Purification and structure determination of four bioactive molecules from a newly isolated Streptomyces sp. TN97 strain

Purification and structure determination of four bioactive molecules from a newly isolated Streptomyces sp. TN97 strain

Process Biochemistry 41 (2006) 1506–1513 www.elsevier.com/locate/procbio Purification and structure determination of four bioactive molecules from a ...

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Process Biochemistry 41 (2006) 1506–1513 www.elsevier.com/locate/procbio

Purification and structure determination of four bioactive molecules from a newly isolated Streptomyces sp. TN97 strain Raoudha Ben Ameur Mehdi a,b, Samiha Sioud a, Lilia Fourati Ben Fguira a, Samir Bejar a, Lotfi Mellouli a,* a

Laboratory of Prokaryotic Enzymes and Metabolites of Sfax-Tunisia, P.O. Box K, 3038 Sfax, Tunisia Laboratory of Applied Chemistry of the Faculty of Sfax, P.O. ‘K’, 3038 University of Sfax, Tunisia

b

Received 6 October 2005; received in revised form 18 January 2006; accepted 20 February 2006

Abstract A new actinomycete strain called TN97 was isolated from Tunisian oasis soil and selected for its antimicrobial activities against bacteria and fungi. Cultural characteristic studies strongly suggested that this strain belongs to the genus Streptomyces. Nucleotide sequence of the 16S rRNA gene (1508 pb) of the TN97 strain exhibited high similarity to the Streptomyces 16S rRNA genes. This similarity reaches 96% with the 16S rRNA gene of Streptomyces lipmanii. In liquid medium, maximal biological activity production was obtained when glycerol or fructose were added at 1% (w/v). Extraction of 10 l fermentation broth of the Streptomyces sp. TN97 strain, grown in tryptic soy broth (TSB) medium supplemented with 1% (w/v) of glycerol, and various separation and purification steps, led to the isolation of four pure bioactive molecules. The chemical structure of these compounds was established on the basis on their IR, ESI-MS and EI-MS, 1H and 13C/APT NMR data and by comparison with reference data from literature. These compounds which are produced simultaneously by the Streptomyces sp. TN97 belong to three different families: two diketopiperazine derivatives [cis-cyclo (Leucyl-Prolyl) and cis-cyclo (L-phenyl, L-prolyl)], an isocoumarin derivative (the 6,8-dihydroxy-3methylisocoumarin) and the N-acetyl-tyramine. # 2006 Elsevier Ltd. All rights reserved. Keywords: Biological activities; Streptomyces sp. TN97; Purification; Structure elucidation

1. Introduction The development of resistance to multiple drugs is a major problem in the treatment of infections by pathogenic microorganisms. This antimicrobial resistance is presently an urgent focus of research and new bioactive compounds are necessary to combat these pathogens. About 70% of all known drugs have been isolated from actinomycetes bacteria of which 75% and 60% are used in medicine and agriculture, respectively [1,2]. This fact has made Streptomyces the most used bacteria in fermentation manufacturing of active pharmaceutical compounds. Streptomyces are Gram-positive bacteria, characterized by a complex morphologic differentiation cycle accompanied by the production of numerous extracellular enzymes as well as, many kinds of bioactive secondary metabolites having great

* Corresponding author. Tel.: +216 74 440 451; fax: +216 74 440 451. E-mail address: [email protected] (L. Mellouli). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.02.010

structural and functional diversity [3], including antibiotics, antifungal, antiviral, anticancer, immunosuppressant agents, insecticides, herbicides, etc. In the course of screening for new bioactive compounds, several research studies are currently oriented towards the isolation of new Streptomyces species from uncommon habitats. To select new Streptomyces species, several methods have been developed to identify this genus of bacteria. These include selective plating technique [4], construction of genetic marker systems [5], a combination of chemical markers, the presence of L,L-diaminopimelic acid and the absence of characteristic sugars in the cell wall [6]. In addition, 16S rRNA sequence data have proved invaluable in streptomycetes systematic, in which they have been used to identify several newly isolated Streptomyces species [7–9]. Bioactive molecules are secondary metabolites nonessential for growth and reproduction but form presumably a defence mechanism to the producer microorganism to compete in nature. Secondary metabolites are synthesized by pathways, which are often connected and influenced by primary metabolism. In fact,

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the intermediate metabolites from primary metabolism, serve as precursors for biosynthesis of secondary metabolites and the composition of the culture medium, closely connected with the metabolic capacities of the producing organism, greatly influences the biosynthesis of the bioactive molecules [8–11]. These active molecules are generally extra cellular and their isolation in highest purity from the complex fermentation broth needs the application of a combination of various separation steps such as solvent extraction, chemical precipitation, ion exchange chromatography, HPLC purification, etc. The present paper describes the isolation of a new actinomycete strain called TN97 having antimicrobial activities from the Sahara of the southern region of Tunisia (Tunisian oasis soil). The identification of this strain and the study of the influence of the composition of the culture medium upon biosynthesis of bioactive molecules are reported. The extraction, purification and structure elucidation of four different bioactive molecules from a liquid culture broth of the Streptomyces sp. TN97 strain are addressed as well. 2. Materials and methods 2.1. Bacterial strains plasmids and primers Bacterial strains, plasmids and primers used in this work are listed in Table 1.

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For the isolation of actinomycetes strains having antimicrobial activities, soil and water samples collected from different regions of Tunisia, were spreaded on solid boiled bran barley medium [15]: 0.2% yeast extract and 2% agar were added to a supernatant of a 4% boiled bran barley. The pH was adjusted at 7. After incubation at 37 8C for several days, colonies showing sporulation and filamentous aspect were picked and propagated on the same solid medium. Obtained strains were tested for their capacity to produce antimicrobial activities. For antibacterial activities determination, indicator microorganisms were grown overnight in LB medium at 30 8C for Micrococcus luteus LB14110 and at 37 8C for E. coli ATCC 8739, then diluted 1:100 in LB medium and incubated for 5 h under constant agitation of 200 rpm at the appropriate temperature. For antifungal activities determination, Verticillium dahliae was grown in potato dextrose agar (PDA) for 7 days at 30 8C. Spores were collected in sterile distilled water then adjusted to a spore density of approximately 104 spores ml1. Strain TN97 was grown in tryptic soy broth (TSB) medium for the preparation of genomic DNA [16]. Cultural characteristics of TN97 strain were compared on the basis of observations made after 7, 14 and 21 days incubation on nutrient agar, Sabouraud agar and yeast malt agar media [17]. To investigate the influence of the medium on bioactive molecules production, spores at 107 ml1 were used to inoculate 1000 ml Erlenmeyer flasks with four indents, containing 200 ml of (TSB) supplemented at 1% (w/v) with one of the five tested carbon sources (starch, fructose, glycerol, glucose and saccharose). After incubation at 30 8C for 72 h in an orbital incubator, biological activities were assayed for each culture supernatant. Influence of magnesium, potassium and trace mineral oligoelements on active molecules production was also investigated. The final magnesium and potassium concentration was 2 and 1 mmol l1, respectively. Oligo elements were provided by adding 1.5 ml of a trace element solution (0.4 g l1 ZnCl2; 2 g l1 FeSO47H2O; 0.065 g l1 H3BO3 and 0.135 g l1 MoNa2O42H2O) to 200 ml of growth medium. For kinetics study of TN97 strain, growth was measured as dry weight of the mycelium.

2.2. Culture conditions 2.3. Biological assay of antimicrobial activities Escherichia coli DH5a was grown on Luria-Bertani (LB) plates supplemented with ampicillin (50 mg ml1) and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (40 mg ml1) when appropriate [14]. Transformation of E. coli DH5a with pIJ2925 derivatives was carried out according to Hanahan [12]. Growth and transformation of TOP10 E. coli strain with the pCR-blunt vector derivative were carried out according to the manufacture’s instructions (Invitrogen).

To isolate new actinomycete strains producing antimicrobial activities, we have used the solid media bioassay test against M. luteus LB 14110 (Grampositive bacteria), E. coli ATCC 8739 (Gram-negative bacteria) and V. dahliae. After incubation of the isolated strains for 7 days at the appropriate growth temperature, plates were covered by 3 ml of top agar containing 50 ml of a 5 h

Table 1 Bacterial strains, plasmids and primers used in this work

Strains Streptomyces sp. TN97 E. coli TOP10 E. coli DH5a E. coli M. luteus V. dahliae Plasmids pIJ2925 pCR-blunt vector pSS1 pSS2 and pSS3

Description

References

New isolated strain selected as a potent antimicrobial activities producer and used as the source of chromosomal DNA to amplify the 16S rRNA gene F-mcrA D(mrr-hsdRMS-mcrBC) f80lacZDM15 DlacX74 deoR recA1 araD139D (ara–leu)7697 galU galK rpsL endA1 nupG (host strain) (F f80 dlacZDM15D(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk , mþ k ) deoR thi-1 susE44 l gyrA96 relA1) (host strain) ATCC 8739 (indicator microorganism) LB 14110 (indicator microorganism) (indicator microorganism for antifungal activities determination)

This work Invitrogen [12]

Olive institute Sfax-Tunisia

Derivative of pUC18 (cloning vector) Col E1 origin (pUC-derived) KnR (cloning vector) Derivative of pCR-blunt vector carrying a 1.5 kb DNA fragment corresponding to the whole 16S rRNA gene of the TN97 strain Derivatives of pIJ2925 carrying respectively the 0.85 and 0.65-kb EcoRI-EcoRI DNA fragments corresponding to the whole 16S rRNA gene of the TN97 strain

[13] Invitrogen This work

Primers used for the amplification of the 16S rRNA Gene of the TN97 strain (P1 and P2): P1: 50 -AGAGTTTGATCCTGGCTCAG-30 P2: 50 -AAGGAGGTGATCCAGCCGC-30

[18]

This work

Primers

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culture of M. luteus LB 14110 or E. coli ATCC 8739 test strains, then incubated overnight at 30 8C for M. luteus and at 37 8C for E. coli. For antifungal activities determination, plates were covered by 3 ml of top agar containing 100 ml of spores’ suspension already prepared from V. dahliae. The cell-free supernatant from 50 ml shake flask culture of the selected TN97 strain at 30 8C for 72 h was concentrated to 20 ml using Rotavapor (Laborata 4000), mixed with 20 ml of ethyl acetate and then shaken slowly at 4 8C for 1 h. The organic fraction was evaporated at 40 8C and the resulting dry extract was recuperated in 10 ml of methanol/n-hexane (v/v). The methanol fraction that possesses biological activities was concentrated to 1 ml. A paper disk was impregnated with 60 ml of the corresponding sample and then laid on the surface of an agar plate containing 3 ml of top agar inseeded by 40 ml of an 5 h old culture of one of the used bacteria for antibacterial tests, M. luteus LB 14110 and E. coli ATCC 8739, and by 100 ml of spores suspension of V. dahliae for antifungal activities. After 2 h at 4 8C, plates containing M. luteus and V. dahliae were incubated at 30 8C and those inoculated with E. coli at 37 8C, all for overnight except V. dahliae for 48 h. Plates were examined for evidence of antimicrobial activities represented by a zone of inhibition of growth of the corresponding indicator microorganisms around the selected strains or the paper disk.

2.4. Extraction and purification of active compounds Spores at 107 ml1 of TN97 strain were used to inoculate 1000 ml Erlenmeyer flasks with four indents, containing 200 ml of TSB supplemented with 1% (w/v) of glycerol. After incubation at 30 8C for 24 h in an orbital incubator with shaking at 250 rpm, this pre-culture was used to inoculate (5%, v/v) a total volume of 10 l culture medium having the same composition of the pre-culture. After 3 days incubation at 30 8C in an orbital incubator with shaking at 250 rpm, the culture broth was filtered to separate mycelium and supernatant which were

treated separately as indicated in Fig. 1. The crude extract (1.5 g), having antimicrobial activities, was subjected to various separation steps (Sephadex LH 20, PTLC, silica gel and HPLC). HPLC purification was accomplished by reverse phase semi-preparative (C18 column 7 mm, 4.6 mm inner diameter  25 cm length), the elution was at a flow rate of 1 ml min1 on isocratic conditions with two solutions: 65% A (methanol) and 35% B (water).

2.5. Spectroscopic measurements 1 H NMR spectra were measured on a Varian Inova (300.135 MHz) spectrometer. 13C NMR spectra were measured on a Varian Inova (75 MHz) ESI-MS was recorded on a Quattro Triple Quadrupole Mass Spectrometer, Finnigen TSQ 7000 with nano-ESI-API-ion source. ESI-HRMS was measured on Micromass LCT mass spectrometer coupled with a HP 1100 HPLC with a Diode Array Detector. Reserpin (MW = 608) and Leucin-Enkephalin (MW = 555) were used as standards in positive and negative mode. EI-MS was recorded on a Finnigan MAT 95 (70 eV). Flash chromatography was carried out on silica gel (230–400 mesh). Thin layer chromatography (TLC) was performed on Polygram SIL G/UV254 (Macherey-Nagel & Co.). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co.). For preparative TLC (PTLC) we have used glass plates, 1.5 mm silica gel 60 F254, Merck, Darmstad, Germany. Size exclusion chromatography was done on Sephadex LH-20 (Pharmacia). cis-Cyclo (L-phenyl, L-prolyl) ‘‘A’’. C14H16N2O2 (244). The Rf = 0.63 (CH2Cl2, MeOH 9%). This compound showed a strong absorption under UV light at 254 nm and gives a violet colouration on spraying with anisaldehyde/sulphuric acid. IR spectrum (KBr, n, cm1) 1600, 1630, 3400 cm1. EI/ MS (70 eV): m/z (%) = 244 (M+, 100), 153 (34), 130 (12), 125 (100), 91 (74), 70 (64), 41 (20); (+)-ESI/MS: m/z (%) = 511 ([2M + Na]+, 100), 267 ([M + Na]+, 65); EI/HRMS = 244.1212 (calc. 244.1191).

Fig. 1. Work-up schema for the purification of the active molecules of the Streptomyces sp. TN97 strain.

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Table 2 1 H and 13C NMR data of the four active compounds

2.6. DNA isolation and manipulation

C-no.

Total DNA preparation was carried out from TN97 strain according to Hopwood et al. [16]. Small-scale plasmid preparations from E. coli were performed as described in [14]. Digestion with restriction endonucleases, separation of DNA fragments by agarose gels electrophoresis, dephosphorilation with alkaline calf intestinal phosphate, ligation of DNA fragments and transformation were done according to Sambrook et al. [14] for E. coli and Hopwood et al. [16] for Streptomyces.

1 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 10 20 30 40 50 60

Chemical shifts (d) A

B

C

D

–; 165.20 –; – 3.60–3.70; 45.80 1.90–2.10; 22.60

–; 166.90 –; – –; 155.70 6.47; 102.10 –; 141.80 6.33; 105.90

–; 130.00 7.02; 129.70 6.73; 115.60 –; 155.00

–; 170.30 –; – 3.64–3.54; 45.50 1.98–2.22; 22.70

6.73; 115.60

–; 167.00 6.30; 103.80 –; 165.00 –; 99.80 2.22; 19.90

7.02; 129.70

1.98–2.22; 38.60 1.75 4.04; 59.00 –; 166.10 6.39 NH; –

1.90–2.10; 28.40 2.30–2.40 4.08; 56.20 –; 169.60 5.57 NH; – 4.27; 59.2 3.60–3.70; 36.90 2.77

–; 135.90 7.20–7.39; 7.20–7.39; 7.20–7.39; 7.20–7.39; 7.20–7.39;

129.40 129.20 127.90 129.20 129.40

4.15; 53.40 2.37; 28.10 1.98–2.22 1.55; 24.70 1.03; 23.30 0.96; 21.20 2.67; 34.60 3.37; 40.80 –; – –; 174.40 1.88; 29.60

A: 1H (CDCl3, 300 MHz); 13C (CDCl3, 50.306 MHz). B: 1H (DMSO-d6, 300 MHz); 13C (CD3OD, 75.478 MHz). C: 1H (CD3OD, 300 MHz); 13C (CD3OD, 125.000 MHz). D: 1H (CDCl3, 300 MHz); 13C (CDCl3, 75.478 MHz). 6,8-Dihydroxy-3-methylisocoumarin ‘‘B’’. C10H8O4 (192.1). The Rf = 0.84 (C6H12 10%, EtOAc). This compound showed a strong absorption under UV light at 254 nm and gives a rose colouration on spraying with anisaldehy/ sulphuric acid. EI/MS: m/z% = 192 (M+, 100), 178 (40), 122 (23), 43 (100), 69 (25); ()-ESI/MS (70 eV): m/z (%) = 191 ([M  H], 100), 382.7 ([2M  Na], 50). p-Hydroxyphenethyl acetamide (N-acetyl-tyramine) ‘‘C’’. C10H13NO2 (179.21). The Rf = 0.45 (CH2Cl2/MeOH 5%). This compound showed UV absorbing zone and stained to violet with anisaldehyde/sulphuric acid. EI/MS (70 eV): m/z (%) = 179.2 ([M]+, 7), 120.1 ([M–(H2N–C O–CH3)]+, 100), 107.1 (30), 91.0 (11), 77.0 (8). cis-Cyclo (Leucyl-Prolyl) ‘‘D’’. C11H18N2O2 (210.27). The Rf = 0.58 (CHCl3/MeOH 5%). This compound give an UV absorbing turned to violet by anisaldehyde/sulphuric acid. EI/MS (70 eV): m/z (%) = 195 (M– CH3, 16), 154 (100), 125 (14), 86 (36), 70 (65), 41 (12). The 1H NMR and 13C NMR data of these four active compounds are shown in Table 2.

2.7. PCR amplification of the 16S rRNA gene of strain TN97 PCR amplification of the 16S rRNA gene of TN97 strain was performed using two primers (P1 and P2) as described in Table 1. Approximately 300 ng genomic template DNA was used with 150 pmol of each primer per 50 ml reaction volume. To improve the denaturation of the DNA, 5% (v/v) DMSO (final concentration) was added to the reaction mixture. Amplification was performed in automated thermocycler (Perkin-Elmer) using 1 U Pfu DNA polymerase (Stratagene) and the recommended buffer system according to the following amplification profile: 94 8C (3 min) followed by 45 cycles of denaturation at 94 8C (30 s), annealing at 60 8C (1 min) and extension at 72 8C (3 min). The PCR reaction mix was analysed by agarose gel electrophoresis and the expected size was purified then cloned into pCR-blunt vector yielding pASS1.

2.8. DNA sequencing and analysis Nucleotide sequences of the 0.65 kb (pSS2) and 0.85 kb (pSS3) EcoRIEcoRI DNA fragments corresponding to the whole 16S rRNA gene (1.5 kb) of TN97 strain were determined on both strands using the dideoxy chain-termination method [19]. Reactions were performed with a Thermo sequenase cycle sequencing kit (Amersham) and specific primers. Homology search was performed using Blast Search algorithm. The nucleotide sequence of the whole 16S rRNA gene of TN97 strain has been assigned GenBank (EMBL) accession number AM040290.

3. Results and discussion 3.1. Isolation and identification of the TN97 strain A new aerobic bacterium designated TN97 isolated from Tunisian oasis soil produced antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi (Fig. 2). Permissive temperature ranges of growth of this strain were 25– 35 8C with an optimum at 30 8C. Culture characteristics of strain TN97 were followed on the basis of observations made after 7, 14 and 21 days incubation on nutrient agar, Sabouraud

Fig. 2. Antimicrobial activities in solid media of strain TN97 against the used indicator microorganisms M. luteus LB14110 (A), E. coli ATCC8739 (B) and V. dahliae (C).

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Table 3 Cultural characteristics of TN97 strain Medium

Growth

Vegetative mycelia

Aerial mycelia

Spores

Nutrient agar Sabouraud agar Yeast malt agar

Poor, dispersed Well, spreading Moderate, elevate

Dispersed, yellowish Abundant, yellowish Dispersed, yellowish

Absence Detachable, pinkish Moderate, brown

Absence Abundant, purple Moderate, white-purple

agar and yeast malt agar (ISP) media [17]. According to the cultural characteristics (Table 3), TN 97 strain grew well on Sabouraud agar medium and the colonies were spreading. The colours of the vegetative and aerial mycelia were yellowish and pinkish, respectively and the spore chains were purple. These characteristics were compared with those of the known species of actinomycetes described in Bergey’s manual of systematic bacteriology [20], and obtained morphological properties suggested strongly that strain TN97 belonged to the genus Streptomyces. This suggestion was confirmed by 16S rRNA gene analysis of the studied strain. This gene was amplified and cloned into pCR-blunt vector yielding pSS1 plasmid. The digestion of this plasmid by different restriction endonucleases shows the presence of a unique EcoRI site in the rRNA 16S gene of the TN97 strain. Taking advantage of this result and also of the presence of two EcoRI sites on both sides of the pCR-blunt vector, we have cloned the two EcoRI-EcoRI DNA fragments corresponding to the whole rRNA gene of TN97 strain into dephosphorylated EcoRI linearized pIJ2925 vector. Obtained plasmids (pSS2 and pSS3) were used for the determination of the complete nucleotide sequence of the 16S rRNA gene of TN97 strain. Total nucleotide sequence of 1508 pb (accession no. AM040290), of the 16S rRNA gene of TN97 strain was determined in both strands. The alignment of this nucleotide through matching with 16S rRNA reported genes sequences in gene bank, provide 100 16S rRNA gene sequences having high similarity with that of TN97 strain. All these 100 16S rRNA genes were from Streptomyces species. The highest sequence similarity (96%) was obtained with the 16S rRNA gene of Streptomyces lipmanii which produces the two b-lactam antibiotics cephamycin C and epithienamycin [21]. Based on the results of cultural characteristics studies of TN97 strain, and on the analysis of the nucleotide sequence of the 16S rRNA gene, we propose the assignment of our strain as Streptomyces sp. TN97 strain.

after 72 h of incubation for all tested carbon sources. However, maximum of growth and the highest biological activities were obtained in cultures supplemented with glycerol or fructose. The inhibition zones of growth of the used indicator microrganisms are always larger with these two carbon sources at different times of incubation. Fig. 3 shows the growth curves of the Streptomyces sp. TN97 strain in the five studied carbon sources and Fig. 4, represents the results of kinetics of growth and the antimicrobial activities against the used indicator microorganisms concerning the glycerol culture. It should be noticed that all kinetics growth and biological tests studies were carried twice and the obtained results were similar. Nevertheless, it has been reported by several works that chemical compounds such potassium, magnesium and trace mineral oligoelements, and other factors including medium volume, oxygen transfer rate, temperature, initial pH of the medium and the incubation time, influence the secondary metabolites production. For the Streptomyces sp. TN97 strain, the three chemical compounds (potassium, magnesium and trace mineral oligoelements) do not affect the production of the active molecules and obtained results, showed that antimicrobial activities against the used indicator microorganisms were similar to those obtained with glycerol or fructose only. However, we have noticed that in our case, agitation rates of 100 and 150 rpm gave a low production of antimicrobial activity and the best result was obtained at 250 rpm. In the case of 200 rpm, biological activity was less than 300 rpm but higher than that of 100 and 150 rpm. Concerning incubation time,

3.2. Influence of the culture conditions on antimicrobial activities production To investigate the effect of carbon sources on antimicrobial activities production, five carbohydrates (starch, fructose, glycerol, glucose and saccharose) were tested separately. Each of these carbon sources was added at 1% (w/v) to the TSB liquid medium. Streptomyces sp. TN97 strain is able to grown and to produce antimicrobial activities in all added carbon sources. Kinetics of growth and active molecules production studies, showed that the secretion of biological activities is closely correlated with the biomass production. Maximum biological activities and biomass production were obtained

Fig. 3. Growth curves of the Streptomyces sp. TN97 strain on TSB medium supplemented with one of the five tested carbon sources.

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Fig. 4. Kinetic of growth and antimicrobial activities production of the Streptomyces sp. TN97 on TSB medium supplemented with glycerol.

antimicrobial activity appeared to be pronounced after 24 h of growth with a maximum at 72 h of incubation. This activity remains stable between 72 and 96 h and then decreases slowly to disappear after 140 h of incubation. 3.3. Extraction, purification and structure elucidation of the active compounds Various separation steps were applied to 10 l culture broth of the Streptomyces sp. TN97 strain grown in TSB medium supplemented with 1% (w/v) of glycerol (Fig. 1). The biological assays show that the fractions F1 and F4 possess antimicrobial activities. The compound cis-cyclo (L-phenyl, Lprolyl) was obtained from the fraction F1. The three other active compounds, 6,8-dihydroxy-3-methylisocoumarin, phydroxyphenethyl acetamide (N-acetyl-tyramine) and the ciscyclo (Leucyl, Prolyl) were obtained from the fraction F4. The

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chemical structure of the four active compounds purified from the Streptomyces sp. TN97 is shown in Fig. 5: cis-Cyclo (L-phenyl, L-prolyl) Fig. 5A. The (+)-ESI/MS spectrum delivered quasi-molecular peaks at m/z 267 ([M + Na]+) and 511 ([2M + Na]+), respectively, which fixed the molecular weight to 244. High resolution at EI ionisation afforded a molecular formula C14H16N2O2. The 1H NMR spectrum showed a singlet at 5.57 ppm assigned at NH proton. Signals from 1 to 4.28 ppm were attributable at CH and CH2 groups and those from 7.2 to 7.39 ppm at aromatic protons. The 13 C NMR spectrum displayed signals of two carbonyls at 165.2 and 169.6 ppm. The 13C signals in the aliphatic region were assigned by APT as four CH2 (28.4; 22.6; 36.9 and 45.8 ppm), and two CH (56.2 and 59.2 ppm) which indicate the presence of two tertiary carbons attributable to (C–H pro) and (C–H phe), respectively. 6,8-Dihydroxy-3-methylisocoumarin Fig. 5B. The 1H NMR spectrum in DMSO exhibited an exchangeable proton at d = 1090 (br s), and the aromatic region indicated only the presence of three sp2 protons at d = 6.47, 6.33 and 6.30 ppm. In the aliphatic region, a methyl signal at d = 2.22 ppm was seen. The 13C NMR spectrum indicated the presence of 10 carbon signals, which were indicated by APT as six quaternary, three sp2 methine and one methyl carbon atoms. The (+)-ESI/MS indicated a pseudo-molecular ion at m/z 215 [M + Na]+. The search in Antibase [23] delivered 6,8-dihydroxy-3-methylisocoumarin. p-Hydroxyphenethyl acetamide (N-acetyl-tyramine) Fig. 5C. The 1H NMR spectrum showed two acidic protons as broad singlets at d 8.73, and 7.51, which could be due to phenolic hydroxyls or amide groups. An 1,4-disubstituted aromatic system was established due to the existence of two doublets each of 2H at d 7.02, and 6.73 ppm. In the aliphatic region, an ethanediyl group attached to NH amide was observed due to the presence of two signals at d 3.37 (q) and 2.67 ppm (t) as two vicinal methylene groups. A 3H singlet of a methyl

Fig. 5. The chemical structures of compounds A–D. (A) cis-Cyclo (L-phenyl, L-prolyl); (B) 6,8-dihydroxy-3-methylisocoumarin; (C) p-hydroxyphenethyl acetamide (N-acetyl-tyramine); (D) cis-cyclo (Leucyl-Prolyl).

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group was observed at d 1.88 ppm, which could be assigned as an acetyl group. The 13C/APT NMR spectra exhibited 8 carbon signals, among them three quaternary sp2 carbon signals were at d 174.40, 155.00 and 130.00 ppm. Two methane signals of double intensity at d 129.70 and 115.60 ppm indicated an 1,4disubstituted aromatic system. The first two quaternary carbon signals (174.40, 155.00 ppm) are characteristic for carbonyl of amide groups and an oxygenated carbon in the aromatic ring, respectively. In the aliphatic region, three sp3 carbon signals were observed, two methylene groups at d 40.80, 34.60 ppm, and one methyl signal at d 29.60 ppm. The EI mass spectrum determined the molecular weight of this compound as 179 Da. The mass ion exhibited an expulsion of acetamide group to give a base peak at m/z = 120. cis-Cyclo (Leucyl-Prolyl) Fig. 5D. The 1H NMR spectrum showed a broad 1H singlet of an amide at d 6.39 ppm, two methane protons at d 4.15 (t), and 4.04 ppm (dd). Furthermore, a 3H multiplet between d 3.54 and 3.64 ppm (3H2, 10Hb), a 1H multiplet at d 2.37 ppm (10Ha), a multiplet between d 198 and 2.22 ppm of (4H2, 5Ha) and a multiplet at d 175 ppm of 5Hb. In addition, a multiplet of 1H at d 1.55 ppm (11H), and two doublets each of 3H as of two equivalent methyl groups were found at d 1.03 and 0.96 ppm, delivering an isopropyl system. The 13C/APT NMR spectra displayed two quaternary carbons of two CO groups at d 170.3 and 166.1 ppm. In the aliphatic region, two methine carbons at d 59.0 and 53.4 ppm linked to hetero atom were visible. Four methylene carbons at d 45.5, 38.6, 28.1, and 22.7 ppm, as well as a third methine carbon at d 24.7 ppm were observed. It showed finally two methyl carbons at d 23.3 and 21.2 ppm of the previously mentioned isopropyl group. The molecular weight was determined as 210 Da by EI mass spectra. The new isolated Streptomyces sp. TN97 strain produces simultaneously four active molecules which belong to three different families:  The cis-cyclo (L-phenyl, L-prolyl) and the cis-cyclo (LeucylProlyl) molecules which are two diketopiperazine (DKP) derivatives. Diketopiperazine molecules constitute a family of secondary metabolites with diverse and interesting biological activities such as antibacterial, fungicidal, herbicidal, immunosuppressor, antitumors, antiviral [22]. The two DKP derivatives, produced by the Streptomyces sp. TN97 strain, were previously described from the North Sea bacterium Cytophaga marinoflava strain Am13,1 and the actinomycete strain A8 for the molecule cis-cyclo (L-phenyl, L-prolyl) [23,24], and from a Norcardia species for the cis-cyclo (Leucyl-Prolyl) [23]. It has been shown by Gruenewald et al. [25] that the ciscyclo (L-phenyl, L-prolyl), could be a compound naturally present in some batches of tryptone. However, it should be noted that in our case, we have purified and characterized the active molecules of other selected strains growing in TSB medium (data not shown), and we did not find the cis-cyclo (L-phenyl, L-prolyl). These results confirm that this active molecule is really synthesized by the Streptomyces sp. TN97 strain.

Table 4 Antimicrobial activities of cis-cyclo (L-phenyl, L-prolyl) ‘‘A’’, 6,8-dihydroxy-3methylisocoumarin ‘‘B’’, N-acetyl-tyramine ‘‘C’’ and cis-cyclo (Leucyl-Prolyl) ‘‘D’’ Test organism

M. luteus LB 14110 E. coli ATCC 8739 V. dahliae

Diameter of inhibition zones (mm) A

B

C

D

11 10 10

ND ND 12

ND ND ND

16 11 12

About 30 mg/platelet, diameter of inhibition zones in mm. ND: activity not detected.

 The third bioactive molecule that produces the Streptomyces sp. TN97 strain is the 6,8-dihydroxy-3-methylisocoumarin. Isocoumarins are a class of natural compounds that have been found to exhibit interesting biological properties including antifungal, anti-inflammatory, anti-allergic, necrotic and antitumor effects [26]. The 6,8-dihydroxy-3-methylisocoumarin compound was previously isolated from the fungus Ceratocystis minor [27], from a cultural broth of Streptomyces sp. (GT061089) [28] and was used also in syntheses. It has been reported that this compound showed strong inhibiting activity on horse radish peroxidase, also exhibited antiviral activity as well as a distinct inhibiting activity on 3ahydroxysteroid dehydrogenase (3a-HSD) [28].  The fourth active compound produced by the studied TN97 strain is the N-acetyl-tyramine that has been patented in 1998 by a Japanese group and it is used as antitumor agent [29]. The antimicrobial activities, against the used indicator microorganisms (M. luteus LB 14110, E. coli ATCC 8739 and V. dahliae), of these four pure compounds are shown in Table 4. Although the four active molecules characterized from the new isolated Streptomyces sp. TN97 strain have been already described from other microorganisms, we think that our strain is very interesting because it produces simultaneously all four active molecules belonging to three different families which can be used in human therapy. In addition, this strain offers a real opportunity for molecular studies to produce new hybrid active molecules especially diketopiperazine derivatives. In fact, the biosynthetic pathways of active diketopiperazine molecules remain largely unexplored especially those produced by the soil bacteria belonging to the genus Streptomyces. The structural elucidation of DKPs derivatives from these bacteria constitutes a large contribution for the understanding of their biosynthesis phenomena and will permit subsequently, the production of new hybrid DKP derivatives with interesting biological proprieties. Actually, our study is focused on the analysis of the biosynthetic pathways of the two DKPs derivatives of the Streptomyces sp. TN97 strain. 4. Conclusion A new aerobic bacterium designated TN97 isolated from Tunisian oasis soil has been selected for its antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. Cultural characteristic studies and analysis of the

R. Ben Ameur Mehdi et al. / Process Biochemistry 41 (2006) 1506–1513

nucleotide sequence (1508 pb) of the 16S rRNA gene (accession no. AM040290) of TN97 strain, strongly suggested that this strain could be an actinomycete bacterium belonging to the genus Streptomyces. We propose the assignment of our strain as Streptomyces sp. TN97 strain. Study of the influence of different nutritional compounds and culture conditions on antimicrobial activities production by the Streptomyces sp. TN97 strain, showed that the highest biological activities were obtained when glycerol or fructose at 1% (w/v) were added in TSB medium after 72 h of incubation at 250 rpm. The application of several extraction and purification steps to the supernatant and the mycelium of 10 l culture broth of the Streptomyces sp. TN97 strain, led to isolation of four pure molecules having biological activities. The chemical structure of these compounds named cis-cyclo (L-phenyl, L-prolyl), ciscyclo (Leucyl-Prolyl), which are two DKPs derivatives, 6,8dihydroxy-3-methylisocoumarin and N-acetyl-tyramine, was established on the basis on their IR, ESI/MS and EI/MS, 1H and 13 C/APT NMR data and by comparison with reference data from literature. Acknowledgments

[10] [11]

[12] [13]

[14] [15]

[16]

[17] [18]

[19]

This work was supported by Tunisian government (Contract Program CBS-LEMP). We are grateful to Prof. Dr. H. Laatsch (Department of Organic Chemistry, University of GoettingenGermany) for his generous help.

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