Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Identification and synthesis of acyl-phosphatidylglycerol in Acinetobacter sp. HO1-N

A minor phospholipid from Acinetobacter sp. HO1-N was identified as acyl-phosphatidylglycerol. Acyl-phosphatidylglycerol synthesis by outer-membrane preparations appeared to be a result of phospholipase A activity.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Activity of glycolysis enzymes in cyclosporine-producing Tolypocladium sp.]

Enzymes of various glycolysis stages, i.e. hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, were detected in cyclosporine-producing organisms belonging to Tolypocladium. The initial activity of the enzymes in the highly active strain was much higher than that in the starting low active strain. During the fermentation the activity of the glycolysis enzymes per 1 mg of mycelium protein in the both strains increased. This was accompanied by a decrease in respiration activity. Therefore, there was a direct correlation between cyclosporine biosynthesis and glycolytic activity of the mycelium in Tolypocladium sp.     

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.     

Acinetobacter marinus sp. nov. and Acinetobacter seohaensis sp. nov., isolated from sea water of the Yellow Sea in Korea

Two Gram-negative, nonmotile, coccobacilli, SW-3T and SW-100T, were isolated from sea water of the Yellow Sea in Korea. Strains SW-3T and SW-100T contained ubiquinone-9 (Q-9) as the predominant respiratory lipoquinone and C18:1 omega9c and C16:0 as the major fatty acids. The DNA G+C contents of strains SW-3T and SW-100T were 44.1 mol% and 41.9 mol%, respectively. A neighbor-joining tree based on 16S rRNA gene sequences showed that the two isolates fell within the evolutionary radiation enclosed by the genus Acinetobacter. Strains SW-3T and SW-100T exhibited a 16S rRNA gene similarity value of 95.7% and a mean DNA-DNA relatedness level of 9.2%. Strain SW-3T exhibited 16S rRNA gene sequence similarity levels of 93.5-96.9% to the validly described Acinetobacter species and fifteen Acinetobacter genomic species. Strain SW-100T exhibited 16S rRNA gene sequence similarity levels of less than 97.0% to the other Acinetobacter species except Acinetobacter towneri DSM 14962T (98.0% similarity). Strains SW-3T and SW-100T exhibited mean levels of DNA-DNA relatedness of 7.3-16.7% to the type strains of some phylogenetically related Acinetobacter species. On the basis of phenotypic, phylogenetic, and genetic data, strains SW-3T and SW-100T were classified in the genus Acinetobacter as two distinct novel species, for which the names Acinetobacter marinus sp. nov. (type strain SW-3T=KCTC 12259T=DSM 16312T) and Acinetobacter seohaensis sp. nov. (type strain SW-100T=KCTC 12260T=DSM 16313T) are proposed, respectively.     

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.     

Induction of the collagenase phorbol ester response element by staurosporine

The indol alkaloid staurosporine is a potent inhibitor of protein kinase C, but has also been shown to have certain effects paradoxically similar to those of protein kinase C–activating phorbol esters. We show here that collagenase mRNA expression is stimulated by 10 nM staurosporine in normal and ras-oncogene–transformed rat fibroblasts. The kinetics of collagenase mRNA induction by staurosporine were slow compared to induction by phorbol ester. Staurosporine induction of the collagenase promoter appeared to be mediated via the TPA response element (TRE). Induction did not involve any increase in jun mRNA expression and did not require expression of c-Jun. Prolonged treatment with phorbol ester to deplete protein kinase C did not inhibit stimulation of the collagenase promoter by staurosporine. Instead, involvement of cAMP-dependent protein kinase (PKA) was indicated by inhibition of staurosporine induction by the PKA inhibitor H-89. In addition, raised levels of cAMP were observed during the first hour of staurosporine treatment. Altogether, our data indicate that staurosporine induces a PKA-dependent pathway leading to c-Jun–independent activation of the collagenase TRE element. © 1994 Wiley-Liss, Inc.     

Degradation of p-benzyloxyphenol by Acinetobacter sp.

Abstract Acinetobacter sp. utilized p -benzyloxyphenol as sole carbon source and degraded it to p -hydroxybenzaldehyde, p -hydroxybenzoic acid, protocatechuic acid and catechol. The intermediates were identified by paper chromatography, TLC, IR, GC and HPLC. Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase during the degradation of p -benzoloxyphenol.