MATERIALS AND METHODS

Transcription

1 MATERIALS AND METHODS Duration and Place of Study This research was carried out from July 2011 to March Extraction, fractionation and purification of antimicrobial compounds were carried out in the laboratory of Microbiology, Department of Biology and Biopharmaca Research Center, IPB, Indonesia. Molecular genetic analysis was carried out in Yohda Laboratory, Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology (TUAT), Japan. The flowchart of method s that used in this study is given in F igure 3. Materials Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province by Abubakar (2009). These bacteria were used for the extraction, fractionation and purification of antimicrobial compounds. Specific primer 63 f (5- CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGGCGGWGTGTACAAG GC-3) was used for analysis of 16S rdna (Marchesi et al. 1998). Degenerate pr imer (f: 5-GCSATGGAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTG SSCYTCSAC-3) for KS domain and degenerate primer (f: 5-AARDSIGGIGSIG SITAYBICC-3); (r: 5-CKRWAICCICKIAIYTTIAYYTG-3) for A domain (Schirmer et al. 2005). Extraction of Antimicrobial Compounds Each of those three isolates was sub-cultured in 500 ml Seawater Complete Broth media (bacto peptone 2.5 g, yeast extract 0.5 g, glycerol 1.5 ml, seawater 375 ml and distilled water 125 ml) and incubated in fluctuate incubator with 100 rpm at 30 0 C until the culture reached the stationary phase. After that, 10% liquid bacterial inocula from previously incubation were cultured in 500 ml SWC broth and incubated in the same condition until reached the stationary phase for secondary metabolite production (Muller et al. 2004).

2 Extraction of antimicrobial compounds was done by modifying the method from Sunaryanto et al. (2010). As much 500 ml of liquid bacterial culture were mixed with 500 ml of ethyl acetate solvent, incubated at room temperature for 24 hours and stirred for 2 hours with 250 rpm. These mixtures were separated and the ethyl acetate layers were evaporated with rotary evaporator until the drying residue was obtained as crude extract. The crude extract of bacterial cells were dissolved with ethyl acetate (pro analyze) to get 100 mg/ml concentration. Extraction of Antimicrobial Compounds DNA Extraction Antimicrobial Activity Test PCR Amplification of Ketos ynthase (KS) and Adenylation (A) Domain Detection of Antimicrobial Compounds Fractionation of Bacterial Crude Extract Cloning of DNA Fragments Encod ing KS and A Domain Sequencing and Bioinformatics Analysis of KS and A Domain Purification of Antimicrobial Compounds Morphological and Molecular Identification Figure 3 Flowchart of procedural steps used in this study. Antimicrobial Activity Test Antibacterial and antifungal activities from crude extracts were tested by using a gar diffusion method a gainst microbial test strains. As much 100 µl bacterial crude extracts dissolved in ethyl acetate (pro analyze) were applied carefully into 6 mm paper disks (Whatman) and at the same time, the disks were dried up using a

3 hairdryer at 40 0 C. After that, the disks were sterilized under UV light for 2 hours and put into agar plate that have been seeded with 1% (v/v) of microbial test strains (conc entration 1x10 6 CFU/ml, OD ). The plate was incubated at 4 0 C for 3 hours to optimize the diffusion of bacterial crude extract into media. This assay was carried out in triplicate. The diameters of inhibition zones were measured in millimeters after incubation for 24 hours at 37 0 C. Control disks were soaked with ethyl acetate solvent and prepared in the same manner (Sudirman 2010). Detection of Antimicrobial Compounds Antimicrobial compounds in each of the bacterial crude extract were detected using bioautography method. As much 10 µl of crude extracts were spotted on TLC plates (MERCK Silica Gel 60 F 254 ) and eluted with vertical chromatography using n-butanol : ethyl acetate solvent mixture with the ratio of 3:7 (v/v). The spots on TLC plate were detected under UV light at 254 nm and 366 nm wave-length. After that, the retardation factor (Rf) values were calculated. The spots on TLC plate were cut off and dried up in room temperature. The developed TLC plates were sterilized under UV light for 1 hour before covered by 15 ml of melting SWC (45 0 C) containing test strains, and incubated at 37 0 C for 24 hours. Diameter of inhibition zone around the chromatogram indicated that the spo t was an active fraction (Sudirman 2005). Fractionation of Bacterial Crude Extract Bacterial crude extract of isolate SAB E-41 was fractionated using semi automated flash chromatography (Buchi Pump Controller C-610). As much 3 g of crude extract was dissolved with chloroform-methanol solvent mixture (90%-10%) and injected into silica gel-column chromatography (column dimension 0.40 x 150 mm, particle size of silica gel 40 x 63 µm). Chloroform-methanol solvent mixture (90%-10%) was flowed into silica gel-column chromatography with the flow rate of 3.5 ml/minutes. The polarity level in the column was increased slowly by changing the methanol concentration from 20%, 30%, 50%, 70% until 90%. Two hundred and five fractions were collected (5 ml/each fraction) from fraction collector and combined into thirty fractions based on the same chromatogram.

4 These fractions were dried up and dissolved with chloroform-methanol-ethyl acetate solvent (1:1:1) to test their antimicrobial activity. Purification of Antimicrobial Compounds Fifteen active fractions were further purified using preparative thin layer chromatography (PTLC) technique. Each of the active fractions was spotted onto silica gel plate (MERCK Silica Gel 60 F 254 ; 0.1 mm thickness) and eluted with n- butanol-ethyl acetate solvent mixture (3:7). The active spots on the silica gel plate were detected under UV light at 254 nm and 366 nm wave-length. The active spots were extracted directly from the silica gel plate and dissolved with chloroformmethanol-ethyl acetate solvent mixture (1:1:1). The active fractions that were purified by preparative TLC were tested for their antimicrobial activity. Morphological and Molecular Identification Morphological characterization of those three isolates was performed using Gram staining p rocedure. Molecular analysis of 16S rdna was done using specific primer, 63f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGG CGGWGTGTACAAGGC-3) (Marchesi et al. 1998). The PCR cycling condition for 16S rdna was carried out under the following condition such as initial denaturation at 94 0 C for 5 min, followed by 30 cycles of denaturation at 94 0 C for 1 min, annealing at 55 0 C for 1 min, elongation at 72 0 C for 1 min and post PCR at 72 0 C for 10 min. PCR products of 16S rdna were purified using GENECLEAN II Kit. These PCR products were sub-cloned into T-Vector pmd20 and transformed into competent E. coli DH5-α using heat shock method (Sambrook & Russell 2001). Afterwards, several steps such as PCR colony, isolation and restriction of recombinant plasmid, PCR sequencing and purification of PCR products were done before the 16S rdna sequence analysis. DNA Extraction Each of bacterial isolates were sub-cultured into SWC broth media and incubated at room temperature for 24 hours. After that, 1.5 ml bacteria isolate was drawn into microtube and centrifuged (18.000xg) for 10 min to obtain bacteria

5 pellet that were used for DNA extraction. The supernatant was discarded and 250 µl Tris-EDTA (TE) buffer was added and centrifuged at 8000 rpm for 10 min. The supernatant was discarded and pellet was re-suspended three times in TE buffer. As much 250 µl TE and 5 µl lysozyme were added together and microtube slowly inverted to allow mixing and incubated at 37 0 C for 30 min. After incubation, the solution was added with 500 µl SDS 10% and 10 µl proteinase K and incubated again at 37 0 C for 60 min. Afterwards, as much 80 µl NaCl was added together with 100 µl CTAB 10% and incubated at 65 0 C for 20 min. After incubation, added again the solution with 650 µl PCI and shake n strongl y then centrifuged at rpm for 10 min. The upper solution was transferred into a new microtube then 650 µl CI was added and centrifuged again in same condition. DNA was precipitated using absolute ethanol (2x vol) and Na acetate 3 M 0.1 vol and incubated overnight in freezer. After that, 1 ml ethanol 70% was added for final washing and centrifuged at rpm for 10 min. The supernatant was discarded and pellet was air dried overnight. After this step, 20 µl of TE was added and the extracted DNA was stored at C for further applications (Sambrook & Russel 2001). PCR Amplification of KS and A Domain KS domain of PKS and A do main of NRPS genes from those three isolates were amplified using PCR primers such as degenerate KS domain (f: 5-GCSATG GAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTGSSCYTCSAC-3) and degenerate A domain (f: 5-AARDSIGGIGSIGSITAYBICC-3); (r: 5-CKRWAICC ICKIAIYTTIAYYTG-3) (Schirmer et al. 2005). The PCR cycling condition for KS domain was carried out in three steps such as initial denaturation at 94 0 C for 5 min, followed by 35 cycles of denaturation at 94 0 C for 1 min, annealing at 50 0 C for 1 min, elongation at 72 0 C for 1 min 10 sec and post PCR at 72 0 C for 10 min. The PCR cycling condition for A domain was the same as for KS domain except for annealing which was carried out at 55 0 C for 1 min. In all cases, the reaction mixtures contained 4 µl dntp mix (2.5 mm), 5 µl 10X Ex Taq buffer, primer forward and reverse (10 µm; each of 5 µl), 2 µl DNA template (500 ng/µl), 1 µl TaKaRa Ex Taq TM (5 units/µl) and 28 µl milli Q. The

6 total volume of reaction mixtures was 50 µl. PCR products were analyzed using agarose gel electrophoresis 1% (b/v). Cloning o f DNA Frag me nts Encoding KS and A Domain Purification of PCR products from KS domain of PKS gene (700 bp) and A domain of NRPS gene (1000 bp) were carried out using GENECLEAN II Kit. Each of the purification prod ucts was sub-cloned into T-Vector pmd20 (TaKaRa) and transformed into competent E. coli DH5α using heat shock method (Sambrook & Russell 2001). Isolation of recombinant plasmid was performed using Mag Extractor TM Quick Plasmid Miniprep kit (Toyobo, Japan). Restriction of recombinant plasmid was conducted using the combination of restriction enzymes such as BamHI (BioLabs) and XbaI (TaKaRa). The reaction mixtures contained 0.2 µl BamHI (5 units/µl), 0.2 µl XbaI (5 units/µl), 1 µl 10X NE buffer 4, 0.5 µl DNA template (500 ng/µl) and 8.1 µl milli Q. The total volume of reaction mixtures was 10 µl. The reaction mixtures were incubated at 37 0 C for 24 hours and the restriction product was analyzed using agarose gel electrophoresis 1% (b/v). Sequencing a nd Bioinformatics Analysis of KS and A Domain DNA fragments that were inserted into plasmid T-Vector pmd20 named pmd20-ks domain and pmd20-a domain were used for the sequencing process. M13 primer RV and M13 primer M4 were used for PCR sequencing. The PCR cycling condition was carried in three steps such as initial denaturation at 96 0 C for 5 min, followed by 25 cycles of denaturation at 96 0 C for 1 min, annealing at 50 0 C for 30 sec, elongation at 60 0 C for 1 min and post PCR at 4 0 C for an unlimited time. The Big Dye X Terminator TM purification kit was used to purify the PCR product before DNA sequencing. The DNA was run in an automated DNA sequencer using ABI 3130 XL Genetic Analyzer. The DNA sequences were compared to the database available at NCBI using BlastN program for 16S rdna and BlastX program for KS and A domain. Construction of phylogenetic tree was carried out using MEGA5 program with neighbor-joining method.