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1.
Mar Drugs ; 18(11)2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33213084

RESUMO

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

2.
Carbohydr Polym ; 250: 117007, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33049875

RESUMO

Laminarans are currently the focus of attention in regard to the selection of prospective agents for the prevention and treatment of cancer. Laminaran from Saccharina cichorioides was aminated to heighten anticancer and radiosensitizing activities and elucidate its molecular mode of action. Aminated laminaran, ScLNH2, was identified as 1,3-ß-d-glucan with -CH2-CH(OH)-CH2-NH2 group at the C6 of branches. ScLNH2 selectively inhibited the viability and colony formation in the MDA-MB-231 cell line of triple negative breast cancer cells. ScLNH2 possessed synergism with radiation, resulting in a decreased number of colonies of MDA-MB-231 cells. The mechanism underling the radiosensitizing effect of ScLNH2 was associated with apoptosis induction via regulation of caspases 9 and 3 and PARP enzyme, preventing the repair of DNA damage in irradiated cells. These findings confirmed that combination therapy by aminated laminaran and radiation might play a role in the optimization of therapy for an aggressive form of human breast cancer.

3.
Carbohydr Polym ; 246: 116635, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32747270

RESUMO

Structure of the fucoidan from Sargassum horneri and products of its enzymatic transformation with molecular weight over 20 kDa were investigated. Fucoidan was hydrolyzed by recombinant fucoidanase FFA1 and its fraction of higher molecular weight was fractionated using anion-exchange chromatography, resulting in three sulphated polysaccharides of various molecular weight (63-138 kDa). Their structures were analyzed using NMR spectroscopy, showing the fucoidan (ShF) to be a branched polysaccharide with the backbone consisting of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment and side chains including the α-l-Fucp-1→2-α-l-Fucp-1→ or α-l-Fucp-1→3-α-l-Fucp(4SO3-)-1→ fragments attached to the main chain at C4. The fragment F3 differing by molecular weight and side chain from other fucoidans fragments possessed the most significant anticancer and radiosensitizing activities.

4.
Mar Drugs ; 18(4)2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32331442

RESUMO

The enzymatic depolymerization of fucoidans from brown algae allowed the production of their standardized derivatives with different biological activities. This work aimed to compare the antiviral activities of native (FeF) and modified with enzyme (FeHMP) fucoidans from F. evanescens. The cytotoxicity and antiviral activities of the FeF and FeHMP against herpes viruses (HSV-1, HSV-2), enterovirus (ECHO-1), and human immunodeficiency virus (HIV-1) in Vero and human MT-4 cell lines were examined by methylthiazolyltetrazolium bromide (MTT) and cytopathic effect (CPE) reduction assays, respectively. The efficacy of fucoidans in vivo was evaluated in the outbred mice model of vaginitis caused by HSV-2. We have shown that both FeF and FeHMP significantly inhibited virus-induced CPE in vitro and were more effective against HSV. FeF exhibited antiviral activity against HSV-2 with a selective index (SI) > 40, and FeHMP with SI ˃ 20, when they were added before virus infection or at the early stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that after intraperitoneal administration (10 mg/kg), both FeF and FeHMP protected mice from lethal intravaginal HSV-2 infection to approximately the same degree (44-56%). Thus, FeF and FeHMP have comparable potency against several DNA and RNA viruses, allowing us to consider the studied fucoidans as promising broad-spectrum antivirals.


Assuntos
Antivirais/farmacologia , Fucus/química , Polissacarídeos/farmacologia , Vírus/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Chlorocebus aethiops , Vírus de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Polissacarídeos/isolamento & purificação , Vírus de RNA/efeitos dos fármacos , Vaginite/tratamento farmacológico , Vaginite/virologia , Células Vero
5.
Mar Drugs ; 18(3)2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32168741

RESUMO

Thе study presents the results of a comparative evaluation of the effect of structural modifications of fucoidans from the brown alga Fucus evanescens (native, highly purified рroduct of fucoidan enzymatic hydrolysis, a new regular 1→3;1→4-α-L-fucan, sulphated mainly at C2 and acetylated at C4 of the fucose residue) on the effector functions of innate and adaptive immunity cells in vitro and in vivo. Using flow cytometry, we found that all examined fucoidans induce the maturation of dendritic cells, enhance the ability of neutrophils to migrate and adhere, activate monocytes and enhance their antigen-presenting functions, and increase the cytotoxic potential of natural killers. Fucoidans increase the production of hepatitis B virus (HBs) specific IgG and cytokine Th1 (IFN-γ, TNF-α) and Th2 (IL-4) profiles in vivo. The data obtained suggest that in vitro and in vivo adjuvant effects of the products of fucoidan enzymatic hydrolysis with regular structural characteristics are comparable to those of the native fucoidan. Based on these data, the products of fucoidan enzymatic hydrolysis can be considered as an effective and safe candidate adjuvant to improve the efficacy of prophylactic and therapeutic vaccines.

6.
Carbohydr Res ; 484: 107776, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421353

RESUMO

Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-ß-d-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553T, inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-ß-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-ß-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-ß-d-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Flavobacteriaceae/enzimologia , Poríferos/química , Esteroides/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavobacteriaceae/efeitos dos fármacos , Fucus/microbiologia , Hidrolases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Estrutura Molecular , Polissacarídeo-Liase/antagonistas & inibidores , Polissacarídeos/química , Esteroides/química , Sulfatos/química
7.
Mar Drugs ; 16(11)2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388774

RESUMO

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-ß(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.


Assuntos
Glicosídeo Hidrolases/química , Oligossacarídeos/química , Feófitas/química , Polissacarídeo-Liase/química , Polissacarídeos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ensaios Enzimáticos , Estabilidade Enzimática , Flavobacterium/química , Flavobacterium/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Polimerização , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Sulfatos/química
8.
Biomolecules ; 8(4)2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30248971

RESUMO

Fucoidans belong to a structurally heterogeneous class of sulfated polysaccharides isolated from brown algae. They have a wide spectrum of biological activities. The complex structures of these polysaccharides hinder structure-activity relationships determination. Fucoidan sulfatases can make useful tools for the determination of the fine chemical structure of fucoidans. In this study, identification and preparation of two recombinant sulfatases able to catalyze the cleavage of sulfate groups from fragments of fucoidan molecules is described for the first time. Two genes of sulfatases swf1 and swf4 of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T were cloned and the proteins were produced in Escherichia coli cells. Sulfatases SWF1 and SWF4 are assigned to S1_17 and S1_25 subfamilies of formylglycine-dependent enzymes of S1 family (SulfAtlas). Some molecular and biochemical characteristics of recombinant fucoidan sulfatases have been studied. Detailed specificity and catalytic features of sulfatases were determined using various sulfated fucooligosaccharides. Structures of products produced by SWF1 and SWF4 were established by nuclear magnetic resonance (NMR) spectroscopy. Based on the obtained data, the enzymes are classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo-3O-sulfatase (SWF4). In addition, we demonstrated the sequential action of sulfatases on 2,3-di-O-sulfated fucooligosacchrides, which indicates an exolitic degradation pathway of fucoidan by a marine bacterium W. fucanilytica CZ1127T.


Assuntos
Polissacarídeos/genética , Sulfatases/genética , Bactérias/química , Bactérias/genética , Escherichia coli/genética , Polissacarídeos/biossíntese , Polissacarídeos/química , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfatases/biossíntese , Sulfatases/química
9.
Carbohydr Polym ; 193: 189-195, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29773371

RESUMO

Enzymatic depolymerization of fucoidans attracts many researchers due to the opportunity of obtaining standardized fucoidan fragments. Fucoidanase catalyzes the cleavage of fucoidan from Fucus evanescens (FeF) to form low molecular weight products (LMP) and a polymeric fraction (HMP) with 50.8 kDa molecular weight and more than 50% yield. NMR spectroscopy shows that the HMP fraction has regular structure and consists of a repeating fragment [→3)-α-l-Fucp2,4OSO3--(1 → 4)-α-l-Fucp2,4OSO3--(1 → 4)-α-l-Fucp2OSO3--(1→]n. The anticancer effects of FeF fucoidan and its derivative (HMP) were studied in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The anticancer activity of the HMP fraction was found to be slightly lower than that of the FeF fucoidan. Research and practical applications of the enzyme include modification of native fucoidans for purposes of regular and easier characterized derivatives acquisition.


Assuntos
Antineoplásicos/farmacologia , Fucus/química , Fucus/enzimologia , Hidrolases/metabolismo , Polissacarídeos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Biocatálise , Configuração de Carboidratos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fucus/metabolismo , Humanos , Hidrolases/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
10.
Carbohydr Polym ; 175: 547-556, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917899

RESUMO

The laminaran SdL and fucoidan SdF were isolated from brown algae Sargassum duplicatum. SdL was 1,3;1,6-ß-d-glucan (1,3:1,6=6:1) with a main chain, represented by 1,3-linked glucose residues, due to NMR spectroscopy data. Single glucose residues could form branches at C6. Unusual structure of fucoidan SdF was studied by chemical and enzymatic methods, NMR spectroscopy of desulfated and deacetylated polysaccharide and mass spectrometry of fucoidan fragments labeled with 18O. Fucoidan was sulfated (31.7%) and acetylated galactofucan (Fuc:Gal∼1:1) with a main chain of 1,4-linked alternating α-l-fucose and ß-d-galactose residues. Side chains were represented by extensive (DP≥5) 1,3-linked 2,4-disulfated α-l-fucose residues with branching points at C2. Fucose residues in the main chain were sulfated at C2 and less at C3, while galactose residues were sulfated at C2, C3, and less at C4, C6. The fucoidan SdF was effective against colony formation of colon cancer cells in vitro.


Assuntos
Antineoplásicos/química , Glucanos/química , Polissacarídeos/química , Sargassum/química , Antineoplásicos/farmacologia , Glucanos/farmacologia , Células HT29 , Humanos , Isótopos de Oxigênio , Polissacarídeos/farmacologia
11.
Carbohydr Polym ; 175: 654-660, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917914

RESUMO

Structure and anticancer activity of fucoidan from Sargassum horneri and from products of its enzymatic transformation were investigated. A gene that encodes fucoidanase ffa1 in the marine bacteria F. algae was identified, cloned and the protein (FFA1) was produced in Escherichia coli. The mass of the gene product FFA1 is 111kDa. Sequence analysis has revealed that fucoidanase FFA1 belongs to the GH107 (CAZy) family. Recombinant fucoidanase FFA1 was used to produce fucooligosaccharides. Structure of 5 sulphated oligosaccharides with polymerization degree 4-10 was established by NMR-spectroscopy. The fucoidan extracted from S. horneri is almost pure fucan. The main chain of the fucoidan is established to consist mostly of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment, with insertions of →3-α-l-Fucp(2,4SO3-)-1→ fragment. Unsulphated side chains with the α-l-Fucp-1→2-α-l-Fucp-1→ structure connect to the main one at the C4 of monosaccharide residue.


Assuntos
Antineoplásicos/química , Polissacarídeos/química , Sargassum/química , Sulfatos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Glicosídeo Hidrolases/metabolismo , Humanos , Polissacarídeos/farmacologia
12.
Glycobiology ; 27(3): 254-263, 2017 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-28031251

RESUMO

A gene that encodes fucoidanase ffa2 in the marine bacterium Formosa algae strain KMM 3553T was cloned, and the protein (FFA2) was produced in Escherichia coli. Recombinant fucoidanase FFA2 was purified, and the biochemical properties of this enzyme were studied. The amino acid sequence of FFA2 showed 57% identity with known fucoidanase FcnA from Mariniflexile fucanivorans. The mass of the gene product FFA2 is 101.2 kDa (918 amino acid residues). Sequence analysis has revealed that fucoidanase FFA2 belongs to the GH107 (CAZy) family. Detailed substrate specificity was studied by using fucoidans from brown seaweeds as well as synthetic fucooligosaccharide with distinct structures. Fucoidanase FFA2 catalyzes the cleavage of (1→4)-α-glycosidic bonds in the fucoidan from Fucus evanescens within a structural fragment (→3)-α-l-Fucp2S-(1→4)-α-l-Fucp2S-(1→)n but not in a fragment (→3)-α-l-Fucp2S,4S-(1→4)-α-l-Fucp2S-(1→)n. Using synthetic di-, tetra- and octasaccharides built up of the alternative (1→4)- and (1→3)-linked α-l-Fucp2S units, the difference in substrate specificity and in the rate of enzymatic selectivity was investigated. Nonsulfated and persulfated synthetic oligosaccharides were not transformed by the enzyme. Therefore, FFA2 was specified as poly[(1→4)-α-l-fucoside-2-sulfate] glycanohydrolase. This enzyme could be used for the modification of natural fucoidans to obtain more regular and easier characterized derivatives useful for research and practical applications.


Assuntos
Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Polissacarídeos/metabolismo , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Oligossacarídeos/química , Oligossacarídeos/genética , Polissacarídeos/química , Conformação Proteica , Especificidade por Substrato
13.
Nat Prod Commun ; 11(9): 1253-1257, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30807014

RESUMO

As a result of the first study on secondary metabolites from the cosmopolitan bioluminescent marine tube polychaete Chaetopterus variopedatus, a new bicyclic guanidine alkaloid, 6-epi-monanchorin (1), along with the previously known monanchorin (2) were isolated. The structure of 1 was elucidated by spectroscopic and chemical methods, including a cleavage of the C1-07 bond to obtain a secondary alcohol (3), which was used to determine the absolute configurations by Mosher's method. It was found that 1 and 2 were mainly accumulated in a secreted mucus special organ of the worm (food net), where green and blue-green microalgae were detected. A biosynthetic pathway to 6-epi-monanchorin and monanchorin from dietary polyeic fatty acid precursors was proposed.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/análise , Guanidinas/análise , Poliquetos/química , Animais , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Metabolismo Secundário , Distribuição Tecidual
14.
Glycobiology ; 26(1): 3-12, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26347522

RESUMO

In recent years, the research of fucoidans has steadily increased. The interest in these substances is due to their various biological activities. Despite a wide range of biological activity and the lack of oral toxicity, fucoidans remain relatively unexploited as a source of medicines because of their heterogeneity. Enzymes that degrade polyanionic polysaccharides are widely used for establishing their structures and structure-activity relationships. Sometimes, to obtain preparations of polysaccharides with standard characteristics, for example, medicines and food supplements, enzymatic treatment can be also applied. Only a few sources of enzymes with fucoidanase activity have been described, and only a few studies regarding the isolation and characterization of fucoidanases have been performed. The data on the specificity of fucoidanases: the type of cleaved glycoside bond, the relation between catalytic activity and the degree of substrate sulfation are scarce. The review summarizes achievements in the research of fucoidanases, mechanisms of enzymatic degradation of fucoidans, as well as of structures of sulfated fucooligosaccharides obtained under the action of fucoidanases.


Assuntos
Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Animais , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Especificidade por Substrato
15.
Mar Drugs ; 11(7): 2413-30, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-23852092

RESUMO

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg2+, Ca2+ and Ba2+ cations strongly activated the enzyme; however, Cu2+ and Zn2+ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.


Assuntos
Organismos Aquáticos/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Fucus/enzimologia , Fucus/metabolismo , Polissacarídeos/metabolismo , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Cátions/metabolismo , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Hidrólise , Sulfatos/metabolismo
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