◆ SpookStack

United States Patent: 6,387,665 Page 7 of 11 * control. The final fermentation pH values of 8.57 and 8.67 after an elapsed fermentation time of ca. 8 hrs were also comparable. The effect of prolonged exposure to these mildly alkaline conditions on cell growth, PA production and subsequent degradation was investigated by repeating the fermentation at a constant pH of 7.50+/-0.05 pH units. This was accomplished using the immersed vessel pH probe and automated additions of 2 N HCl or 1 N NaOH. The results shown in Table 1 demonstrate that there was no clear effect of constant pH on any of the parameters evaluated. SDS-PAGE analysis of the fermentation time points sampled for PA production also revealed no significant differences. The final fermentation presented in Table 1 was a noncontinuous fed-batch trial during which 1/10 volume of a 10-fold concentrate of sterile-filtered tryptone was added after 5 hrs or an O.D..sub.600nm of 7.5. The result suggested that such fed-batch fermentations provide possible protocols for improvement to increase yield and decrease proteolysis. Harvest conditions: Fermentations were allowed to proceed until no further increase in O.D..sub.600nm was observed. At this point, the fermentor was cooled to 10.degree. C. and the protease inhibitors phenylmethylsulfony] fluoride (PMSF), 1,10-phenanthroline (OP) and ethylenediamine tetraacetate (EDTA) were added to final concentrations of 0.1, 0.05 and 2 mM, respectively. The cells were then pumped from the fermentor vessel at room temperature using an Amicon DC10L concentrator equipped - with a 10-ft.sup.2 0.1 .mu.polysulfone hollow-fiber cartridge. The fermentor liquor was diluted 1:1 with 25 mM diethanolamine (DEA), 50 mM NaCl, 2 mM EDTA, 0.1 mM PMSF adjusted to pH 8.9 with HCl. The filtrate was collected at an operating pressure of less than 20 psi and transferred directly to a second Amicon DCIOL equipped with two 30 kDa cutoff 10-ft.sup.2 wound spiral cellulosic cartridges. The filtrate was concentrated approximately 10-fold before being subjected to diafiltration at an operating pressure of less than 30 psi against the same buffer. The conductivity of the retentate was monitored with an Amber Sciences conductivity meter and platinum immersion pencil-type electrode. The diafiltration step generally required 20 liters of buffer, but was considered complete only after the conductivity of the concentrated retentate was equivalent to that of the starting buffer. Quantitation of 83 kDa PA in crude fermentation liquor: The fermentation liquor was sampled using a sterile port at regular intervals throughout the fermentation process. The samples for PA determination were filtered through syringe type 0.2.mu. cellulose acetate filters, 0.1 mM PMSF, 2 mM EDTA, 50 .mu.M OP and 20 mM HEPES pH7.3 were added and the samples were frozen at -70.degree. C. The samples were defrosted on ice and concentrated using Amicon Centricon 30 concentrators at 4500.times.g. The samples were concentrated approximately 10-fold, diluted to the original volume with 10 mM TRIS pH8.0, 0.1 mM PMSF, 2 mM EDTA, 0.05 pM OP and concentrated again. The concentrated sample was desalted again using the same buffer, frozen and finally lyophilized using a Speed-Vac. The dried samples were dissolved in 25 .mu.l of the TRIS buffer described above and diluted 1:1 with a 2.times.SDS solubilization buffer consisting of 50 mM Na.sub.2 CO.sub.3, 4% (w/v) SDS, 12% (v/y) glycerol, 2% (v/v) 2-mercaptoethanol and 0.01% (w/v) Bromphenol Blue prior to heating at 95.degree. C. for 5 min. The fermentation samples containing varying amounts of PA 83 kDa were solubilized as described above and run on a Daiichi 4-20% gradient TRIS/TRICINE gel to approximate total yield of PA. Two hundred to 2000 ng samples of purified PA were solubilized in the same buffer and loaded onto the gel in constant total volume of 3 .mu.l., Three or four appropriate dilutions of the fermentation samples determined from the first gel were loaded onto the gel with the standards and electrophoresed at 100 V initially and 140 V once the samples entered the separating gel and until the Bromphenol Blue dye reached the bottom edge of the separating gel. The gel was then fixed in 10% (v/v) acetic acid 20% (v/v) MeOH for 10 min, rinsed with MQ H.sub.2 O and stained with Coomassie Brilliant Blue 0.05% (w/v) in 10% (v/v) acetic acid for a minimum of 16 hrs to allow complete and uniform staining. The stained gel was then destained in 10% (v/v) acetic acid until the background contained no visible residual dye. The gel was then scanned on a laser densitometer (LKB, Ultrascan XL Laser Densitometer). Representative portions of the gel without protein were randomly http://patft.uspto.gov/netacgi/nph-Parser?Sectl1=PTO2&Sect2=HITOFF&u=/netahtml/sear... 6/28/2005