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.     

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.     

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.     

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.     

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.     

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.     

Specificity of Thermophilic Streptomyces Alkaline Proteinase

The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe²⁴-Phe²⁵, Leu¹⁵-Tyr¹⁶ and Leu¹¹-Val¹²) and additionally at six others (Leu⁶-CySO₃H⁷, Gln⁴-His⁵, Leu¹⁷-Val¹⁸, His⁵-Leu⁶, Glu¹³-Ala¹⁴, Asn³-Gln⁴).

Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr⁴-Ile⁵ and Phe⁸-His⁹ bonds.     

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.     

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.     

提高碱性蛋白酶生产效益的措施

综述了提高碱性蛋白酶生产效益的可能途径：菌种的及时复壮,菌种的诱变选育,菌种的代谢调控选育,工艺条件的控制。     

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.     

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.     

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).     

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.     

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.     

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.     

地衣芽孢杆菌产生碱性蛋白酶的动力学研究

应用自动控制发酵设备,首先进行分批发酵试验摸索了地衣芽孢杆菌2709生长与代谢的基本规律。然后采用补料分批发酵方法限制生长基质浓度,测定了一系列(S_I,μ_I)、(μ_j,q_pj)数据,获得K_S、μ_max、α、β等参数的值,并且推导出了细胞生长与产物合成的动力学公式,从而证明了用Monod方程描述地衣芽孢杆菌2709生长速率与基质浓度关系的合理性和合成碱性蛋白酶的发酵属于生长部分关联型。     

Isolation and Identification of Microorganism,Producing Microbial Alkaline Proteinase Inhibitor (MAPI)

In the screening of actinomycetes’ culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MAPI, abbreviation of microbial alkaline proteinase inhibitor.Judging from the morphological and physiological properties of the actinomycetes which produced MAPI, this strain was identified as Streptomyces nigrescens.

For the production of MAPI, this strain was aerobically cultured at 25 ~ 27%C in a jar fermentor which contained an optimum medium consisting of polypepton 3 %, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄·nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21 ~ 24 hr cultivation.

MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

An Aminopeptidase of Aspergillus sojae

An aminopeptidase was purified from Aspergillus sojae X–816. The molecular weight of the enzyme was estimated to be 220,000. The isoelectric point was at pH 5.3. The optimum pH for l-leucylglycylglycine was 7.5. The enzyme was stable up to 37°C against temperature treatment for 15 min. Some chelating agents inhibited the enzyme activity. The Km value for l-leucylglycylglycine at pH 7.5 and 37°C was 45 mm. The Km value for l-leucyl-β-naphthylamide at pH 7.0 and 37°C was 2.2 mm.