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1.
Katarzyna Struszczyk Mirosława Szczęsna-Antczak Marta Walczak Emilia Pomianowska Tadeusz Antczak 《Carbohydrate polymers》2009,78(1):16-24
This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca2+ and Mg2+ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan. 相似文献
2.
Simone Laska Arnulf Kletzin 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,737(1-2)
A hydrogenase–sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC). All chromatographic steps were performed in the presence of 0.5% ε-aminocaproic acid resulting in the elution of the SR complex as a sharp peak. In contrast, chromatography using buffers without ε-aminocaproic acid, or in the presence of detergents, were not successful. The purified A. ambivalens SR complex consisted of at least four subunits with relative molecular masses of 110 000, 66 000, 39 000 and 29 000, respectively. A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively. 相似文献
3.
The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase. 相似文献
4.
《Biocatalysis and Biotransformation》2013,31(2-3):60-70
AbstractThe goal of the present work was to compare the partitioning behavior of chitinase and laminarinase (from Trichoderma spp.) in soya lecithin liposomes at different temperatures and examine the activity of the resulting microencapsulated enzymes against Fusarium oxysporum. In both cases the partition coefficients (Ko/w) were greater than 1, indicating affinity of the enzymes for microencapsulation in liposomes. Enthalpy calculations indicated that the process was endothermic in the case of laminarinase and exothermic in the case of chitinase. Soya lecithin liposomes were stable for more than 20 days. The stability of the immobilized enzyme was increased in the case of chitinase, but was not changed in the case of laminarinase. Although the antifungal effects of individual immobilized preparations decreased after microencapsulation compared with non-immobilized enzymes, they were increased by the synergistic effect of both encapsulated enzymes. The application of free or immobilized enzymes was also shown to enhance the inhibitory effect of the chemical fungicide, thiabendazole, on F. oxysporum. 相似文献
5.
Richard T. Mayer T. G. McCollum R. P. Niedz C. J. Hearn Roy E. McDonald E. Berdis H. Doostdar 《Planta》1996,200(3):289-295
Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 and isoelectric points 10.3–10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-d-glucosamine oligosaccharide (GlcNAc)3. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8–100% acetylated chitosans and (GlcNAc)4–6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)2–6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.Mention of a trademark, warranty, propriety, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations Ab
antibody
- BCLVC
basic chitinase/lysozyme cv. Valencia callus
- BCVC
basic chitinase cv. Valencia callus
- CE
capillary electrophoresis
- CM-chitin-RBV
carboxymethyl-chitin-remazol brilliant violet
- GlcNAc
N-acetyl-d-glucosamine
- HEWL
hen egg-white lysozyme
- Mr
relativemolecular mass
- pI
isoelectric point
- PLC
potato leaf chitinase
- PR
pathogenesis-related
- SEC
size exclusion chromatography
We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner. 相似文献
6.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
7.
A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito. 相似文献
8.
Sheng-Hsiang Li Yee-Hsiung Chen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,726(1-2)
This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF1 and LF2, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF3, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF2 antibody that was prepared to give no crossreaction with serum albumin. Both LF1 and LF2 showed a Mr 74 000 band while LF3 gave a Mr 70 000 band on reducing SDS–PAGE. All of them were reduced to a Mr 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF2/LF3 in LF1. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF2 antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle. 相似文献
9.
Hitoshi Saito Hiromi Yamada Yoshiomi Kato 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,119(4):625-630
A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects. 相似文献
10.
S. P. Gautam A. K. Gupta R. Shrivastava M. Awasthi 《World journal of microbiology & biotechnology》1996,12(1):99-100
Two thermostable enzymes produced by the thermophilic fungus Paecilomyces varioti, a chitinase and laminarinase, were used to isolate protoplasts of a thermophilic fungus, Malbranchea sulfurea. The frequency of protoplast regeneration observed (35%) was considerably higher than that obtained using commercial lytic enzymes. 相似文献
11.
Rawdkuen S Benjakul S Visessanguan W Lanier TC 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2006,144(4):544-552
A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner. 相似文献
12.
Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin
Su Jing Yang Simon J. T. Mao 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,731(2):379
Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a Mr (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine Mr 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine Mr 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin. 相似文献
13.
Klomklao S Kishimura H Yabe M Benjakul S 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2007,147(4):682-689
Two pepsins (A and B) were purified from the stomach of pectoral rattail (Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 °C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0–6.0 but were unstable at the temperatures greater than 40 °C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0–30%). The enzymes had high affinity and activity toward hemoglobin with Km and Kcat values of 98–152 μM and 32–50 S− 1, respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins. 相似文献
14.
Uchida D Hirose H Chang PK Aranishi F Hirayabu E Mano N Mitsuya T Prayitno SB Natori M 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,127(4):525-532
Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by
-mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml−1; it amounted to 10.3% of the total serum protein. 相似文献
15.
G. E. Aktuganov A. V. Shirokov A. I. Melent'ev 《Applied Biochemistry and Microbiology》2003,39(5):469-474
The specific nature of the chitosanase activity of the strain Bacillus sp. 739 was determined. Maximum enzyme activity was observed in a medium containing biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity by chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. The temperature and pH optima of the purified chitosanase were in the ranges 45–55°C and 6.0–6.5, respectively. Time to half-maximum inactivation of the enzyme at 50°C was equal to 1 h. With colloidal chitosan as the substrate, the value of K
of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin. 相似文献
16.
The components and structure of the cell wall of Rhizopus delemar were investigated using purified lytic enzymes, protease and chitosanase from Bacillus R-4 and chitinase II from Streptomyces orientalis. When these enzymes were used individually they only partially lysed the cell wall, but when allowed to react on the cell wall together, a complete lysis was achieved by cooperative action. These modes of action on the cell wall and the chemical and morphological data suggested that the cell wall structure was different in Rhizopus delemar of Zygomycetes from filamentous fungi of Euascomycetes and that its wall structure might be composed mainly of chitin fibers cemented by chitosan and protein or peptides scattered in a mosaic manner. 相似文献
17.
Chitosanase and chitinase activities in tomato roots during interactions with arbuscular mycorrhizal fungi or Phytophthora parasitica 总被引:6,自引:0,他引:6
Pozo M; Azcon-Aguila C; Dumas-Gaudot E; Barea J 《Journal of experimental botany》1998,49(327):1729-1739
New chitosanase acidic isoforms have been shown in Glomus
mosseae-colonized tomato roots and their induction, together
with the previously described mycorrhiza-related chitinase isoform, has
been further corroborated in plants colonized with another
Glomus species (G.
intraradices),as well as in tomato roots colonized in
vitro by Giaspora rosea. The induction of
these chitosanase isoforms appears as a specific response to the arbuscular
mycorrhizal (AM) symbiosis, and does not correspond to unspecific defence
mechanisms, since these isoforms were not induced by the pathogen
Phytophthora parasitica. Analysis by
isoelectrofocusing showed two closely migrating chitinase isoforms,
specific to mycorrhizal plants colonized either with G.
mosseae or G. intraradices, and their
isoelectric points were estimated to be 4.5 and 4.7. The estimated
molecular mass of chitosanases was 20 kDa, and after isoelectrofocusing,
the chitosanase activities were detected along the acidic pH range
(6.5-3.5). Constitutive and induced isoforms were also investigated during
a time-course study. In some experiments, chitin and chitosan were embedded
together as substrates in polyacrylamide gels with the aim of studying the
capacity of some isoforms to display both chitinase and chitosanase
activities. In extracts from plants colonized with either G.
mosseae or G. intraradices, some
constitutive chitinases and the previously described mycorrhiza-related
chitinase isoform, appeared to display chitosanase activity, while this
bifunctional character was not found for the chitinases from
non-mycorrhizal tissue, nor in Phytophthora-infected
plants. These results suggest some diversity in the chitinase activities
concerning substrate specificity in mycorrhizal plants. The possible
implications of these observations in the functioning of the symbiosis is
discussed.Key words: Arbuscular mycorrhizas, chitinases, chitosanases,
Phytophthora parasitica, tomato,
Lycoperiscon esculentum.
相似文献
18.
A chitosanase and a protease were purified from the culture supernatant of Serratia sp. TKU016 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of the chitosanase and protease determined
by SDS–PAGE were approximately 65 and 53 kDa, respectively. The chitosanase was inhibited completely by Mn2+, but the protease was enhanced by all of tested divalent metals. The optimum pH, optimum temperature, pH stability, and thermal
stability of the chitosanase and protease were (pH 7, 50°C, pH 6–7, <50°C) and (pH 8–10, 40°C, pH 5–10, <50°C), respectively.
SDS (2 mM) had stimulatory effect on TKU016 protease activity. The result demonstrates that TKU016 protease is SDS-resistant
protease and probably has a rigid structure. Besides, TKU016 culture supernatant (2% SPP) incubated for 2 days has the highest
antioxidant activity, the DPPH scavenging ability was about 76%. With this method, we have shown that shrimp shell wastes
can be utilized and it’s effective in the production of enzymes, antioxidants, peptide and reducing sugar, facilitating its
potential use in biological applications and functional foods. 相似文献
19.
Pinus taeda 《Plant Physiology and Biochemistry》2000,38(12)
Two proteins having quinate dehydrogenase (QDH, quinate:NAD(P)+-oxidoreductase, EC 1.1.1.24) and shikimate dehydrogenase (SDH, shikimate:NADP+-oxidoreductase, EC 1.1.1.25) activities were purified about 3 000-fold from young loblolly pine (Pinus taeda L.) needles. A combination of ammonium sulfate solubilization, and chromatographies on DEAE-cellulose, 2′, 5′ ADP-Sepharose and Mono-Q was used. Throughout all purification steps, the QDH activity consistently co-purified with the activity of the first of three forms of SDH, and the ratio of QDH/SDH was constant (variation from 1.63 to 1.89). These data indicate that both QDH and SDH activities are catalyzed by a single broad-specificity quinate (shikimate) dehydrogenase. Gel chromatography on Superdex 75 was used to estimate the native molecular mass of two forms of the enzyme as 35 and 53 kDa. 相似文献
20.
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