilize any other common sugats oF other substrates hhaving a terminal fructofuranose group (eg. fructose phosphate, ‘mothylructeside and inulin), Lavan yield from raffinose is ebout one- {third of that fom sucrose, The amounts of sucrose consumed are Account for entirely as lovan and rodicinyg sugars. When ralfinose (galglustry) is sed as a substrate, the products ae leva, melbiose (falta), and fructose, but not sucrose and galactose. A. sors of lucoso-ended, 26-inkod polysaccharides can bo usod in synthesis of the fovan molecule (Halve, 1951). However, neithor levanbioso, le ise, nor lovantetraosois found tosorve a an accaplor substrate In ‘Addition, levansuerase from B. subtilis appoared ta have some alii for glucose but nat for fructose. . Innere aie Aeration ‘Tho formation oflevan from sucrose by lovansucras is inhibited by various sugars and sugar alcohols (Table Il. Inhibition was caused by both sugars and glycosides, for example, by glucose, moths alycoside, and others that have a configuration about carbon slow 2 similar to that of glucose, such as galactose, maltose, {ose (Hestrin and Avineri-Shapiro, 1948). However, no appreciable Inhibition occurred for products like O-mannose, o-fruclose, oF ‘mannitol, fr which configuration or structure et carbon tom 2 difrs from thal of wglucose. The inhibition of levansuerase activity by gl ‘ose is function of glucose concentration, and complete inhibition of levan production from 1% sucrose occurs t 16% glucose concentration, ‘The inhibitory effect diminished a tho glucose concentration fel 1 tiveto that of sucrose. Thus, t appears that glucose inhibition is caused "Seema st Suan Oar (arto Aalto Wan ad a Siby eampotition with the glacase moiety of sucrase for tho enzyme (Tkachenko and Toitsyanskaya, 1976) Poyeliylono glycol of M, 4000 ‘hasan activating effect, increasing the maximums voloity by a factor of Uheee (Dodonder, 1960). D. HHvemyass Many lovanfonning microorganisms also produce hydol xymoe lovanasos, that daprade the lovan (osttin and Golds ‘Avigad, 1965), Certain stains of Psoulomonas, Azolobactor, Aorobac- ter, Serratia, Bacillus, Clostridium produce exocellular lovanase (Fu che, 1959). Lovansuorasoitzll is suspected to cause tho hydrolysis under certain conditions (Rapoport and Dedonder, 1063). Although Indirect evidence ofthe reversal of onzymatic syathosisoflovan can bo ‘observed litte fs known about the nature of such enzyme degradation. Smith (1976) showed that f-fructofuranosidase prosont in fall fescue Alograded lovan by reioving one fructose rosiduo at a time until a tolecul of sucrose remained. Lovansucrase of B. subilis has shydo- Iie effect on small lovans (Dodonder, 1960). This hydrolytic action stops at branchpoints. Neither inulin, inulobioso, inulotrioss, nor ‘mathyl »fructofuranoside is hydrolyzed, although these substrates are hhydrolyzed by inulinse and yeast invertaso. This hydrolytic activity may be responsible for the appearance of heterogeneous short-cha polysaccharides, rather than uniform high molecular weight polymers, |W, Chemical Structure and Properties ‘A. Srmucrune Lvans ere polymers of p-fuctose attached by (2 6) linkages that carry # glucosyl residue at the end of the chain. Thoy constitute @ series of homologous oligasaccharides and polysaccharides, which ean bbe considered derivatives of sucrose (Hist, 1957: Tanaka etal. 1981). Figure 2 shows the chemical structures of levan and inulin. They are usualy representod by the formula C-F-(}, where G-F denotesa sucro- ‘yl group, F the fructose, and nthe nummbor of fructose units present im the molecule. Although the structure of lovan is represonted by 4 straight chain of (2 ~ 6), linkagee of many bacterial lovans are branched through (2+ 1} bonds (Feingold and Gaia, 1957; Avigad and Feingold, 1965; Dawos et, Lindberget al, 1973: Marshall and We ‘usually short and sometimes consist eal in (F2-1F2-1F 2-12-16 ) ERE Levan ( F2-8F2-6F2-6F2-16 ) Pe: 2 Choma autre of iain od (a, ‘The structures of levans synthesized in call-foe enzyme systoms a similar to those produced by whole-cell systems, but thoy differ in length. The average chain longth of lovans producod by the enzyme systom was reported to be 10-12 monomeric units, whereas that by ‘whol call was much longer sometimes the molecular waight exccedod. several million (Evans and Hibbert, 1946; Marshall and Weigel, 1980; Stivala and Zwoig, 100%; Tanaks o ol, 1963; Ponti and Dol Campill 1985). In gonoral, fovans produced by diferont organisms have similar ‘raclures The difference may bo a vatying dogres of polymerization ofthe repeating u ‘Lovans ere one othe fow natural polymors in which thocarbohyérate Sault cl, oA), Moreover the enbancd flexibility ofthe fersnose ing in comparison with tho rolatively rig pyranceo ring of ity of reserve polysaccharide givos additional flexibility to the whole fructan molecule. The linear 2+ 6linkod levan moleculos are axblenl tend tbe Heft twist whore tho ois of iain is ght hand Feonch, 10M}. ‘The composition and propartis of lovan depend yreatly an the envi onmontal factors i which ti amis ar grown (Stivala and 1982}. Tho goneral propertis of levans rscrabe those of dextrans, Lovans are lovaroitory, amorphous oF microcrystalline, of ‘varying solubility in cold water very soluble in hot water and insoluble inabsalutcthyl alcohol. Levans are generally nore soluble than inalin, Wwihich is almost insoluble (<0.5%) in water at room tomperature (Phelps, 1965) The high solubility ofl bea charactorstic of {2+ 6) linkage compared to B(2—+ 1) linkage. nay bs only a suppor factor. Lovans ao nonroducing, not hydrolyzed by yeast inver- {ase and amylaso ection, but vory susceptible to hydeolysis Ly acid ‘They ate not colored by iodine, but hydrogen chloride imparisa purple colar that distinguishes leva from other polysaccharides not con ng fructose (Ponts and Del Campillo, 1985). The molocular welght Viscosity of levans vary doponding on the organisms used. Goncraly, thor viscosity n aqueous solution increases sharply when various salts ‘represent, but it decreases drastically with asmallincreaseintempora- tur (Kang and Cottroll, 1979} ‘Certain biological proponies of lovan, such as promotion of infection and necrosis (Feingold and Gohatia, 1987; Shilo, 1959), tumor ink tion and stimulation (Loibovil and Stark, 1984; Yavetr et o.. 1965: Kelbovict et el. 1986: Stark and Lelbovicl, 1986). and increase in cell permeability for a cytotoxic agent (Leibovict and Stark, 1985) have attacied attention. These elfects are partly caused by suppression of normal inflammatory response. Only levan with M, >10" promotes infection: the effect is lost when the polymer degrades. Levan given intravenously to mice greatly incrases the virulence of intrapert ‘oncalyinjocted bacteria. This pally caused by th intravenous lovan sealing the vascular lining, thus affecting its permeability and pro- venting escape of blood constituent into the peritoneal cavity. The fendothalial sealing of lovan may have practical importance. Natural levans are serologically active and elicit antibody production, but p> ‘fed levan preparations sre not antigenic, © Comrosmow ano Prorcenes or Bocillus polymyxa Levan ‘The levan produced by a strain of B. polymyxa (NRRL B-18475) ‘consisted of ~08% fructose when analyzed by high-porformance liquid cKOHIAL LEVAN mi chromatography (HPLC) ofthe acd hydrolysate (Han and Clarke, 1990) ‘The product wae readily solublo in witor (upto ~S%) a ows tempers ture, bat farther product was very suscepliblo to hydrolysis when heated for 15 minutos |n059% oxalic ati. The easy hydrolyse typical ofthe fuctolursno structure (Feingold and Cohatia, 1957). Bocauco tho intial molecule in levan fermation is suctoso, a torn bo prosont in emia groupe vo significant amount of glucaue ws ‘A'5% aquoous solution of erudolovan, after dilysis tough a morn: brane with 12,000 Da cut off, gave one sharp clean peak jst below 2 10" Da on Sophacryl $500 (Clarke eto, 1908) This poak was sharpor (oprocenting a narrower rango of molecular mse) than those of the ‘commercially available dextrans used as gel permeation chromatog phy (GPC) standards. The uniformity ofthe product was perhaps caused by long formentation (<10 days) and absence of hydrolytic ectivity in thecnzyme system. Thecompound was slabe in aqueous solution at pt 45 for =36 hours. The B.polymyxa (NRRL B-18475) leven has specific ‘optical rotation fal of 42.07, wheroas most other lovan preparations Were reported to be 44 = 4° (Schlubach and Berndt, 1964; Murphy. 1952; Dedonder and Peaud-Lenoel, 1957; Barker et al. 1955). The 8. polymyea levan was nonhygrescopic: lyophilized shoos of levan have been maintained under atmospheric conditions for <6 months. ‘mn Fig. 3, carbon-13 nuclear magnetic resonance (C-NMR) poaks ie 3. OAMH pat fin ad (lore pect by Sey)me vow w as from levan are compared to peaks from inulin, The MENMR spec ra es at 108.2, 80:5, 77.0, 75.7, 61.6, and 60.7 ppm. which is almost Wentieal othe peak positions for levan pre- viously kientified by S 1a. (1987) The poak positions vary slightly from values in the literature, but the relative spacings botwoon the values obtained fom experimenial levan and those frm the ture are similar. The anomeric peak (C-2) ty carons[C-1 and C6) are more closely grouped in i Ting carbons (C-3. C-4, and C5) are more closely grouped i his is characteristic ofthe dilferoncesbetwoon (Seymour et al. 1979). Data clearly show the polysaccharide produced by he B. polymyxa (NKR 8.18475) 0 be levan type with tl (2 + 6)fructofuranoside (Table il}. tra of bacterial Iovan and inulin, The infrared specra of the bacterial Tevan were similar to inulin but the bactei lowed a character {stically weaker peak at dt (cin (Barkar snd Stophons, 1954), Lasher (1976) oported that Levan had infrared absorption peaks (em-*) at 919, ‘860, and 803, whereas inulin had peaks at 933, 872, and 13. ‘Methylation analysis revealed thatthe B. poiymyxa levan was mado of 71% l2 —> 6} linkage (1.3.4ctrimethyl-pdructose, 13% terminal {group at 1 oF 2 postion (13.4.6-0F 2.3.45 otramahyl0-ructoee, 2% branching at 1.2, oF 6 position (3:4 dimethyl-ofructse), and 4% free fructose (Clarke al 1988) The branches occurred in the C-1 postion with a f(t + 2) linkago, with side chains of (2 ~ 6)linod residues. Murphy (1952) also reported a similar composition for B. plymyra ‘Tame tn (user Srtarox “CNR Sacra oe rooics Beto poms NRL Cartons _Tnlin® tevan® ke = § wo| I" ae 4 Inara spat fl intin ad (evan pcb N Moi lovan, The degree of branching in other microbial evans i reported to bbe 520% (Lindborg eta, 1973) ‘The B. polymyxa polysaccharides exhibit relatively high viscosity and high pseudoplasticity (Kang eof, 1983). The viscosity ofthe prod ct is highest when grown an sucrose or glucose and lowest on xylose, ‘Des and Maddon (1985) report that B: polymyxa (NCIB 12429) polysac-mma win we 6 teat yield-strese solution ths cncantation Y. Analytical Methods A. lavas Levan may be determined by using any ofthe colorimetric specific for kelove an exptessed as thee fructose content (Lo ee 1978). The thiobarbituric ac method! (Percheron, 1962) or the seid resorcinol color eaysn! metho (Roc et al. 1943; Arsen, hiydoalysis(.4.0.5% oxalic i 100°C. 19 minutes) may also be used, ‘The amouat of fructose In ino hydrolysate ca be determined by the Teva in the fermentation ure through a column of x Matrex Collafin, GH25 Danvers, Massachusets). A small amount of a ‘isture of levan and dextran was analyzed by passing tho sample through a polarimeter and usingan automated resorcinol thiourea test (Manly and Cormier 1270 ‘A dire! method of measuring lvan is by HPLC. Lavan and free sugars were easily separated onan anion exchange column (eq. Aminex HPX- {7C, Biorad, Richmond, California} (Han and Clarke 1989). Paper and thindayer chromatography (TLC) can be usod for determination of short-chain componcats in levan-producing systems or levan hydroly- Sate. Small quantities of the noamobile levan ean be separated from other Keto sugars by TLC. The position of lovan can be identified by Spraying reagents specific for levan, such as reeorcinol HC and nap- thoresorcinol HCI (Forsyth, 1950: Yapho and Arsenault, 1965), urea HCI ‘and urea metaphosphoric acid (Dedonder. 1966). and dimedon ((5.3- dimethy-1.s-cyclehoxanedions)phasphoric acid) (Adachi, 1968). Le van in fermentation broth ean be approximated by wefghing the precipi te formed by adding ethanol or isopropanol. The precipliate can he fled in advance by redissolving in water, dialyzing. and freeze drying “The structur of lovan can be studied by instrumental analysis. n- {tared spectroscopy is widely used to investigate polymer structure and {oanalyze functional proups, The infrared absorption spectrom oflevan has (ypical bands, which can help in is analysis end differentiate fr 1976). cits fructofuransidos anil partly decompese during derivatization to fori stable for analysis, MC-NMAK spin scopy fs usofl method to determine the linkage type of polyec (Shimanairs ee, 1947: Seymour ot, 1979, The MC-NMI spi ff levan an lin produc sx main resonances, and ea Auli and other polysaccharides (stein etl, 1994: Lashe ha classic! methylation analysis, in which polysaccharides aro «x hhaustively methylaod, hyerolsed. derivatizad with aldol acetate, andthe methy! eer obtained is analyzed by gas-liquid chromatogrs Phy (Hakomor, 1964) Thesa studies provide information onthe links exten! of branching. but cannot show he ents of side ‘of geval importance in siudying immunochemical peo His (Linalborg eta. 1973), Immunoassay mtd utilizing antifructan activity of some myelan fmmunoglobuline have Leen proposed (Glaudemans, 1975) Some ae specific for Iovan (Cisae et al, 1874). whareas others are specific for {Inulin (Sioofkark and Glaudemans, 1977) The precipitate formed with concanavalin A differentiate levan and inulin (Goldstein and So 1965). Although no attempts have Deon made to quantity levan by this ‘method, the method may makeit possible tadetermine lovan ina highly specific way. B, Levanta Lovancueraso is an enzyme responsible for forming lovan from su rose and accumulating glucose in the mensiruum. Therefore, levan- {veraso asay is gonerally bazod on the dotermination of foe glucoso in the reaction minture (Chamber ef al, 1974: Tanaka et af, 1970). The reaction conditions aro eot 0 thal the volocily of glucose formation ‘depends only on the amount of enzyme (2r0-0rdr reaction}. The rea: tion is stopped by dilution in boiling butler: the fre glucose is dt mined by the glucose oxidase method or the Somogi-Nelson method (Coska, 1971). This mothod can bo used for Iovansucrase in cultural broth, call extract, o ntact cells. However this method eannot bo usod to test for activity in cultures grown on glucose as substeate. The Addition of lovan primer tothe reaction mixture provides more consis- {ent conditions forthe assay during purification. Because conditions of the reaction produce significant variations in levan yields, any mathods based on lovan determination cannot be used to asray the enzyme100 oun was activity. Such a determination way be ws Ton conditions for the study of the ree VI. Production of Levan Allkxown production of xopolysaceharidesisby aerobic submerged fermentation. Microbial production of polysaccharides requires fe mentation and handling of a highly viscous solution, which Is not volved in many nonviscous fermentation processes, Unique fermen- tation parameters of aeration, agitation, and pH and temperate con- Uwols should be established foreach polysaccharide fermentation. The conditions for producing levan by grossing cultures of baceria vary according the mieroorganisins used. Figuse5 slows the flow diagram ‘of levan production by B. polymyxa. Bacillus polymyxa (Strain NARE,0-16479) produces a large quantity of extracellular polysaccharides when grown on 4=10% sictose 80 lution. Table 1 list the composition of he medium for levan produ tion, Yeast extact enhances the levan yield. The highest level of evan A ose cute ‘Ae rein, 1 a 1 . om ! | | it recy Pe: 3. Mow cagamet evar podctin by ci plymaa stale NRALIBKS. was obtained in media contining 815% sucrose. The organistn caw. verted sucrose (S| to levan {L] and accumulated a sil ammount of elucose (G) in the growth medium (Pi. 6), During fermentation, the sucrose levels dropped and levan started to appear in 2 days hereate, sucrose lovels gradually deersed as levan increased, Glucose was the ‘najor by-product. & small amaust of fructose (F) and unidentified fermentation producis smaller in molecular weight wore alo observed ‘The pit of the growth modivin fll from 7.0 (0.4.7 because of acid th | ke or Lille | Like | Lik eft tn oman ding rnntsinn rey cir payee en