Neutral Proteinases I and II of Aspergillus sojae: Some Physicochemical Properties and Amino Acid Composition

Some physicochemical properties of neutral proteinases I and II, zinc-containing metalloenzymes, from Aspergillus sojae were investigated.

Neutral proteinase I: The enzyme protein had a sedimentation coefficient of 3.90S, an intrinsic viscosity of 0.0315 dl/g, and a partial specific volume, calculated from the amino acid and carbonhydrate composition, of 0.715 cm³/g. The molecular weight was 42,200 from the Yphantis’ procedure, and was 42,500 from the calculation according to the Scheraga-Mandel-kern’s formula. The integral numbers of amino acid residues per molecule calculated on the basis of 42,200 as molecular weight were as follows; Lys₁₆, His₆, Arg₁₃, Trp₈, Asp₅₆, Thr₂₅, Ser₂₃, Glu₃₁, Pro₁₈, Gly₄₀, Ala₃₃, l/2Cys₄, Val₁₁, Met₆, Ile₁₅, Leu₂₅, Tyr₂₀, Р?е₁₀, (amide-ammonia)₂₉, in addition to mannose₆, galactose₁, hexosamine₃.

Neutral proteinase II: The enzyme protein had a sedimentation coefficient of 2.32S, an intrinsic viscosity of 0.0270 dl/g, and a calculated partial specific volume of 0.714 cm³/g. The molecular weight was 16,800 from the Yphantis’ procedure, and was 18,000 from the sedimentation and intrinsic viscosity. The following amino acid compositions was calculated on the basis of 16,800 as molecular weight; Lys₈, His₃, Arg₃, Asp₁₉, Thr₁₇, Ser₁₁, GIu₂₃, Pro₅, Gly₉, Ala₂₄, l/2Cys₄, Val₅, Ile₃, Leu₁₃, Tyr₁₀, Phe₃, (amide-ammonia)₁₅. In the enzyme preparation, neither methionine nor tryptophan was detected and carbohydrate was also absent.

In both neutral proteinases I and II, no free SH group was detected by the PCMB-titration in the presence of 8 M urea.     

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.     

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.     

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.     

Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

Molecular Conformation of Alkaline Proteinase of Aspergillus sojae in Aqueous Solution

The molecular conformation of the alkaline proteinase of Aspergillus sojae in aqueous solution was investigated by the optical rotatory dispersion, Cotton effects, infra-red absorption spectra (amide I and V bands), ultraviolet difference spectra, etc. It is concluded that; (1) there are about 10 to 15% of the α-helix and a small amount of the β-structure in the enzyme molecule, but most parts of the peptide chain are present as the disordered structure; (2) the enzyme molecule is compactly folded even in the disordered parts; and (3) the two tryptophan residues involved in the peptide chain are burried in the interior of the molecule.     

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.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

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.     

Successive Isolation of Proteinase Hyperproductive Mutants of Aspergillus sojae

Hydrochloric acid treatment of methyl 3-(4-isobutylphenyl)-3-methylglycidate and methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate, a rearrangement product of the former, in acetic acid gave 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-isobutylphenyl)-pro-panal. The same treatment of 2-hydroxy-3-(4-isobutylphenyl)-3-butenoic acid gave 2-(4-isobutylphenyl)-propanal. Both 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-iso-butylphenyl)-propanal were oxidized to 2-(4-isobutylphenyl)-propionic acid.     

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.     

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.     

Hyperproduction of Proteinase and Some Hydrolytic Enzymes by Mutants of Aspergillus sojae

Mutant strains of Aspergillus sojae exhibited coordinate increases of acid proteinase, α-amylase, and cellulase and a decrease of pectin trans-eliminase accompanied with the hyperproduction of alkaline proteinase in wheat bran koji culture. The production of these enzymes in the wheat bran solid medium, liquid wheat bran-defatted soybean medium, and liquid glucose-peptone medium were surveyed. The analyses on the production patterns of these enzymes under the different cultural conditions suggest that mutation in these mutants producing elevated levels of the above enzymes is due to a more complex alteration than a single gene mutation.     

Relating Trypsin Enzymatic Properties with Amino Acid Composition

In this paper, the DNA fragment of trypsin genes from eight crustaceans were cloned and sequenced. The amino acid composition of the 24 deduced and 42 selected trypsin sequences were compared. Low arginine, methionine, and proline content and high aspartic acid, glutamic acid, and isoleucine content attributed to the distinct catalytic efficiency of crustacean trypsins.     

Physicochemical Properties and Amino Acid Sequence of Sheep Brain Galectin-1

A beta-galactoside-specific soluble 14-kD lectin from sheep brain was isolated, sequenced, and compared with similar galectins from other species. Percent identities of amino acid sequence and the carbohydrate recognition domain (CRD) revealed that the isolated galectin shares all the absolutely preserved and critical residues of the mammalian galectin-1 subfamily. The isolated sheep brain galectin (SBG) showed more than 90% amino acid sequence (92%) and carbohydrate recognition domain identity (96%) with human brain galectin-1. Conformational changes were found induced by interaction of the protein with its specific disaccharide and oxidizing agent (hydrogen peroxide). Upon oxidation a drastic change in the environment of aromatic residues and conformation of the galectin was observed with the loss of hemagglutination activity, while no significant change was observed upon addition of D-lactose (Gal(beta1-4)Glc) in the far-UV and near-UV spectra, suggesting no significant modification in the secondary as well as tertiary structures of sheep brain galectin. But the functional integrity of the CRD is found to be affected in the presence of oxidizing agent, indicating intramolecular disulfide bonds and requirement of complete polypeptide chain for functional integrity of the carbohydrate recognition domain.     

Chemical and Physicochemical Properties of Phytase from Aspergillus terreus

Some chemical and physicochemical properties of the purified phytase preparation produced by Asp. terreus were investigated. From the results of the examination of amino acid analysis, it was suggested that there existed some components other than amino acids in the purified enzyme. Examination of the neutral sugar analysis, therefore, was made by gaschromatography, and it was found that the purified enzyme preparation contained mannose, galactose and a small amount of inositol.

The molecular weight of the enzyme was found to be 214,000 by the Archibald method, and 2.2~2.3×10⁵ by gel-filtration on a Sephadex G–200 column. It was found that by guanidine hydrochloride or by urea, the purified enzyme preparation was dissociated into only one kind of subunit. The native enzyme was supposed to be a homohexamer of the subunits whose molecular weight is 37,000.     

Neutral Proteinase II of Aspergillus sojae: An Enzyme Specifically Active on Protamine and Histone

To identify the compounds in machine cutting-fluid emulsion that have an obnoxious odor, we separated volatile components from the emulsion using both steam distillation with a Nickerson–Likens apparatus and vacuum distillation. These components were analyzed by gas chromatography and gas chromatography-mass spectrometry using a fused silica capillary column coated with cross-linked 5% phenylmethyl silicone. 2,6-Dimethyl-3-methoxypyrazine was detected. The main odorous compounds were dimethyl disulfide, dimethyl trisulfide, and 2-butene-1-thiol, the last compound being tentatively identified by its mass spectrum.