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 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 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 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 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 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.     

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.     

Neutral Proteinases I and II of Aspergillus sojae

Epimerization of (?)-isodihydrocarvone (I) to (?)-dihydrocarvone (II) by Pseudomonas fragi IFO 3458 was studied. I was easily epimerized to II by the growing cells, the resting cells or the cell-free extracts.

An epimerase catalyzing the conversion of I to II was partially purified from the bacterial extracts about 56-fold with heat-treatment, ammonium sulfate precipitation and DEAE-Sephadex A-50 column chromatography. By the action of this epimerase, the ratio of I to II becomes about 25: 75 (K=3). It appeared that the epimerase is very stable to heat; the activity of epimerization remains 66 and 36% after treatment at 97°C for 60 and 120 min, respectively.     

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.     

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.     

Some Properties of Neutral Proteinases I and II of Aspergillus sojae as Zinc-containing Metalloenzyme

Some experiments were carried out with purified neutral proteinases I and II of Aspergillus sojae in relation to their characteristics as metalloenzyme.

The both enzymes contained one gram atom of zinc and about two gram atoms of calcium per mole (molecular weights of 41,700 for I and 19,800 for II were estimated by gel filtration) of enzyme protein, and the zinc was essential for the activity. Some metal-chelating agents, such as ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, 8-hydroxyquinoline and α,α′-dipyridyl, inhibited the activity of the both enzymes. In the inactivation of neutral proteinase II by EDTA a distinct pH-dependency was observed. The EDTA-inactivated enzymes were reactivated fully or partially by the addition of some metal ions such as Zn²⁺, Co²⁺, Mn²⁺, Cu²⁺ (only neutral proteinase II) and Ni²⁺. Zinc-free apo-enzymes were prepared from the native enzymes by the dialysis against EDTA solution. The apo-enzyme of neutral proteinase I still contained calcium, while that of neutral proteinase II did not. The apo-enzymes restored their activity for the most part either by the addition of excess amount of zinc or by mixing with a stoichiometric amount of zinc in the presence of calcium at an alkaline condition.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

Purification and Properties of Intracellular Proteinase from Streptococcus cremoris

Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl₂ in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, ethylenediaminetetraacetate, and sodium lauryl sulfate. The K_m value of the enzyme towards each casein fraction was almost the same, and the V_max of the enzyme towards α_s-casein was smaller than those towards the other casein fractions.     

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.     

Purification of Peptidases of Aspergillus oryzae and Some Properties of the Purified Peptidases

The purification of Aspergillus oryzae peptidases was attempted by the fractional precipitation with acetone, ammonium sulphate, and by starch zone electrophoresis. We, thus, achieved a great success in the separation of dipeptidase free from aminopolypeptidase and proteinase as well as in the separation of aminopolypeptidase free from dipeptidase and proteinase.

The specific activity (C₀) of the former (leucylglycine hydrolysis) was 7000 and that of the latter (leucylglycylglycine hydrolysis) 22000.

The leucylglycine dipeptidase was remarkably activated by Zn⁺⁺, and Co⁺⁺. Some other enzyme properties were also found and are discussed.     

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.