Biochem. J. (1984) 220, 213-220Printed in Great Britain

213

The extraction and mechanism of a novel isomaltulose-synthesizing enzymefrom Erwinia rhapontici

Peter S. J. CHEETHAMTate and Lyle Group Research and Development, Philip Lyle Memorial Research Laboratory, P.O. Box 68,

Whiteknights, Reading RG6 2BX, U.K.

(Received 17 November 1983/Accepted 6 February 1984)

The single enzyme that mediates the bioconversion is demonstrated to belocated in the cells' periplasmic space, a site that facilitates its use as an industrialbiocatalyst, and to be a previously undescribed hexosyltransferase with four novelfeatures. The enzyme is sucrose-specific, and has an intramoleular mechanism inwhich both glucose and fructose residues appear to be enzyme-bound. Thirdly, it isreaction-non-selective, forming'simultaneously isomaltulose and a second hithertouncharacterized a-(l -l 1)-linked disaccharide (trehalulose), by hydrolysis of sucrose

followed by reaction of glucose with the C-6 and C-1 positions of the fructofuranoserespectively. Finally, on extended incubation an unusual recycling mechanism causedthe concentration of isomaltulose, the kinetically preferred product, to reach a

transient maximum concentration and then fall, and the concentration of trehalulose,the thermodynamically favoured product, to rise slowly.

Isomaltulose (6-O-ac-D-glucopyranosyl-D-fructo-furanase), a functional isomer of sucrose com-monly referred to as palatinose, is a reducing di-saccharide (60% of glucose), of about one-third thesweetness of sucrose, that is very resistant tohydrolysis by acid and invertase (fl-D-fructofurnao-sidase). It has very similar physical properties tosucrose, is found naturally in small quantities inhoney (Siddiqui & Furgala, 1967) and has potentialindustrial uses either as itself or in a hydrogenatedform (Bucke & Cheetham, 1981, 1982; Cheethamet al., 1982; Munir, 1982; Kutzbach et al., 1982;Shimizu et al., 1982; Takazeo et al., 1982;Lanterno, 1983; see also British Patent Specifica-tion 1 429 334 assigned to the South German SugarCo.).

Historically the first mention of isomaltulosederived from a microbial source was by Stodola etal. (1952) and Sharpe et al. (1954), who describedit as a by-product of leucrose (5-O-o-D-gluco-pyranosyl-D-fructopyranose) and dextran forma-tion by Leuconostoc mesenteroides, although theformation of reducing disaccharides by the actionof this organism, and by Streptococcus bovis actingon sucrose, was first mentioned by Hehre (1951).Bailey & Bourne (1959) later noted that a reducingdisaccharide similar to isomaltulose was producedby Saccharomyces bovis, and Stodola et al. (1956)

and Sharpe et al. (1960) confirmed the formation ofisomaltulose by enzymes derived from L. mesen-teroides. Later an ca-glucosidase from Saccharo-myces italicus was found to form isomaltulose as atransglucosylation product (Halvorsen, 1966).More recently, Fuji et al. (1983) have studied theoligosaccharides formed by the action of Serratiaplymuthica on sucrose. They found isomaltulose tobe the major product and the other sugars to be 1-O-a-D-glucosylfructose, isomaltose and isomelezi-tose (6F-a-D-glucosylsucrose).

Isomaltulose can be produced in high yields byusing very stable continuous columns of immobi-lized Erwinia rhapontici (Cheetham et al., 1982;Bucke & Cheetham, 1982). The enzyme activitywas stabiliz'ed by immobilizing in alginate ratherthan other support materials, by using structurallyintact but non-growing cells rather than isolatedenzyme, disrupted cells or growing cells, by usingconcentrated pure sucrose as substrate and bymaintaining complete conversion of the sucroseinto isomaltulose. The present study demonstratesthe value of investigating the inter-relationshipsbetween the physiological state of the cells,conditions of immobilization and use, and theactivity and stability of the immobilized cells.The present paper describes the enzyme that is

responsible for this microbial bioconversion,

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P. S. J. Cheetham

which is an example of the growing exploitation ofmicrobial enzymes in the form of immobilized cells(Abbott, 1977; Cheetham, 1980), the most impor-tant of which is glucose isomerase (Bucke, 1983).

Methods

Erwina rhapontici (N.C.P.P.B. 1578) cells weregrown, viable cell counts were made and enzymeactivity was assayed at its optimal pH of 7.0, itsoptimal temperature of 300C and with a substrateconcentration of 1.6M as described previously(Cheetham et al., 1982). In high-pressure-liquid-chromatographic analysis the assumption wasmade that sucrose, isomaltulose and trehalulosesolutions of the same concentration have the samerefractive index. Cells were osmotically shockedby resuspending 1.5g wet wt. of cells in 150ml ofice-cold deionized water for 30min. Cells weresonicated as a 10% (wet wt./vol.) suspension indeionized water at 2.8A for 340min with the use ofa probe with a tip diameter of 2.5 cm and coolingthe sample at 5 min intervals. Cells were disruptedby mixing 2g wet wt. of cells with 2g of dry sandand 2ml of deionized water and shaking atmaximum amplitude for 20min at room tem-perature in a Mickle shaker (Laboratory Engineer-ing Co., Gomshall, Surrey, U.K.). Cells weretreated with detergent by resuspending 0.5g in10ml of 0.15% (w/v) Triton X-100 solution for 3h.Cells were shocked at 700C for 3 min in a waterbath or by adjusting to pH 2.0 or pH 10.0 with acidor alkali before being adjusted back to neutral pH.After each treatment the cell suspension wascentrifuged at 17000g for 30min at 30°C, and thepellet of cells or cell debris and the supernatantwere assayed.

In the radiometric assays enzyme was obtainedand assayed at 30°C with 1.6M-sucrose, pH7.0, asdescribed above except that unlabelled glucose orfructose (both 0.5M) and very small quantitiesof radioactively labelled sucrose, glucose and/orfructose (Amersham International, Amersham,Bucks., U.K.) were added to give working radio-activities of 10Ci/ml. Then 2M' samples ofsolutions diluted to 2% (w/v) were chromato-graphed on Merck Silica gel 60 plates in butanol/methanol/water (5:3:2, by vol.) for 16 h. Theplates were then incubated in contact with X-rayfilm (Kodak, Hemel Hempstead, Herts., U.K.) forseveral days before developing. The original or aduplicate plate was then stained for free or com-bined fructose with carbazole stain (Adachi, 1965).Results could be quantified by scraping off eachradioactive spot and counting its radioactivity in atoluene-based scintillant containing 2,5-diphenyl-oxazole (4g/1), 1,4-bis-(5-phenyloxazol-2-yl)ben-

zene (0.1 g/l) and Fison's mix no. 1 emulsifier withthe use of a 10000c.p.m. external standard.The experiments described above that utilize

labelled sugars do not allow the usual proceduresfor determining whether the reaction proceeds viaan odd or even number of steps depending onwhether the glycosidic bond of the product isinverted or not with respect to the substrate. This isbecause sucrose can be regarded both as 3-D-fructofuranosyl-a-D-glucopyranose and as a-D-glucopyranosyl-fi-D-fructofuranose, and so is hydro-lysed by both' invertases and a-glucosidases.Measurement of the isomaltulose/trehalulose

ratio is one of the few methods of directly deter-mining the reaction mechanism. This is because itis not possible to use standard methods (Fersht,1977). Measurement of the ratios of the productsproduced from different substrates cannot becarried out since the enzyme is substrate-specific.Moreov.er, measurement of the rates of formationof the two products with various ratios of acceptorto donor molecules cannot be employed since noequilibration of exogenous monosaccharides withthe reactants inside the active site take place (Fig.1). Furthermore, this reaction is not amenableto spectrophotometric detection or to I3C-n.m.r.analysis because isomerization of the fructoseshould take place over the same time periods thatare required for structural analysis.

ResultsExtraction of the enzymeEnzyme could be extracted by osmotic shocking

(Table 1), together with periplasmic markerenzymes such as acid phosphatase (35 iM-p-nitro-phenyl phosphate hydrolysed/min per ml of ex-tract), but without any significant cytosolic en-zymes. Other methods of extraction were lesssuccessful (Table 1), and permeabilization in-creased activity only slightly (latency 10-15%). Nosucrose or isomaltulose could be detected in thecontents of the cells as liberated by Mickle-shakingor sonication, further indicating that the enzymethat forms isomaltulose is not located intra-cellularly and showing that isomaltulose does notfunction as a compatible solute as in Dunaliellaspecies (Brown, 1974). The amount of enzymesolubilized was not related to the degree of celldisruption as measured by the viable cell count orto the amounts of protein released, and could notbe enhanced by sonication or detergent treatmentof the cell debris producing by milling. The solubleextract obtained by osmotic shocking containedten protein bands on polyacrylamide-gel electro-phoresis.

Further proof for a periplasmic rather than an

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Isomaltulose synthase

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intracellular location was provided by the fact thataddition of a non-lethal dose of the antibiotic linco-mycin (10mg/l) to the growth medium enhancedthe cells' activity by 60%. Lincomycin is known toinhibit proteinases that limit the synthesis ordegrade other periplasmic proteins (Levner et al.,1977).

Properties of the enzymeThe enzyme had no ion or cofactor require-

ments, and possible intermediates such as UDP-glucose or polysaccharide were not observed. It isnot a phosphorylase, as no sugar phosphates couldbe detected and sucrose was not rapidly hydrolysedin the presence of enzyme and arsenate. Noevidence for a regenerated co-substrate was found,as in some transferases (EC 2.7.5. group). Theenzyme is absolutely substrate-specific towardssucrose (Km 0.28 M), since a variety of sugarsincluding maltose, lactose, raffinose, galactose,arabinose and mannose proved to be completelyunreactive when tested alone or with sucrose. Suchabsolute substrate-specificity for both donor andacceptor is surprising, because sucrose is a smallmolecule and because other isomaltulose-producing organisms readily use mannose,arabinose and glucose as acceptors (Mauch& Schmidt-Berg-Lorenz, 1964). Furthermoreglucosyltransferases are usually substrate-non-specific and use fructose only when it is present inhigh concentrations relative to the preferredacceptor. The reaction is essentially irreversible(less than 0.03M-sucrose remaining), indicating afree energy of about -1 1.8 kJ/mol compared witha AG value of -28kJ/mol for the hydrolysis ofsucrose. The activation and enthalpy of thereaction are calculated to be 17.00 and 14.5 kJ/molrespectively.

Mechanism of the enzymeThe enzyme converted labelled sucrose into

isomaltulose (approx. 85% yield). Isomaltuloseformed from specifically labelled sucrose (samples5-8, Fig. 1) could be hydrolysed with acid to yieldglucose and labelled fructose. The labelled or non-radioactive isomaltulose or trehalulose prepared asabove could also be purified by chromatography ona 115cm x 2.2cm diam. column of Dowex AGWX40 (200-400 mesh; K+ form), or by preparativehigh-pressure liquid chromatography with aWaters Prep LC/system 500A, with four PrepPak500/C 18 columns used in series, both with water asthe eluate. Purified isomaltulose and trehaluloseformed from universally or specifically labelledsucrose had very similar specific radioactivities.Smaller concentrations of a second disaccharidewere also formed (Fig. 1), for which the structures1 - 0 - a - D - glucosylpyranosyl - ,B- D- fructopyranose

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P. S. J. Cheetham

Glucose/fructose

; jjz- _ ~~~~~~Sucrose

Isomaltulose

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...: .. .. ... .. .. ::.... ... ...:

:..: :. ....... ----O rig in

- Gic Fru Suc - Glc Fru Suc Suc - Suc

IU-14C]Suc ([U-_4C]Fru]Suc )U-14C]Glc [U-14C]Fru(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

Fig. 1. Autoradiography of the products of the reaction of radioactively labelled sugars with soluble enzyme obtainedfromosmotically shocked E. rhapontici cells

The samples illustrated are (left to right) enzyme incubated with [U-14C]sucrose alone (1), [U-14C]sucrose supple-mented with glucose or fructose (2 and 3 respectively) and non-radioactive sucrose (4). Samples (5)-(8) are sucrosespecifically labelled in the [U-_4C]fructose moiety supplemented as in samples (184). Samples 9 and 10 are [U-14C]-glucose incubated alone and supplemented with non-radioactive sucrose, and samples (11) and (12) are [U-14C]-fructose incubated alone and supplemented with non-radioactive sucrose. Because of the relatively short incubationtimes used in this experiment, the amounts of trehalulose formed were insufficient to be easily detected visually as inFig. 2, but could be easily quantified by scintillation counting as described in the Methods section.

(major form, 66%) and 1-O-a-D-glucosylpyranosyl-P-D-fructofuranose (33%) were determined by 13Cn.m.r. The neologism trehalulose is proposed, byanalogy with lactose/lactulose, for this new ex-ample of a (1 -.1)-linked disaccharide. Little freeglucose or fructose (0.07M and 0.1OM respectively)and no oligosaccharides were formed (Fig. 1),showing that water and the disaccharide productsare poor acceptors. Isomaltulose could not beconverted non-enzymically into trehalulose, andvice versa, and no sucrose was formed when eitherpurified isomaltulose or trehalulose was incubatedwith enzyme.An intramolecular (direct or monomolecular)

transglycosylation mechanism, with both di-saccharides being formed via a common inter-mediate, is presumed. The reaction is not mediatedby closely associated but distinct enzymes, or evenby one protein possessing two active sites withdifferent specificities (Yourno et al., 1970), be-cause little monosaccharide is formed, becauseexogenous labelled monosaccharides are not incor-porated into either disaccharide product whetherprovided alone or with sucrose (Fig. 1), and be-cause these monosaccharides did not affect therates of product formation and sucrose con-sumption. An enzyme-linked glucose intermediateis indicated, since if free glucose were presentsugars such as trehalose, isomaltose, maltose,maltulose and maltotriose could be formed. Similar

logic implicated the involvement of enzyme-linkedfructose (Scheme la). Furthermore the enzyme'sabsolute substrate-specificity and its non-reaction

al-6 al-6(a) G-F(f) G-F(f) + E

(t 1-2p a 1 -2,BE + G-F(f) G-F(f) G F(f)

'JN~N~1,~,wJw al-1 al-1G-F(f) -sG-F(f) + E

al-1G-F(p)

al-6(b) G-F(f + E

a1-2fiE + G-F(f)--G + F(f

w:\l al-1G-F(f) + E

F(p) G -F(p)+ Eal-1

Scheme 1. Proposed mechanisms for the formation ofisomaltulose and trehalulose from sucrose by the soluble

enzyme from E. rhaponticiKey: G, glucopyranose; F(f), fructofuranose; F(p),fructopyranose; E or /77777777, enzyme. Enzyme-catalysed reactions are denoted by continuous linesand non-enzymic isomerization by broken lines.The direction of reaction is roughly indicated by therelative thicknesses of the lines. Note that atequilibrium both free fructose (Furcke et al., 1979)and the fructose component of trehalulose existpredominantly (approx. 66%) in the pyranose form.

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Isomaltulose synthase

with and non-production of monosaccharides alsoimplies multiple attachment of the substrate to theenzyme (Scheme la).The following mechanism is proposed, involving

binding of sucrose to the enzyme, its hydrolysis andthen the synthesis of a-(I -*6)- or a-(I -1I)-bondsby non-selective reaction of glucose with the C-6and C-l secondary hydroxy groups of the fructo-furanose (Scheme la). Surprisingly, isomaltuloseformation requires rotation of the fructose residuethrough 1800, whereas trehalulose, which is formedin much lower yields, requires no such rotation andis presumably formed by reaction before theenzyme-linked fructose residue can rotate. Eitherthe glucose or fructose moiety can be regarded asthe leaving group.A less likely alternative mechanism involving

only enzyme-bound glucose can also be envisaged(Scheme lb) in which reaction of glucose with boththe furanose and pyranose forms of fructose maytake place. However, attempts to channel exclu-

sively the 6'-blocked substrate 6'-chlorosucroseinto trehalulose were unsuccessful. Also, borate(0.3M), which should selectively form a complexwith the free fructofuranose, did not inactivatethe enzymes, unlike other isomaltulose-forming en-zymes (Mauch & Schmidt-Berg-Lorenz, 1964). Iso-merization to fructopyranose with subsequentreaction to trehalulose (Scheme lb) is unlikely,since glycosylation should occur before mutarota-tion can occur, since no f-linked disaccharidessuch as gentiobiose are formed, and also beforefructose can diffuse out of the active site, as littlefructose is formed. Perhaps the fructose is held inthe furanose configuration by association withreactive amino acids in the active site of theenzyme. Also, unless isomerization takes placemuch more rapidly in the active site than in freesolution, the ratio of isomaltulose to trehaluloseformed should vary during the reaction (Fig. 2). Inaddition, reaction via open-chain fructose isunlikely, as n.m.r. shows that very little is present,

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Fig. 2. Measurement ofthe ratio ofisomaltulose to trehalulose (@)formed during a typical reaction ofthe soluble enzymefromE. rhapontici with sucrose

The percentage conversion of sucrose into products is also depicted (0). Thus 1% conversion of the 1.6M-sucroseused as the substrate in this experiment results in the generation of approx. 0.01 39M-isomaltulose and 0.0021 M-tre-halulose. Extraction and assay of samples and immobilization ofenzymes and cells were as described in Table 1 andCheetham et al. (1982). Isomaltulose and trehalulose were determined either by high-pressure liquid chromato-graphy or by t.l.c. (see inset) followed by scraping the radioactively labelled spots offthe t.l.c plate and counting theirradioactivity.

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217

P. S. J. Cheetham

and also the reaction was not inhibited by xylitol ormannitol (up to 0.25M), which should form com-plexes with acyclic fructose (Ananichev et al.,1980).

Reaction non-selectivityA surprising feature was the simultaneous rather

than sequential formation of two disaccharidesfrom pure sucrose (Fig. 2). Simultaneous formationis an intrinsic property of the enzyme, since theratio of isomaltulose to trehalulose produced wasconstant throughout the early stages of the reactionand under a variety of conditions (Fig. 2), whereasin a sequential reaction an early peak in the con-centration of the intermediate should be apparent.Very similar isomaltulose/trehalulose ratios, indi-cating that isomaltulose and trehalulose are formedin parallel, were obtained when antimicrobialagents such as chloramphenicol or benzylpenicillinwere added, proving that trehalulose is not formedby the action of contaminant micro-organisms,with samples of cells harvested during growthphase, intact non-growing cells, disrupted cells,partially purified enzyme or cells that had beenpH- or temperature-shocked, and under a varietyof experimental conditions. For instance, the ratiodid not vary significantly, when pulses of labelledsucrose were supplied, with the pH, temperature orsubstrate concentration used, when cells or en-zyme were immobilized by a variety of methods(Cheetham et al., 1982; Bucke & Cheetham, 1981),or throughout the useful life of the immobilizedcells, with the proviso that lower values were ob-served in batch reactions and when immobilizedbiocatalysts were used, presumably owing to theinfluence of diffusional restrictions on producttransfer. Also, under the conditions found optimalfor E. rhapontici, immobilized Erwinia carotovoracells formed roughly equal proportions of tre-halulose and isomaltulose throughout its half-life(975 h), the cells having an initial activity of5.85pmol of trehalulose formed/min per g wet wt.of cells.

Note also that other isomaltulose-producingorganisms also form trehalulose (Mauch &Schmidt-Berg-Lotenz, 1964; Kutzbach et al., 1982;Munir, 1982; Shimizu et al., 1982), and an evenmore marked non-selectivity is probably exhibitedby E. carotovora (Lund & Wyatt, 1973).

Changes in product concentrations after prolongedincubationsOn extended incubation, once all the sucrose had

been consumed a very slow but continuous declinein the initial concentration of isomaltulose andcorresponding increases in that of trehalulose

occurred (Fig. 2). In an extreme case, in which flowthrough a column of immobilized cells was stoppedfor 10 days, the isomaltulose/trehalulose ratio inthe syrup fell to 1.15:1 (0.83M and 0.72M respec-tively). This low rate of reaction is consistent withthe observation that isomaltulose is much morestable to acidic hydrolysis than is sucrose. Confir-mation was provided by the very slow formation oftrehalulose by the enzyme working 'in reverse'when the enzyme or cells were incubated withpurified isomaltulose, whereas pure trehaluloseproved to be completely inert over the time periodsused in these experiments. Thus isomaltulose ismetastable and its formation is slowly reversible,whereas trehalulose is formed virtually irreversibly(Fig. 2), perhaps because it is not re-admitted backinto the active site.

Virtually irreversible enzymes, such as manyenzymes with a regulatory role in metabolism, areusually characterized by a relatively large AGvalue. Thus the low AG of about -12kJ/molcalculated for isomaltulose synthase and the obser-vation that the heats of hydrolysis of both di-saccharides are both small because yields aretemperature-invariant (4-65°C) can only be ac-counted for by isomaltulose having a greaterentropy of hydrolysis than sucrose. Thus overlong time periods isomaltulose is slowly recycledthrough the enzyme with a proportion sequesteredas trehalulose on each passage (Scheme 1). In orderto minimize trehalulose formation it is thusimportant for the immobilized enzyme to have lowdiffusional limitations, and also to be permanentlyimmobilized because even low concentrations ofsolubilized enzymes in the column eluate couldform excessive trehalulose and decrease the yieldsof pure isomaltulose recovered by crystallization.

Although little is known about whether the sub-strate-specificity ofenzymes differs when acting inthe 'normal' or 'forward' direction and the thermo-dynamically less favoured 'reverse' direction, it isof note that the enzyme described in the presentpaper appears to be absolutely substrate-specificfor sucrose in the 'forward' direction and forisomaltulose when acting in the 'reverse' direction,trehalulose, the other product of the forwardreaction, being unreactive.

Discussion

Many industrially important enzymes have notbeen subjected to detailed biochemical study.However, in the present paper a new peri-plasmically located sucrose-specific hexosyltrans-ferase is described that illustrates the advantage ofscreening for micro-organisms possessing novel

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Isomaltulose synthase

enzymes. Such novel enzymes are especially usefulwhen new products are required.

This periplasmic location is advantageous whenthe enzyme is to be used associated with its parentcells. This is because, by comparison with intra-cellular enzymes, it is probably less liable toproduct inhibition, because substrate transfer andproduct export are facilitated without deliberatepermeabilization and because it will be less subjectto physiological turnover mechanisms. Thus forbiotechnological applications it may prove to beadvantageous to modify the location of normallyintracellular proteins by the attachment of appro-priate signal sequences (Inouye & Beckwith, 1977;Randall et al., 1978).

This enzyme has a novel intramolecular non-selective mechanism that allows the formation ofkinetically or thermodynamically preferred pro-ducts and has a high activity in a low-water-activity environment, thus allowing a high processintensity consistent with large-scale use to beachieved. The mechanism is similar to the forma-tion of allolactose from lactose (Hall, 1982), al-though in that case other sugars are also formed byindirect transglucosylation. By contrast, with theE. rhapontici enzyme other isomaltulose-producingenzymes readily incorporate monosaccharides(Mauch & Schmidt-Berg-Lorenz, 1964). The en-zyme appears to be specific for primary hydroxygroups, catalysing reactions involving the C-6 andC-1 hydroxy groups of fructose, forming iso-maltulose and trehalulose respectively. Despitebeing absolutely substrate-specific, the enzyme isactually reaction-non-selective, non-selectivitybeing a characteristic of most chemical catalystsbut of very few enzymes (Jones et al., 1976; Dixon& Webb, 1979). Two enzymes that have beenreported to display this effect, aconitase andalcohol hydrogenase, only catalyse the formationof different constitutional isomers, citrate andisocitrate and axial and equatorial alcohols respec-tively, presumably because each substrate can bindto the active site in two alternative ways (Bentley,1969). Also, malate dehydrogenases (EC 1.1. 1.38-40) and phosphogluconate dehydrogenase (EC1.1.1.44) can also act as decarboxylases.The enzyme bears some similarities to the

enzyme (EC 2.4.1.18) that introduces the branchesinto amylopectin, but can be best classed as anintramolecular isomerase (EC 5.4.-.- group), inwhich case it would be the first example of such amutase acting on a sugar. The trivial nameisomaltulose synthase is proposed. Alternatively, itcould be the first glycosyltransferase (EC 2.4.1.-group) to be shown to have a true intramolecularmechanism.

In many respects isomaltulose would appear tobe a secondary metabolite. Trehalulose formation

can be regarded as a 'luxury feature' of the enzyme,tolerated because both disaccharides have similaradaptive roles. That is, they enable cells thatcannot grow owing to an absence of nitrogenous orother nutrients to sequester sucrose into a carbonand energy store that is unavailable to potentialcompetitors, rather than metabolizing it wastefullyand unproductively. Thus it is obviously advanta-geous for such a process to be mediated by a peri-plasmically rather than an extracellularly locatedenzyme. This role is in agreement with the conceptthat the periplasmic space of Gram-negativebacteria has an organelle-like role possessingspecialized metabolic functions and containingenzymes that in Gram-positive organisms areextracellularly located. Thus the isomaltulose syn-thase described in the present paper can beregarded as a form of extracellular enzyme,although compartmentalized within the outermembrane of the cell.The marked stabilization of the enzyme by

sucrose (Cheetham et al., 1982) is an advantageousfeature, as it enables the enzyme to transformefficiently the large quantities of sucrose en-countered in the cells' natural environment. Thisstabilization could also, of course, be due to iso-maltulose acting as an inducer of the isomaltulosesynthase. This transformation may be a means ofthe cell avoiding 'substrate-accelerated death'(Dawes, 1976), whereby cell viability is rapidlylost when glucose, for instance, is supplied to aquiescent cell culture. Protection would thus beachieved by maintaining a balanced metabolismby excluding excess carbon source from the cellwhen nitrogenous and other nutrients are absent,and by providing an energy reserve that preventsthe consumption of endogenous metabolic re-serves, especially as the long-term survival of cellshas been related to a low rate of endogenousmetabolism (Dawes, 1976). Another example maybe the way in which Pseudomonas aeruginosaaccumulates gluconate and 2-oxogluconate whenexposed to high concentrations of glucose. Theenzymes involved, glucose dehydrogenase andgluconate dehydrogenase, are both orientated inthe cytoplasmic membrane, so that direct oxida-tive metabolism takes place in the periplasmicspace and most of the products do not enter thecell. Only when glucose is limited does the cellswitch its metabolism so that the glucose and 2-oxogluconate are rapidly transported into the cell,phosphorylated and metabolized (Whiting et al.,1976a,b). Such potentially useful non-growth-associated activities may be best searched for inspores and other forms of resting micro-organisms,which are naturally adapted to environmentswhere nutrients are available in small quantitiesand/or infrequently.

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220 P. S. J. Cheetham

I thank Dr. H. B. F. Dixon, University of Cambridge,Cambridge, U.K., for his advice concerning the classifi-cation of the enzyme described in the present paper.

ReferencesAbbott, B. J. (1977) Annu. Rep. Ferment. Processes 1,

205-235Adachi, S. (1965) J. Chromatogr. 17, 225-229Ananichev, A. U., Ulezlo, I. V., Egorov, A. M.,

Bezorodov, A. M. & Berezin, I. U. (1980) Biokhimiya45, 992-998

Bailey, R. W. & Boume, E. J. (1959) Nature (London)194, 904-905

Bentley, R. (1969) Molecular Asymmetry in Biology, vols.1 and 2, Academic Press, New York

Brown, A. D. (1974) J. Bacteriol. 118, 769-777Bucke, C. (1983) in Microbial Enzymes in Biotechnology

(Fogarty, W. M., ed.), pp. 93-129, Applied Science,London and New York

Bucke, C. & Cheetham, P. S. J. (1981) Br. Patent Applic.2066639A

Bucke, C. & Cheetham, P. S. J. (1982) U.S. Patent4359531

Cheetham, P. S. J. (1980) Top. Enzyme Ferment. Bio-technol. 4, 189-328

Cheetham, P. S. J., Imber, C. E. & Isherwood, J. (1982)Nature (London) 299, 628-631

Dawes, E. A. (1976) Symp. Soc. Gen. Microbiol. 26, 19-54Dixon, M. & Webb, E. C. (1979) Enzymes, 3rd edn., pp.

668-669, Longmans, LondonFersht, A. R. (1977) Enzyme Structure and Mechanism,W. H. Freeman, San Francisco

Fuji, S., Kishihasa, S., Komoto, M. & Shimizu, J. (1983)Nippon Shokuhin Kogyo Gakkaishi 30, 339-344

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