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1.
Int J Biol Macromol ; 149: 754-766, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31987953

RESUMO

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.


Assuntos
Bivalves/enzimologia , Água Doce , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Acetilgalactosamina , Resinas Acrílicas , Animais , Estabilidade Enzimática , Hexosaminidase A/química , Hexosaminidase A/metabolismo , Hexosaminidase B/química , Hexosaminidase B/metabolismo , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Peso Molecular , Temperatura
2.
World J Microbiol Biotechnol ; 35(2): 18, 2019 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-30656484

RESUMO

Chitosan-degrading fungal strain, Penicillium sp. IB-37-2A, produced mainly extracellular chitosanolytic enzymes under submerged agitating cultivation in presence of soluble chitosan or colloidal chitin as main carbon source. Significant N-acetyl-ß-D-glucosaminidase activity (8-18 × 103 U·ml-1) was also detected in culture filtrate of the fungal strain. Alone major exo-chitosanase from culture filtrate of Penicillium sp. IB-37-2A was purified in 46-fold using ultrafiltration, affinity sorption on colloidal chitosan and hydrophobic chromatography on Phenyl-Sepharose CL 4B and characterized. Molecular weight of the exo-ß-1.4-glucosaminidase is 41 kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55 °C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55 °C. Activity of the enzyme is strongly inhibited by 1 mM Hg2+ and Ag+, in less degree-10 mM Cu2+, Zn2+, Ni+ and Fe2+, slightly activated-with 1 mM Mg2+, 10 mM Ca2+, tween-80 (10 mM) and Triton X-100 (1 mM). Viscosimetric assay confirmed reported earlier exo-splitting manner of the enzyme activity. Soluble chitosan (deacetylation degree (DD) 80-85%) is most rapidly hydrolyzed by the enzyme (Vmax = 7.635 µM × min-1 × mg-1, KM ~ 0.83 mg/ml). Purified exo-chitosanase also degraded laminarin, ß-glucan, colloidal chitin and showed significant chitobiohydrolase activity (V ~ 50 µM × ml-1 × min-1 for pNP-GlcNAc2) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo-ß-1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.


Assuntos
Quitosana/química , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Penicillium/crescimento & desenvolvimento , Cromatografia DEAE-Celulose , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Penicillium/enzimologia , Temperatura , Ultrafiltração
3.
Microb Cell Fact ; 16(1): 16, 2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-28137256

RESUMO

BACKGROUND: Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase-named Chi18H8 and belonging to family 18 glycosyl hydrolases-was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 µg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. RESULTS: We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. CONCLUSIONS: Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.


Assuntos
Antifúngicos/farmacologia , Quitinases/metabolismo , Quitinases/farmacologia , Escherichia coli/genética , Hexosaminidases/metabolismo , Hexosaminidases/farmacologia , Microbiologia do Solo , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Reatores Biológicos , Quitina/metabolismo , Quitinases/genética , Quitinases/isolamento & purificação , Clonagem Molecular , Escherichia coli/metabolismo , Fusarium/efeitos dos fármacos , Biblioteca Gênica , Hexosaminidases/genética , Hexosaminidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão/enzimologia , Ácido Láctico/metabolismo , Metagenoma , Metagenômica/métodos , Filogenia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Rhizoctonia/efeitos dos fármacos
4.
Appl Biochem Biotechnol ; 181(2): 650-666, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27639392

RESUMO

A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett-Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box-Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with ß-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments.


Assuntos
Acetilglucosamina/biossíntese , Bacillus licheniformis/classificação , Bacillus licheniformis/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Solanum tuberosum/microbiologia , Acetilglucosamina/isolamento & purificação , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Especificidade da Espécie
5.
Carbohydr Polym ; 93(2): 615-21, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23499103

RESUMO

An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-ß-D-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95kDa; the optimum pH and temperature for activity are 6.0 and 45°C, respectively. The purified exo-ß-D-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-D-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern confirmed using real-time mass spectrometry shows that exo-ß-D-glucosaminidase cleaves the glycosidic bonds between GlcN-GlcN and GlcN-GlcNAc but not between GlcNAc-GlcN or GlcNAc-GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-ß-D-glucosaminides were obtained, indicating that exo-ß-D-glucosaminidase is a retaining enzyme.


Assuntos
Proteínas Fúngicas/isolamento & purificação , Hexosaminidases/isolamento & purificação , Paecilomyces/enzimologia , Acetilação , Quitina/metabolismo , Quitosana/metabolismo , Meios de Cultura/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Indução Enzimática , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Glicosilação , Hexosaminidases/biossíntese , Hidrólise , Micélio/enzimologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Estereoisomerismo , Especificidade por Substrato
6.
Appl Microbiol Biotechnol ; 96(1): 143-51, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22234531

RESUMO

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.


Assuntos
Enterococcus faecalis/enzimologia , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Programas de Rastreamento/métodos , Polissacarídeos Bacterianos/metabolismo , Vibrio/metabolismo , Hidrólise , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos Bacterianos/química
7.
J Basic Microbiol ; 52(3): 324-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21953631

RESUMO

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-ß-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 µM ml(-1) and 0.119 µM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.


Assuntos
Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Trichoderma/enzimologia , Carbono/metabolismo , Cátions Bivalentes/metabolismo , Fracionamento Químico , Cromatografia em Gel , Cromatografia por Troca Iônica , Análise por Conglomerados , Meios de Cultura/química , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Ativadores de Enzimas/metabolismo , Estabilidade Enzimática , Hexosaminidases/química , Concentração de Íons de Hidrogênio , Cinética , Metais/metabolismo , Dados de Sequência Molecular , Peso Molecular , Filogenia , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura , Trichoderma/crescimento & desenvolvimento , Trichoderma/isolamento & purificação
8.
Acta Biochim Pol ; 58(2): 261-3, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21666887

RESUMO

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-ß-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.


Assuntos
Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Clostridium/enzimologia , Trato Gastrointestinal/microbiologia , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/metabolismo , Anaerobiose , Proteínas de Bactérias/isolamento & purificação , Quitinases/isolamento & purificação , Clostridium/crescimento & desenvolvimento , Técnicas de Cultura/métodos , Glicosídeo Hidrolases/isolamento & purificação , Hexosaminidases/isolamento & purificação , Humanos
9.
Bioprocess Biosyst Eng ; 34(6): 681-6, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21293880

RESUMO

The production of chitinases and hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and type of carbon source. Crude enzyme obtained from solid-substrate culture presented activities of exochitinases (32 and 51 kDa), endochitinases (26 kDa), ß-N-acetylhexosaminidases (61, 80, 96 and 111 kDa). Additionally, submerged cultures produced exochitinases (32 and 45 kDa), endochitinases (10 and 26 kDa) and ß-N-acetylhexosaminidases (61, 96 and 111 kDa). ß-N-acetylhexosaminidases activity determined in solid-substrate culture with added chitin was ca. threefold (7.58 ± 0.57 U mg(-1)) higher than submerged culture (2.73 + 0.57 U mg(-1)). Similarly, hydrophobins displayed higher activities in solid-substrate culture (627.3 ± 2 µg protein mL(-1)) than the submerged one (57.4 ± 4.7 µg protein mL(-1)). Molecular weight of hydrophobins produced in solid-substrate culture was 7.6 kDa and they displayed surface activity on Teflon.


Assuntos
Quitinases , Proteínas Fúngicas , Hypocreales/enzimologia , Quitina/metabolismo , Quitinases/química , Quitinases/isolamento & purificação , Quitinases/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Microscopia Eletrônica de Varredura , Politetrafluoretileno/química , Esporos Fúngicos/crescimento & desenvolvimento , Especificidade por Substrato , beta-N-Acetil-Hexosaminidases/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismo
10.
Glycobiology ; 21(4): 503-11, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21098515

RESUMO

A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-ß-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-ß-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-ß-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-ß-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.


Assuntos
Hexosaminidases/biossíntese , Photobacterium/enzimologia , Proteínas Recombinantes/biossíntese , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Dissacarídeos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura
11.
Artigo em Inglês | MEDLINE | ID: mdl-20208168

RESUMO

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of approximately 105 kDa (Gls93-F1) and approximately 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 A, and diffracted to 1.8 A resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 A, and diffracted to 2.4 A resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.


Assuntos
Hexosaminidases/química , Trichoderma/enzimologia , Cristalização , Cristalografia por Raios X , Expressão Gênica , Hexosaminidases/genética , Hexosaminidases/isolamento & purificação
12.
Carbohydr Res ; 344(8): 1046-9, 2009 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-19393602

RESUMO

An exo-beta-D-glucosaminidase gene was cloned from Aspergillus sp. CJ22-326 and expressed in Escherichia coli. The purified protein showed an exo-chitosanase activity in a viscosimetric assay and TLC analysis. This is the first report on cloning of a gene encoding an Aspergillus sp. exo-beta-D-glucosaminidase.


Assuntos
Aspergillus/enzimologia , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Escherichia coli/genética , Hexosaminidases/genética
13.
Clin Biochem ; 40(7): 467-77, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17321512

RESUMO

OBJECTIVES: Hexosaminidase activity is present in lysosomes, plasma membrane and cytosol of many human cells. Plasma membrane and cytosolic hexosaminidase is not well characterized, particularly as regards their isoenzyme forms and their relationship with the lysosomal ones. DESIGN AND METHODS: Erythrocyte hexosaminidase isoforms were chromatographically separated, characterized and compared to those in the plasma of healthy individuals and in the erythrocytes of a Tay-Sachs patient. RESULTS: Hexosaminidase isoenzymes were found in plasma membrane and cytosol and were composed of the same alpha- and beta-subunits as the lysosomal and plasma hexosaminidase A and B isoenzymes, though with some structural and kinetic differences. In addition, the cytosol contained a hexosaminidase that is a specific N-acetyl-beta-D-glucosaminidase, the one involved in the removal of N-acetylglucosamine residues O-linked to proteins, named O-GlcNAcase. CONCLUSIONS: This work provides an additional step in the characterization of hexosaminidases helping better understand their role in non-lysosomal compartments and their involvement in physiological or pathological situations.


Assuntos
Citosol/enzimologia , Membrana Eritrocítica/enzimologia , Eritrócitos/enzimologia , Hexosaminidases/metabolismo , Adulto , Cromatografia por Troca Iônica , Cromatografia Líquida , Feminino , Hexosaminidase A , Hexosaminidases/sangue , Hexosaminidases/isolamento & purificação , Humanos , Isoenzimas/sangue , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Doença de Tay-Sachs/enzimologia , beta-N-Acetil-Hexosaminidases/sangue , beta-N-Acetil-Hexosaminidases/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismo
14.
Biotechnol Lett ; 28(20): 1655-60, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16912928

RESUMO

An exo-beta-D-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-beta-D: -glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90 degrees C (over 20 min) and pH 6. It had a half-life of 9 h at 90 degrees C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg(2+), Mn(2+), Co(2+), Ca(2+), Sr(2+), Ni(2+) (at up to 10 mM).


Assuntos
Hexosaminidases/biossíntese , Pyrococcus horikoshii/enzimologia , Cátions Bivalentes , Clonagem Molecular , Dimerização , Dimetil Sulfóxido/química , Ditiotreitol/química , Estabilidade Enzimática , Escherichia coli/metabolismo , Guanidina/química , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Dodecilsulfato de Sódio/química , Temperatura , Ureia/química
15.
Protein Expr Purif ; 45(1): 125-31, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16289917

RESUMO

An extracellular 104 kDa exo-beta-d-glucosaminidase was purified and characterized from the culture supernatant of Aspergillus fumigatus S-26, which showed exceptionally strong chitosanolytic enzyme activity. The purified enzyme showed optimum pH of 3.0-6.0 and optimum temperature of 50-60 degrees C, and was stable between pH 2.0 and 10.0 and under 35 degrees C. The Km, Vmax, and kcat were determined to be 1.0 mg chitosan/ml, 7.8x10(-8) mol/s/mg protein, and 28.3 s-1, respectively. The exo-beta-D-glucosaminidase was severely inactivated by Cu2+ and Hg2+ at 10 mM. 2-Hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, and p-chloromercuribenzoic acid inhibited the enzyme. The enzyme did not degrade chitin, cellulose, and starch. The exo-beta-D-glucosaminidase did not reduce the viscosity of chitosan solutions at early stage of reaction, suggesting the exo-type of cleavage in polymeric chitosan chains. The exo-beta-D-glucosaminidase liberated only GlcN from chitosan, and GlcN plus the one-residue shortened oligomers from (GlcN)2-7. The exo-beta-D-glucosaminidase exhibited transglycosylation activity, resulting in the one-residue elongated oligomers.


Assuntos
Aspergillus fumigatus/enzimologia , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Quitosana/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Hexosaminidases/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Metais Pesados/farmacologia , Peso Molecular , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura , Fatores de Tempo , Viscosidade
16.
Appl Microbiol Biotechnol ; 65(6): 694-702, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15322771

RESUMO

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80 degrees C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.


Assuntos
Acetilglucosaminidase/isolamento & purificação , Quitina/metabolismo , Quitinases/isolamento & purificação , Regulação Enzimológica da Expressão Gênica , Hexosaminidases/isolamento & purificação , Thermococcus/enzimologia , Acetilglucosaminidase/química , Acetilglucosaminidase/metabolismo , Anaerobiose , Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Celulose/metabolismo , Quitinases/química , Quitinases/metabolismo , Meios de Cultura/química , Estabilidade Enzimática , Hexosaminidases/química , Hexosaminidases/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Temperatura , Thermococcus/crescimento & desenvolvimento , Thermococcus/metabolismo
17.
Protein Expr Purif ; 32(2): 309-16, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14965778

RESUMO

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.


Assuntos
Clostridium perfringens/enzimologia , Hexosaminidases/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sistema ABO de Grupos Sanguíneos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Hexosaminidases/genética , Hexosaminidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , alfa-N-Acetilgalactosaminidase
18.
Mikrobiol Z ; 65(6): 13-23, 2003.
Artigo em Ucraniano | MEDLINE | ID: mdl-15077544

RESUMO

A scheme has been developed for isolation and purification of the enzyme with alpha-N-acetylgalactosaminidase and alpha-galactosidase activities which included fractionation by ammonium sulphate and chromatography on TSK-gels Toyopearl HW-60 and Fractogel DEAE-650-s and Sepharose 6B. The enzyme was purified 600 times with the yield of 28%. The enzyme preparation did not contain fucosidase, invertase and proteolytic activities. Molecular mass of the enzyme from the data of gel-filtration on Sepharose 6B was 430 kDa, according to the data of electrophoresis in DS-PAAG--70 kDa. It is shown that acidic and hydrophobic aminoacids prevail in the enzyme molecule, the carbohydrate component containing galactose, mannose, glucosamine and two nonidentified hexosamines is also present there. The enzyme preparation is stable during 48 hours at 20 degrees C; its pH-optimum is at pH 3.5-4.1. Michaelis constants concerning n-nitrophenyl-alpha-N-acetylgalactopyranoside and n-nitrophenyl-alpha-D-galactopyranoside were 1.18 and 1.25 mM, respectively.


Assuntos
Aspergillus niger/enzimologia , Glicosídeo Hidrolases/isolamento & purificação , Sulfato de Amônio , Cromatografia em Gel , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/química , Hexosaminidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Peso Molecular , Polímeros , Sefarose , Temperatura , alfa-Galactosidase/química , alfa-Galactosidase/isolamento & purificação , alfa-N-Acetilgalactosaminidase
19.
Biochemistry (Mosc) ; 67(6): 689-95, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12126478

RESUMO

An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.


Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Proteínas de Bactérias/metabolismo , Eritrócitos/imunologia , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Hexosaminidases/metabolismo , Sequência de Carboidratos , Hexosaminidases/isolamento & purificação , Cinética , Dados de Sequência Molecular , alfa-N-Acetilgalactosaminidase
20.
J Appl Microbiol ; 91(3): 498-505, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11556916

RESUMO

AIMS: To purify and characterize the chitinolytic activity of Penicillium janthinellum P9 and to evaluate possible uses of the purified enzymes in the control of fungal growth and spore germination. METHODS AND RESULTS: The chitinolytic activity of P. janthinellum P9 was associated to two beta-N-acetyl-hexosaminidases (CHI1 and CHI2) that were purified by preparative isoelectric focusing and preparative electrophoresis and partially characterized. Treatment of test fungi with purified enzyme solutions caused reduced spore germination, reduction of hyphal length and mycelial damage. The combined action of the two enzymes and a systemic fungicide completely inactivated pests and food-spoiling moulds such as Fusarium solanii, P. canescens and Cladosporium cladosporioides. Treatment with the two enzymes increased germination of freeze-dried fungal spores. CONCLUSION: The chitinolytic activity of P. janthinellum P9 is associated with two extracellular beta-N-acetyl-hexosaminidases that can cause damage to the cell walls of other fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This appears to be the first report on the characterization of extracellular chitinolytic enzymes produced by a Penicillium strain. The results of this study might have some impact in the applied research field.


Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Quitina/metabolismo , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Penicillium/enzimologia , Antifúngicos/farmacologia , Eletroforese em Gel de Poliacrilamida , Microbiologia de Alimentos , Conservação de Alimentos , Liofilização , Hexosaminidases/farmacologia , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Focalização Isoelétrica , Penicillium/metabolismo , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/fisiologia , Especificidade por Substrato
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