Motility was determined using sulfide-indole-motility medium Fat

Motility was determined using sulfide-indole-motility medium. Fatty acid methyl esters were extracted and analyzed by the Sherlock Microbial Identification system (MIDI, Newark, DE) according to the manufacturer’s instructions. All assays were performed in triplicate. The 16S rRNA gene of strain B7 was amplified by PCR with the universal AZD8931 order primers 27F and 1541R and sequenced [16]. Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony algorithm within MEGA4 [17]. The DNA-DNA hybridization between B7

and Paenibacillus ehimensis IFO 15659T was performed using the thermal denaturation method [14]. Production and purification of active compounds Strain B7 maintained on nutrient agar slants was inoculated into 50 mL of nutrient broth and cultivated at 30°C for 24 h. The seed culture of strain B7 was transferred

to a 2L Erlenmeyer flask that contained 500 mL of the KL medium. The culture was incubated on a rotary shaker (200 rpm) at 30°C for 3 d. After centrifugation at 4500 g for 30 min at 4°C, the cell-free Dinaciclib research buy supernatant was loaded onto a column packed with Amberlite XAD-16 resin (Sigma, St. Louis, MO). The column was washed with distilled water prior to elution with stepwise gradients of aqueous methanol (30, 60, and 100%, v/v). Each fraction was concentrated and assessed for activity using the paper disc method. The PLEKHB2 active fraction was evaporated and dried before being redissolved in acetonitrile. The concentrated solution was then applied to a C18 SPE column (Hardwee, Germany). The column was washed with five bed volumes of distilled water, followed by five bed volumes of an acetonitrile/water mixture (20:80, v/v). The fraction that contained the active

compounds was eluted from the column by washing with three bed volumes of an acetonitrile/water mixture (68:32, v/v). Further purification was performed using a preparative HPLC system (Dalian Elite, Dalian, China) that was equipped with an YMC-pack DOS-A C18 (5 μm, 250 × 20 mm) column. The mobile phase consisted of Milli-Q water that contained 0.02% trifluoroacetic acid and acetonitrile. A linear gradient of 15% to 55% acetonitrile (40 min) was used for elution at a flow rate of 10 mL/min. UV detection was performed at a wavelength of 210 nm. Fractions from multiple runs were collected and combined for the Epacadostat subsequent antimicrobial activity assays. The active fractions were passed through the HPLC column two consecutive times. Amino acid analysis Approximately 300 μg of the purified compound in 0.4 ml of 6 M HCl with 0.1% phenol was hydrolyzed at 110°C for 16 h. Amino acid analyses was performed using ion-exchange chromatography with a Hitachi L-8900 amino acid analyzer (Tokyo, Japan) according to the method described by Qian et al. [18].

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