Studies on the respiratory system of Aspergillus oryzae I. Development of respiratory activity during germination in the presence and absence of antimycin A

Active growth of Aspergillus oryzae was observed when conidiawere inoculated into a medium containing antimycin A. Immediatelyafter adding antimycin A, to young mycelia germinated in itsabsence, growth stopped, but began again after several hours.This restored growth was antimycin A-insensitive. Percentagegermination was the same in the presence and absence of thisdrug. It seems that drug-resistant germination and growth donot result from selection of resistant cells but result frominduction of antimycin A-insensitive mitochondria in the wholepopulation. Endogenous respiration of cells germinated in theabsence of antimycin A was inhibited by this drug, whereas thatof cells grown in the presence of antimycin A was completelyinsensitive. Antimycin A-sensitivity of cellular respirationseems to determine the effect of this drug on mycelial growth.Mitochondria were isolated from mycelia grown in the presenceand absence of this drug. The difference in antimycin A-sensitivityin endogenous respiration was attributed to a difference inproperties of the mitochondrial respiratory systems. ¹Present address: Department of Chemistry, Institute of MedicalScience, University of Tokyo, Tokyo, Japan (Received December 21, 1969; )     

Investigation of Endomyces magnusii respiratory system. Characteristics of mitochondria from cells grown in the presence of antimycin A]

Mitochondria from AA-cells are a unique model with separately finctioning 1st and 3rd points of energy coupling and with completely blocked 2nd coupling point. In vivo the terminal segment of the respiratory chain does not operate, cytochromes c and a+a3 remain oxidized, and dominating terminal oxidase appears to be a component inhibiting by salicylhydroxamate and bound with the respiratory chain via cytochromes b or ubiquinone pool. The blocking of the 2nd coupling point is due to firm and, probably, quantitative binding of AA with cytochromes b, especially with b559, and also to a partial denaturation of the complex III.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Studies on the Autolysis of Aspergillus oryzae

An intracellular nuclease inhibitor was 1270 times purified from a heat treated cell free extract of fresh mycelia of Aspergillus oryzae, by ammonium sulfate fractionation and chromatographies using DEAE-cellulose and Sephadex G-75. The purified sample of the inhibitor showed a UV absorption curve typical for protein, and it was inactivated by proteases such as chymotrypsin. The inhibitor stoichiometrically inactivated nuclease O (an intracellular nuclease of Asp. oryzae), forming an enzyme-inhibitor complex. But, it did not affect nuclease S₁, RNase T₁, RNase T₂ or pancreatic RNase. The inhibitor was insensitive to 10^?5m p-chloromercuribenzoate or 10^?4m Pb²⁺. Molecular weights estimated by the method of Andrews were 23,000 for the inhibitor, 47,000 for nuclease O, and 82,000 for the enzyme-inhibitor complex. The nuclease activity was recovered from the inactive complex by the action of chymotrypsin.

Nuclease O of Asp. oryzae was purified and crystallized from 113.5 kg of wet mycelia and 2 kl of culture filtrate, by salting out with ammonium sulfate and by chromatographies on CM-Sephadex C-50 and Sephadex G-100. The purified nuclease showed a single peak with apparent sedimentation constant 2.9S in an ultracentrifuge. The molecular weight measured by short column method was 64,000. The nuclease was completely inhibited by the specific nuclease inhibitor obtained from Asp. oryzae. The nuclease was activated by 0.1 mm Mg²⁺ and Mn²⁺, and completely inhibited by 1 mm EDTA. Optimum pH for activity was 7.6 for RNA and 7.4 for DNA. The nuclease degraded polyadenylic acid, polyuridylic acid and polycytidylic acid without forming detectable amount of mononucleotides. And, the main product from RNA was oligonucleotides. The enzyme showed no nonspecific phosphodiesterase activity.     

Studies on the Autolysis of Aspergillus oryzae

In was found that an intracellular ribonuclease was present as an inactive form in the fresh mycelium of Asp. oryzae. It was about 3 times activated either by 3 m urea or by the autolysis of mycelium at 30°C for 20 hr. The optimum pH of the ribonuclease activity was 8.3. It was heat sensitive (60°C, 10 min), and completely inhibited by 5 mm EDTA. It was activated by 1 mm Mg²⁺ and inhibited by Zn²⁺, Ca²⁺, Cd²⁺, Co²⁺ and Cu²⁺.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.     

Studies on the Autolysis of Aspergillus oryzae

Crystalline nuclease O obtained from autolyzed Aspergillus oryzae hydrolyzed heat-denatured calf thymus DNA 19 times faster than native DNA. Digestion of the heat-denatured DNA with an exess of the enzyme produced mono-, di- and tri-nucleotides with 5′-terminal phosphate, which amounted 3.4, 58.3 and 38.2%, respectively, of total degradation products. Hydrolysis of the native DNA with a sufficient amount of nuclease O produced mono-, di-, tri- and tetra-nucleotides with 5′-terminal phosphate, which amounted 1.9, 47.9, 36.7 and 13.6%, respectively, of total degradation products. Although nuclease O showed no strict base specificity on the native and heat-denatured DNA, di-and tri-nucleotides in the digests were resistant to further hydrolysis by nuclease O. Native γDNA was hydrolyzed by nuclease O through the mechanism of single strand break, which was shown by neutral and alkaline sucrose density gradient centrifugations.     

Studies on the Autolysis of Aspergillus oryzae

Properties of nuclease O, a new intracellular enzyme which was partially purified from autolyzate of Asp. Oryzae,¹⁾ are described in this paper. The purified enzyme preferentially depolymerized RNA and heat denatured DNA, but apparently did not attack native DNA. It was activated by 0.1 mm Mg²⁺ or Mn²⁺, and inactive in the presence of EDTA. Optimum pH of the activity were 7.7 for DNA and 8.2 for RNA. By heat treatment (60°C, 10 min at pH 6) the nuclease completely lost its activity for RNA and DNA. Optimum concentration of Tris buffer for enzymatic activity was 0.15~0.2m.     

Studies on Peptidases of Aspergillus oryzae

Screening experiments for dipeptidase and aminopolypeptidase from 40 strains of molds were conducted using Leu-Gly, Gly-Leu, Ala-Gly and Gly-Gly-Leu as substrates.

The strains of Aspergillus oryzae RO-0129 A-2, IAM-2600 and IAM-2616 showed strong activities of both dipeptidase and aminopolypeptidase.

Further, optimal conditions for making culture as well as those for the extractions of the peptidases from the mycerial mats were investigated.     

Studies on Peptidases of Aspergillus oryzae

The homogeneity of Aspergillus dipeptidase prepared according to the standard method established by us was ascertained by ultracentrifugation and some characteristic properties of the enzyme was further investigated.

Hydrolysis of various dipeptides by the purified dipetidase was tested in the presence of divalent metal ions such as Co⁺⁺ or Zn⁺⁺, and the characteristics of greatest interest may be enumerated as follows:

1. The homogeneous dipeptidase requires Zn⁺⁺ for activation in the case of the hydrolysis of leucylglycine, leucylalanine leucylleucine, etc.

2. The homogeneous dipeptidase requires Co⁺⁺ for activation in the case of the hydrolysis of glycylleucine, glycylleucine, glycylglycine, glycylphenylalanine, etc.

3. In the case of the hydrolysis of alanylglycine, alanylleucine, valylglycine, etc., this enzyme does not require any metal ions.

     

Studies on the Protease Constitution of Aspergillus oryzae

In order to elucidate the protease constitution of Aspergillus oryzae, systematic separation of proteases was elaborated by sequential chromatography on Amberlite CG–50, DEAE-Sephadex A–50 and CM-cellulose. As the results, three kinds of proteases, that is, acid-, neutral- and alkaline proteases were isolated and purified in crystalline form except neutral one. Purified neutral protease could not be crystallized, but was confirmed to be homogeneous by ultracentrifugal analysis. Besides these proteases, a new protease which was unknown up to the present in the constitution of Asp. oryzae proteases, was first isolated and designated as “semi-alkaline protease” according to its optimal pH.     

Studies on the respiratory properties of mitochondria isolated from developing winter wheat seedlings

Mitochondria isolated from shoots of 2 days, light- and dark-grown winter wheat (Triticum aestivum L. cv. Rideau) seedlings oxidize alpha-ketoglutarate and l-malate with good respiratory control and ADP: O ratios. The efficiency of oxidative phosphorylation, and respiratory control are both reduced significantly when succinate or NADH is employed as substrate. Respiratory control values and ADP: O ratios show a general decline in mitochondria from seedlings of increasing age, whether grown in light or dark. In light-grown seedlings, the decrease in respiratory control with aging is due principally to a decrease in the rate of state 3 respiration, while in dark-grown material, the decrease appears to be due mainly to an increased rate of state 4 respiration. In both light- and dark-grown seedlings, oxygen consumption during state 3 respiration is severely inhibited by oligomycin. During state 4 respiration, 2,4-dinitrophenol stimulates oxygen uptake to a level approximately two-thirds the normal ADP-stimulated rate.