Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides

For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.     

Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases

The hydrolysis reaction of chitosan using immobilized chitosanases with regard to the composition of its products and the yield of the intermediate target products, pentamer and hexamer of chitosan oligosaccharides, was investigated. Chitosanase was immobilized onto agar or agarose gel particles by the multipoint attachment method. In batch experiments, surface enzyme density, support particle size, temperature, agitator speed, and initial substrate concentration significantly affected the composition of the oligosaccharides produced. It was believed that these factors all related to the reaction rate and mass transfer rate at the surface of the support materials immobilizing the enzymes. These effects were summarized as a correlation with Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The result showed that the reaction conditions that give a low value of Da provide a high yield of pentamer and hexamer oligosaccharides.     

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis,HPLC, and real-time ESI-MS

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from − 3 to − 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.     

Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases

The continuous production of chitosan oligosaccharides using a packed-bed enzyme reactor was investigated as to the effects of the operation conditions on the yield of pentamers and hexamers of chitosan oligosaccharides. A column reactor packed with immobilized chitosanases prepared by the multipoint attachment method was used for continuous hydrolysis of chitosan. In this reactor, the decrease of the yield of the target intermediate oligosaccharides due to axial mixing was negligible. The surface enzyme density of the support and flow rate of the substrate solution significantly affected the maximum yield of pentamers and hexamers. These effects were summarized as a correlation with the Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The optimum condition was determined based on Da. Under the optimized condition (Da = 0.12), pentamers and hexamers could be produced continuously for a month with a yield of over 35% (7 kg/m(3) in concentration).     

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.     

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.     

Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisk impeller

An immobilized enzyme bioreactor consisting of an agar gel-coated multidisk impeller was developed for the hydrolysis of highly viscous chitosan solutions, and the operating conditions for the production of physiologically active chitosan oligosaccharides (pentamers and hexamers) were investigated. Chitosanase was directly immobilized on the agar gel-coated multidisk impeller by a multipoint attachment method. The high stability of the immobilized enzyme was confirmed by means of five repetitions of a batch hydrolysis reaction. When the enzyme activity at the support surface was relatively high, the yield of the target products was higher at an impeller speed of 2 s⁻¹ than at a speed of 1 s⁻¹. However, no significant increase in yield was observed at impeller speeds higher than 2 s⁻¹ in reactions at either of the two substrate concentrations tested (5 and 20 kg/m³). When the surface enzyme activity was low, the impeller speed did not affect the yield of the target products. The maximum yield of pentamers and hexamers increased as the surface enzyme activity decreased, and high yields (>30%) were obtained at activities below 160 U/m². From the viewpoint of productivity, the optimal surface-enzyme activity was about 340 U/m², and at that activity, the yield of target products was 22%. This yield was higher than that reported for conventional acid hydrolysis. To maximize both the productivity and the yield of the target products, the surface area for the immobilized enzyme should be increased. Our results suggest that it may be possible to obtain high yields of pentamers and hexamers of chitosan oligosaccharides from highly viscous chitosan solutions with this reactor.     

Determination of angiotensin converting enzyme inhibitory activity by high-performance liquid chromatography/electrospray-mass spectrometry

A sensitive and rapid method for determination of angiotensin converting enzyme (ACE) inhibitory activity was developed based on a combination of enzymatic reaction followed by high performance liquid chromatography/electrospray-mass spectrometry (HPLC-ESI-MS) determination of its product. The most commonly used substrate hippuryl-histidyl-leucine (HHL) or hippuryl-glycyl-glycine (HGG) hydrolysis catalyzed by purified rabbit lung ACE or human plasma ACE was investigated in the presence of benazeprilat. The incubation time was 8 min for purified lung ACE, and 16 min for human plasma ACE. The produced hippuric acid (HA) was separated from substrate HHL or HGG by HPLC on a C(18) column with isocratic elution within 6.5 min, and quantified by electrospray ionization mass spectrometry (ESI-MS) with p-phthalic acid as an internal standard (IS). The limit of detection of HA was 6.0 ng/ml. HHL or HGG hydrolysis catalyzed by purified lung ACE displayed excellent accuracy and reproducibility. The small total reaction volume, the low concentration of substrate, and the simple treating procedures present the advantages of the new method. Furthermore, the total time of the whole procedure for one sample with the novel method is less than 1/2 of that of the conventional HPLC or spectrophotometry method, while the accuracy and the precision of the new method are almost the same as the conventional HPLC method with UV detection.     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Purification and characterization of a chitinase from Trichoderma viride

Usukizyme, a commercial enzyme preparation from Trichoderma viride, showed multiple chitin- degrading activities. One of these was purified to homogeneity by sequential DEAE Sepharose CL-6B, Q-Sepharose FF, and Sephacryl S-100 HR column chromatographies. The purified enzyme showed optimum activity at pH 3.5 and 50 degrees -55 degrees C and was stable in the pH range of 3.5-6.0 and up to 45 degrees C. It showed higher activity toward chitosan-7B, a 62% deacetylated chitosan, as opposed to highly deacetylated chitosan substrates. Products of degradation of a 1% (w/v) solution of partially deacetylated chitin (PC-100) were purified on CM-Sephadex C-25 and analyzed by HPLC, exo-glycosidase digestion, and nitrous acid deamination. The enzyme was unable to split the GlcN-GlcN linkages in the substrate. It produced mainly (GlcNAc)(2) and (GlcNAc)(3) along with mixed oligosaccharides. When subjected to nitrous acid degradation, some of the mixed oligosaccharides produced mainly 2-deoxyglucitol, implying the presence of GlcN at the reducing end of the oligosaccharides.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Development and application of a model for chitosan hydrolysis by a family 18 chitinase

Hydrolysis of partially deacetylated chitosans by ChitinaseB (ChiBeta) from Serratia marcescens results in mixtures of oligosaccharides typically between 2 and 20 sugar residues. The amounts of different oligomer fractions depend on the degree of acetylation of the starting chitosans. We have used experimentally determined distributions of hydrolysis products to develop a model for chitosan hydrolysis by ChiB. Important elements of the model include interaction parameters for acetylated/deacetylated units in each of the six subsites in the active cleft and degree of processivity (multiple attack). The hydrolysis reaction is described as a chemical reaction with an activation barrier that depends on the substrate sequence presented to the enzyme subsites. Using a Monte Carlo approach, the interaction parameters were refined by minimizing the difference between observed and predicted amounts of hydrolysis products obtained upon degradation of chitosan with a degree of acetylation of 65%. The final model can accurately predict complex patterns of oligosaccharides produced in the hydrolysis of chitosans with various degrees of acetylation, as well as patterns observed during reactions with chito-oligosaccharides. The behavior of a ChiB mutant with a mutation in subsite +2 (Gly188Asp), which reduces the affinity for an acetylated sugar, could be predicted correctly by introducing one single change in the model parameters (the interaction energy for an acetylated unit in the +2 subsite). The proposed model may be used to explore degradation products for different enzyme-substrates combinations and to optimize conditions for preparation of specific oligosaccharides. In addition, the model provides insight into subsite interaction parameters and the degree of processivity, which complements previous experimental studies on the mode of action of ChiB.     

Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for the enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55 degrees C and the enzyme to substrate ratio of 1:400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol: distilled water) yielded fractions with molecular weights in the range 3.2-26.4 kDa.     

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Depolymerization of High-Molecular-Weight Chitosan by the Enzyme Preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55°C and an enzyme to substrate ratio of 1 : 400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol:distilled water) yielded fractions with molecular weights in the range 3.2–26.4 kDa.     

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation

Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M _r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls.     

Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis

Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.     

Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.