Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

Purification of Alkaline Proteinase from Aspergillus Candidus

An alkaline proteinase of Aspergillus Candidus was purified from wheat bran solid culture by batchwise treatment with Amberlite IRC–50 and sequential chromatography on DEAE-cellulose, hydroxylapatite and Sephadex G–100 gel. This purification results in a 18-fold increase of proteolytic activity and the enzyme preparation was homogeneous in sedimentation analysis of the ultracentrifuge and polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be about 23,000 by gel glltration and 22,000 by calculation from the amino acid composition. The enzyme consisted of Lys₁₄, His₄, Arg₃, Asp₂₅, Thr₁₅, Ser₂₃, Glu₁₅, Pro₇, Gly₂₂, Ala₂₄, Met₂, Val₁₆, Ile₁₁, Leu₁₀, Tyr₆, Phe₇, Trp₂ and amide ammonia₁₄ and did not contain cysteine or cystine.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Crystallization and Some Properties of Alkaline Proteinase from Aspergillus sulphureus

An alkaline proteinase of Aspergillus sulphureus (Fresenius) Thorn et Church has been purified in good yields from wheat bran culture by fractionation with ammonium sulfate, treatment with acrynol, and DEAE-Sephadex A-50 column chromatography. The crystalline preparation was homogeneous on sedimentation analysis and polyacrylamide gel zone electrophoresis. The molecular weight was calculated to be 23,000 by gel filtration. The amino acid composition of the enzyme was determined. The enzyme did not precipitate with acrynol. Optimum pH for the hydrolysis of casein was 7 to 10 at 35°G for 15 min. Optimum temperature was 50°C at pH 7 for 10 min. The enzyme was highly stable at the range of pH 6 to 11 at 5°C, whereas relatively stable at pH 6 to 7 at 35°C. Metalic salts tested did not affect activity. Chelating agents, sulfhydryl reagents, TPCK, and oxidizing or reducing reagents tested, except iodine, had no effect on the activity. Diisopro-pylfluorophosphate and N-bromosuccinimide almost completely inactivated the proteinase.     

Physicochemical Properties and Amino Acid Composition of Alkaline Proteinase from Aspergillus sojae

Some physicochemical properties and amino acid composition of alkaline proteinase from Aspergillus sojae were found to be as follows: The isoelectric point was at pH 5.1. The molecular weight was 25,500 using the Sheraga-Mandelkern’s formula, based upon the values of the sedimentation coefficient $(s_{20,w}^{°}=\text{?}2.82\text{?}\text{S})$, the intrinsic viscosity ([η] = 0.027 dl/g), and the partial specific volume $({\displaystyle \overline{V}}\text{?}=\text{?}0.726\text{?}\text{ml}/\text{g})$. The enzyme contains 16.8% of nitrogen and is composed of 250 residues of amino acid; Asp₃₁ Glu₁₉, Gly₂₇, Ala₃₂, Val₁₈, Leu₁₄, Ile₁₄, Ser₂₈, Thr₁₈, ${\text{(}{\displaystyle \stackrel{}{{\displaystyle \stackrel{?}{\text{Cys C}}}}}\text{ys})}_1$, Met₂, Pro₆, Phe₇, Tyr₈, Trp₂, His₅, Lys₁₄, Arg₃, (amide-NH₃)₂₀.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal.     

Purification and Properties of Acid Carboxypeptidase II from Aspergillus oryzae

Acid carboxypeptidase II from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The optimum activity of the enzyme lay at pH 3.0 for carbobenzoxy-L-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, α, α′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 105,000.     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

Purification and Characterization of Alkaline Proteinase from AlkalophilicBacillussp.

Alkaline proteinase was purified from Bacillussp. isolated from soil. The pH optimum was 11.5 at 37°C. Calcium divalent cation was effective in stabilizing the enzyme, especially at higher temperatures. The proteolytic activity was inhibited by the specific serine proteinase inhibitor PMSF (phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, and Hg. The enzyme was stable in the presence of detergents, such as Triton-X100, Tween-80, SDS (sodium dodecyl sulfate), and EDTA (ethylenediaminetetraacetic acid), at pH 11.5 and 37°C for 30 min. The optimum pH was 11.5 at 37°C, and the optimum temperature was 62°C at pH 11.5.     

Purification and properties of glutaminase from Aspergillus oryzae

Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l-glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.     

Isolation of Alkaline Proteinase from Aspergillus sojae in Homogeneous Form

The alkaline proteinase of Aspergillus sojae was isolated in gram quantities as a homogeneous form. The purification procedures were, (1) batchwise-treatment with ion exchange resin Duolite CS 101, (2) fractional precipitation with ammonium sulfate, (3) precipitation with acetone, (4) column chromatography on DEAE-cellulose, and (5) gel filtration with Sephadex G-100. The recovery of the activity was about 12%. The purified enzyme preparation was found to be homogeneous by several criteria such as ultracentrifugation, paper and moving-boundary electrophoreses, etc. Any kinds of carbohydrate and phosphorus were not detected in this preparation, suggesting that this enzyme is a simple protein.     

Properties of Arylamidase from Aspergillus oryzae

α-Amylase-like proteins were synthesized in a heterologous cell-free protein synthesizing system prepared from Escherichia coli. The proteins were precipitable with anti-α-amylase serum and detected only when RNA extracted from α-amylase producing Bacillus subtilis cells was used as messenger. The in vitro α-amylase-like products seemed to consist of two components having molecular weights of 30,000 and 13,000.     

Purification of an Aspergillus oryzae Metallo-proteinase by Talopeptin-aminohexyl-Sepharose and Its Properties

Simple and speedy purification of Aspergillus oryzae metallo-proteinase was performed using Talopeptin-aminohexyl-Sepharose The properties of the metallo-proteinase were: optimum pH 6.5; pH stability, pH 5~11; optimum temperature,50°C; and molecular weight 42,000 (SDS electrophoresis). These results were similar to those of neutral protease I from Aspergillus oryzae reported by Nakadai et al. This metallo-proteinase was compared with others from microbes using the metallo-proteinase inhibitors FMPI, PLT, and Talopeptin. The metallo-proteinase is unique in the point at which FMPI and PLT gave nearly stoichiometrical inhibition.     

Studies on an Alkaline Proteinase of Aspergillus sydowi

An alkaline proteinase of Aspergillus sydowi (Bainier et Sartory) Thom et Church has been purified approximately 4.5-fold from a culture filtrate by fractionation with ammonium sulfate, treatment with acrynol and Alumina gel C_γ, and DEAE-Sephadex column chromatography. The purified proteinase obtained as needle crystals was monodisperse in both the ultracentrifuge and the electrophoresis on polyacrylamide gel.

The optimum pH and temperature for the activity were 8.0 and 40°C, respectively. Fifty per cent of the activity was lost at 45°C within ten minutes and 95% at 50°C. At 5°C, the enzyme was highly stable at the range of pH 6 to 9. None of metallic salts tested promoted the activity, but Zn⁺⁺, Ni⁺⁺ and Hg⁺⁺ were found to be inhibitory. Sulfhydryl reagent, reducing and oxidizing reagents tested except iodine had no effect on the activity, but potato inhibitor, DFP and NBS caused a marked inhibition.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca⁺⁺ in the enzyme solution. The protective effect of Ca⁺⁺ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca⁺⁺ for the protective effect at pH 7.1, 7.5 and 7.8 were 10^?2, 10^?3 and 10^?4 M, respectively. Conversely, at higher pH values such as 9.0, Ca⁺⁺ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution.

The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2 m trichloroacetic acid solution, with 14~17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chrysogenum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slightly on casein hydrolyzed by the above-mentioned proteinases.     

Comparison of Alkaline Proteinase from Hyperproductive Mutants and Parent Strain of Aspergillus sojae

Physico-chemical properties of alkaline proteinase from the parent strain were compared with those from hyperproductive mutants of Aspergillus sojae. All the results on behavior of enzyme protein to ion exchange resin and celluloses, gel filtration, ultracentrifugal sedimentation, disc electrophoresis and isoelectrofocusing on polyacrylamide gel column, specific activity, substrate specificity, and kinetic constants provided evidence in favor of the conclusion that the parent and mutant strains produced the chemically identical enzymes and that superactivity of alkaline proteinase in culture extracts or filtrates of mutant strains was not attributed to alteration of catalytic property of the enzyme, but to hyperproduction of the identical enzyme resulting from the genetic change in the regulatory mechanism of enzyme synthesis.     

Purification of Two Acid-Proteases of Aspergillus oryzae from Takadiastase

Two acid-proteases, shown previously to be present in Takadiastase and tentatively named as SE- and DEAE-protease, were purified. These two enzymes did not differ in enzymatic properties investigated but differed in some physical properties, such as isoelectric points.

Though the SE-protease showed only a single peak in ultracentrifugal sedimentation patterns, it was separated into two or three components in electrophoretic patterns. On the contrary, the DEAE-protease showed only a single peak in either sedimentation patterns or electrophoretic patterns.