RESUMEN
This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T1 (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S1 (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man10GlcNAc2 and Man11GlcNAc2 were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.
Asunto(s)
Aspergillus oryzae/enzimología , Biotecnología , Fermentación , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Ácido Aspártico Endopeptidasas/metabolismo , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Glucano 1,4-alfa-Glucosidasa/aislamiento & purificación , Glucano 1,4-alfa-Glucosidasa/metabolismo , Humanos , Japón , Ribonucleasa T1/aislamiento & purificación , Ribonucleasa T1/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/aislamiento & purificación , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Tripsinógeno/metabolismoRESUMEN
Aspergillus oryzae protyrosinase (pro-TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro-TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro-TY and acid-activated tyrosinase (acid-TY) were 266 and 165 kDa, respectively, as estimated by gel-filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro-TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8-anilino-1-naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid-TY. The dimerization of acid-TY involving the intermolecular disulfide bond is essential for the activity.
Asunto(s)
Disulfuros/química , Proteínas Fúngicas/química , Precursores de Proteínas/química , Naftalenosulfonatos de Anilina/química , Cromatografía en Gel , Dicroismo Circular , Cisteína/química , Dimerización , Disulfuros/metabolismo , Activación Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Peso Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Dodecil Sulfato de Sodio/química , Espectrometría de FluorescenciaRESUMEN
Aspergillus saitoi 1,2-alpha-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T(m) values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 degrees C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.
Asunto(s)
Aspergillus/enzimología , Cisteína , Calor , Manosidasas/química , Disulfuros , Estabilidad de Enzimas , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Manosidasas/genética , Manosidasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Desnaturalización ProteicaRESUMEN
The pepstatin-insensitive carboxyl proteinase grifolisin was purified from fruiting bodies of the fungus Grifola frondosa, a maitake mushroom. The enzyme had an optimum pH of 3.0 for the digestion of hemoglobin and 2.8 for milk casein digestion. Its molecular mass was determined to be 43kDa by SDS-PAGE and 40kDa by gel chromatography on Superose 12, and its isoelectric point was found to be 4.6 by isoelectric focusing. The enzyme hydrolyzed four major bonds in the oxidized insulin B-chain: Phe1-Val2, Ala14-Leu15, Gly20-Glu21 and Phe24-Phe25 at pH 3.0. The first 15 amino acid residues in the N-terminal region were AVPSSCASTITPACL, and the coding region of the grifolisin gene (gfrF) has a 1960-base pair cDNA. The predicted mature grifolisin protein consisted of 365 residues and was 26% identical to that of sedolisin from Pseudomonas sp. 101 and 34% identical to that of aorsin from Aspergillus oryzae. Grifolisin is a member of the sedolisin S53 family and is not inhibited by pepstatin.
Asunto(s)
Carboxipeptidasas/química , Carboxipeptidasas/metabolismo , Grifola/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Carboxipeptidasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.
Asunto(s)
Calor , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Mutagénesis Sitio-Dirigida , Zinc/metabolismo , Secuencia de Aminoácidos , Aspergillus/enzimología , Aspergillus/genética , Secuencia de Bases , Catálisis , Dicroismo Circular , Disulfuros , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Punto Isoeléctrico , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Penicillium/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrofotometría Atómica , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
An intracellular aspartic proteinase obtained from the hepatopancreas (liver) of Japanese common squid (Todarodes pacificus) was purified to homogeneity. The molecular mass of the enzyme was 36,500 Da on SDS-PAGE, and the isoelectric point was 8.29 by isoelectric focusing. The enzyme activity was optimal at pH 3.5, pH 2.2 and pH 3.0 for the substrates acid-denatured hemoglobin, acid-denatured casein, and MOCAc-GKPILFFRLK(Dnp)-D-R-NH2, respectively. Enzyme activity decreased rapidly at 50 degrees C. The Km and kcat values of the enzyme were estimated to be 3.2 mM and 46 s(-1) with MOCAc-GKPILFFRLK(Dnp)-D-R-NH2, and 1.7 mM and 1.1 s(-1) with MOCAc-SEVNLDAEFRK(Dnp)RR-NH2. The enzyme activity was strongly inhibited by pepstatin A, but only partially inhibited by DAN and EPNP. The Ki values for pepstatin A, DAN and EPNP were 0.5 nM, 0.5 mM and 0.2 mM, respectively. A cDNA encoding the enzyme was cloned by RT-PCR and subjected to nucleotide sequencing. The entire open reading frame was 1179 bp and coded for a protein of 392 amino acid residues. The mature enzyme consisted of 334 amino acids. The deduced amino acid sequence of the enzyme showed a high degree of identity to the sequences of cathepsins D found in various species.
Asunto(s)
Catepsina D/genética , Decapodiformes/enzimología , Hepatopáncreas/enzimología , Animales , Secuencia de Bases , Catepsina D/aislamiento & purificación , ADN Complementario , Inhibidores Enzimáticos , Cinética , Datos de Secuencia Molecular , Péptidos/metabolismo , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A. oryzae was already cloned and the protein structures of its catalytic and copper binding domains were investigated. However, our recent results revealed that the melO gene was highly expressed in submerged culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state culture. Another tyrosinase gene was screened using the expressed sequence tag (EST) library. One redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular cloning showed that the gene carried six exons interrupted by five introns and had an open reading frame encoding 616 amino acid residues. This gene was designated as melB. The deduced amino acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinases but the sequences of the copper binding domains were highly conserved. When the melB gene was expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase. These results indicated that the melB gene encodes a novel tyrosinase associated with melanization in solid-state culture.
RESUMEN
The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic acyl-peptide-4-methylcoumaryl-7-amides (peptide-MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be g(xx) = 5.20, g(yy) = 4.75, and g(zz) = 2.24; the metal center was a divalent cobalt ion in a high spin state.
Asunto(s)
Aspergillus oryzae/enzimología , Cobalto/química , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Zinc/metabolismo , Amidas/química , Amidas/metabolismo , Secuencia de Aminoácidos , Aminoácidos Básicos/metabolismo , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Cobalto/metabolismo , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Estabilidad de Enzimas , Histonas/metabolismo , Calor , Cinética , Datos de Secuencia Molecular , Especificidad por Sustrato , Timo/químicaRESUMEN
The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.
Asunto(s)
Aspergillus/enzimología , Calcio/metabolismo , Manosidasas/química , Manosidasas/genética , Western Blotting , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Temperatura , Factores de TiempoRESUMEN
A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.
Asunto(s)
Aspergillus oryzae/enzimología , Serina Endopeptidasas/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.