Influence of nutritional factors on the protease production by Leuconostoc oenos from wine

Leuconostoc oenos X₂L isolated from wine secretes two proteases (I and II) into the medium. Growth and protease production were found to be dependent on the composition of the medium. Both proteases were subject to control by ammonium ions and amino acids that repressed their production and the effectiveness was higher on the enzyme II synthesized at the end of bacterial growth. The enzymes were also differently affected by the inclusion of sodium phosphate in the basal medium.     

Amylase production by three Lactobacillus strains isolated from chicken crop

Three amylolytic Lactobacillus strains designated LEM 220, LEM 207 and LEM 202 were isolated from the chicken crop. They belonged to the subgenus Thermobacterium. Strain LEM 220 resembled Lact. acidophilus. Amylase production was more abundant in cells grown in media containing amylopectin or starch than in media containing glucose or maltose. Optimum pH and temperature of the amylase were 5.5 and 55°C respectively. Hydrolysis of amylopectin gave maltose, maltotriose and small amounts of glucose. Strain LEM 207 also resembled Lact. acidophilus , but differed from strain 220. It had a lower amylase activity. Optimum pH and temperature of the amylase were 6.4 and 40°C, respectively, and hydrolysis of amylopectin gave maltose, maltotriose and carbohydrates higher than maltopentaose. Strain LEM 202 was similar to Lact. vitelinus. It had the lowest amylase activity which was increased only in presence of maltose. Amylase properties were similar to those of LEM 220.     

Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

Effect of cultivation conditions on proteinase production byLactobacillus murinus

Proteinase production byLactobacillus murinus was influenced by temperature, glucose concentration, initial pH and nitrogen sources. Maximum proteinase production occurred at 45°C, pH 6.6 and with 0.5 % (W/V) glucose. Tryptone, peptone and gelatin inhibited it. Member of theScientific Researcher’s Career of theConsejo Nacional de Investigaciones Cientificas y Técnicas (Conicet), Argentina.     

Arginine metabolism inLactobacillus leichmannii

Lactobacillus leichmannii ATCC 4797 metabolizes arginine via the arginine dihydrolase pathway producing ornithine, ammonia, carbon dioxide, and ATP. The specific activities of arginine deiminase and ornithine transcarbamylase were two-or threefold lower (stationary growth phase) in galactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. When glucose was virtually exhausted from the medium, maximum activities of both enzymes were achieved. The formation of two first enzymes of the arginine dihydrolase pathway inL. leichmannii ATCC 4797 seems to be under the control of two processes: induction by arginine and repression of the induced synthesis by glucose.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Purification and partial characterization of Oenococcus oeni exoprotease

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.