Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2--glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families.

However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications.

Initial interest in the enzymes hydrolyzing dextran arose from studies that aimed to elucidate the structure of dextran and to obtain partially hydrolyzed dextran polymers produced by Leuconostoc mesenteroides for infusion purposes (80). Dextranases also have other important industrial applications since these enzymes can depolymerize various troublesome microbial dextran deposits. The presence of dextran in harvested sugar canes and dextran formation by microbes in sugar factories lead to lowered sucrose yield. The fact that dextran is a component of dental plaque, which is considered to contribute to the development of dental caries, has been one of the main driving forces to investigate dextran-hydrolyzing enzymes. Dextran can be modified by dextranases to be used in many biotechnological applications.

Since the first reports on Cellvibrio fulva dextranase in the 1940s, more than 1,500 scientific papers and more than 100 patents have been issued on dextran-hydrolyzing enzymes found in a number of microbial groups, fungi being the most important commercial source of dextranase. Higher organisms also possess dextran-hydrolyzing activities, but relatively few studies focusing on such enzymes have been published. The present paper aims to present relevant data on microbial dextranases published thus far. Since this is the first larger overview into the field, earlier literature is also cited rather widely. The enzymatic properties of dextran-hydrolyzing enzymes from different microbial sources, existing nomenclature, cloning and sequence analysis of dextranase genes, methods for measuring dextran-hydrolyzing activity, and potential applications of dextranases are discussed. Because of the increasing importance of glycobiology in biosciences, it is possible to predict that dextran and the enzymes involved in its synthesis, modification (e.g., through transglycosylation), and hydrolysis will have increasing significance in the future. To comprehend the special nature of dextran-degrading enzymes, a brief outline of the structure and properties of dextran polymer and dextran-synthesizing enzymes is also presented.

CLASSIFICATION OF DEXTRAN-HYDROLYZING ENZYMES:
Dextran-degrading enzymes form a diverse group of different carbohydrases and transferases. These enzymes have often been classified as endo- and exodextranases based on the mode of action and commonly called dextranases (49, 50, 231). According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) and the types of reactions catalyzed and product specificity, these enzymes were classified as dextranases (EC3.2.1.11), glucan-1,6--D-glucosidases (EC3.2.1.70), glucan-1,6--isomaltosidases (EC3.2.1.94), dextran 1,6--isomaltotriosidases (EC3.2.1.95), and branched-dextran exo-1,2--glucosidases (EC3.2.1.115) (45). Cycloisomaltooligosaccharide glucanotransferase (CITase) also produces hydrolyzed dextran as one of its reaction products (148) (Table 1). Moreover, an unrelated enzyme, -glucosidase (EC3.2.1.20), catalyzes reactions similar to those of exodextranases (EC3.2.1.70) (85).
  
In another classification system glycosylhydrolases and glycosyltransferases have been divided into families on the basis of the similarities in the amino acid sequences (61, 62, 63, 64; http://afmb.cnrs-mrs.fr/CAZY/). The Carbohydrate Active Enzymes (CAZy) database describes the families of structurally related catalytic and carbohydrate-binding modules (functional domains) of enzymes that degrade, modify, or create glycosidic bonds. An analogous classification system for dextran-hydrolyzing enzymes with dextranases divided into four families has also been presented (5), but it is not presented in this review. In contrast to the IUB-MB system, the CAZy database was designed to integrate both structural and mechanical features of these enzymes; enzymes with different substrate specificities can be placed in the same family, and enzymes that hydrolyze the same substrate are sometimes placed in different families (16, 64).

According to sequence similarities, dextran-glucosidases (EC3.2.1.70) have been included in glycosylhydrolase families13 and 15 (http://afmb.cnrs-mrs.fr/pedro/CAZY/ghf.html)Isomaltodextranase (EC3.2.1.94) and isomaltotriosidase(EC3.2.1.95) have different structures and have been included in glycosylhydrolase families 27 and 49, respectively. Endodextranases are found in glycosylhydrolase families 49 and 66, with no sequence similarities between the two families. Also, Bacillus circulans CITase possessing a high homology with endodextranases is classified in family glycosylhydrolase 66.

SOURCES, MAIN PROPERTIES, INDUCTION, AND MECHANISM OF ACTION
The enzymes capable of hydrolyzing dextrans are found in various microbial groups (Table 1), in animal and human tissues (51, 161, 170), and in coleoptiles of the genus Avena (65, 66). Dextranase activity is also demonstrated in soil samples (40). In general, dextran-hydrolyzing enzymes have high substrate specificity. The physicochemical properties of a number of purified dextranases are presented in Table 1.

Endodextranases (EC3.2.1.11) from Fungi:
Mold dextranase, 1,6--D-glucan 6-glucanohydrolase (EC3.2.1.11), is an enzyme, which catalyzes endohydrolysis of -(1,6)-D-glycoside linkages in random sites of dextran. Isomaltose, isomaltotriose, and a small amount of D-glucose, together with traces of higher oligomers, are the main reaction products. However, a variation in the reaction products and substrate specificities of dextranases from different sources are evident. For example, endodextranase from a Penicillium sp. degrades cyclodextrans to isomaltose and glucose (143). All mold dextranases (EC3.2.1.11) can degrade the cross-linked dextran Sephadex (Table 1).

Molds are the commonest source for the extracellular endodextranases (EC3.2.1.11) and exhibit a higher enzyme activity than dextranases from bacteria and yeasts. There is only one report on intracellular mold dextranase found in Penicillium lilacinum NRRL 896 and Penicillium funiculosum NRRL 1132 (72). The hydrolysis products of Penicillium notatum dextranase are isomaltose and isomaltotriose with a small amount of glucose, as in most fungal dextranases. Reducing sugars are released from dextran much faster and in larger amounts by random attack of endodextranases compared to terminal end-group attack of exoenzymes (225). Thus, a 67 to 74% conversion of dextran to sugar syrup is attained by P. notatum dextranase. The dextranases of two Penicillium species (Penicillium lilacinum NRRL-896 and Penicillium funiculosum NRRL-1132) produce, in addition to oligosaccharides that contain one glucose unit joined by an -1,4 linkage to a glucose unit of a homolog to isomaltose, small proportions of D-glucose, isomaltose, and isomaltotriose from -1,4-branched dextran of L. mesenteroides NRRL-1415 (1, 2, 191).

Mold dextranases can also hydrolyze oligosugars. D-Glucose is released from isomaltotriose and from higher homologs up to isomaltoheptaose by Penicillium lilacinum NRRL-896 dextranase. The hydrolysis occurs at the first linkage from the reducing end. The enzyme also catalyzes an extremely slow, concentration-dependent degradation of isomaltose. This may occur via condensation to isomaltotetraose followed by hydrolysis of the first linkages to give D-glucose and isomaltotriose (218). A similar phenomenon has been found with dextranases from Aspergillus carneus and Penicillium luteum (68) (Table 1).

The mode of action of Aspergillus carneus has been investigated with a series of isomaltodextrins and their derivatives as the substrates. The enzyme readily hydrolyzes these substrates, and the reaction products are similar to those of fungal dextranases. In these studies, the degree of dextran T2000 hydrolysis by A. carneus dextranase was about 40% (213). This value is lower than that of P. luteum (55%) when the reducing sugars liberated are calculated as glucose (Table 1). This difference was considered to be due to the difference in their hydrolytic abilities toward isomaltotriose, which was readily split by the latter enzyme but very slowly by the former. In contrast, L. mesenteroides IAM 1046 dextran, containing 66% -1,6-, 19% -1,4-, and 15% -1,3-glucosidic linkages, was hydrolyzed slowly and to a lesser extent by both enzymes. The amino acid compositions of these two enzymes are closely similar (47, 67, 68, 213).

A strain of Penicillium aculeatum produces large quantities of dextranase in its culture broth. The crude enzyme was highly stable (117) (Table 1). About 90% of the substrate dextran was converted to isomaltose in a 4-h period at 40°C. No D-glucose was observed, and thus the results differed from those obtained with the Penicillium luteum and Penicillium funiculosum enzymes (117). Chaetomium gracile produced endodextranases, while the maximal dextran hydrolysis to glucose was 55% (60).

Typically, dextranase synthesis by several fungi is induced in the presence of dextran (47, 48, 49, 59, 117). However, with Sporothrix schenckii a tenfold increase in the dextranase production was achieved without cell mass increase when soluble bacterial dextrans were substituted with glucose as the substrate (6). When Penicillium minioluteum HI-4 was grown on minimal medium supplemented with different carbon sources, dextran but not starch, glucose, glycerol, lactose, or sorbitol induced high dextranase expression. Quantitation of mRNA indicated that dextran affected dextranase expression at the transcriptional level. When fungi were cultivated in the presence of both dextran and glucose or glycerol, dextranase expression was repressed at the transcriptional level. In the 5' noncoding region of the dexA gene there are several sequences similar to those involved in binding of CreA, important in the D-glucose-mediated carbon catabolite repression of several genes (39). The putatively conserved nature of this regulatory mechanism in fungi suggests that dexA may be under the control of a CreA homolog.

The ability of yeasts to synthesize dextranases has mainly been observed in the genus Lipomyces (Table 1). The characteristics of the Lipomyces starkeyi (ATCC 20825) dextranase (EC3.2.1.11) regarding the effects of pH and temperature on activity and stability are very similar to those of the Chaetomium and Penicillium enzymes (NRRL 1768; Table 1). The purified enzyme shows the same Km values as reported for the IGC 4047 dextranase but is not regulated through product inhibition, in contrast to the latter enzyme (105, 106) (Table 1). The Lipomyces starkeyi dextranase is a glycoprotein containing 8% sugar. The Penicillium funiculosum (34) and Chaetomium (60) dextranases are also glycoproteins. The specificity of Lipomyces starkeyi dextranase is similar to that of fungal dextranases, the final hydrolysis products being isomalto-oligosaccharides from glucose to isomaltotetraose (105).

In recent studies, a novel glucanohydrolase from mutant Lipomyces starkeyi strain KSM 22 has been shown to possess either dextranolytic or amylolytic activity depending on reaction conditions (207) (Table 1). Competition studies with different amounts of dextran and starch as substrates showed consistency with the hypothesis that hydrolysis of dextran and starch occurs at two independent active sites (95, 114, 172).

Endodextranases from Bacteria:
Endodextranases can be obtained from several bacterial genera, including Pseudomonas, Brevibacterium, Streptococcus, Bacteroides, and Bacillus (Table 1). Bacterial endodextranases, like fungal endodextranases, show distinct patterns of action for each specific microorganism. An enzyme extract from the cellulose-degrading bacterium Cellvibrio fulva hydrolyzes dextran mainly into comparatively large fragments. Apparently no D-glucose or disaccharides from the ends of the molecule are formed (80). For example, the extracellular endodextranase of anaerobic Lactobacillus bifidus produces a mixture of oligosaccharides but no glucose or isomaltose when incubated with essentially unbranched dextran of Streptomyces bovis and branched Leuconostoc dextran (7, 8).

Most of the bacterial dextranase producers have the ability to synthesize several -glucosidases with different subcellular localizations and substrate specificities simultaneously (29, 30, 205). Two extracellular dextranases (D1 and D2) and one intracellular dextranase (D4) from Pseudomonas sp. strain UQM 733 have been isolated. Both extra- and intracellular dextranases are induced in the presence of dextran. D1 and D2 differ in their physicochemical properties, which is possibly attributable to the presence of two proteins in D2 while D1 produces much higher yields of low-molecular-weight oligosaccharides from dextran (164). No activity of D1 appeared with potato starch, amylopectin, amylose, glycogen, or sucrose. However, the enzyme was capable of slowly attacking the -1,6 linkages in pullulan. Based on studies with reduced and tritiated oligosaccharides, a model for three active sites of the enzyme was postulated (165). The intracellular dextranase, D4, was very similar to D1 in molecular weight, pH, and temperature optima as well as mode of action (Table 1). Dextranase activity has also been detected in both intra- and extracellular fractions of Bacteroides oralis Ig4a obtained from human dental plaque (205) (Table 1).

Extracellular isomaltotriose-producing dextranases occur in Streptococcus mutans K1-R and Flavobacterium sp. strain M-73 with a strict specificity for consecutive -1,6-glucosidic linkages. The final yields of isomaltotriose produced from clinical dextran by the endo-action of these two enzymes were 63% for the Flavobacterium sp. and 99 to 100% for S. mutans dextranase. Dextranase-producing bacterial strains in the genus Bacillus have also been isolated from soil samples. One strain, determined by 16S RNA analysis as Paenibacillus illinoisensis exhibiting a stable dextranase activity, was characterized (89). The chromatography of products from dextran T-500 with crude enzyme suggested a random endo-type hydrolysis resulting in liberation of long-chain oligomers together with glucose and isomaltose units (Table 1).

The production and characteristics of thermostable dextranases have been reported (69, 227, 228). Among the isolates, Thermoanaerobacter sp. strain Rt364 produces dextranase with a high thermostability. The production of endodextranases inducible by dextran has been found in two Arthrobacter strains, Arthrobacter globiformis T-3044 (147) and Arthrobacter sp. strain CB-8 (155).

Glucose-Forming Exodextranases:
Exodextranases, such as glucodextranase (EC3.2.1.70; glucan 1,6--glucosidase), catalyze stepwise hydrolysis of the reducing terminus of dextran and derived oligosaccharides to yield solely ß-D-glucose; i.e., hydrolysis is accompanied by inversion at carbon-1 in such a way that new reducing ends are released only in the ß-configuration. Only few bacteria and yeasts are known to produce glucodextranases. Dextran-inducible extracellular glucodextranase occurs in Arthrobacter globiformis strains I42 (Table 1) and T-3044 (146, 147). Although glucodextranase I42 releases glucose from dextran and isomaltose and also from starch, maltose, nigerose, and kojibiose, its activity to -1,4-glucosidic linkages is much less than to -1,6-glucosidic linkages (134, 153). This might indicate that glucodextranase and glucoamylase activities are due to enzymes functioning differently in different conditions (153). Besides that, glucodextranase I42 converts - and ß-D-glucosyl fluorides to ß-D-glucose and hydrogen fluoride, providing additional evidence for the functional flexibility of the catalytic groups of the carbohydrases (97). Glucodextranase T-3044 exhibits properties, pH optimum, and mass similar to those of glucodextranase I42 (147).

Intracellular dextran glucosidases (EC3.2.1.) producing -D-glucose from dextran exist in several strains of Streptococcus mitis (115, 216, 217). S. mitis ATCC 903 exoglucanase was purified 925-fold, and some properties were studied (Table 1). The enzyme was active with isomaltose and dextran but nonactive with substrates of -1,1, -1,2, -1,3, and -1,4 glucosidic linkages. Sucrose, fructose, and mannose had no effect on the activity, while 400 mM glucose almost completely inhibited the enzyme (115). The S. mitis 439 intracellular enzyme has an activity pattern closely similar to that of glucodextranase (EC3.2.1.70) but the glucose residues released from isomaltopentaose and dextran by the action of this enzyme are in the -configuration, demonstrating that it is a glucosidase (216). S. mitis 439 dextran glucosidase acts on molecules with a glucose joined through -1,6 bonds to either a maltosaccharide or an isomaltosaccharide and acts more readily on panose than on isomaltose. A comparable intracellular dextran glucosidase, DexB, in S. mutans LT11 releases free glucose from the -1,4,6 branch points in panose (226). The growth of the strain on panose-induced medium and the rate of hydrolysis of panose were equivalent to those of isomaltotriose and higher than those of isomaltose (226).

Exoenzyme activity that releases glucose from dextran has been detected in animal tissues and in bacteria (51, 161, 170) (Table 1). An enzymatic complex capable of hydrolyzing dextrans to D-glucose as the sole or major product has been found in intestinal anaerobic bacterium of the Bacteroides genus. This complex evidently contains two different dextranases active at pH 5.0 to 5.5 (186). Exodextranases have also been isolated from extra- and intracellular fractions of Bacteroides oralis IG4 (205) (Table 1).

Inoculation of dextran-containing medium with a soil sample resulted in accumulation of several Bacillus species, which were isolated and characterized as Bacillus subtilis and Bacillus megaterium. The cleavage mechanism of the cell-bound exodextranase of Bacillus species involved endwise cleavage of D-glucose residues from the terminal groups, leaving the rest of the dextran molecule intact. Isomaltodextrins were hydrolyzed at a higher rate than dextrans of 100 kDa and 2,000 kDa under the same conditions (231).

Three intracellular glucosidases (G1, G2, and G3) from Pseudomonas sp. strain UQM 733 have also been described (Table 1). The action of purified G1, G2, and G3 on pure isomaltooligosaccharides shows that the glucosidases have optimal activity on isomaltotetraose and are, therefore, classified as oligoglucanases (30). Glucosidases G1 and G2 exhibit general properties different from those of glucosidase G3 (29) (Table 1).

Pig spleen acid -D-glucosidase, possessing dextranase activity, is an exoglucanase with broad specificity (161). The enzyme of 106 kDa was purified over 2,000-fold. It hydrolyzed reducing -D-glucosyl disaccharides and almost completely degraded dextrans that contain -1,3 and -1,6 linkages. The pH optimum of dextran glucosidase activity was pH 4.8 to 5.0. Studies with various pHs, temperatures, and inhibitors caused changes in the activity of the -D-glucosidase against oligo- and polysaccharide substrates, suggesting that the enzyme has multiple substrate-binding sites (161).

Isomaltose-Forming Exodextranases:
The soil bacterium A. globiformis T6 isomaltodextranase (EC3.2.1.94; 1,6--D-glucan isomaltohydrolase) is a novel extracellular exoenzyme capable of hydrolyzing dextran by removing successive isomaltose units from the nonreducing ends of the dextran chains (177, 179) (Table 1). The properties of the enzyme are unusual for it is able to split not only -1,6 linkages of glucooligosaccharides but also -1,2-, -1,3-, and -1,4-links to yield isomaltose (211); it can split dextran so that the -configuration of the anomeric carbon atoms is retained in the hydrolysis products (150); it has transfer and condensation activities on isomaltose to produce isomaltotetraose in concentrated solutions (94, 178); and it can split -1,4-glucosidic linkage of panose and -1,6-glucosidic linkage of isomaltotriose and pullulan as well (151, 206). It was concluded that there is a single active site on the enzyme molecule for hydrolysis of -1,6- and -1,4-glucosidic linkages responsible for both the isomaltodextranase and isopullulanase activity (206). This isomaltodextranase hydrolyzes 13 dextrans to various extents (11 to 64%, 13 days) at initially high but gradually decreasing rates.

Dextran B-1355 fraction S, unlike the other dextrans, has been found to be hydrolyzed initially at the lowest rate among the dextrans used, but the rate was maintained for a long period of time with little decrease in a manner that 85% of dextran was converted within 13 days (181). Extracellular isomaltodextranase (optimal pH 5.0) from the actinomycete Actinomadura sp. strain R10 and that from Arthrobacter demonstrate similar modes of action on dextran, but the enzyme is more active on the 1,6--D-glucopyranosidic linkages while the relative activity increases within the degree of polymerization. In contrast, the relative activity of the actinomycete enzyme is almost constant throughout the same series of substrates and much higher on 1,3-, and 1,4- linkages than the Arthrobacter enzyme (182).

Isomaltotriose-Forming Exodextranases:
Exoisomaltotriohydrolase (EC3.2.1.95) is produced by Brevibacterium fuscum var. dextranolyticum (Table 1). The enzyme is a glycoprotein that removes isomaltotriose from the nonreducing ends of dextran and reduced isomaltodextrins (200). Isomaltotriodextranase does not hydrolyze other than -1,6-glucosidic linkages. The purified recombinant enzyme shows the same optimum pH, lower specific activity, and a similar hydrolytic pattern to the native enzyme (133) (Table 1).

Debranching Exodextranase:
Branched dextran exo-1,2--glucosidase (EC3.2.1.115) was found in the culture supernatant of the soil bacterium Flavobacterium sp. strain M-73 by Mitsuishi et al. (131). The general properties of dextran 2-glucohydrolase were examined with an electrophoretically homogeneous preparation (Table 1). The enzyme had a strict specificity for 1,2--D-glucosidic linkage at the branch points of dextrans (containing 12 to 34% of 1,2- linkages) and related polysaccharides producing free D-glucose as the only reducing sugar. The enzyme did not hydrolyze disaccharides or oligosaccharides containing linear 1,2--glucosidic bonds (100, 131, 132, 202).

Cycloisomalto-oligosaccharide Glucanotransferase:
Cycloisomalto-oligosaccharide glucanotransferase (CITase) is a novel enzyme that catalyzes the conversion of dextran to cyclodextran by intramolecular transglycosylation (cyclization). CITase has been purified to homogeneity from the culture filtrate of Bacillus circulans T-3040 (Table 1). CITase produces three cyclic isomaltooligosaccharides (cycloisomalto-heptaose, -octaose, and -nonaose) with a total yield of about 20%, wherein cycloisomalto-octaose is the main product. Coupling, disproportionation, and hydrolytic reactions are also observed. The enzyme does not act on amylopectin and pullulan (143, 144). Immobilization of CITase and its application in the production of cycloisomalto-oligosaccharides from dextran have been studied more recently (87). Cyclodextrans almost equally inhibit both reducing sugar and dextran producing activities of the dextransucrase reaction. The inhibition is dependent on the cyclodextran concentration (104).

Since the general characteristics of CITase and cyclomaltodextrin glucanotransferase (EC2.4.1.19) resemble each other, the enzymatic mechanism of CITase can be postulated. First, the main domain (A) of cyclomaltodextrin glucanotransferase closely resembles the structure of -amylase. Second, the starch-binding "groove" on domain A contains a similar catalytic Asp-Glu residue pair as in -amylases. Finally, cyclomaltodextrin glucanotransferase contains the starch-anchoring domain E as well as domain B that partially protect the catalytic Asp-Glu dyad from the attack of water molecules. Whenever the average length (and concentration) of the starch is high, the hydrolytic function dominates, but when the length is decreasing the transglycosylation reaction becomes prevalent (see discussion in ref. 124). Decreasing water activity by the addition of organic solvents shifts the equilibrium towards cyclization (37, 125). It is also possible to predict that CITase and endodextranase (glycosylhydrolase family 66) have related structural relationship similar to what has been detected between amylase and cyclomaltodextrin glucanotransferase.

BIOLOGICAL FUNCTION OF DEXTRANASES:
Role of Dextranases in Dextran-Producing Microorganisms:

It is reasonable to believe that the biological role of dextrans, for the benefit of microbes that produce them, is not only to provide protective and adhesive effects, but also to provide sugar storage for those microbes that are capable of depolymerizing them. Interestingly, certain extracellular exodextranases have special domains for anchoring dextrans into the cell surface from where the glucose units can be economically delivered to the cell (see discussion in reference 134). The ability to maintain food storage outside the cell is especially favorable in conditions when microbes are within reach of vast amounts of oligosugars (e.g., sucrose in mouth). In such conditions dextran is apparently synthesized fast by extracellular enzymes (probably already specifically bound to dextran polymers), while the monosugars generated from the transglycosylation are consumed immediately for metabolism. From the viewpoint of a microbe or a microbial association, optimization of the dextraneous environment in respect of the synthesis of polymerization-depolymerization activities and specificities is complex.
Sucrose is the major constituent of the human diet and both water-soluble and water-insoluble glucans are synthesized from it by oral streptococci (Streptococcus mutans, S. sanguis, S. sobrinus, S. cricetus, and S. rattus). They are believed to be responsible for the formation of dental plaque and the induction of caries on the surface of teeth and have, therefore, been a subject of numerous studies (11, 23, 46, 55). The glucan-producing streptococci S. sanguis, S. bovis, and S. mutans are also the most frequent organisms associated with endocarditis in humans. The chemical and physical properties of these glucans distinguish them from each other (43, 58, 140). Both soluble and insoluble glucans are important in cell-cell and cell-surface adhesive interactions in dental plaque (25, 55, 230). On the cleaned tooth surface, S. sanguis, S. mitis, S. oralis, and S. gordonii predominate among the first colonizing bacteria, and it is believed that these species help to establish conditions for development of the plaque biofilm (25, 108, 116, 155).

S. sanguis, which synthesizes little or no dextranase, produces not only soluble glucans but also large amounts of -1,6-linked insoluble glucans that are subject to extensive hydrolysis by exogenous dextranase (13, 54, 220). Strains of S. mutans serotype d produce water-insoluble glucans that are resistant to further hydrolysis by exogenous dextranase (56, 220). It has been demonstrated that -1,6-linked side chains allow the insoluble glucan to adhere to the surface of teeth, while the -1,3 regions render the glucan insoluble in water and contribute to the resistance to exogenous dextranases (41).

The total amount of glucans and their structures are influenced not only by the activities of the glycosyltransferases but also by extracellular dextranases. Oral streptococci are predominant producers of the dextranases (24, 46, 55, 220). Strains of S. mutans constitutively produce both endo- and exodextranases (162, 171, 220), whereas S. sobrinus synthesizes only endodextranase (10, 222). Endodextranase activity is present in the culture filtrates, while dextran glucosidase is predominantly cell associated (220). Endodextranase may regulate glucan synthesis by altering the ratio of -1,6 to -1,3 linkages and modify the glucan substrate to a firmer form, hence influencing its solubility and adhesive properties (25, 46, 220). Therefore, dextranase activity influences sucrose-dependent adherence of bacterial cells.

Dextranase-deficient mutants of S. mutans (DexA–) are more adherent to a smooth surface than the parent strain, but no difference in sucrose-dependent cell-cell aggregation has been observed (24). Endodextranase activity evidently provides primer or branch points for glucosyltransferases and thus contributes to the complexity of the glucan structure (25, 44). Possible intermediates in glucan synthesis could also be the products of exodextranase activity, which have been determined in intra- and extracellular extracts of oral strains of S. mitis (115, 215, 216, 217).

The current knowledge of sugar metabolism of S. mutans strains combined with genomic data suggest that this organism is capable of metabolizing a wider variety of carbohydrates than any other gram-positive organism, and thus, carbohydrate metabolism is the key survival strategy for S. mutans (4, 226). The dextranase of S. mutans breaks down glucans to isomalto-oligosaccharides, which are then transported into the cell via the products of the multiple sugar metabolism (msm) operon. In the cell, the oligosaccharides are further degraded to glucose by the products of the dexB gene, a dextran glucosidase (24). S. sobrinus and S. salivarius do not have such a mechanism and are unable to utilize dextrans or isomaltosaccharides as the sole carbon source (44, 112).

The second role of dextran glucosidase is in facilitating the total degradation of glycogen-like intracellular polysaccharide storage (IPS) to glucose by removing the -1,6 glucose stubs. IPS is believed to be of significance in the absence of dietary carbohydrates. The third possible function suggested for the dextran glucosidase is in the metabolism of -limit dextran products from the degradation of extracellular starch by human salivary -amylase or plaque-derived amylase (226).

The dextranase produced by S. sobrinus appears to be regulated in an entirely different way than the dextranase of S. mutans, exemplifying a different kind of strategy within dextran-producing microbes. A heat-stable, glucan-binding protein called Dei, which has the ability to inhibit dextranase activity with high specificity, has been detected in S. sobrinus but not in S. mutans. This inhibition causes the accumulation of water-soluble glucan, which inhibits plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus can only inhibit dextranase from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus serotype h), and S. macacae (serotype h) (201). Under conditions of carbohydrate limitation of S sobrinus, Dei levels are high and little active dextranase can be detected. When growth rates increase, the relative proportions and binding of dextranase and Dei alter and free dextranase becomes available (25). This finding suggests that Dei exists in some serotypes of mutans group of streptococci and participates in sucrose metabolism through its interaction with dextranase (201).

Role of Dextranases in Non-Dextran-Producing Microorganisms:
The majority of the non-dextran-producing microorganisms use dextrans either as the sole or as a secondary carbon source. Typically, the dextran-degrading enzyme synthesis of several fungi and soil bacteria is induced when grown in the presence dextran (29, 30, 47, 48, 59, 117, 147, 155). As mentioned above, most of the bacterial producers are able to synthesize simultaneously a few -glucosidases with different subcellular localizations (Table 1). A wide range of bacterial species, such as Bacteroides spp., Bifidobacterium spp., and Fusobacterium spp. associated with dental plaque, produce inducible dextran-hydrolyzing enzymes (26, 76, 86, 196, 205).

Three D-glucan-hydrolyzing enzymes from Bacteroides oralis Ig4a have been found. Extracellular endodextranase hydrolyzes polysaccharides in dental plaque to produce oligosaccharides that are small enough to enter the cells. The others, cytoplasmic exodextranase and mutanase hydrolyze the oligosaccharides to monosaccharides, thus permitting the use of dental plaque polysaccharides for microbial growth (205). Interestingly, an enzyme identical to dextranase (EC3.2.1.11) is also associated with the cell walls of growing coleoptiles of a plant, Avena. The enzyme plays a prominent role in the growth process, hydrolyzing certain cell wall components and providing necessary plasticity to the cell walls to extend (66).

STRUCTURE-FUNCTION ANALYSIS OF DEXTRANASES:
At present, there are 13 annotated sequences of endodextranases from species of Streptococcus, Arthrobacter, Paenibacillus, and Penicillium in the databanks (EMBL/GenBank and SWISS-PROT). Only one crystal structure of an endodextranase from Penicillium minioluteum (Dex49A) and one from glucodextranase (exodextranase; iGDase) from Arthrobacter globiformis have been published. There have been some attempts to solve three-dimensional structures by computer modeling using sequence data and three-dimensional structures of homologous proteins. Compared to the knowledge of structural relationships and mechanisms of action of enzymes involved in synthesis and degradation of starch, cellulose, and chitin, the data on dextran metabolism are still elusive. However, it seems plausible that the basic understanding of the structures and mechanisms of other carbohydrate enzymes may be applicable to the structure-function analysis of dextranases.

The sizes of dextranase genes and their protein products are highly divergent (Table 2). This is exemplified by aligning the 13 dextranase protein sequences that are currently available in the protein data banks. Multiple sequence alignment for proteins was created using ClustalW (version 1.82) (http://www.ebi.ac.uk/clustalw/) using default values. The phylogram (Fig. 1) indicates sequences that are related, but due to the great difference in sequence length, the alignment as such is not well presented. This indicates that dextranases form a group of proteins that possess similar enzyme activities even though they have highly different primary protein structure.

APPLICATIONS OF DEXTRANASES:
The dextrans themselves are polydisperse and as such mostly not suitable for technological applications. However, enzymatically processed fractionated dextrans possess a significant commercial interest in cosmetics, drug formulations, and vaccines, as cryoprotectants, and as stabilizers in the food industry. Selected dextran fractions in combination with polyethylene glycol solutions form a two-phase system. In addition to using dextranases for processing dextrans, the enzymes themselves are increasingly important in the food, dental, and detergent industries (see below). Finally, dextran-hydrolyzing enzymes are important for elucidating the fine structure of dextran and certain other polysaccharides (1,2, 15, 28).

Clinical Applications of Dextran and Dextranases:
Initial interest in dextranases was raised in regard to their possible application in commercial production of clinical dextran, i.e., a sterile solution of dextran of a specific molecular weight to be used to restore blood volume in patients suffering shock as a result of blood loss (80, 90, 113, 127). Relatively low-molecular-weight clinical dextrans have previously been produced from dextran by controlled acid hydrolysis followed by organic solvent fractionation. However, the yields are low (10 to 12%) due to losses during hydrolysis and fractionation. The enzymatic method seemed to have potential for replacing the acid hydrolysis for clinical dextran production and such processes were patented in the 1950s. The enzymatic method needs less energy and simpler equipment and results in a more uniform product with a 25% to 52% yield (21, 141, 142).

The highest yield of clinical dextran, 94% of total dextran produced, has been obtained in mixed-culture fermentation of Lipomyces mesenteroides and a constitutive dextranase mutant of Lipomyces starkeyi in the presence of sucrose. A simple industrial fermentation was developed to produce controlled-size dextrans with a small polydispersity index (36, 90, 93). Dextrans of molecular weights between 900 and 1,800 were considered less likely to cause anaphylactic reactions than the higher-molecular-weight dextrans (214). Due to the extremely strict regulatory demands of the intravenously administered clinical dextrans and their stagnant market, significant technological progress has not yet managed to overcome the traditional chemical processes.

The advantages of processed dextrans for biomedical applications are the biocompatibility, slow biodegradability, and feasibility of incorporation of molecules into the matrices formed by dextrans (99, 127, 193). Dextran hydrogels and their chemical modifications have been evaluated as carriers for controlled release of drugs to targeted organs by slow dextranase hydrolysis. Remarkably, biodegradable dextran hydrogels containing polyethylene glycol have exhibited regulated insulin release (138). A substantial number of pharmacokinetic studies on dextran conjugates with therapeutic and imaging agents have been carried out in animals (31, 42, 127, 138, 139).

Dextranase can be used as universal targeting method for therapeutic agents (57). In the case of cancer, for example, a bispecific antibody has been created against cancer antigen and dextranase. The bispecific antibody is first injected into the blood circulation, where it binds to cancer cells. Dextranase is then injected and subsequently captured by the antibody-bound cancer cells. Finally, a cytotoxic therapeutic agent conjugated to dextran is injected into the bloodstream, and the conjugate is cleaved by the action of dextranase to release the cytotoxic drug selectively into the cancer cells (57).

In endocarditis, an exopolysaccharide product from viridans streptococci (glycocalyx, composed predominantly of dextran) has been associated with a delayed antimicrobial efficacy in cardiac vegetations. Enzymatic digestion of the glycocalyx by dextranase has been shown to enhance the antibiotic activity of penicillin and temafloxacin (33, 128).

Dextrans also contribute to human health since they are resistant to mammalian digestive enzymes in the small intestine but are readily fermented in the large intestine, particularly by probiotic bacteria belonging to the genera Lactobacillus and Bifidobacterium. Prebiotic oligosaccharides, including isomalto-oligosaccharides, are believed to promote the growth and proliferation of these microbes most efficiently. Immobilized dextransucrase with soluble dextranase has been used for synthesis of prebiotic oligosaccharides (109).

Applications of Dextranases in Treatment of Dental Plaque:
Dental plaque, the bacterial film adhering to tooth surfaces, is composed of closely packed bacteria and noncellular material. Roughly 20% of the dry weight of dental plaque is water-insoluble glucans (121). Degradation and removal of these glucans have been suggested to prevent oral diseases such as dental caries. Dextranase can inhibit the synthesis of insoluble glucans (121, 183, 196, 215) as well as the adherence of streptococci (183, 184). Simultaneous use of several enzymes, such as dextranase and mutanase, could be advantageous (140, 219). A novel glucanhydrolase, DXAMase from Lipomyces starkeyi, appears to be effective in reducing synthesis of insoluble glucans, inhibiting sucrose-dependent adhesion to glass, and removing bacterial films previously formed in the presence of sucrose. These in vitro properties of DXMase are considered propitious for dental plaque agent (172).

For the treatment of dental plaque, various compositions that comprise enzymes hydrolyzing or inhibiting glucans have been proposed (95, 96, 188, 212). Another approach to the control of dental caries would be genetic engineering of oral commensal organisms to antagonize the cariogenicity of S. mutans strains. The genes encoding dextranase and mutanase have been cloned and expressed in oral streptococci (110, 122). The transformant S. gordonii has been found to repress the firm adherence of water-insoluble glucan in a cocultivation experiment with cariogenic bacteria in the presence of sucrose (110). However, it has not yet been demonstrated that such a strategy is effective in vivo. A novel transformant technique, resident plasmid integration for cloning of foreign DNA in oral streptococci, has been used to clone the gene coding for cycloisomalto-oligosaccharide glucanotransferase (CITase) that produces cycloisomalto-oligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferases. CITase has been isolated from the Bacillus circulans T-3040 chromosome (145) and transferred into S. gordonii, and the gene product was secreted into the culture medium at low levels (190).

Use of Dextranases in the Sugar Industry:
One of the major industrial applications of dextranases is the reduction of sliming in sugar production processes. The growth of Leuconostoc and Lactobacillus spp. is the most important factor in contributing to the postharvest deterioration of cane sugar and frost-damaged beet sugar (18, 113, 207). Problems caused by dextran in raw sugar include sucrose loss, increased viscosity of process syrups, and poor recovery of sucrose due to inhibition of crystallization. Dextranases are used in various analytical methods for measuring glucan content in sugar juices and in raw sugar (18, 19, 167, 194, 197). Cane dextran isolated from deteriorated cane juices and raw sugars possesses an average molecular mass of 5,000 kDa and are polydisperse by nature. Dextrans isolated from various sugar cane products possess a very similar structure, 95% -1,6 linkages and 5% branching, probably through -1,3 bonds (18).

The majority of the methods used to remove dextran from sugar solutions rely on its enzymatic hydrolysis. Dextranases reduce the molecular mass and therefore the viscosity of juices (22, 32, 81, 118, 233). Trademarks like Novo dextranase of P. lilacinum (Denmark) and dextranase Hutten DL-2 of Chaetomium gracile (Japan) have been successfully used to treat dextran-contaminated sugar process streams (169, 233). Even at relatively low levels of dextran in raw juice (i.e., 75 mg/liter) the filtration rate is markedly dropped and, consequently, the slicing capacity is decreased by 50%. A dosage of 10 ppm dextranase NOVO 50 L enzymes to the extraction is sufficient to restore slicing to 90% of the nominal capacity (17).