Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

[This corrects the article on p. 507 in vol. 30.].     

Purification and Characterization of Beauveria bassiana Proteinases

Two chymotrypsin‐like serine proteinases are produced by B. bassiana 278 when grown on different carbon and nitrogen sources. By employing acetone precipitation, gel filtration and ion‐exchange chromatographies, the enzymes were separated from the culture filtrate after propagation of the fungus on medium enriched either with ground larvae of Apis mellifera (Proteinase I) or porcine blood plasma (Proteinase II). The purified enzymes with a molecular mass of approximately 32 kDa hydrolyzed natural protein substrates: casein, hide powder azure (HPA), azocoll and much less elastin Congo Red and collagen. They differ from each other in the optimum pH value, amino acid composition, Michaelis constant and susceptibility to natural chymotrypsin inhibitors. Both proteinases hydrolyze suc‐Ala‐Ala‐Pro‐Phe‐p‐NA with an apparent K_m of 2.03 × 10^—3 M and 1.04 × 10^—4 M, respectively. The turkey ovomukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Proteinase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be very high (4.11 × 10⁹ M^—1).     

Preparations of Extracellular Proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.     

Purification and characterization of endo-beta-N-acetylglucosaminidase of Aspergillus oryzae

An endo-beta-N-acetylglucosaminidase which hydrolyzes the N,N'-diacetylchitobiosyl linkage in asparagine-linked oligosaccharides was purified from the enzyme product of Aspergillus oryzae. Its substrate specificity was similar to that of endo-beta-N-acetylglucosaminidase H from Streptomyces griseus with respect to the relative activities toward the glycopeptides obtained from ovalbumin and bovine IgG. The present endoglycosidase exhibited a broad optimum pH range and was relatively stable. Metal ions, chelating agents and D-mannose did not have a significant effect on the enzyme activity.     

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.     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

Production,Rapid Purification and Catalytic Characterization of Extracellular Phytase from Aspergillus Ficuum

Abstract

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakis-phosphate 3-phosphohydrolase, EC 3.1. 3.8). At pH 5.0 and 60°C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2nM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 × 10^?3M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

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 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 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.     

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 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.     

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 Characterization of Intracellular Proteinases in Pleurotus ostreatus Fruiting Bodies

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL.

ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn²⁺-depleted ProB can regain the activity with Zn²⁺, Co²⁺, or Mn²⁺. Specific cleavage of Pro₂₉-LYS₃₀ in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues.

ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn²⁺ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.