The wild-type strain, click here A. sobria 288, grown in 3  mL NB (0.5), was collected by centrifugation and the cells suspended in 0.3  mL distilled water. The suspension was

heated in boiling water for 10  min. The suspension was centrifuged to separate the precipitates from the supernatant. The supernatant was used as the source of the DNA template in PCR amplification and each set of oligonucleotides was used as a primer. The length of the DNA fragment amplified by the first and second sets was the 2251  bp and 1569  bp band, respectively. Subsequently, the amplified DNA in the reaction mixture was purified by treatment with phenol. The nucleotide sequence of each DNA was then determined by the dideoxy chain termination method. The protein

investigated in this study was shown to be a lipase and its amino acid sequence was deduced. Antiserum against the lipase was prepared by injecting the peptide GGDDNKGDTTSSLDYC-NH2, which is a keyhole limpet hemocyanin conjugate and composed of the 15 amino acid residues at the amino terminal end of the protein under investigation, into rabbits. Preparation of the antiserum was entrusted to the Peptide Institute (Mino, Osaka, Japan). A portion of overnight preculture of A. sobria 288 (asp−, amp−) (1  mL) was inoculated into 100  mL  NB (0.5). Bacteria were grown at 37°C with shaking at 140  r.p.m. At 6  hrs, 12  hrs, and 24  hrs, 20  mL of culture liquid was removed and the cells separated from the culture supernatant by centrifugation. https://www.selleckchem.com/products/nivolumab.html BCKDHB Proteins in the

culture supernatant of A. sobria 288 (asp−, amp−) were precipitated by treatment with TCA as follows: TCA solution was added to 1.0  mL of culture supernatant to reach a concentration of 10%. The mixture was left for 30  min at room temperature and the insoluble materials yielded collected by centrifugation. After rinsing with ethanol, the precipitates were suspended in  100 μL Tris-HCl buffer (pH 7.4). The cells recovered by centrifugation were suspended in 2  mL of 10  mM Tris-HCl buffer (pH 7.5). The cell suspension was divided equally into two tubes (1  mL/tube). A periplasmic fraction of the cells was prepared by treatment with polymyxin B (22). Polymyxin B solution (1  mL) was added to a tube containing cell suspension. The mixture was incubated at 4°C for 15  min. The concentration of polymyxin B in the mixture was 6500 U/mL. After incubation, the mixture was centrifuged (12,000 g for 15  min). The supernatant obtained was used as the periplasmic fraction. An outer membrane fraction of the cells was prepared by treatment with sodium lauryl sarcosinate by the method of Filip et al. (23). Briefly, the cells of another tube were broken by sonication, and the insoluble materials precipitated by centrifugation at 10,000 g for 20  min. To solubilize the cytoplasmic membranes selectively, the precipitates were suspended with sodium lauryl sarcosinate solution.