RESUMEN
Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell-1 d-1 ). After two purification steps, the active enzyme was recovered (2.4 × 106 U mg-1 ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.
Asunto(s)
Reactores Biológicos , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Lentivirus/genética , Proteínas Recombinantes , alfa-Galactosidasa , Animales , Células CHO , Cricetinae , Cricetulus , Enfermedad de Fabry , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , alfa-Galactosidasa/genética , alfa-Galactosidasa/aislamiento & purificación , alfa-Galactosidasa/metabolismoRESUMEN
An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60-80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-D-galactopyranoside, α-D-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-D-galactoside and 12 mM versus α-D-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg(2+) and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of -SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Polyporales/enzimología , alfa-Galactosidasa/química , alfa-Galactosidasa/aislamiento & purificación , Secuencia de Aminoácidos , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Polyporales/química , Polyporales/genética , Polyporales/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismoRESUMEN
α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4-7. Maximal activities were determined at 60, 55, and 50 °C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50 °C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg²âº, Agâº, and Cu²âº. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes.
Asunto(s)
Aspergillus/enzimología , Manipulación de Alimentos/métodos , Glycine max/química , Leche de Soja/metabolismo , alfa-Galactosidasa , Aspergillus/química , Fibras de la Dieta/metabolismo , Dispepsia/prevención & control , Estabilidad de Enzimas , Galactanos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Cinética , Mananos/metabolismo , Melibiosa/metabolismo , Metales Pesados/farmacología , Oligosacáridos/metabolismo , Gomas de Plantas/metabolismo , Rafinosa/metabolismo , Leche de Soja/química , Especificidad por Sustrato , Temperatura , alfa-Galactosidasa/química , alfa-Galactosidasa/aislamiento & purificación , alfa-Galactosidasa/metabolismoRESUMEN
Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.
Asunto(s)
Fabaceae/enzimología , Semillas/enzimología , alfa-Galactosidasa/aislamiento & purificación , alfa-Galactosidasa/metabolismo , Germinación , Concentración de Iones de Hidrógeno , Peso Molecular , Oligosacáridos/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by alpha-galactosidases that cleave alpha-1,6-linkages of alpha-galactoside residues. The objectives of this study were the purification and characterization of extracellular alpha-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration and anion exchange chromatographies presented an Mr value of 60 kDa and the N-terminal amino acid sequence YENGLNLVPQMGWN. The Km values for hydrolysis of pNP alphaGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The alpha-galactosidase presented absolute specificity for galactose in the alpha-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and polymers. The enzyme was noncompetitively inhibited by galactose (Ki = 2.7 mM) and melibiose (Ki = 1.2 mM). Enzyme treatments of soy milk for 4 h at 60 degrees C reduced the amounts of stachyose and raffinose by 100%.
Asunto(s)
Ascomicetos/enzimología , Oligosacáridos/metabolismo , Rafinosa/metabolismo , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Flatulencia , Hidrólisis , Oligosacáridos/análisis , Rafinosa/análisis , Alimentos de Soja , Leche de Soja/química , alfa-Galactosidasa/química , alfa-Galactosidasa/aislamiento & purificaciónRESUMEN
Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.
Asunto(s)
Glycine max/enzimología , Oligosacáridos/química , alfa-Galactosidasa/metabolismo , Unión Competitiva , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Flatulencia/prevención & control , Humanos , Hidrólisis , Cinética , Rafinosa/química , Semillas/enzimología , Especificidad por Sustrato , Termodinámica , alfa-Galactosidasa/aislamiento & purificaciónRESUMEN
The characterization and properties of a beta-galactanase and alpha- and beta-galactosidases as well as heparan sulfate and chondroitin sulfate degrading enzymes which appear during the 15 days of the embryonic development of the mollusc Pomacea sp. is reported. The beta-galactanase, which appears around day 7 of development, was separated from alpha- and beta-galactosidase which emerge at day 1 and 4 after oviposition, respectively. The galactanase seems to be responsible for the degradation of an acidic beta-galactan (which is also synthesized by the eggs around day 5) to galactose and di- and tri-galactosides. Heparan sulfate appears around day 10 of development together with a heparan sulfate endoglucuronidase responsible for the degradation of its N-acetylated region. An alpha-N-acetylglucosaminidase and a beta-glucuronidase which act upon the N-acetylated fragments formed from heparan sulfate emerge around day 4 of development. Chondroitin sulfate and a chondroitin sulfate sulfatase emerge around day 9 of development whereas a beta-N-acetylgalactosaminidase and the beta beta-galactan, heparan and chondroitin sulfate, respectively. The possible role of these elements in the migration of mesenchymal cells, in the processes of cell-cell recognition and control of cell growth is discussed.