Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764. Page: 18
viii, 44 p. : ill.View a full description of this thesis.
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in Amicon Ultra 10K centrifugal filters (Millipore).
Gel filtration chromatography. Fractions exhibiting Nit activity with FMN following the
second round of anion exchange chromatography were pooled and desalted prior to
being loaded (approximately 5 mg) onto Superdex 200 column (bed volume 24 ml).
The column was equilibrated in 20mM NaH2PO4-Na2HPO4 buffer (Na-Pi) (pH 7.0), and
proteins eluted at a linear flow rate of 0.5 ml/min. Determination of the approximate
molecular weight of eluting proteins was accomplished by comparing the elution
volumes (times) to that of standard proteins (apoferritin, 443 kDa; alcohol
dehydrogenase, 150 kDa; albumin, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome
C, 12.4 kDa) eluted under identical conditions. To those fractions exhibiting Nit
activity, dithiothreitol (1 mM) and glycerol (1%) were added prior to concentration (~
40X) in Amicon Ultra 10K centrifugal filters (Millipore). Concentrated proteins were
stored at -800C.
Gel Electrophoresis
Discontinuous SDS-PAGE (12 or 16%) was carried out with a Mini-Protean
(Bio-Rad) apparatus using either 12 or 16% acrylamide gels supported with a 5%
stacking gel. A 12% gel (8.4 x 5.3 cm), was prepared by mixing (in order) 4 ml of 30%
acrylamide/bis stock solution, 2.5 ml of 1.5 M Trizma buffer (pH 8.8), 100 pl of 10%
SDS, 2.05 ml diH20, 50 pil of 10% ammonium persulfate, and 5pl TEMED (N,N,N,N-
tetramethylenediamine). The gel was covered with isopropyl alcohol and the gel
allowed to polymerize for 45 minutes before washing off the isopropyl alcohol and
layering the separating gel with a 5% stacking gel. The stacking gel was prepared by18
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Chou, Chia-Ni. Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764., thesis, December 2010; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc33224/m1/28/: accessed May 6, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .