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Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

Elvira Khalikova,^1,2 Petri Susi,^1, and Timo Korpela^1*

Joint Biotechnology Laboratory, Department of Chemistry, University of Turku, 20520 Turku, Finland,¹ Institute of Biology, Department of Biotechnology, 450054 Ufa, Russia²

SUMMARY

INTRODUCTION

STRUCTURE AND PROPERTIES OF DEXTRAN

CLASSIFICATION OF DEXTRAN-HYDROLYZING ENZYMES

SOURCES, MAIN PROPERTIES, INDUCTION, AND MECHANISM OF ACTION

Endodextranases (EC3.2.1.11) from Fungi

Endodextranases from Bacteria

Glucose-Forming Exodextranases

Isomaltose-Forming Exodextranases

Isomaltotriose-Forming Exodextranases

Debranching Exodextranase

Cycloisomalto-oligosaccharide Glucanotransferase

BIOLOGICAL FUNCTION OF DEXTRANASES

Role of Dextranases in Dextran-Producing Microorganisms

Role of Dextranases in Non-Dextran-Producing Microorganisms

STRUCTURE-FUNCTION ANALYSIS OF DEXTRANASES

Sequence Comparison Studies

Dextranase Isoforms

Secondary and Tertiary Structures of Dextranases

METHODS FOR MEASURING DEXTRAN-HYDROLYZING ACTIVITY

APPLICATIONS OF DEXTRANASES

Clinical Applications of Dextran and Dextranases

Applications of Dextranases in Treatment of Dental Plaque

Use of Dextranases in the Sugar Industry

CONCLUSIONS AND FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Dextran is a homoglycan of -D-glucopyranose molecules coupled primarily with -1,6 linkages. Due to diverse branching of the glucose backbone chain, dextran polymers have a remarkable diversity in chain length and in physicochemical properties. The degradation of dextrans entails a number of glycosyl hydrolases with different specificities and modes of action. Initially, these enzymes were called endo- and exodextranases. However, the divergence has obliged us to specify them in more detail, taking into account the structures of substrates and reaction products. A novel classification system based on amino acid sequence similarities links dextranases to other families of glycoside hydrolases.

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.

STRUCTURE AND PROPERTIES OF DEXTRAN

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Dextran is a collective name for high-molecular-weight polymers composed of D-glucose units connected with -1,6 linkages and various amounts of side branches linked with -1,2, -1,3, or -1,4 to the main chains. The enzymes that synthesize these glucans from sucrose are known by the generic term dextransucrase (1,6--D-glucan-6--glucosyltransferase, EC2.4.1.5.). They are glucansucrases produced by various Leuconostoc and Streptococcus species (135, 136, 191) and by the mold Rhizopus spp. (175). Other dextran-producing bacteria, Acetobacter capsulatus (renamed Gluconobacter oxydans) and Acetobacter viscous, produce dextrin dextranase (EC2.4.1.2) that converts dextrins to dextran (192, 229). The Leuconostoc mesenteroides strains are inducible and require sucrose in the medium for the biosynthesis of dextrans with the exception of recently isolated constitutive enzyme mutants, e.g., strains B-512 FMC, B-742, B-1142, B-1299, and B-1355 (28, 91, 92, 98, 136, 168). Streptococcus species are generally constitutive and do not require sucrose in the growth media for enzyme expression (43, 55, 191).

Dextransucrase catalyzes the synthesis of glucan, which contains 50% or more -1,6 glucosidic bonds within the main chain (136). The structures and properties of bacterial dextrans vary between microbial strains and according to growth rate and reaction conditions (27, 84, 91, 96, 156, 191, 221). The position of the branch linkages, the degree of branching, the length of branch chains, and molecular weight distribution affect the physicochemical properties of dextrans (1, 2, 127, 187). For example, the degree of solubility in water decreases when the degree of branching is increased. In fact, dextrans with >43% branching through 1,3- linkages have been considered water insoluble (127).

The most extensively studied dextransucrase from the commercially important strains NRRL B-512 and B-512F of L. mesenteroides synthesizes a soluble linear -1,6-linked dextran with about 5% randomly distributed -1,3-branched linkages of up to 50 to 100 residues (168, 176, 191). The long branches are important factors for properties of the dextran (209). On the other hand, other strains of Leuconostoc are known to produce several dextransucrases resulting in synthesis of glucans composed of water-soluble fraction and water-insoluble or less-soluble fraction (14, 27).

Leuconostoc mesenteroides strain B-1299 produces both extracellular and intracellular dextransucrases that synthesize two kinds of dextrans: fraction L, which precipitates in 38% ethanol, has 27% of -1,2-, and 1% of -1,3-branch linkages; and fraction S, which precipitates in 40% ethanol, has 35% of -1,2-branch linkages (14, 15, 38, 92). L. mesenteroides B-742 also expresses two kinds of dextransucrases, producing fraction S with 50% -1,6-, 50% -1,3-, and no -1,4-branch linkages, and fraction L, which contains, in addition to -1,6 linkages, 14% -1,4- and about 1% of -1,3-branch linkages (91).

Dextrans with low degree of branching can be hydrolyzed by Penicillium endodextranase, but highly branched dextrans produced by B-1142(S), B-742(S), and B-1299(S) are resistant to endodextranase hydrolysis (92). L. mesenteroides NRRL B-1355 produces two distinct glucans. Fraction L is similar to B-512 dextran, but fraction S has an altered sequence of -1,6 (53%) and -1,3 linkages and has been named alternan (27, 130, 136).

Detailed chemical structures and properties of glucans produced by streptococci have been reported in a number of publications (23, 43, 55, 84, 126, 191, 203). In earlier investigations water-insoluble polysaccharides, synthesized by streptococcal mutansucrases (EC2.4.1.5), were described as typical dextrans (55, 84, 191). Several Streptococcus strains form two groups of glucosyltransferases: those that synthesize water-soluble glucans, primarily consisting of -1,6 bonds, which classifies them as dextrans, and those that synthesize water-insoluble glucans, primarily having more than 50% of -1,3-bonds with small proportions of -1,6 and -1,4 linkages (52, 136, 223). Water-insoluble glucan from Streptococcus mutans strain OMZ176 contains up to 90% of -1,3-glucosidic linkages. In contrast, gelatinous glucan from Streptococcus sanguis strain 804 has equal amounts of -1,3 and -1,6-glucosidic linkages (54, 58).

The degree of polydispersity of dextrans significantly affects their in vivo behavior (127). Native dextrans isolated from culture filtrates of L. mesenteroides strain B-512F are often extremely polydisperse and contain molecules of all sizes from oligomers to molecular weights of several hundreds of millions. The range of molecular sizes may be reduced (degree of polydispersity 2) by either partial acid or enzymatic hydrolysis. Dextrans with average mass of 4 to 2,000 kDa and polydispersity of 1.5 are commercially available for research purposes from Amersham, Sweden. Solutions containing small (40 kDa) or large (70 kDa) preparations are called clinical dextrans (127).

CLASSIFICATION OF DEXTRAN-HYDROLYZING ENZYMES

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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).

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

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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.

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 K_m 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).

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).

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).

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).

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

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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).

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

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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.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Neighbor-joining tree showing phylogenetic clustering of representative collection of divergent group of dextranases shown in Table 2.

cDNA clones expressed in dextran-induced cultures of Penicillium minioluteum HI-4 have been identified by differential hybridization (48). Analysis of selected clones revealed nonhomologous cDNAs corresponding to four different genes, dexA, dexB, dexC, and dexD. One of these cDNAs (dexA) encodes endodextranase (EC3.2.1.11). According to the structure prediction of DexA, the enzyme has a mechanism of hydrolysis with net inversion of anomeric configuration and it is, therefore, included in glycosylhydrolase family 49 (63, 159, 160). The enzyme shows 29% and 33% sequence identity with Arthrobacter sp. strain CB-8 dextranase and Brevibacterium fuscum var. dextranlyticum strain 0407 isomaltotriodextranase, respectively (48, 133).

The dexA cDNA sequence comprises 2,109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues that might correspond to a signal peptide. The predicted molecular mass is 64 kDa, which is somewhat smaller than that estimated for the native enzyme (67 kDa). The dexB (1.6 kb) gene shows sequence homology to sugar transporter proteins, whereas the dexC (1.8 kb) gene belongs to glycosylhydrolase family 13, showing high sequence identity (51%) to oligo-1,6-glucosidase of Bacillus cereus. The dexD (1 kb) product is homologous to -amylase proteins according to structural comparisons. Taking into account that the dex genes (with the exception of dexD) are grouped in the same 9-kb BamHI fragment, it is tentative to speculate that the genes necessary for dextran assimilation and hydrolysis are clustered in the genome of P. minioluteum (50, 111).

The gene encoding extracellular isomaltotriodextranase, an exo-type enzyme (EC3.2.1.95), designated DexT, has been cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranolyticum strain 0407 and expressed in Escherichia coli. A single open reading frame (ORF) consisting of 1,923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and mature protein of 604 amino acids (M_r 68,300) was found. The primary structure of DexT had significant similarity (78.5% and 63.0%) with two other reported endo-type dextranases isolated from two Arthrobacter strains, CB-8 and T-3040, respectively (82, 133).

Isomaltodextranase (EC3.2.1.94), other than DexT, from A. globiformis T6 is an exo-type enzyme (Table 1). However, it possesses an entirely different structure, and isomaltodextranase is, therefore, classified in glycosylhydrolase family 27. It contains enzymes having a mechanism of hydrolysis with an overall retention of anomeric configuration. The imd gene is 1,689 bp in length and encodes a protein with a molecular mass of 66 kDa. Isomaltodextranase has weak homology with the C terminus of -galactosidase from Cyamopsis tetragonoloba and practically no homology with dextranases from Arthrobacter sp. strain CB-8 (155) and S. mutans (171), which implies that the isomaltodextranases have different specificities (82).

Glucose-producing exo-type dextranases have been included in glycosylhydrolase families 13 and 15. Glycosylhydrolase family 13 comprises enzymes responsible for the hydrolysis of -1,2, -1,3, -1,4, and -1,6 glucosidic linkages. Few sequences of dextran glucosidase genes from Streptococcus and Arthrobacter species are available. Isomaltodextranase from Arthrobacter globiformis T6 belongs to glycosidehydrolase family 27, but this isomaltodextranase has poor sequence homology to this family. The enzymes cleave the glycosidic bond of the substrate by either retaining or inverting the anomeric configuration. It is believed that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active-site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomaltodextranase active site that catalyze the hydrolysis reaction of dextran have been identified, and the roles of nine amino acid residues in this isomaltodextranase were studied by site-directed mutagenesis. Out of 15 enzymes that were mutagenized, eight had reduced dextran-hydrolyzing activities. Aspartic acid-227 and Asp-342, which are part of the apparent catalytic site, are essential for hydrolase activity toward dextran (210).

The gene encoding dextran glucosidase from Arthrobacter globiformis T-3044 has been cloned and expressed in Escherichia coli (147). The enzyme gene consists of a unique ORF of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and six internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggests that the enzyme is translated from mRNA as a secretory precursor with a signal peptide of 28 amino acid residues, whereas there was no evidence for a signal peptide in S. mutans Ingbritt dextran glucosidase (dexB) (147, 171). The deduced amino acid sequence of the mature glucodextranase enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 Da that showed about 38% sequence identity to that of glucoamylase from Clostridium sp. strain G0005. Although the glucodextranase that was produced from the transformant was shorter than the authentic enzyme by two amino acid residues at the N terminus, its enzymatic properties were practically the same as those of the authentic enzyme.

A comparison of the deduced amino acid sequence of Streptococcus mutans Ingbritt dextran glucosidase encoded by dexB with the sequences of other proteins revealed regions of local similarity that correspond to highly conserved regions of -amylases (171). These regions are also found in enzymes that attack the -1,6 interchain linkages in starch and pullulan: pullulanase, isoamylase, and neopullulanase. It is believed that these regions are involved in substrate binding and catalysis of these carbohydrases (82). In addition, DexB and isoamylase have a common region in their C-terminal ends. The highest degree of homology was found between DexB and dextranase (DexS) from Streptococcus suis with 34% identity and 74% similarity, as well as a significant amount of overall sequence similarity between DexB and cyclodextrin glucanotransferase of Klebsiella pneumoniae (171, 185). An analysis of the dexB ORF revealed that it codes for a 536-amino-acid protein with a predominantly hydrophilic character and a predicted mass of 62,000 Da. There was no evidence for the existence of a signal peptide. Furthermore, the size of DexB detected by SDS-PAGE in recombinant E. coli was 62 kDa, close to that predicted from the sequence data (62,103 Da) (171). DexB is intracellular and does not undergo any posttranslational modification (171).

The primary amino acid sequence of the dexB gene product of S. mutans strain LT11 showed significant similarity to Bacillus oligo-1,6-glucosidase. dexB is a a member of the multiple sugar metabolism (msm) operon, which contains genes for both transport and subsequent breakdown of products from hydrolysis of extracellular polysaccharides (226). Structure prediction and hydrophobic cluster analysis have also shown that S. mutants LT11 dextran glucosidase and Bacillus sp. oligo-1,6-glucosidase have similar domain structures, with a catalytic (ß/)₈-barrel and a smaller C-terminal domain (226).

A glucodextranase (iGDase; EC3.2.1.70) from Arthrobacter globiformis I42 was classified in glycosidehydrolase family 15. The iGDase gene was cloned and the primary structure was deduced. A homology search with other proteins revealed that dextran glucosidase from A. globiformis T-3044 was the most homologous protein (80% similarity) over the primary structures. Apart from this homology, the N- and C-terminal parts of iGDase are individually homologous with different kinds of proteins. The N-terminal region showed high similarity to bacterial glucoamylases (52% similarity), whereas the C-terminal region showed 29% identity to the S-layer homology domain of pullulanase (134).

The bacterial endodextranases from the Steptococcus species and cycloisomalto-oligosaccharide glucanotransferase from a Bacillus sp. showed significant similarity and were classified in glycosylhydrolase family 66. Genes encoding extracellular dextranases were cloned from S. mutans, S sobrinus, S. salivarius, S. suis, S. downei, and S. rattus. A single copy of the dextranase (dex) gene was detected in S. mutans (24), S. sobrinus (10), and S. salivarius (112).

The dextranase gene (dexA) from S. mutans Ingbritt (serotype c) encoded a dextranase (Dex) protein consisting of 850 amino acids with a molecular mass of 94.5 kDa. Thus, it was smaller than the SDS-PAGE-estimated masses of the native dextranase (120 kDa) produced by S. mutans Ingbritt and the recombinant DexA (133 kDa) produced by E. coli cells (74, 75). The same phenomenon was observed in S. sobrinus UAB66 (serotype g) dextranase, where the deduced molecular mass of Dex from the nucleotide sequence (143 kDa) was smaller than the molecular mass of native Dex (175 kDa). The molecular mass of purified enzyme from clone pYA902 was estimated to be 130 kDa. Since this size is exactly half of that obtained by gel filtration (260 kDa), native Dex might exist in a dimeric form (171, 222).

Recombinant S. sobrinus dextranase (Dex) is mainly transported into the periplasmic space of E. coli cells, whereas S. mutans recombinant dextranase is located in the cytoplasm. The deduced N-terminal amino acid sequences of extracellular dextranases of S. mutans Ingbritt (dexA) and S. sobrinus UAB66 (dex) showed 57.8% homology. No cross-reactivity exists between the dextranase of S. sobrinus UAB66 and surface protein antigen A (SpaA), which appears to be one of the proteins necessary for sucrose-independent adherence (222). The ORF of a dextranase gene from S. rattus is 2,760 bp and it encodes a protein consisting of 920 amino acids with a mass of 100,163 Da and pI of 4.67. The physicochemical properties of S. rattus dextranase purified from recombinant E. coli cells are similar to those of S. mutans and S. sobrinus dextranases (79).

The dextranase-encoding gene from S. salivarius strain M-33 has been cloned and sequenced. One of the clones is a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds to that of native Dex (86 kDa) from the S. salivarius M-33 culture supernatant (149). A comparison of the S. salivarius M-33 Dex sequence with that of S. mutans Ingbritt dextran glucosidase (DexB) (171) and Arthrobacter sp. CB-8 Dex (155) reveals no homology between these proteins (149). Another gene encoding extracellular endodextranase was cloned from S. salivarius strain PC1, and its native product was recovered from culture media as a single 110-kDa polypeptide whereas the recombinant strain produced a 190-kDa protein and two lower-molecular-mass polypeptides (90 and 70 kDa) (112).

DNA fragments encoding the S. downei dextranase were amplified by PCR and inverse PCR based on comparisons between the dextranase gene sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex gene was determined. A 3,891-bp ORF encodes dextranase protein consisting of 1,297 amino acids with a molecular mass of 139,743 Da and isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex has homology to those of S. sobrinus, S. mutans, and S. salivarius Dex in the conserved region. A DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus but not to the other species of S. mutans. The recombinant plasmid, which harbored the dex ORF of S. downei, produced a recombinant Dex enzyme in E. coli cells. An SDS-PAGE analysis of the recombinant enzyme indicated that there are multiple active dextranase forms (77). Comparison of the amino acid sequences of the Dex proteins and glucosyltransferases indicated that the amino acid sequences of the Dex enzymes produced by S. mutans, S. sobrinus, S. salivarius, and S. downei were similar to those of the catalytic sites of glucosyltransferases of mutans streptococci (78, 137).

Observations suggest that the higher-molecular-mass forms of dextranase gradually lose activity during storage, while the activities of the smaller forms remain unaffected (171). This loss of activity can be prevented by protease inhibitors. The very large variability of molecular masses of dextranases from different microbial sources and the common existence of catalytically active isoforms suggest exceptional, not yet identified structural/functional features that deserve further attention from the scientific community.

Based on common sequence patterns in the structural core region between DexC (glycosylhydrolase family 13) of P. minioluteum and Bacillus cereus oligo-1,6-glucosidase, a three-dimensional structure was predicted. Even though dexA and dexC have no significant sequence similarity and the predicted three-dimensional structures are different, their products catalyze chemically equivalent reactions (50).

Computer modeling studies indicated that S. rattus dextranase has two variable regions, an N-terminal signal peptide and a C-terminal cell wall-sorting signal. The main molecule contains two functional domains, a catalytic domain (12 amino acids) and a substrate-binding domain (120 amino acids residues) at the C-terminal side. This structural organization is quite similar to the dextranases from S. mutans, S. sobrinus, and S. downei (79). Asp385 of the Dex of S. mutans Ingbritt is essential for enzyme activity, and the catalytic and substrate-binding sites are located at different sites within the Dex molecule (78, 137). Replacement of Asp385 of DexA from S. mutans Ingbritt results in complete disappearance of enzymatic activity, while the enzyme retains its ability to bind dextran (78). Deletion of the N terminus abolishes enzyme activity but does not affect dextran-binding ability, while deletion of the C-terminal 120 amino acids fully abolished the ability to bind dextran (137).

The dextranases from S. mutans, S. sobrinus, and S. downei have a putative cell wall-anchoring region at the C terminus (75, 77, 222). However, the cell wall-bound form of dextranase has not been detected, and the enzyme has always been reported as extracellular. The presence of a cell wall-anchoring region in Dex may suggest that it is temporarily cell wall associated and then released extracellularly by an unknown modification(s) (77).

The crystal structure of a glycosylation-free mutant form of endodextranase from P. minioluteum (termed Dex49A) has been solved in unliganded (at 1.8 Å resolution) and product-bound (at 1.65 Å resolution) forms (111). The enzyme forms 10 right-handed parallel ß-helix domains that are connected to an N-terminal ß-sandwich domain (Fig. 2). In the structure of the product-bound form, isomaltose was found to bind in a crevice on the surface of the enzyme. The nonreducing end sugar forms a hydrogen bond to the Asp395 carboxylate, which is the plausible catalytic acid for the 1,6-glycosidic bond. Asp395 is conserved within glycosylhydrolase family 49, as are Asp376 and Asp396. The latter two aspartyl residues are hydrogen bonded to a water molecule, which is suitably positioned for nucleophilic attack. The structures most similar to DexA are the galacturonases found in glycosylhydrolase family 28, which is suggestive of a new glycosylhydrolase clan for glycosylhydrolase families 28 and 49.

View larger version (33K): [in this window] [in a new window]   FIG. 2. Crystal structure of endodextranase Dex49A from Penicillium minioluteum (111). The right-side structure is turned 90o counterclockwise around the vertical axis. Reprinted from reference 111 with permission from Elsevier.

METHODS FOR MEASURING DEXTRAN-HYDROLYZING ACTIVITY

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To measure the enzyme activity of a dextran-hydrolyzing enzyme can sometimes be difficult because of the large variability of available substrates and because the reaction product is an undefined mixture of sugar polymers. This also makes it laborious to assess the validity of the reaction conditions. Since the selection of the assay method is a compromise between factors such as convenience, speed, and accuracy, we give here an overview of the spectrum of methods used. Because there are no commonly accepted methods in the field, it is hard to compare enzyme activities between different investigations. Hence it would be desirable to put more effort into developing standardized and kinetically valid methods with generally acceptable formulations of the enzyme units. In particular, a correlation of a method of choice against high-pressure liquid chromatography data when analyzing one or more of the end products would be informative (119).

The first methods to be used for measuring dextranase activities were based on viscosimetric analysis (59, 71, 80). One unit of viscosity-reducing activity was defined as the amount of enzyme which reduced the specific viscosity of the mixture by half in 10 min. The nephelometric method defines dextranase activity as a dextran solution's loss of opalescence (7). These methods are apparently suitable when dextranase cleaves the dextran molecule at random to produce long oligosaccharides. Solutions of high-molecular-weight dextrans show a strong apparent UV absorption at 220 nm (102), which allows direct determination of the amount of high-molecular-weight dextrans produced differently by endo- and exodextranases (103).

In saccharogenic methods, one unit of enzyme has been defined as the amount of enzyme producing 1.0 µmol of glucose (35), 1.0 µmol of isomaltose (158), 1.0 mg of isomaltose monohydrate (20), or 1 mg of isomaltotriose (7, 8) per unit time under the assay conditions. The liberated reducing sugars in a reaction mixture are frequently analyzed with the Somogyi assay (44, 195) with 3,5-dinitrosalicylic acid reagent (49, 129), thiourea borax-modified O-toluidine color reagent (35), or alkaline potassium ferricyanide solution (225). In principle, the measurement of total reducing sugars is universal and allows comparisons between different methods. Unfortunately, however, there is no common substrate to assay the activity. Dextran T2000 (47, 68, 76), T-260 (3), T110 (158), and T-40 (196) have been widely applied. In addition, the incubation time has varied from 10 min (47, 67) to 2 h (20, 225). The hydrolytic activity of exo- and endodextranases has also been monitored with p-nitrophenyl--D-glucopyranosides as the substrates (115, 121). The method is simple but needs strict verification for the absence of other interfering enzymes.

Endodextranase activity can be measured spectrophotometrically by using chromogenic substrates with negligible interference from endogenous glucose or isomaltose (107, 120, 123). The assay is fast, sensitive, quantitative, and especially suitable for enzymes releasing relatively long dextran oligomers. The more sensitive fluorometric assay using amino-dextran-70 coupled with the fluorescent dye BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) has been described (232). Other assay procedures include the use of chemically modified insoluble substrates and measure of the release of soluble chromo- or fluoro-coupled fragments with sizes different from that of the substrate (70, 88, 174). Soluble and insoluble substrates are easy to separate, but the conditions of the mass transfer from solid to liquid phase must be kept constant.

Solid-phase (plate) assays using agar gels may be useful for screening dextranase activities. In short, a suspension of Sephadex in a buffer is supplemented with agar, sterilized, and poured onto petri dishes. Small wells are punched and filled with the test solutions, followed by incubation. The extent of clearance around the hole due to the opalescence of Sephadex provides an est