CONCLUSION

In this thesis 39 strains of North Sea bacteria (Ruegeria sp., Halomonas sp., Mesorhizobium sp., Kokuria sp., actinomycetes, etc.) from the project of marine biotechnology were studied.

Of these 38 strains were screened by biological and chemical methods. Four strains were studied in detail under defined conditions. One strain was studied with regard to its biosynthetic ability to produce peptides using a nonribosomal peptide synthetase (NRPS).

1. Chemical and biological screening of the North Sea bacteria

1) Through systematic and broad variation of the fermentation conditions (temperature, duration and cultivation medium) it was understood that marine bacteria can grow at normal temperature (15 °C − 32 °C) and duration (12 h − 6 days), contrary to the hypothesis that marine bacteria grow more slowly than streptomycetes. But the yield of secondary metabolites is lower (0.01 – 1 mg/L) and can not be raised by changing temperature and duration.

2) The yield of secondary metabolites can be raised by directed supplementing of precursors or addition of adsorption resins (XAD).

3) The culture broth was extracted under different conditions to obtain three crude extracts.

The extraction of acidified filtrate proved to be an important step for marine bacteria.

4) All extracts were screened by biological (agar test against E. coli, B. Subtilis, S. aureus, C.

albicans, three microalgae or interaction screening), chemical (TLC developed with different solvent systems and stained with different staining reagents), physicochemical (HPLC-DAD) approaches. Due to the low productivity of marine bacteria, the HPLC-DAD screening proved to be helpful because of the comparison of the UV-spectra with the database.

5) The biosynthetic potential of the North Sea bacteria was appraised. Most of them produce small aromatic metabolites in low yield.

6) According to the screening results four strains were chosen for further investigation.

2. Metabolites of strain T5

1) The cultivation condition of the strain T5 was optimized for the scale-up fermentation and thereby led to a good yield and reproducible production of tropodithietic acid (11).

2) The 6-fold increase of yield of tropodithietic acid (11) was carried out by the supplementation of phenylalanin and histidine to the medium of strain T5 (from 1 mg/L to 6 mg/L).

3) The isolation and purification procedure of tropodithietic acid (11) has been simplified.

Thereby the strong adsorption of this compound on silica gel and Sephadex LH-20 has been avoided.

4) The structure of tropodithietic acid (11) was elucidated by X-ray analysis and spectroscopic data. In this case it was avoided to arrive at a wrong conclusion (thiotropocin 13) due to their similar physicochemical data.

5) The solvent complex effect of DMSO with tropoditietic acid (11) was observed and documented.

6) As a minor component hydroxytropodithietic acid (0.04 mg/L) was isolated. Its structure was elucidated by X-ray analysis.

7) Tropodithietic acid (11) was biologically tested against bacteria, fungi, different tumor cell lines, saline shrimps, nematode and ion channel activities. It showed activities in all assays except for the ion channel.

8) The biosynthetic pathway for tropodithietic acid (11) is supposed.

9) The biosynthetic potential of strain T5 was studied by variation of nutrients. Anthranilic acid and its ethyl ester were isolated as major components from peptone medium supplemented with tryptophan and histidine.

3. Metabolites of strain RK377

1) From the shaking culture on the MB medium, isatin (33) and 3-(3-hydroxy-2,3-dihydro-1H-indol-3-yl)-2-oxo-propionic acid (34) were isolated and characterized by spectroscopic methods and X-ray analysis, respectively.

2) From 40 L-fermentation of strain RK377 in the MB medium in the artificial seawater, three new natural products, namely 3-(4’-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (35), 3,4-di(4’-hydroxyphenyl)pyrrole-2,5-3-(4’-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (36), 7-hydroxy-2H-benzo[1,4]thiazin-3-one (37), were identified. Additional four indole derivatives, namely acetic acid (41), carboxylic acid (39), indole-3-carboxaldehyde (40), 3-hydroxyacetylindole (38), and also phenylacetic acid (42), bis (2-ethylhexyl)phthalate (43) were isolated and characterized by spectroscopic methods and X-ray analysis.

3) From 40 L-fermentation of strain RK377 on SJ medium, two new imidazol and pyrimidin derivatives, namely glusun I (44) and gluson II (46) were isolated and characterized by spectroscopic methods. Because of the low yields the structures are proposals only.

4) All isolated compounds of strain RK377 were tested against E. coli, B. subtilis, S. aureus, and C. albicans. No activities have been observed so far.

5) The putative biosynthesis approaches of 3-(4’-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (35) and 3,4-di(4’-hydroxyphenyl)pyrrole-2,5-3-(4’-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (36) were discussed.

6) The biosynthetic potential of strain RK377 was proved by the isolation of different metabolites from the culture broth using different mediums.

4. Investigation of strain RK2207

From a 10 L-fermentation of strain RK2207 the new bacteriopheophytin aL (51) and two bacteriocarotines (52 and 53) were isolated and characterized by spectroscopic methods.

5. Investigation of strain H260, which was selected by genetic screening for NRPS

1) Using the OSMAC approach strain H260 was cultivated in different mediums and variation of the cultivation duration. The extracts of their mycelium, culture filtrate and acidified culture filtrate were screened by biological assays against E. coli and three different microalgae, and chemical screening and HPLC-ESI-MS-MS experiments.

2) The cell extract from a peptone medium supplemented with or without glucose showed the strongest activities against E.coli and microalgae.

3) The detection of polar peptides using a chemical screening approach was not very effective, because the detection was disturbed by the constituents of the cultivation mediums.

4) The HPLC-ESI-MS-MS method was used to prove the presence of peptides. Six diketopiperazines were characterized, namely cyclo-(Pro-Val) (54), cyclo-(Pro-Leu) (55, two stereoisomers), cyclo-(Phe-Pro) (56, two stereoisomers), cyclo-(Tyr-Pro) (57).

5) A bioactive component was assigned to the zone on the TLC by bioautography and to the peak of the HPLC chromatogram by the “peak survey” method. It is known that this bioactive component is very polar and unstable.

6) From 50 L-fermentation of this strain one peptide was isolated and characterized by spectroscopic methods. Additionally the strain produces indoleacetic acid as the major component.

7) The production of the peptide has proved the presence of NRPS in strain H260. This result shows that the NRPS-screening is a valuable tool to find producers of NRPS.