Aspergillus Saitoi Research Articles

Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with water-soluble carbodiimides was studied. 2. Gluc M1 was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide (CMC) at pH 4.5. 3. Inactivation of Gluc M1 with [14C]CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M1. 4. The CD spectrum of CMC-modified Gluc M1 (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained. 5. In the presence of maltose, when Gluc M1 was incubated with [14C]CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M1 modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M1 was inactivated completely with the incorporation of ca. 4 CMC moieties. 6. The logarithm of the half-life of the inactivation of Gluc M1 by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M1. 7. The protection by maltose of Gluc M1 from inactivation and the increase in K1 values for maltose of CMC-modified Gluc M1's suggested that a crucial carboxyl group(s) was located near or on subsites 2 and 3.

Purification and characterization of a minor glucoamylase from Aspergillus saitoi.

Purification and characterization of a minor glucoamylase from Aspergillus saitoi.

Purification of an acidic α- d-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-α- d-mannosidic linkage in yeast mannan

An acidic α- d-mannosidase (α- d-mannoside mannohydrolase, EC 3.2.1.24) has been isolated from culture filtrate of Aspergillus saitoi. The extracellular α-mannosidase was homogeneous in polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 51 000 and the isoelectric point pH 4.5. The purified enzyme has a pH optimum of 5.0, a K m of 0.45 mM with baker's yeast mannan and has no activity towards p- nitrophenyl-α- d-mannoside . The mode of action of the enzyme has been studied with baker's yeast mannan and saké yeast mannan. The enzyme cleaves specifically the 1.2-α-linked side chain, producing free mannose.

The Hydrolysis of Gelatin by an Immobilized Acid Protease. I. Immobilization and Hydrolysis Kinetics

An acid protease from <i>Aspergillus saitoi</i> was immobilized on agarose beads of approximately 100 micrometers diameter. Solutions of gelatin were prepared in a saturated potassium bitartrate buffer and contacted with the immobilized enzyme. The Michaelis-Menten constant was determined to be 7.8 x 10^-5 M at 25°C and pH of 3.5. The maximum velocity of hydrolysis corrected for bed voidage was found to be 6.8 x 10^-7 M per second under the same conditions.

Purification and characterization of a glucoamylase from Aspergillus saitoi.

1. A major glucoamylase [EC 3.2.1.3] of Aspergillus saitoi was purified by ultrafiltration followed by successive chromatography on DEAE-Sephadex, Ultrogel AcA 44 and SP-Sephadex. The purification achieved was 23-fold from crude extract with a yield of 21%. The purified enzyme, named Gluc M1, was proved homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, ultracentrifugation, and also from the absence of the glycosidase activities detected in crude extract. 2. Gluc M1 was a glycoprotein containing 18% neutral sugar and 0.77% glucosamine, and its molecular weight was estimated to be about 90,000 by SDS-polyacrylamide gel electrophoresis and amino acid composition. The N-terminal amino acid was identified as alanine. 3. The pH optimum of Gluc M1 was 4.5 with soluble starch as a substrate. The enzyme was stable between pH 2.5 and 7.5 and retained full activity at temperatures up to 50 degrees C. The enzyme activity was inhibited by Hg2+ and, to a lesser extent, by Pb2+ and Mn2+. 4. The Km value for malto-oligomer markedly decreased with increasing chain length of substrate in glucose unit (n) and the Vmax value increased with n, thus resulting in the increase in the Vmax/Km value with n. The kinetic parameters for other substrates such as soluble starch, glycogen and isomaltose as well as the K1 values for some saccharides were also determined.

An α-mannosidase purified from Aspergillus saitoi is specific for α1,2 linkages

The substrate specificity of an α-mannosidase purified from Aspergillus saitoi was studied in detail. This enzyme hydrolyzes yeast mannan partially but does not act on p-nitrophenyl α-mannopyranoside. Survey of the action of the enzyme on various oligosaccharides liberated from glycoproteins indicated that the enzyme hydrolyzes Manα1→2Man linkage but not Manα1→3Man and Manα1→6 Man linkages at all. All Manα1→2 residues in intact bovine pancreatic ribonuclease B were removed completely by incubation with the α-mannosidase.

Carboxamidomethylation of a ribonuclease from Aspergillus saitoi.

1) The inactivation of a RNase from Aspergillus saitoi (RNase Ms) was studied to obtain information on its active site. 2) Inactivation of RNase Ms by iodoacetamide was greater at an alkaline pH, and was protected more by 2',(3')-AMP than by 2',(3')-GMP. 3) Analysis of the hydrolysis products with 6 N HCl and alkaline treatment of carboxamidomethylated RNase Ms showed that the sites of reaction were one carboxyl group and one histidine residue. 4) Since the incorporation of a carboxamidomethyl group into carboxylic acid was not protected by 2',(3')-AMP, it was concluded that the formation of N1-carboxamidomethylhistidine was responsible for the loss of enzymatic activity of RNase Ms.

PH-profiles of the kinetic parameters of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active site of a ribonuclease from Aspergillus saitoi, the pH-profiles of kinetic parameters of RNase Ms were measured. (1) The pH dependences of kinetic parameters were measured using 2′, 3′-CCMP and 2‘, 3’-cUMP as substrates. Analyses of the results indicated the presence of three functional groups having pKa values of ca. 2.7 4.5, and 6.75 in the active site of RNase Ms. (2) The pH dependence of the inhibition constant of 2′, (3′)-AMP gave a pattern similar to the pK1-pH profiles of the substrate described above, again indicating the involvement of three functional groups in the active site. (3) The inhibition constant of adenosine changed only slightly with change in pH, and the functional group having pKa about 6.75 was not observed. The results indicate the interaction of this group with the phosphate moiety of the nucleotide. (4) A similar dependence of kinetic parameters of RNase Ms Using three substrates of the cyclization step was obtained. (5) By analyzing the pH-profiles of pKm and pK1, it was concluded that the active site of RNase Ms contains at least three functional groups, a histidine pKa about 6.75), a histidine or carboxyl group (pKa 4.0–4.5) and a carboxyl group (pKa about 2.7).

The effect of elimination of amino acids from the carboxyl-terminal of the minor ribonuclease from Aspergillus saitoi by carboxypeptidase A on the enzymatic activity.

In order to investigate whether carboxyl-terminal amino acid is involved in the active site of a base non-specific ribonuclease from Asp. saitoi (RNase Ms), removal of carboxyl-terminal amino acid residues by digestion with carboxypeptidase A was investigated. By 24 hours digestion, about 3 serine residues and 1.0 alanine residue were removed from RNase Ms and its activity decreased to about 70% of the native enzyme so far as measured with ribonucleic acid (RNA) as a substrate. The pH optimum and Km of carboxypeptidase treated RNase Ms (CP-RNase Ms) were very similar to those of native RNase Ms so far as measured with RNA as a substrate. However, Km of CP-RNase Ms using ApC as a substrate seemed to be larger than that of native RNase Ms. The large increase in Km value was not observed in the early stage of digestion where about 3 serine residues were removed. The gross structure of CP-RNase Ms was quite similar to that of the native RNase Ms by judging from circular dichroism spectrum at wavelength between 230-205 nm. From the results described above, it was concluded that carboxyl-terminal amino acid was not involved directly in the active site of RNase Ms.

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity

The specificity and mode of action of an acid proteinase ∗∗ ∗∗ Acid proteinase activity is expressed in the unit, katal (symbol kat) [12] (1 kat = 6 · 10 7 U). One katal of the acid proteinase is defined as the amount of enzyme which yields the color equivalent of 1 mol of tyrosine · s −1) with the Folin-Ciocalteau phenol reagent, using casein as substrate at pH 2.7 and 30°C (Enzyme Nomenclature (1972) IUPAC and IUB recommendations, pp. 26–27, Elsevier, Amsterdam). (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N, O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, lecuine and isoleucine in the P′ 1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogen-activating acid proteinase from Asp. saitoi.

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.

C-Terminal peptidyl-L-proline hydrolase activity of Aspergillus acid carboxypeptidase.

Aspergillus saitoi acid carboxypeptidase hydrolyzed C-terminal peptidyl-L-proline bonds and released the C-terminal proline from Z-Gly-Pro-Leu-Gly-Pro and Z-Gly-Pro at pH 3.3. Proline liberated by the enzymic reaction was measured by a sensitive colorimetric ninhydrin method in glacial acetic acid at 513 nm. A Km value of 1.0 mM and a kcat value of 0.09 s-1 for Z-Gly-Pro-Leu-Gly-Pro hydrolysis, and a Km value of 5.0 mM and a kcat value of 0.0045 s-1 for Z-Gly-Pro hydrolysis were calculated from Lineweaver-Burk plots.

Acid phosphatase of Aspergillus saitoi purification and properties.

Aspergillus saitoi produced a several forms of acid phosphatases. Fraction III-A, the largest fraction in them, was purified and proved to be homogenious by ultracentrifugal analysis and polyacrylamide disc electrophoresis. The molecular weight of the enzyme was 12.2×104, and the isoelectric point was pH 4.4. It was a typical acid phosphatase having the activity in only an acidic range. Optimum pH was 3.0. Other properties of this enzyme were also examined. Other four fractions were partially purified and their properties were examined.

Further studies on the specificity of the minor ribonuclease from Aspergillus saitoi.

In order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25 degrees. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The Ki values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribonucleosides was in the order dT, dA, dG, and dC.

Purification and Properties of a New Ribonuclease from Aspergillus saitoi

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.

Some properties of ribonucleases from Aspergillus saitoi and seminal vesicles immobilized on sepharose 4B activated by cyanogen bromide.

Some properties of ribonucleases from Aspergillus saitoi and seminal vesicles immobilized on sepharose 4B activated by cyanogen bromide.

Submerged Production, Purification, and Crystallization of Acid Carboxypeptidase from Penicillium janthinellum IFO-8070

Penicillium janthinellum IFO-8070 produced an acid carboxypeptidase of molecular weight 51,000 in a liquid medium at 25 C. Maximum enzyme concentration was obtained within 3 to 6 days in a medium containing 2% wheat bran, 1% defatted soybean, and 1% KH(2)PO(4); the initial pH was 2 to 4. When submerged aerobic conditions were used, a 51,000-molecular-weight acid carboxypeptidase was produced and no detectable amounts of 160,000-molecular-weight acid carboxypeptidase were produced. Acid carboxypeptidase with a molecular weight of 51,000 was purified 330-fold from koji culture to yield a crystalline protein which was demonstrated by disc electrophoresis to be homogeneous. The purification method included ammonium sulfate fractionation, Amberlite CG-50 chromatography, acetone fractionation, Amberlite CG-50 rechromatography, and concentration in a collodion bag. The specific activity of the enzyme was about three times more than that of the acid carboxypeptidase from Aspergillus saitoi.

Production and Some Properties of a New Type of Acid Carboxypeptidase of Penicillium Molds

Among some 38 strains of the genus Penicillium we investigated seven wild-type strains (P. daleae IFO-6087, P. frequentans AHU-8328, P. funiculosum IAM-7013, P. janthinellum IFO-8070, IAM-7026, P. lividum IAM-7200, and P. oxalicum AHU-8336) that were found to be excellent strains for a new type of acid carboxypeptidase production in a surface koji culture at 25 C. The production of acid carboxypeptidase was determined in various culture conditions in a koji culture. The maximum yields of acid carboxypeptidase were obtained by P. janthinellum IFO-8070. Partial purification and isolation of the acid carboxypeptidase from strains of Penicillium were performed with gel filtration on Bio-Gel P-100. Characterization studies indicate that the acid carboxypeptidases from P. daleae IFO-6087, P. funiculosum IAM-7013, P. janthinellum IFO-8070, and P. oxalicum AHU-8336 have some properties similar to those of the enzyme of Aspergillus saitoi with regard to the hydrolysis of several peptides at acidic pH range but have other slightly different properties with regard to stability, pH optima, inhibitors, and molecular weights.

Comparative specificity of microbial acid proteinases for synthetic peptides. III. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala) m , Z-Lys-Leu-(Ala) 2, and Z-(Ala) n-Lys-(Ala) 3 (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala) 2-Lys-(Ala) 3 was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.

Comparative specificity of microbial acid proteinases for synthetic peptides: I. Primary specificity

The specificity of various acid proteinases from mold and yeast such as Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp. were comparatively determined using with ▪ (X = various amino acid residues) as substrates. Pepsin was used in a comparative study. Since the peptides were susceptible to these enzymes at the peptide bonds indicated by the arrows, except for the ones from both Aspergillus species and Rhodotorula, we could examine their specificity with respect to the amino acid residue on both sides of the splitting point. The results indicated that the microbial enzymes were specific for aromatic, or bulky and hydrophobic amino acid residues on both sides, as had been observed with pepsin. The specificity of the enzymes from Aspergillus and Rhodotorula was not determined because of lack of hydrolysis of the peptides.