Antibacterial Activity Composition of the Fermentation Broth of Streptomyces djakartensis NW35

The new compound Z-4-2 was isolated from the fermentation broth of Streptomyces djakartensis NW35, together with the known compound N-acetyltryptamine (Z-9-2) by bioassay-guided fractionation. Its chemical structure was elucidated as (E)-2-methoxy-1,4 naphthoquinone-1-oxime (Z-4-2) mainly by NMR analyses and MS spectral data. Their antibacterial activities against bacteria were evaluated by the filter paper method. The results of indicated that these compounds possess significant antibacterial activities.


Introduction
Soil actinomycetes are still important sources of novel antibiotics. The vast majority (70%) of the known antibiotics were first isolated from actinomycetes [1,2]. In the past decades, although many species which produced biologically active metabolites have been obtained from soil samples, the chance of isolating a new actinomycete strain from a common terrestrial habitant has reduced markedly [3,4]. To meet the increased demands for the discovery of new bioactive compounds, researchers have to look for novel microorganisms in unusual environments [5]. Chemically polluted soil is one such sort of unusual environment. In fact, chemical pollutants, especially some pesticides, could be mutagens, so mutant strains might be produced by induced mutations. Some of the mutant OPEN ACCESS strains might give rise to increased productivity of bioactive metabolites, or even produce new bioactive compounds. Based on this thinking, the NW35 strain of Streptomyces djakartensis which was isolated from a pesticide-polluted soil sample has been investigated. In this paper, we report the antibacterial composition of the fermentation broth of S. djakartensis NW35.

Bioactivity
The antibacterial activities of these two compounds against several strains of bacteria were evaluated by the filter paper method. The results indicated that compound Z-4-2and Z-9-2 showed significant antibacterial activities (Table 2). Note: All values were means of three replicates, "+" means visible; "++" means clear; "+++" means transparent; "-" means no inhibitory ring or no inhibition activity.

General
Melting points (uncorrected) were taken on a Fisher-Johns melting point apparatus. IR spectra were acquired using a Bruker TENSOR2 spectrophotometer. The NMR data were recorded on a Bruker Avance 500 instrument (500 MHz for 1 H and 125 MHz for 13 C) in CDCl 3 with TMS as internal standard. HR-ESI-MS data were obtained by Bruker Daltonics APEX II 49e (ESI) mass spectrophotometer. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd, Qingdao, China) was used for chromatographic separations.

Extraction and Isolation
The culture of 90 L was filtered through cheesecloth to separate the medium and culture liquid at 25 °C, pH 7.0. The filtrate was absorbed onto HPD100 macroporous resin (Baoen Co., Ltd., Cangzhou, Hebei, China), and then eluted with H 2 O and MeOH in sequence. The MeOH fraction was evaporated in vacuum. The concentrate was subjected to column chromatography and eluted with EtOAc and MeOH in sequence. The antimicrobial fraction was concentrated under vacuum, and further purified on a Waters 600E HPLC apparatus (Waters Co., Ltd., Milford, MA, USA) equipped with a Hypersil ODS-BP (20 × 250 mm, 10 μm) reverse phase column, using methanol-water as the mobile phase, flow rate of 3.0 mL/min, monitored by UV detector at 230 nm to afford two compounds Z-4-2 (20 mg) and Z-9-2 (10 mg).

Antibacterial Activity
The standard bacterial strains A clinical isolate of MRSA was obtained from Nanjing Medical University. Ampicillin (Sigma, Shanghai, China) was used as positive control. The antibacterial activities of compounds against eight strains of bacteria were evaluated by the filter paper method [7], Mueller-Hinton (Hangzhou Microbial Reagent Co. Ltd., Hangzhou, China) agar was used as an assay medium. The medium at 45 °C was mixed with the pathogen bacterial suspension containing approximately 10 8 cfu·mL −1 . Next, the mixture was poured on 9 cm Petri dishes. The tested compounds were dissolved in acetone at the concentration of 1,000 ppm, The filter paper (5 mm in diameter) were impregnated with 10 μL/disc of each compound, then were absolutely dried and placed on the inoculated agar. The inoculated plates were incubated at 37 °C for 24 h. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism. Experiments were run in triplicate. The results indicated that Z-4-2 and Z-9-2 could effectively inhibit Gram-positive bacteria, such as Bacillus cereus, B. subtilis, whereas they were inactive towards Gram-negative bacteria ( Table 2).