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Masahiko Okada - One of the best experts on this subject based on the ideXlab platform.
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synthesis of a novel Chitin Derivative having oligo ɛ caprolactone side chains in aqueous reaction media
Macromolecular Chemistry and Physics, 2001Co-Authors: Sirinat Detchprohm, Keigo Aoi, Masahiko OkadaAbstract:A Chitin-based graft copolymer, Chitin-graft-oligo(e-caprolactone) (2), was synthesized via ring-opening graft polymerization of (e-caprolactone (e-CL) to ca. 50% partially deacetylated Chitin 1 catalyzed by tin (II) 2-ethylhexanoate in the presence of water as a swelling agent. The graft copolymer with ca. 40 wt.-% poly(e-CL) content was obtained by the reaction using the catalyst of 0.17 mol-% and water of 130 mol-%, respectively, to the e-CL monomer at 100°C for 20h. The chemical structure of 2 was characterized by IR, 1 H and 13 C NMR spectroscopies. The poly(e-CL) contents by IR were in accordance with those determined by 1 H NMR analysis. T 1 measurements of an aqueous solution of 2 suggested that the molecular motion of the hydrophobic poly(e-CL) side chains is restricted to some extent. On the other hand, it was demonstrated by 13 C CP/MAS NMR that the mobility of the Chitin skeleton of 2 in the solid-state is higher than that of the partially deacetylated Chitin. X-ray diffraction diagrams showed that 2 is amorphous, indicating that the crystallinity due to the Chitin main chain was reduced by introducing the oligo(e-CL) side chains.
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synthesis of a novel n selective ester functionalized Chitin Derivative and water soluble carboxyethylChitin
Macromolecular Chemistry and Physics, 2000Co-Authors: Keigo Aoi, Masahiko Okada, Hiroaki Sato, Hajime Ohtani, Shin Tsuge, Taizo Seki, Shinichi Mizutani, Yoshiki ShiogaiAbstract:A novel Chitin detivative having a pendant ester function, 2-N-(2-ethoxycarbonylmethyl)chain (2), was synthesized by a Michael-type nucleophilic addition of an amino group of partially deacetylated Chitin (1) to ethyl acrylate in phosphate buffer/methanol (5:3, v/v) at 40°C. N-Selective monosubstitution occurred exclusively in the polymer reaction, which was supported by a reaction of methyl 2-ammo-2-deoxy-D-glucopycanoside with ethyl acrylate to afford methyl 2-N-(2-ethexycarbonylethyl)-2-amino-2-deoxy-D-glucopyranoside. The degrees of substitution (DSs) of 2 were determined by 1 H NMR spectroscopy. T1 analysis of 2 was carried out in other to clarify differences of signal intensities of the pendant ester protons and the pyranose ring pronons. The result of the T 1 measurement suggested a relatively restricted molecular motion of the Chitin backbone in comparison with the flexible pendant ethyl ester groups. Furthermore, 2-N-(2-carboxyethyl)Chitin sodium salt (3) was synthesized from ethyl aorylate and 1 by the Michael addition followed by hydrolysis in 0,1 N NaOH aq, as 40°C, The DSs of 3 were varied from 0.26 to 0.88, which were almost controlled by the reaction period of the Michael reaction from 6 to 168 h. 3 showed good solubility in water. Viscosity measured on a cone-plate viscometer for the 1.0 wt.-% aqueous solution of 3 (DS, 0,26) was 0.074 Pas.sec.
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new Chitin based polymer hybrids 4 soil burial degradation behavior of poly vinyl alcohol Chitin Derivative miscible blends
Journal of Applied Polymer Science, 1999Co-Authors: Akinori Takasu, Keigo Aoi, Maki Tsuchiya, Masahiko OkadaAbstract:Soil burial degradation behavior of miscible blend systems of poly(vinyl alcohol) (PVA)/partially deacetylated Chitin (1), PVA/Chitin-graft-poly(2-methyl-2-oxazoline) (2), and PVA/Chitin-graft-poly(2-ethyl-2-oxazoline) (3) was investigated in comparison with the case of a pure PVA film. The degradation of the blend films was followed by the weight changes, scanning electron microscopic observation, Fourier transform infrared spectroscopy, 1H-NMR, and size exclusion chromatography analyses. The rate of weight decrease in these PVA/Chitin Derivative hybrids was higher than that of control PVA in the soil burial test. Fourier transform infrared spectra of the recovered samples of the blends showed an apparent increase of the absorption intensity due to β-diketone structure in PVA, which reflects the progress of biodegradation of PVA by PVA-oxidizing enzymes. Scanning electron microscopic observation revealed that these blend films were degraded by bacteria and actinomycetes. The triad tacticity and number-average molecular weight of PVA in the hybrids after soil burial determined by 1H-NMR and size exclusion chromatography, respectively, were almost the same as those before soil burial. These results suggested that enzymatic degradation of the hybrid films occurred mainly on the surface and that degradation of the PVA-based samples in the soil was accelerated by blending the Chitin Derivatives. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1171–1179, 1999
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characterization of Chitin based polymer hybrids by temperature programmed analytical pyrolysis techniques 1 Chitin graft poly 2 methyl 2 oxazoline poly vinyl chloride blends
Macromolecules, 1997Co-Authors: Hiroaki Sato, Akinori Takasu, Keigo Aoi, Hajime Ohtani, Shin Tsuge, Masahiko OkadaAbstract:Chitin-graft-poly(2-methyl-2-oxazoline)/poly(vinyl alcohol) (PVA) blends were characterized by means of analytical pyrolysis techniques such as temperature-programmed pyrolysis−mass spectrometry (TPPy−MS), TPPy−gas chromatography (TPPy−GC) and Py−GC mainly in terms of their miscibility and the site of the intermolecular interaction. The thermal degradation of the Chitin Derivative/PVA blends took place apparently in two stages reflecting the degradation of both constituent polymers. However, peak-top temperatures for both degradation stages changed as a function of blend composition; the first degradation stage, mostly corresponding to the dehydration of PVA, shifted to higher temperatures when the Chitin Derivative content is increased in the blend. Although the yields of 2-butenal from the PVA moiety increased by blending the Chitin Derivative, those of acetamide and N-ethylacetamide from the Chitin Derivative moiety exhibit an opposite trend. These temperature shifts and the variations in the yields of...
Vincent G H Eijsink - One of the best experts on this subject based on the ideXlab platform.
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aromatic residues in the catalytic center of Chitinase a from serratia marcescens affect processivity enzyme activity and biomass converting efficiency
Journal of Biological Chemistry, 2009Co-Authors: Henrik Zakariassen, Svein J Horn, Morten Sorlie, Kjell M Varum, Vincent G H EijsinkAbstract:The processive Serratia marcescens Chitinases A (ChiA) and B (ChiB) are thought to degrade Chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp167, Trp275, and Phe396 in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward β-Chitin and the soluble Chitin-Derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward Chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.
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costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Svein J Horn, Pawel Sikorski, Jannicke B Cederkvist, Gustav Vaajekolstad, Morten Sorlie, Bjornar Synstad, Gert Vriend, Kjell M Varum, Vincent G H EijsinkAbstract:Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and Chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, Chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites +1 and +2) led to reduced processivity and a reduced ability to degrade crystalline Chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble Chitin-Derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.
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endo exo mechanism and processivity of family 18 Chitinases produced by serratia marcescens
FEBS Journal, 2006Co-Authors: Svein J Horn, Pawel Sikorski, Morten Sorlie, Bjornar Synstad, Kjell M Varum, Audun Sorbotten, Vincent G H EijsinkAbstract:We present a comparative study of ChiA, ChiB, and ChiC, the three family 18 Chitinases produced by Serratia marcescens. All three enzymes eventually converted Chitin to N-acetylglucosamine dimers (GlcNAc2) and a minor fraction of monomers. ChiC differed from ChiA and ChiB in that it initially produced longer oligosaccharides from Chitin and had lower activity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could convert GlcNAc6 directly to three dimers, whereas ChiC produced equal amounts of tetramers and dimers, suggesting that the former two enzymes can act processively. Further insight was obtained by studying degradation of the soluble, partly deacetylated Chitin-Derivative chitosan. Because there exist nonproductive binding modes for this substrate, it was possible to discriminate between independent binding events and processive binding events. In reactions with ChiA and ChiB the polymer disappeared very slowly, while the initially produced oligomers almost exclusively had even-numbered chain lengths in the 2-12 range. This demonstrates a processive mode of action in which the substrate chain moves by two sugar units at a time, regardless of whether complexes formed along the way are productive. In contrast, reactions with ChiC showed rapid disappearance of the polymer and production of a continuum of odd- and even-numbered oligomers. These results are discussed in the light of recent literature data on directionality and synergistic effects of ChiA, ChiB and ChiC, leading to the conclusion that ChiA and ChiB are processive Chitinases that degrade Chitin chains in opposite directions, while ChiC is a nonprocessive endoChitinase.
Keigo Aoi - One of the best experts on this subject based on the ideXlab platform.
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synthesis of a novel Chitin Derivative having oligo ɛ caprolactone side chains in aqueous reaction media
Macromolecular Chemistry and Physics, 2001Co-Authors: Sirinat Detchprohm, Keigo Aoi, Masahiko OkadaAbstract:A Chitin-based graft copolymer, Chitin-graft-oligo(e-caprolactone) (2), was synthesized via ring-opening graft polymerization of (e-caprolactone (e-CL) to ca. 50% partially deacetylated Chitin 1 catalyzed by tin (II) 2-ethylhexanoate in the presence of water as a swelling agent. The graft copolymer with ca. 40 wt.-% poly(e-CL) content was obtained by the reaction using the catalyst of 0.17 mol-% and water of 130 mol-%, respectively, to the e-CL monomer at 100°C for 20h. The chemical structure of 2 was characterized by IR, 1 H and 13 C NMR spectroscopies. The poly(e-CL) contents by IR were in accordance with those determined by 1 H NMR analysis. T 1 measurements of an aqueous solution of 2 suggested that the molecular motion of the hydrophobic poly(e-CL) side chains is restricted to some extent. On the other hand, it was demonstrated by 13 C CP/MAS NMR that the mobility of the Chitin skeleton of 2 in the solid-state is higher than that of the partially deacetylated Chitin. X-ray diffraction diagrams showed that 2 is amorphous, indicating that the crystallinity due to the Chitin main chain was reduced by introducing the oligo(e-CL) side chains.
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synthesis of a novel n selective ester functionalized Chitin Derivative and water soluble carboxyethylChitin
Macromolecular Chemistry and Physics, 2000Co-Authors: Keigo Aoi, Masahiko Okada, Hiroaki Sato, Hajime Ohtani, Shin Tsuge, Taizo Seki, Shinichi Mizutani, Yoshiki ShiogaiAbstract:A novel Chitin detivative having a pendant ester function, 2-N-(2-ethoxycarbonylmethyl)chain (2), was synthesized by a Michael-type nucleophilic addition of an amino group of partially deacetylated Chitin (1) to ethyl acrylate in phosphate buffer/methanol (5:3, v/v) at 40°C. N-Selective monosubstitution occurred exclusively in the polymer reaction, which was supported by a reaction of methyl 2-ammo-2-deoxy-D-glucopycanoside with ethyl acrylate to afford methyl 2-N-(2-ethexycarbonylethyl)-2-amino-2-deoxy-D-glucopyranoside. The degrees of substitution (DSs) of 2 were determined by 1 H NMR spectroscopy. T1 analysis of 2 was carried out in other to clarify differences of signal intensities of the pendant ester protons and the pyranose ring pronons. The result of the T 1 measurement suggested a relatively restricted molecular motion of the Chitin backbone in comparison with the flexible pendant ethyl ester groups. Furthermore, 2-N-(2-carboxyethyl)Chitin sodium salt (3) was synthesized from ethyl aorylate and 1 by the Michael addition followed by hydrolysis in 0,1 N NaOH aq, as 40°C, The DSs of 3 were varied from 0.26 to 0.88, which were almost controlled by the reaction period of the Michael reaction from 6 to 168 h. 3 showed good solubility in water. Viscosity measured on a cone-plate viscometer for the 1.0 wt.-% aqueous solution of 3 (DS, 0,26) was 0.074 Pas.sec.
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new Chitin based polymer hybrids 4 soil burial degradation behavior of poly vinyl alcohol Chitin Derivative miscible blends
Journal of Applied Polymer Science, 1999Co-Authors: Akinori Takasu, Keigo Aoi, Maki Tsuchiya, Masahiko OkadaAbstract:Soil burial degradation behavior of miscible blend systems of poly(vinyl alcohol) (PVA)/partially deacetylated Chitin (1), PVA/Chitin-graft-poly(2-methyl-2-oxazoline) (2), and PVA/Chitin-graft-poly(2-ethyl-2-oxazoline) (3) was investigated in comparison with the case of a pure PVA film. The degradation of the blend films was followed by the weight changes, scanning electron microscopic observation, Fourier transform infrared spectroscopy, 1H-NMR, and size exclusion chromatography analyses. The rate of weight decrease in these PVA/Chitin Derivative hybrids was higher than that of control PVA in the soil burial test. Fourier transform infrared spectra of the recovered samples of the blends showed an apparent increase of the absorption intensity due to β-diketone structure in PVA, which reflects the progress of biodegradation of PVA by PVA-oxidizing enzymes. Scanning electron microscopic observation revealed that these blend films were degraded by bacteria and actinomycetes. The triad tacticity and number-average molecular weight of PVA in the hybrids after soil burial determined by 1H-NMR and size exclusion chromatography, respectively, were almost the same as those before soil burial. These results suggested that enzymatic degradation of the hybrid films occurred mainly on the surface and that degradation of the PVA-based samples in the soil was accelerated by blending the Chitin Derivatives. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1171–1179, 1999
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characterization of Chitin based polymer hybrids by temperature programmed analytical pyrolysis techniques 1 Chitin graft poly 2 methyl 2 oxazoline poly vinyl chloride blends
Macromolecules, 1997Co-Authors: Hiroaki Sato, Akinori Takasu, Keigo Aoi, Hajime Ohtani, Shin Tsuge, Masahiko OkadaAbstract:Chitin-graft-poly(2-methyl-2-oxazoline)/poly(vinyl alcohol) (PVA) blends were characterized by means of analytical pyrolysis techniques such as temperature-programmed pyrolysis−mass spectrometry (TPPy−MS), TPPy−gas chromatography (TPPy−GC) and Py−GC mainly in terms of their miscibility and the site of the intermolecular interaction. The thermal degradation of the Chitin Derivative/PVA blends took place apparently in two stages reflecting the degradation of both constituent polymers. However, peak-top temperatures for both degradation stages changed as a function of blend composition; the first degradation stage, mostly corresponding to the dehydration of PVA, shifted to higher temperatures when the Chitin Derivative content is increased in the blend. Although the yields of 2-butenal from the PVA moiety increased by blending the Chitin Derivative, those of acetamide and N-ethylacetamide from the Chitin Derivative moiety exhibit an opposite trend. These temperature shifts and the variations in the yields of...
Shiro Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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enzymatic polymerization to an alternating n phthaloyl Chitin Derivative catalyzed by Chitinase
Chemistry Letters, 2011Co-Authors: Masashi Ohmae, Kazuhiro Kurosaki, Akira Makino, Shiro KobayashiAbstract:A Chitin Derivative with an alternating N-phthaloyl group was prepared via Chitinase-catalyzed polymerization. A chitobiose oxazoline Derivative with a N-phthaloyl group at the C2′ position was des...
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Chitinase catalyzed synthesis of an alternatingly n sulfonated Chitin Derivative
Biomacromolecules, 2007Co-Authors: Akira Makino, Masashi Ohmae, Hideaki Nagashima, Shiro KobayashiAbstract:An alternatingly N-sulfonated Chitin Derivative (2) was synthesized via ring-opening polyaddition of an N-sulfonated chitobiose oxazoline Derivative (1) catalyzed by Chitinases from Bacillus sp. and Serratia marcescens. The polymerization proceeded homogeneously, providing 2 as a water-soluble polysaccharide in good yields with total control of regioselectivity and stereochemistry. Mn of 2 reached 1900 and 4180 by use of Chitinases from Bacillus sp. and Serratia marcescens, which correspond to 8−10 (n = 4−5) and 18−20 (n = 9−10) saccharide units, respectively. These results indicate that Mn of 2 is controllable by selecting Chitinases from different origins. It is considered that the C-2 position of the nonreducing unit in the oxazoline-type monomer is not deeply involved in the catalysis of Chitinase.
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Chitinase catalyzed copolymerization to a Chitin Derivative having glucosamine unit in controlled proportion
Polymer Journal, 2006Co-Authors: Akira Makino, Masashi Ohmae, Shiro KobayashiAbstract:Chitinase-catalyzed copolymerization of an N,N′-diacetylchitobiose oxazoline monomer (1) with an N-acetylchitobiose oxazoline monomer (2) has been investigated. Catalysis of Chitinase derived from Bacillus sp. showed close polymerizability of these two comonomers, giving rise to the corresponding copolymer. The degree of N-deacetylation (DDAc) of the product was controlled by varying the comonomer feed ratio. In contrast, catalysis of Chitinase from Serratia marcescens showed different reactivity of the comonomers. Thus, homopolymerization of monomer 2 was preferentially occurred at the beginning of the reaction and copolymer was hardly obtained.
Svein J Horn - One of the best experts on this subject based on the ideXlab platform.
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aromatic residues in the catalytic center of Chitinase a from serratia marcescens affect processivity enzyme activity and biomass converting efficiency
Journal of Biological Chemistry, 2009Co-Authors: Henrik Zakariassen, Svein J Horn, Morten Sorlie, Kjell M Varum, Vincent G H EijsinkAbstract:The processive Serratia marcescens Chitinases A (ChiA) and B (ChiB) are thought to degrade Chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp167, Trp275, and Phe396 in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward β-Chitin and the soluble Chitin-Derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward Chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.
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costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Svein J Horn, Pawel Sikorski, Jannicke B Cederkvist, Gustav Vaajekolstad, Morten Sorlie, Bjornar Synstad, Gert Vriend, Kjell M Varum, Vincent G H EijsinkAbstract:Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and Chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, Chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites +1 and +2) led to reduced processivity and a reduced ability to degrade crystalline Chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble Chitin-Derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.
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endo exo mechanism and processivity of family 18 Chitinases produced by serratia marcescens
FEBS Journal, 2006Co-Authors: Svein J Horn, Pawel Sikorski, Morten Sorlie, Bjornar Synstad, Kjell M Varum, Audun Sorbotten, Vincent G H EijsinkAbstract:We present a comparative study of ChiA, ChiB, and ChiC, the three family 18 Chitinases produced by Serratia marcescens. All three enzymes eventually converted Chitin to N-acetylglucosamine dimers (GlcNAc2) and a minor fraction of monomers. ChiC differed from ChiA and ChiB in that it initially produced longer oligosaccharides from Chitin and had lower activity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could convert GlcNAc6 directly to three dimers, whereas ChiC produced equal amounts of tetramers and dimers, suggesting that the former two enzymes can act processively. Further insight was obtained by studying degradation of the soluble, partly deacetylated Chitin-Derivative chitosan. Because there exist nonproductive binding modes for this substrate, it was possible to discriminate between independent binding events and processive binding events. In reactions with ChiA and ChiB the polymer disappeared very slowly, while the initially produced oligomers almost exclusively had even-numbered chain lengths in the 2-12 range. This demonstrates a processive mode of action in which the substrate chain moves by two sugar units at a time, regardless of whether complexes formed along the way are productive. In contrast, reactions with ChiC showed rapid disappearance of the polymer and production of a continuum of odd- and even-numbered oligomers. These results are discussed in the light of recent literature data on directionality and synergistic effects of ChiA, ChiB and ChiC, leading to the conclusion that ChiA and ChiB are processive Chitinases that degrade Chitin chains in opposite directions, while ChiC is a nonprocessive endoChitinase.