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The action of colchicine solutions on a diploid Saccharomyces vini culture used in viniculture yielded three polyploid strains. The strains differ from the parent culture in their cell morphology and a higher endopolygalacturonase activity.
After a screening performed on pectinolytic micro-organisms, a strain of B. subtilis was isolated. This strain has a pectic enzyme capable of beta-elimination on highly methylated pectin (degree of esterification = 85%), without the action of a pectinesterase. This activity corresponds to that of a pectin-lyase.
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A group of enzymtic preparations, mainly of the Soviet make were investigated for the presence of antibiotic substances admixtures. Altogether 25 preparations obtained from molds, actinomycetes, bacteria and yeast were studied. The results showed that 18 preparations contained antibiotic substances in an amount of 0.6-1300 U, equivalent to oxytetracycline units per 1 g. Most often antibiotics were present in preparations of the pectinase and protease group. In the group of glucoamylase the antibiotic activity was uncovered in a single preparation. Procedures employed in determining the antibiotic activity of enzymatic preparations is described and the possible role of antibiotics in the manifestations of their toxic properties discussed.
Several strains of Pseudomonas aeruginosa were investigated for pathogenicity to plant tissue. Inoculations into the intercellular spaces of leaves of Vicia faba (28 degrees C) and Vinca rosea (36 degrees C) showed negative results. In leaves of Nicotiana tabacum (32 degrees C) the bacteria multiplied; the tissue reacted to form partial lesions and necrosis. Neither in culture filtrates of the bacteria nor in eluates of inoculated leaves pectolytic and cellulolytic enzymes or breakdown products of these could be obtained. Whilst the proteolytic activity was high, symptoms for effects on the plant tissue, however, were not observed. Although this bacterium may be able to multiply after injection into tobacco leaf, the general question about pathogenicity to plants must be denied.
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Polysaccharases release microorganisms from their natural seat, marine sediments for example. The enzymatic activity works both on the microbial adherence polysaccharides and on the support surfaces (cellulose, pectine, etc.). Dosages of glucose confirm polysaccharase activity. An association of bacitracine, thiophenicol and a few enzymes: cellulase, pectinase, amyloglucosidase, alpha amylase, hyaluronidase, release a considerable number of bacteria. The culture on specific mediums confirm the specificity of this release. E. coli polyresistant strain where isolated by amylo-glucosidase, glucuronidase association in a mixture of thiophenicol and bacitracine. Bacillus and other Gram positive bacteria are frequently isolated by this method. The number of colonizer microorganisms on solid media are considerably higher with sediments treated by enzymes, or by enzyme, antibiotic mixtures, than with untreated ones.
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The production of exohydrolases (alpha-amylase and pectinase) by fungi belonging to the genus Aspergillus was studied in the course of batch cultivation and, if immobilized cells were used, in the semicontinuous regime of growth. The cells were immobilized on a fixed filtering plate and on floating, in the growth medium, polyhedrons. Such a cultivation of immobilized microbial cells in the semicontinuous regime of growth on submerged polyhedrons freely floating in the nutrient medium makes it possible to cultivate the cells for 1.5 months with the active production of exocellular hydrolases. Under these conditions, Aspergillus oryzae 3-9-15 produces more alpha-amylase and A. awamori synthesizes more pectinases.
The ability of the two cultures Penicillium sp. and Fusarium sp. to synthesize extracellular pectolytic enzymes was investigated. The cultivation conditions providing a high level of the biosynthesis of these enzymes were identified. The methods of isolating the enzymes by means of organic solvents were developed. The pectinase from Penicillium sp. showed a higher thermostability whereas that from Fusarium sp. displayed a greater acid resistance. Using glutaraldehyde and titanium salts, active immobilized forms of pectinases on silica carriers were prepared.
Immobilization of pectawamorine G10x on silochromes, using cyanuric chloride, 2,4-toluylene diisocyanate, glutaric dialdehyde, thionyl chloride, phosphorus tribromide, titanium tetrachloride, zirconium oxychloride and hafnium oxychloride was studied. The use of glutaric dialdehyde assured the strongest binding and the preatest stability of activity. Properties of the native pectawamorine G10x and immobilized preparations were studied on a comparative basis. Pectawamorine G10x immobilized by means of hafnium oxychloride showed increased stability when stored at 5 degrees C and used repeatedly. In every case, except for cyanuric chloride and glutaric dialdehyde, maximum activity was at a temperature 10 degrees C higher than for the native enzyme, and optimum pH varied for the preparations with different binding reagents.
The localization of acid invertase (AI, EC 3.2.1.26) in tomato fruits was studied. AI was localized in the intercellular fraction (cell wall fraction). A cDNA encoding a wall-bound form of AI from tomato fruits was cloned and its nucleotide sequence was determined. The cloned cDNA was 2363 base pairs long and contained an open reading frame of 1908 base pairs which encoded a polypeptide of 636 amino acids. RNA blot analysis indicated that the mRNA for the acid invertase was about 2.5 kb in length. The levels of the mRNA were low at the mature green stage but increased during ripening of fruit.
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