14200-67-0Relevant academic research and scientific papers
Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase
Slámová, Kristyna,Krejzová, Jana,Marhol, Petr,Kalachova, Lubica,Kulik, Natallia,Pelantová, Helena,Cva?ka, Josef,K?en, Vladimír
, p. 1941 - 1950 (2015/06/02)
Abstract The synthesis of oligosaccharides using mutant glycosidases has been dynamically developing due to the need for novel carbohydrate-based materials. Chitooligomers (β-1→4-linked oligomers of N-acetylglucosamine) are bioactive compounds applicable in many industrial and pharmacological areas; however, their accessibility is still rather low. In this work, GH20 β-N-acetylhexosaminidase from the fungus Talaromyces flavus was engineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-times suppressed hydrolytic activity. Thus, we have prepared the first GH20 transglycosidases. In the reactions catalyzed by these mutant β-N-acetylhexosaminidases we were able to easily prepare and isolate both natural and modified chitooligomers in sufficient amounts for their complete spectral characterization and possible further application. The presented method for the synthesis of chitooligomers with aglycones suitable for linking to other biological structures is simple and robust enough to be easily scaled up.
Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group
Yabushita, Mizuho,Kobayashi, Hirokazu,Kuroki, Kyoichi,Ito, Shogo,Fukuoka, Atsushi
, p. 3760 - 3763 (2015/12/08)
Chitin, a polymer of N-acetylglucosamine units with β-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53 % yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70 %. Our process can greatly reduce the use of acid compared to the conventional process.
An ammonium sulfate sensitive chitinase from Streptomyces sp. CS501
Rahman, Md. Arifur,Choi, Yun Hee,Pradeep,Yoo, Jin Cheol
, p. 1522 - 1529 (2015/03/30)
A chitinase from Streptomyces sp. CS501 was isolated from the Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase (Ch501) was purified to 4.60 fold with yield of 28.74 % using Sepharose Cl-6B column. The molecular mass of Ch501 was approximately 43 kDa as estimated by SDS-PAGE and zymography. The enzyme (Ch501) was found to be stable over a broad pH range (5.0-10.0) and temperature (up to 50 °C), and have an optimum temperature of 60 °C. N-terminal sequence of Ch501 was AAYDDAAAAA. Intriguingly, Ch501 was highly sensitive to ammonium sulfate but it's completely suppressed activity was recovered after desalting out. TLC analysis of Ch501 showed the production of N-acetyl d-glucosamine (GlcNAc) and Diacetylchitobiose (GlcNAc)2, as a principal hydrolyzed product. Ch501 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis, which can be used for the biological control of fungus. As has been simple in purification, stable in a broad range of pH, ability to produce oligosaccharides, and antifungal activity showed that Ch501 has potential applications in industries as for chitooligosaccharides production used as prebiotics and/or for the biological control of plant pathogens in agriculture.
Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC
Umemoto, Naoyuki,Ohnuma, Takayuki,Mizuhara, Mamiko,Sato, Hirokazu,Skriver, Karen,Fukamizo, Tamo
, p. 81 - 90 (2013/01/16)
A tryptophan side chain was introduced into subsite +1 of family GH-18 (class V) chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC, respectively) by the mutation of a glycine residue to tryptophan (G74W-NtChiV and G75W-AtChiC). The specific activity toward glycol chitin of the two mutant enzymes was 70-71% of that of the wild type. Using chitin oligosaccharides, (GlcNAc)n (n = 4, 5 and 6), as the substrates, we found the transglycosylation reaction to be significantly enhanced in G74W-NtChiV and G75W-AtChiC when compared with the corresponding wild-type enzymes. The introduced tryptophan side chain might protect the oxazolinium ion intermediate from attack by a nucleophilic water molecule. The enhancement of transglycosylation activity was much more distinct in G75W-AtChiC than in G74W-NtChiV. Nuclear magnetic resonance titration experiments using the inactive double mutants, E115Q/G74W-NtChiV and E116Q/G75W-AtChiC revealed that the association constant of (GlcNAc)5 was considerably larger for the latter. Amino acid substitutions at the acceptor binding site might have resulted in the larger association constant for G75W-AtChiC, giving rise to the higher transglycosylation activity of G75W-AtChiC. The Author 2012. Published by Oxford University Press. All rights reserved.
Development of innovative technologies to decrease the environmental burdens associated with using chitin as a biomass resource: Mechanochemical grinding and enzymatic degradation
Nakagawa, Yuko S.,Oyama, Yasuhiro,Kon, Nobuko,Nikaido, Mitsuru,Tanno, Koichi,Kogawa, Jun,Inomata, Shoji,Masui, Ayano,Yamamura, Akihiro,Kawaguchi, Mitsuaki,Matahira, Yoshiharu,Totani, Kazuhide
experimental part, p. 1843 - 1849 (2011/09/14)
The production of N-acetylglucosamine (GlcNAc, chitin monosaccharide) from crab or shrimp shells generally requires numerous steps and the use of deleterious substances. One goal for researchers is the direct production of GlcNAc and NN′-diacetylchitobiose [(GlcNAc)2, chitin structural dimeric unit] from both chitin and crab shells. The present study reports the development of an intensive ball "converge" mill for the rapid mechanochemical conversion of chitin or crab shells into amorphous, chitinase-sensitive microparticles. Optimal crab shell grinding parameters were determined, and close to 100% direct degradation of chitin from crab shell to GlcNAc was achieved. This is the first report of using a mechanochemical process with enzymatic degradation to decrease the environmental burdens associated with GlcNAc production from chitin.
Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method
Synstad, Bjornar,Vaaje-Kolstad, Gustav,Cederkvist, F. Henning,Saua, Silje F.,Horn, Svein J.,Eijsink, Vincent G. H.,Sorlie, Morten
, p. 715 - 723 (2008/09/19)
In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher kcat and 15 fold-lower Km than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-β-D-N- N′-N″-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of β-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)2 (main product), GlcNAc, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.
Kinetics of hydrolysis of chitin/chitosan oligomers in concentrated hydrochloric acid
Einbu, Aslak,Grasdalen, Hans,Varum, Kjell M.
, p. 1055 - 1062 (2008/02/03)
The kinetics of hydrolysis in concentrated hydrochloric acid (12.07 M) of the fully N-acetylated chitin tetramer (GlcNAc4) and the fully N-deacetylated chitosan tetramer (GlcN4) were followed by determining the amounts of the lower DP oligomers as a function of time. A theoretical model was developed to simulate the kinetics of hydrolysis of the three different glycosidic linkages in the tetramers. The model uses two different rate constants for the hydrolysis of the glycosidic bonds in the oligomers, assuming that the glycosidic bond next to one of the end residues are hydrolysed faster than the two other glycosidic linkages. The two rate constants were estimated by fitting model data to experimental results. The results show that the hydrolysis of the tetramers is a nonrandom process as the glycosidic bonds next to one of the end residues are hydrolysed 2.5 and 2.0 times faster as compared to the other glycosidic linkages in the fully N-acetylated and fully N-deacetylated tetramer, respectively. From previous results on other oligomers and the reaction mechanism, it is likely that the glycosidic bond that is hydrolysed fastest is the one next to the nonreducing end. The absolute values for the rate constants for the hydrolysis of the glycosidic linkages in GlcNAc4 were found to be 50 times higher as compared to the glycosidic linkages in GlcN4, due to the catalytic role of the N-acetyl group and the presence of the positively charged amino-group on the N-deacetylated sugar residue.
Improving glycopeptide synthesis: A convenient protocol for the preparation of β-glycosylamines and the synthesis of glycopeptides
Hackenberger, Christian P. R.,O'Reilly, Mary K.,Imperiali, Barbara
, p. 3574 - 3578 (2007/10/03)
(Chemical Equation Presented) Herein we apply a recently introduced protocol using ammonium carbamate in methanol to the amination of crude chitobiose leading to 1,β-aminochitobiose. This simple, one-step procedure allows a facile preparation of unstable glycosylamines in contrast to the commonly implemented ammonium bicarbonate based amination of water-soluble carbohydrates. The new amination protocol leads to an improved synthesis of the key chitobiosyl-asparagine building block for the SPPS of glycopeptides. The utility of the method is demonstrated with the synthesis of a 39-amino acid glycoprotein.
An efficient synthesis of argifin: A natural product chitinase inhibitor with chemotherapeutic potential
Dixon, Mark J.,Andersen, Ole A.,Van Aalten, Daan M. F.,Eggleston, Ian M.
, p. 4717 - 4721 (2007/10/03)
The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product.
Enzymatic synthesis of 3-O-methylated chitin oligomers from new derivatives of a chitobiose oxazoline
Sakamoto, Junji,Kobayashi, Shiro
, p. 698 - 699 (2007/10/03)
Regiospecifically 3-O- and/or 3′-O-methylated derivatives of a chitobiose oxazoline have been synthesized as new substrate monomers and subjected to a chitinase catalysis, leading to the first synthesis of 3-O-methylated chitin oligomers via enzymatic oligomerization. (Graph Presented).
