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

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

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 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 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.     

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex

The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB^R321A mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.

Structured summary:

MINT-7385426:Dlp1 (uniprotkb:Q86YH6) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385083, MINT-7385058:IspB (uniprotkb:P0AD57) and IspB (uniprotkb:P0AD57) bind (MI:0407) by blue native page (MI:0276)MINT-7385413:Dlp1 (uniprotkb:O13851) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385024:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dps1 (uniprotkb:O43091) by pull down (MI:0096)MINT-7385041:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dlp1 (uniprotkb:O13851) by pull down (MI:0096)MINT-7385388:IspB (uniprotkb:P0AD57) and Dps1 (uniprotkb:O43091) physically interact (MI:0915) by blue native page (MI:0276)     

Studies on Amylases of Aspergillus oryzae Cultured on Rice

α-Amylase was purified from a culture of Aspergillus oryzae on steamed rice by means of ion exchange chromatography on DEAE-Sephadex, and the purified enzyme was crystallized with ammonium sulfate. The preparation was found to be homogeneous by means of sedimentation and disc electrophoretic analyses. The enzyme was revealed to have strong α-amylase activity by the dinitrosalicylate method and the iodine color method. Large single crystals of the enzyme were prepared by making the concentrated enzyme solution to 0.41 saturation of ammonium sulfate at pH 5.0. A brief communication on the preliminary X-ray crystallography was also presented.     

Induced Autolysis of Aspergillus oryzae (A. niger group): IV. Carbohydrates

The examination of substances formed during induced autolysis by Aspergillus niger was continued in this work, which dealt in particular with carbohydrates. The autolysate contained a large amount of d-glucose (14 to 20% dry wt) and traces of glycolic aldehyde, dihydroxyacetone, ribose, xylose, and fructose. It also contained glycopeptides (about 10% dry wt), which were split from the cell wall during autolysis and which differed from one another in their level of polymerization and their composition. They were constituted by glucose and mannose, glucose and galactose, or mannose, glucose, and galactose (mannose being the most abundant in this case), and amino acids (chiefly alanine, serine, glutamic acid, and aspartic acid). During autolysis, only a part of the cell wall was dissolved, since it retained its shape. Upon further chemical hydrolysis, it produced mostly glucose and glucosamine, and smaller amounts of mannose, galactose, and amino acids. Presumably, glucomannoproteins and glucogalactoproteins were present in the intact cell as a macromolecular complex, constituting, together with chitin, the major part of the cell wall of Aspergillus.     

Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

Farnesyl diphosphate synthase localizes to the cytoplasm of Trypanosoma cruzi and T. brucei

The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes.     

The half-lives of dolichol and dolichyl phosphate in rat liver

Rat liver dolichol and dolichyl-P were labeled by injection of [³H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in -unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T_1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T_1/2 of 32 h. Injected dolichol was recovered in the lysomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.     

Studies on the Arylsulphatase and phenol sulphotransferase activities of Aspergillus oryzae.

1. Crude extracts of Aspergillus oryzae grown under conditions of sulphur limitation possess high arylsulphatase activity. 2. This activity can be greatly enhanced by the inclusion of tyramine or a number of other phenols in the assay medium. 3. The arylsulphatase activity of these extracts can be resolved into three distinct fractions by chromatography on DEAE-cellulose. 4. The effect of tyramine is restricted to one of these fractions only. 5. Evidence is presented which indicates that this effect is the consequence of a phenol sulphotransferase activity, which shows no requirement for 3'-phosphoadenosine 5'-phosphate as a cofactor, and which will not transfer sulphate from 3'-phosphoadenosine 5'-sulphatophosphate to potential phenolic acceptors. 6. The three enzymes differ also in their molecular weights and substrate specificities.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.