Two criteria suggest that most of the proteinase of Streptococcus lactis is localized in the cell wall. (i) Intact cells possess proteinase activity when incubated with a high-molecular-weight substrate. (ii) Most of the cell-bound proteinase activity is released during spheroplast formation under conditions which result in the release of only 1% of the intracellular enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase. The solubilized cell wall, plasma membrane, and cytoplasm fractions contained 84, 0, and 16%, respectively, of the total proteinase activity with casein as substrate. The physiological role of a surface-bound proteinase in this organism is discussed.

Localization of Proteinase(s) near the Cell Surface of Streptococcus lactis

Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii.

Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2

A physical model of high-pressure disruption of bakers' yeast cells

The highly radioresistant Rubrobacter radiotolerans, contains red pigments. Since the pigments could not be extracted by usual methods, a new method was developed in which the pigments were extracted with organic solvents after addition of 10 N KOH to the intact cells, followed by neutralization. These pigments were also extracted after treatment with achromopeptidase, but not with lysozyme. The extracted pigments separated into two main spots by TLC (48.6% and 22.6%), and were confirmed to be carotenoids by chemical tests. The two major pigments had 13 conjugated double bonds as determined from the main maximum wavelength of the light absorption spectra. Their molecular weights were determined to be 740 and 722 by mass spectrometry. The mass spectra of their TMS-derivatives revealed that they contained four and three tertiary OH groups, respectively. Confirming their identical light and IR spectra, these pigments were determined to be bacterioruberin and monoanhydrobacterioruberin, respectively, the characteristic carotenoids of halophilic bacteria. The existence of these pigments in bacteria other than halobacteria provides interesting new evidence on the distribution of these compounds.

Pigments of Rubrobacter radiotolerans

The release constant, k, of brewers yeast sonicated at powers up to 200 W at 20 kHz has been shown to be independent of cell concentration up to values of 60 g made up to 100 ml. It is inversely proportional to the volume of the treatment vessel in the range 75 to 450 ml, and almost proportional to the input acoustic power from 60 to 195 acoustic watts. A flow system is described and a relationship linking protein release, flow rate, and the protein release constant, determined from batch experiments, is derived. Good agreement between the theoretical prediction of protein release and experimental results with the flow system was obtained.

Kinetics of protein release from yeast sonicated in batch and flow systems at 20 kHz

Publisher Summary This chapter describes physical and chemical methods for disintegrating microbes for preparing biologically active fractions. Direct measurement of cell disintegration can be sometimes dispensed with and the criteria of yield of the desired enzyme activity be relied on, especially when one of the chemical methods of extraction is used. In any comparative work and where control or location is studied by means of a physical method of disruption or with spheroplasts, some method of assessing disintegration is essential. Estimation of cell disintegration by measuring the release of typical cell components can be made quantitatively more precise than direct counting. There is some difficulty in interpretation and in obtaining a normal zero time and 100% standards. For instance, the measurement of the release of soluble protein is often used. This depends partly on the degree of disruption, but also on the method used to separate soluble protein from insoluble debris.

Chapter I The Disintegration of Micro-organisms

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Chapter I The Disintegration of Micro-organisms