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1.
Characteristics of a Streptomyces coelicolor A3(2) Extracellular Protein Targeting Chitin and Chitosan
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Akihiro Saito Kiyotaka Miyashita Goran Biukovi Hildgund Schrempf 《Applied microbiology》2001,67(3):1268-1273
Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets α-chitin, β-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described. 相似文献
2.
Saito A Miyashita K Biukovic G Schrempf H 《Applied and environmental microbiology》2001,67(3):1268-1273
Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described. 相似文献
3.
Schrempf H 《Antonie van Leeuwenhoek》2001,79(3-4):285-289
Chitin is the second most abundant renewable polysaccharide, as it is a component of the exoskeleton of many organisms and of the cell walls of numerous fungi. Most streptomycetes secrete a number of chitinases, hydrolyzing chitin to oligomers, chitobiose or N-acetylglucosamine which can be utilized as carbon or nitrogen source. The chitinases of several streptomycetes have been shown to have a modular arrangement comprising catalytic, substrate binding as well as linker domains. Moreover, during growth in the presence of chitin-containing substrates, many Streptomycesstrains have been shown to secrete formerly unknown, small (about 200 aa) chitin binding proteins (CHBs) which lack enzymatic activity and specifically target and invade chitin. Several motifs, including the relative location and spacing of four tryptophan residues, are conserved in the investigated CHB types, CHB1 and CHB2. The affinity of CHB1 to crab shell chitin is two times higher than that of CHB2. Comparative studies of various generated mutant CHB1 proteins led to the conclusion that it is one of the exposed tryptophan residues that directly contributes to the interaction with chitin. On the basis of immunological, biochemical and physiological studies, it can be concluded that the CHBs act like a glue with which streptomycetes target chitin-containing samples or organisms. The ecological implications of these findings are discussed. 相似文献
4.
Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system may be exploited to improve the insecticidal system of Bt-crops. A
nucleotide fragment of 1368 bp from B. thuringiensis serovar konkukian S4, containing the complete coding sequence of the chitin binding protein Cbp50, was cloned and sequenced. Analyses have shown the protein to contain a modular structure consisting of an N-terminal CBM33 domain, two copies of a
fibronectin-like domain and a C-terminal chitin binding domain classified as CBM5. The Cbp50 protein was heterologously expressed
in Escherichia coli, purified and assessed for chitin binding activity. A deletion mutant (CBD-N; containing only the N-terminal CBM33 domain)
of Cbp50 was produced to determine the role of C-terminal domains in the binding activity of the protein. The full-length
Cbp50 was shown to bind β-chitin most efficiently followed by α-chitin, colloidal chitin and cellulose. The polysaccharide
binding activity of CBD-N was drastically decreased. The data demonstrate that both the N-terminal and C-terminal domains
of Cbp50 are essential for the efficient binding of chitin. The purified Cbp50 showed antifungal activity against the phytopathogenic
fungus Fusarium oxysporum and the opportunistic human pathogen Aspergillus niger. This is the first report of a modular chitin binding protein in bacteria. 相似文献
5.
R. Jayakumar V.V. Divya Rani K.T. Shalumon P.T. Sudheesh Kumar S.V. Nair T. Furuike H. Tamura 《International journal of biological macromolecules》2009,45(3):260-264
Chitin is a novel biopolymer and has excellent biological properties such as biodegradation in the human body and biocompatible, bioabsorable, antibacterial and wound healing activities. In this work, α- and β-chitin membranes were prepared using α- and β-chitin hydrogel. The bioactivity studies were carried out using these chitin membranes with the simulated body fluid solution (SBF) for 7, 14 and 21 days. After 7, 14 and 21 days the membranes were characterized using SEM, EDS and FT-IR. The SEM, EDS and FT-IR studies confirmed the formation of calcium phosphate layer on the surface of the both chitin membranes. These results indicate that the prepared chitin membranes were bioactive. Cell adhesion studies were also carried out using MG-63 osteoblast-like cells. The cells were adhered and spread over the membrane after 24 h of incubation. These results indicated that the chitin membranes could be used for tissue-engineering applications. 相似文献
6.
Fan Y Fukuzumi H Saito T Isogai A 《International journal of biological macromolecules》2012,50(1):69-76
Water dispersions of TEMPO-oxidized α-chitin nanowhisker (TOChN), partially deacetylated α-chitin nanowhisker/nanofiber mixture (DEChN), HCl-hydrolyzed chitin nanowhisker (HHChN) and squid-pen β-chitin nanofiber (SQChN) were prepared, and the properties of nano-dispersions and their cast films were characterized between the four chitin nano-samples. Because SQChN has the highest aspect ratio, its 0.1% dispersion had the highest shear stress and viscosity at the same shear rate in the four chitin nano-samples, and showed gel-like behavior in the whole shear rate range from 10−3 to 103 s−1. AFM images of the self-standing films showed that film surfaces consisted of characteristic chitin nano-elements with different morphologies and degrees of orientation between the four chitin samples, whereas all chitin nanowhisker/nanofiber films had similar thermal degradation points at ∼200 °C. The DEChN film had the highest tensile strength of ∼140 MPa, elongation at break of ∼10% and light-transmittance of 87% at 400 nm. In contrast, the SQChN film had the lowest tensile strength, Young's modulus and light-transmittance. All chitin nanowhisker/nanofiber films had similar oxygen permeabilities of ∼1 mL μm m−2 day−1 kPa−1, which was clearly lower than that (184 mL μm m−2 day−1 kPa−1) of a poly(lactic acid) film. 相似文献
7.
Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101 总被引:2,自引:0,他引:2
Shimosaka M Fukumori Y Zhang XY He NJ Kodaira R Okazaki M 《Applied microbiology and biotechnology》2000,54(3):354-360
A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101. The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of
chitosans having a low degree of acetylation (0–30%). The enzyme hydrolyzed glucosamine oligomers larger than a pentamer,
but did not exhibit any activity toward N-acetyl-glucosamine oligomers and colloidal chitin. The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined. B. gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues. Analysis of the N-terminal amino acid sequence of the purified
chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain. The deduced
amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases.
Received: 30 July 1999 / Revised revision: 17 February 2000 / Accepted: 25 February 2000 相似文献
8.
Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa 总被引:1,自引:0,他引:1
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One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D). 相似文献
9.
Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi 总被引:1,自引:0,他引:1
Jonathan E. Urch Ramon Hurtado‐Guerrero Damien Brosson Zhanliang Liu Vincent G. H. Eijsink Catherine Texier Daan M. F. van Aalten 《Protein science : a publication of the Protein Society》2009,18(6):1197-1209
The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de‐N‐acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline β‐chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite. 相似文献
10.
Fu-Pang Lin Hsu-Han Chuang Yi-Hsuan Liu Chia-Yu Hsieh Pei-Wen Lin Hsu-Yang Lin 《Archives of microbiology》2009,191(3):265-273
C-Terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region
of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606)
included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and
hydrolysis abilities. The overall catalytic efficiency, k
cat/K
M, of AcD1ChiAK606 with the 4MU-(GlcNAc)2 and the 4MU-(GlcNAc)3 chitin substrates was 15–26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative
substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble α-chitin substrate. Both fluorescence
and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal
of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown
function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided
evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent
on the putative C-terminal chitin-binding domain and the function-unknown A region. 相似文献
11.
Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 μM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues. 相似文献
12.
Yang HJ Zhou F Malik FA Bhaskar R Li XH Hu JB Sun CG Miao YG 《Archives of insect biochemistry and physiology》2010,75(4):221-230
The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc. 相似文献
13.
The shape and overall dimensions of the recently discovered Streptomyces alpha-chitin-binding protein, CHB1, were investigated by synchrotron radiation X-ray solution scattering. The radius of gyration and the maximum size of CHB1 were determined to be 1.75 +/- 0.03 nm and 6.0 +/- 0.2 nm, respectively. Using two independent ab initio approaches the low-resolution shape of the protein was found to consist of two domains, an elongated main globule with a length of about 4 nm and a foot-like domain of about 2 nm width. The structural and functional properties of CHB1 depend strongly on the presence of disulfide bonds; upon their reduction, the protein loses its affinity to chitin. 相似文献
14.
Hitoshi Sashiwa Shizu Fujishima Naoko Yamano Norioki Kawasaki Atsuyoshi Nakayama Einosuke Muraki Mongkol Sukwattanasinitt Rath Pichyangkura andSei-ichi Aiba 《Carbohydrate polymers》2003,51(4):391-395
N-Acetyl-
-glucosamine (GlcNAc) was produced from chitin by use of crude enzyme preparations. The efficient production of GlcNAc by cellulases derived from Trichoderma viride (T) and Acremonium cellulolyticus (A) was observed by HPLC analysis compared to lipase, hemicellulase, and pectinase. β-Chitin showed higher degradability than α-chitin when using cellulase T. The optimum pH of cellulase T was 4.0 on the hydrolysis of β-chitin. The yield of GlcNAc was enhanced by mixing of cellulase T and A. 相似文献
15.
Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta
E. Brunner H. Ehrlich P. Schupp R. Hedrich S. Hunoldt M. Kammer S. Machill S. Paasch V.V. Bazhenov D.V. Kurek T. Arnold S. Brockmann M. Ruhnow R. Born 《Journal of structural biology》2009,168(3):539-547
The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state 13C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40–100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state 13C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The 13C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of α-chitin than β-chitin. 相似文献
16.
Daraghmeh N Rashid I Al Omari MM Leharne SA Chowdhry BZ Badwan A 《AAPS PharmSciTech》2010,11(4):1558-1571
A co-processed excipient was prepared from commercially available crystalline mannitol and α-chitin using direct compression
as well as spray, wet, and dry granulation. The effect of the ratio of the two components, percentage of lubricant and particle
size, on the properties of the prepared co-processed excipient has been investigated. α-Chitin forms non-hygroscopic, highly
compactable, disintegrable compacts when co-processed with crystalline mannitol. The compaction properties of the co-processed
mannitol–chitin mixture were found to be dependent upon the quantity of mannitol added to chitin, in addition to the granulation
procedure used. Optimal physicochemical properties of the excipient, from a manufacturing perspective, were obtained using
a co-processed mannitol–chitin (2:8, w/w) mixture prepared by wet granulation (Cop-MC). Disintegration time, crushing strength, and friability of tablets, produced
from Cop-MC using magnesium stearate as a lubricant, were found to be independent of the particle size of the prepared granules.
The inherent binding and disintegration properties of the compressed Cop-MC are useful for the formulation of poorly compressible,
high-strength, and low-strength active pharmaceutical ingredients. The ability to co-process α-chitin with crystalline mannitol
allows chitin to be used as a valuable industrial pharmaceutical excipient. 相似文献
17.
Purification, Characterization, and Antifungal Activity of Chitinase from Streptomyces venezuelae P10 总被引:1,自引:0,他引:1
Streptomyces venezuelae P10 could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C.
The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography
and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of
the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against
phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed
products were N-acetylglucosamine and N, N’-diacetylchitobiose. 相似文献
18.
Ju Hee Kuk Woo Jin Jung Gyung Hyun Jo Kil Yong Kim Ro Dong Park 《World journal of microbiology & biotechnology》2006,22(2):135-139
Summary
N,N′-diacetylchitobiose was produced from chitin as a major hydrolytic product by controlling the ratio of β-N-acetylglucosaminidase to N,N′-diacetylchitobiohydrolase activities in the crude enzyme preparation of Aeromonas sp. GJ-18. When the enzyme preparation was preincubated at 50 °C, β-N-acetylglucosaminidase was nearly inactivated, while the N,N′-diacetylchitobiohydrolase was still active. Thus, the composition of chitin oligosaccharides depended on the preincubation temperature of the crude enzyme preparations. Typically, after 7 days of incubation with the substrate chitin, 78.9 and 56.6% of N,N′-diacetylchitobiose yields were obtained from swollen α-chitin and powdered β-chitin, respectively, with enzyme preparations that had been pretreated at 50 °C for 60 min. 相似文献
19.
Ming-Bo Qu Shao-Peng Sun Yuan-Sheng Liu Xiao-Rui Deng Jun Yang Qing Yang 《Insect Science》2021,28(3):692-704
The insect group II chitinase (ChtII, also known as Cht10) is a unique chitinase with multiple catalytic and chitin-binding domains. It has been proven genetically to be an essential chitinase for molting. However, ChtII's role in chitin degradation during insect development remains poorly understood. Obtaining this knowledge is the key to fully understanding the chitin degradation system in insects. Here, we investigated the role of OfChtII during the molting of Ostrinia furnacalis, a model lepidopteran pest insect. OfChtII was expressed earlier than OfChtI (OfCht5) and OfChi-h, at both the gene and protein levels during larva–pupa molting as evidenced by quantitative polymerase chain reaction and western blot analyses. A truncated OfChtII, OfChtII-B4C1, was recombinantly expressed in Pichia pastoris cells and purified to homogeneity. The recombinant OfChtII-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface as evidenced by scanning electron microscopy. It synergized with OfChtI and OfChi-h when hydrolyzing insoluble α-chitin. These findings suggested an important role for ChtII during insect molting and also provided a strategy for the coordinated degradation of cuticular chitin during insect molting by ChtII, ChtI and Chi-h. 相似文献
20.
The content of glucosamine in the walls of daughter (without bud scars) and mother (multiscar) cells ofSaccharomyces cerevisiae was examined in a control and after treatment with dilute alkali, acid and buffer. The occurrence of chitin in the bud and birth scars is discussed. The results of IR and X-ray analysis of cell-wall fractions indicate the presence of α-chitin which is a part of the chitin-glucan complex. The size of the crystallite of α-chitin in this complex is about 60 Å. 相似文献