Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae cavara. II. Purification and properties of a beta-glucosidase.

Three components (GA, GB-1, and GB-2) of beta-glucosidase were detected in the culture filtrate of Pyricularia oryzae grown in a cellulose or cellulose derivative medium. Among them, GB-1 was induced most strongly. Purified GB-1 was homogeneous on polyacrylamide gel electrophoresis and showed an approximately 1,400-fold increase of specific activity over the starting material. The molecular weight was determined to be 240,000 by sodium dodecyl sulfate-gel electrophoresis. A similar value was also obtained by sucrose density gradient centrifugation. The enzyme contained a high proportion of acidic amino acids and mannose, and the isoelectric point of the enzyme was pH 4.15. The enzyme had a pH optimum of 5.5 and a temperature optimum at 55 degrees C. beta-Glucosidase activity was inhibited by Mn2+, Cu2+, Hg2+, p-chloromercuribenzoate, and glucono-delta-lactone. The enzyme split off glucose units one by one from the nonreducing ends of not only beta-glucooligosaccharides but also some beta-glucans, such as carboxymethylcellulose, laminaran, pustulan, and zeagallan. The affinity for cello- and laminari-oligosaccharides tended to increase in parallel with the chain length.     

Membrane Permeability of Conidia of Bipolaris oryzae

Germinability of the conidia of B. oryzae, after an efflux of substances — both electrolytes and non-electrolytes — was tested in sterilized solutions of plasmolytica and osmotica, ranging from molarity to 10^?12 M. Maximum percent germination was recorded at 10^?8 M concentration regardless of the exogenous supplement in the order KCl >D-glucose >Mannitol >NaCl. Germination was very poor or non-existent in other, higher or lower molarities tested except at 10^?7 M wíth c. 50% germination. In nature the conidia, though nutrient independent, are subject to leaching and likely to lose viability; hence the possible role of the proper osmoticum, basing on the observed fact, in maintenance of membrane equilibrium and germinability of the leached conidia has been discussed.     

米根霉孢子凝聚条件的研究

米根霉是食品工业中的一种重要微生物。米根霉能产生多种用于食品工业的代谢产物,其菌丝形态对产物的积累有重要影响。传统上,米根霉的菌丝球形成方式是非凝聚型。本研究通过改变培养基的pH值、糖含量等条件,探讨菌丝球的形成方式。利用不同生长状态下的米根霉孢子,测定相对应的电荷和疏水值,得到控制米根霉孢子凝聚的条件。研究结果表明培养基pH 3.0、葡萄糖320 g/L时,在34℃、200 r/min的培养条件下,米根霉孢子达到了最高的凝聚率88.62%。本研究改变了米根霉菌丝球形成的方式,为米根霉等丝状真菌菌丝球的研究提供了一定的基础和依据。     

Taka-Amylase A in the Conidia of Aspergillus oryzae RIB40

A study of Taka-amylase A of conidia from Aspergillus oryzae RIB40 was done. During the research, proteins from conidia and germinated conidia were analyzed using SDS–PAGE, 2-D gel electrophoresis, Western blot analysis, MALDI-TOF Mass spectrometry, and native-PAGE combined with activity staining of TAA. The results showed that TAA exists not only in germinated conidia but also in conidia. Some bands representing degraded products of TAA were detected. Conidia, which formed on starch (SCYA), glucose (DCYA), and glycerol (GCYA) plates, contained mature TAA. Only one active band of TAA was detected after native-PAGE activity staining. In addition, TAA activity was detected in cell extracts of conidia using 0.5 M acetate buffer, pH 5.2, as extraction buffer, but was not detected in whole conidia or cell debris. The results indicate that TAA exists in conidia in active form even when starch, glucose, or glycerol is used as carbon source. TAA might belong to a set of basal proteins inside conidia, which helps in imbibition and germination of conidia.     

Conidia of Aspergillus oryzae as Naturally Immobilized Phosphatases

The following enzymes were detected in conidia of Aspergillus oryzae var. No. 13 that were collected from a soy sauce factory: phosphatases acting nonspecifically on nucleotides; ribosidase(s) acting on adenosine, inosine, guanosine and xanthosine; and deaminases acting on guanine, adenosine, guanosine, cytidine, AMP and GMP. Most phosphatase activity was found to be bound firmly to the conidia. Thus, when a nucleotide solution was passed through a conidia-containing column, the corresponding nucleoside was recovered as a clear and colorless solution. The bound phosphatases were rather stable at pH 4 to 9 and at temperatures below 50°C. The optimum conditions for activity were at about 45°C and at pH about 5 and 8. When UMP·Na₂ solution was passed through a column containing 5 g of the conidia, about 200 mg of substrate was hydrolyzed per 1 hr. Conidia of molds belonging to the genus Aspergillus may be industrially employed as naturally immobilized phosphatases.     

Substrate specificity of Aspergillus oryzae family 3 beta-glucosidase

Among glycoside hydrolases, beta-glucosidase plays a unique role in many physiological and biocatalytical processes that involve the beta-linked O-glycosyl bond of various oligomeric saccharides or glycosides. Structurally, the enzyme can be grouped into glycoside hydrolase family 1 and 3. Although the basic ("retaining, double-displacement") mechanism for the catalysis of family 3 beta-glucosidase has been established, in-depth understanding of its structure-function relationship, particularly the substrate specificity that is of great interest for developing the enzyme as a versatile biocatalyst, remains limited. To further probe the active site, we carried out a comparative study on a family 3 beta-glucosidase from Aspergillus oryzae with substrates and competitive inhibitors of different structures, in attempt to evaluate the site-specific spatial and chemical interactions between a pyranosyl substrate and the enzyme. Our results showed the enzyme having a strict stereochemical requirement (to accommodate beta-d-glucopyranose) for its "-1" active subsite, in contrast to its family 1 counterpart.     

Ribosomes in Dormant and Germinating Conidia of Aspergillus oryzae

Ribosomes were isolated from dormant and germinating conidia of Asp. oryzae No. 13. The ribosomes which consisted of 80 S were easily dissociated into 40 S and 60 S in low Mg^{+ +} buffer. Polyribosomes were not found in dormant conidia, but were found in germinating conidia. Ribosomes in Aspergillus fungi consisted of almost equal amount of RNA and protein, and the base compositions of RNA were alike, as compared as ribosomal RNA between dormant and germinating conidia.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Studies on the bacterial spore coat. 6. Effects of alkali extraction on the spore of Bacillus thiaminolyticus

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three distinct layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and then the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.