N.B.IannucciI; F.J.WolmanI; S.A.CamperiI; A.A.N.CaizoI; M.GrasselliII; O.

CasconeI ICtedra de Microbiologa Industrial y Biotecnologa, Facultad de Farmacia y Bioqumica , UBA Junn 956, 1113, Buenos Aires, Argentina IIDepartamento de Ciencia y Tecnologa, Universidad Nacional de Quilmes, Argentina

Power point by : Satri Yanto (0810412058)

Source : Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632 Braz. J. Chem. Eng. vol.20 no.1 So Paulo Jan./Mar. 2003

http://dx.doi.org/10.1590/S0104-66322003000100006

Method

: Immobilised metal ion affinity chromatography (IMAC) Instruments : High-performance Affinity Chromatography Column : PD-10 columns. Eluent : Theres different eluents were assayed. * Ethyleneglycol and isopropanol only eluted less than 5% of adsorbed protease. * 1 M NaCl in 20 mM sodium acetate buffer, pH 5.0, eluted more than 90% of adsorbed protease on the three dye membranes. Detector System : UV absorbance wavelength 280 nm

Membran

yang berisi Cibacron Biru F3G-A, Red HE-3B dan Kuning 4R-DIA untuk afinitas kromatografi menggunakan membran mikrofiltrasi polietilen berlubangserat, nominal 0,33 pM diameter pori internal dan porositas 70% nominal. Diameter dalam dan luar adalah 0,6 dan 1,2 mm.
Membran

IDA dibuat dari polisulfon berlubang-serat membran mikrofiltrasi, memiliki nominal 0,65 pM diameter pori internal dan persen nominal porositas 80. Diameter dalam dan luar adalah 0,75 dan 1,25 mm.

Figure 1 shows the isotherms for adsorption of the neutral protease contained in Flavourzyme on dye-affinity membranes at pH 5.0. From these isotherms, qms of 19090, 12930 and 13870 U/ml for Yellow 4R-HE, Red HE-3B and Cibacron Blue F3G-A, respectively, were calculated. Kds were 18, 15 and 17 U/ml, respectively. The productivity of the process was 1900 U/ml.min 89% of electrophoretically pure enzyme was recovered

Figure 2 shows the pattern obtained. Ninety-nine per cent of the PE activity was retained by the chromatographic matrix and eluted quantitatively with 20 mM sodium phosphate buffer, 0.1 M EDTA, pH 7.0, thus indicating that the fractionation procedure can be successfully scaled up. The time of the fractionation process was far shorter than when working with chelating soft gel (Camperi et al., 1996), where lower flow rates must be used to allow mass transfer. The better hydrodynamic properties of the membranes resulted in enormous savings of time and a higher productivity: 750 PE U/ml.min compared with that previously obtained working with chelating soft gels, 52 U/ml.min