The inulinase-active whole cells of yeast Kluyveromyces marxianus were immobilized by entrapment into agar gel with 76% retention of the enzymatic activity. The enzymatic properties of the immobilized cells were investigated and compared with those of free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation. The optimum pH of both free and immobilized cells was 6. The optimum temperature for inulin hydrolysis with the immobilized cells was 5°C higher than that with the free cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 25.62kJ/mol and 13.27kJ/mol, respectively. Km values were 8.3mM inulin for the free cells and 10.5mM for the immobilized cells. Free and immobilized cells showed fairly stable activities between pH 4 to 7 but free cell inulinase was more labile at other pH values compared to the immobilized counterpart. Thermal stability of the enzyme was improved by immobilization. There was no loss of activity of the immobilized cells on storage at 4°C for 30 days.
Hydrolysis of inulin from Jerusalem artichoke tuber extract by cells of the yeast Kluyveromyces marxianus immobilized in agar has been studied in batch and packed bed bioreactor systems. In a batch reactor using around 43 units of enzyme activity, about 94% hydrolysis of the total potential sugars was achieved in 10hr. The product consisted of about 90% fructose and 10% glucose. The same immobilized cells were used repeatedly for 9 cycles starting with fresh juice at the beginning of each cycle. The activity of the immobilized cells remained more or less constant during the first 5 cycles but beyond that it started to decrease. The maximum conversion rate obtained after 9 batch cycles was about 48% of the original. In column reactors, packed with 103 units of enzyme activity, the conversion rate remained almost constant at dilution rates lower than 0.34hr-1 but decreased beyond 0.34hr-1. The productivity at dilution rates D=1.1hr-1 and 0.61hr-1 was calculated to be 46g•l-1 hr-1 and 31g•l-1•hr-1 respectively and the corresponding conversion rates were 76 and 95%. Continuous operation of the column at a constant dilution rate of D=0.61hr-1 for 15 days at 45°C, resulted in 43% loss of the original activity. The half life of the bioreactor was estimated to be 17 days.
A taxonomic study of the genera Lipomyces LODDER et KREGER-VAN RIJ and Myxozyma VAN DER WALT, WEIJMAN et VON ARX was made by comparing the electrophoretic patterns of nine enzymes. The enzymes studied were glucose-6-phosphate dehydrogenase (EC 126.96.36.199), glutamate dehydrogenase (EC 188.8.131.52), malate dehydrogenase (EC 184.108.40.206), lactate dehydrogenase (EC 220.127.116.11), fumarase (EC 18.104.22.168), tetrazolium oxidase, catechol oxidase (EC 22.214.171.124), esterase (EC 126.96.36.199), and glutamate-oxaloacetate-transaminase (EC 188.8.131.52). Lipomyces lipofer LODDER et KREGER-VAN RJI and Lipomyces tetrasporus NIEDWDORP, BOS et SLOOFF had a uniform pattern within each species. On the other hand, Lipomyces kononenkoae NIEUWDORP, BOS et SLOOFF and Lipomyces starkeyi LODDER et KREGER-VAN RIJ each varied considerably in their enzyme patterns. Though about 50% similarity was observed between L. starkeyi and L. tetrasporus, five Lipomyces species, Lipomyces anomalus BABJEVA et GORIN, L. lipofer, L. kononenkoae, L. starkeyi, and L. tetrasporus, differed from each other in their enzyme patterns. Myxozyma melibiosi (SHIFRINE et PHAFF) VAN DER WALT, WEIJMAN et VON ARX and Myxozyma mucilagina (PHAFF, STARMER, MIRANDA et MILLER) VAN DER WALT WEIJMAN et VON ARX each had a uniform pattern which differed from the five Lipomyces species. The enzyme patterns of six Myxozyma sp. strains were dissimilar to those of two Myxozyma species. One strain of Myxozyma sp. and two strains of L. starkeyi showed about 60% similarity. The enzyme patterns of other Myxozyma sp. strains were dissimilar to those of the five Lipomyces species.
Methods for increasing the productivity of thermostable amylase of Bacillus licheniformis were investigated. Cells of B. licheniformis NYK 74 were treated with N-methyl-N′-nitro-N-nitrosoguanidine resulting in mutants which were resistant to D-cycloserine. These were selected first and among them amylase hyper producers were selected. By a series of the same treatments, except that the concentration of D-cycloserine was increased step by step, a strain was developed which produced 2, 000 times more amylase than the original one.
The effects of different concentrations of nickel (2.1, 4.2, 5.04μM) and silver (0.013, 0.026, 0.052μM) on survival, growth, carbon fixation and acetylene reduction of Nostoc muscorum has been studied. All concentrations of the heavy metals except 2.1μM Ni were inhibitory. At 2.1μM, nickel chloride stimulated carbon fixation and nitrogenase activity. Silver seems to be much more toxic than nickel because concentrations as low as 0.026μM silver chloride inhibited approximately 90% of the carbon fixation. EDTA protected carbon fixation and calcium ameliorated the nitrogenase activity in the test alga. However, none of these ameliorative agents were so effective against silver toxicity. This study suggests that (i) calcium plays an imporatnt role in nitrogen fixation, (ii) like hydrogen production and uptake hydrogenase, nickel seems to have some role in regulating nitrogenase activity, and (iii) carbon fixation is a more sensitive parameter than growth and nitrogensase in evaluating metal toxicity.
The phosphorylation of a cellular protein of 46, 000 daltons was stimulated by the addition of adenosine 3′, 5′-cyclic monophosphate (cAMP) in the sexually fertile dikaryotic and Amut Bmut strains of Coprinus cinereus, but not in the sexually sterile Amut, Bmut, and wild-type monokaryons. Cyclic AMP-dependent inhibition of glutamate dehydrogenase was observed only in the dikaryotic and Amut Bmut strains, but not in the Amut, Bmut, and monokaryotic strains. Extremely low levels of ATP and cAMP were sufficient to bring about half maximal inactivation of glutamate dehydrogenase. The results indicate that glutamate dehydrogenase is an endogenous substrate for cAMP-dependent protein kinase which is regulated by the incompatibility factors.
Immunological cross-reactivities among intracellular proteins, sulfite reductase and hydrogenase, and somatic antigens of Desulfovibrio strains were investigated. Antisera were prepared against purified sulfite reductases of Desulfovibrio vulgaris, strain Miyazaki K (MK), and strain Miyazaki F (MF). In double diffusion test, these two antisera (γ-globulin fractions) gave a single fused precipitin line or sometimes with a spur, or crossing. These variants occurred with crude extracts from D. vulagaris, strains MK, MF, Miyazaki Y (MY), and HildenboroughT (subsp. vulgaris), D. desulfuricans subsp. desulfuricans, strains Essex 6T, NRC 49001, and Norway 4, D. gigas NCIB 9332T, D. africanus BenghaziT, and D. salexigens British GuianaT, as well as with the purified enzymes from MK and MF. The relationship among these sulfite reductases is discussed considering the pattern of spur formation. Sulfite reductase activity in crude extracts from these strains was decreased by adding the two antisera, though the levels of decrease varied with strains. Antiserum against hydrogenase from MK cross-reacted with hydrogenases from strains Hildenborough and NRC 49001 but not with those from the other strains. Agglutination tests with antisera prepared against somatic antigens of the three strains, MK, Hildenborough, and MF, revealed that cross-reaction among strains was rather restricted. The three strains, MK, Hildenborough, and NRC 49001, shared common somatic antigens and the two strains, MF and MY, had others in common, but the cells of the other strains were not agglutinated with these antisera.
Effects of inhibitors of protein synthesis on sporulation in Saccharomycescerevisiae were analyzed kinetically to determine whether the sporulation requires the synthesis of short-lived proteins (labile proteins). Pulse-treatment with cycloheximide at low concentrations (0.1-10μM) caused marked delay in spore formation without any significant effect on sporulation synchrony. The delay in spore formation greatly exceeded the length of pulses. A similar effect on spore formation was caused by treatment with puromycin. The delay in spore formation was brought about only by cycloheximide pulses carrried out during meiosis I. In the pulse-treated cells, meiotic progression during meiosis I was markedly delayed. The subsequent meiotic progression to telophase II proceeded without any significant delay, but the duration of spore wall formation after the progression to telophase II was prolonged. The increase in activities of fumarase and malate synthase which occurred during meiosis I was not affected by the cycloheximide pulse, and the levels of those activities were retained rather more stably in pulsetreated cells than in untreated cells. These results suggest that labile proteins are synthesized during meiosis I and play some roles during meiosis 1 in the progression of meiosis and in the events preparatory for spore wall formation.
The slime streamers from acid mine drainage water were examined using both scanning and transmission electron microscopes. The acid streamers were composed of innumerable chains of bacterial rods which had a multilayered cell envelope characteristic of gram-negative bacteria. The slime matrix was made up of densely interwoven microfibrils of extracellular acid polysaccharides which stained positively with ruthenium red. The microfibrils varied considerably in thickness and were woven in various patterns in the electron micrographs of thin sections. Characteristic architecture such as lattice structure of electron-dense dots and surface projections were observed in the bacterial cell envelopes. Bacteriophages occurred in the slime matrix of the acid streamers.
Forty nine strains of 12 species of purple nonsulfur phototrophic bacteria (Rhodospirillaceae) and one strain of facultatively methylotrophic bacteria, Protomonas extorquens were investigated with regard to their quinone systems and cellular fatty acid composition. These bacteria had the ubiquinone systems. Some strains had in addition a small amount of menaquinone system, which had the same isoprenoid chain length as the major ubiquinones. The strains in the genera Rhodobacter, Rhodomicrobium, Rhodospirillum, Rhodopseudomonas except for Rhodopseudomonasviridis, and Protomonas extorquens had Q-10 as a major component. Out of the above mentioned strains, the strains of Rhodopseudomonasacidophila also had MK-10 concurrently. The strains of Rhodopila globiformis and Rhodopseudomonas viridis had Q-9 and MK-9. Most of the Rhodocyclus gelatinosus strains had Q-8 and MK-8. The strains of Rhodocyclus gelatinosus contained large amounts of C16:0 acid and C16:1 acid. The strains in the genera Rhodopseudomonas except for Rhodopseudomonasacidophila, Rhodomicrobium, Rhodobacter, and Rhodopila, and in Protomonas extorquens, contained a large amount of C18:1 acid. The strains of Rhodospirillum rubrum, and Rhodopseudomonas acidophila contained large amounts of C16:0 acid, C16:1 acid, and C18:1 acid. Based on these results along with information on other taxonomic properties such as morphology and phylogeny, the relationship of the facultatively methylotrophic bacterium Protomonas extorquens to the species of Rhodospirillaceae genera is discussed.