Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764. Page: 16
viii, 44 p. : ill.View a full description of this thesis.
Extracted Text
The following text was automatically extracted from the image on this page using optical character recognition software:
Preparation of cell extracts. Frozen cell pellets were suspended in Na-K phosphate
buffer (pH 7.0 (2 ml/1 g cells) containing 1mM dithiothreitol, 1% glycerol and 50pg/ml
DNase. Cells were broken at 4C in a French Press (20,000 psi) followed by low-
speed centrifugation at 30,000 x g. The resulting supernatant was then subjected to
ultracentrifugation at 150,000 x g for 90 minutes and the supernatant (HSS, high-
speed supernatant) retained.
Anion exchange chromatography. A Source 30Q (GE healthcare) anion exchange
column (bed volume 55 ml) was equilibrated with 20 mM Piperazine buffer (pH 10.0)
(running buffer) for 3 column volumes (flow rate 3 ml per min) before applying
approximately 200 mg of HSS protein (~ 13 ml HSS equilibrated with 2 equal volumes
of running buffer). Non-binding proteins were removed by applying an additional 3
column volumes of running buffer before eluting bound proteins with a linear gradient
of 0-0.5 M Na2SO4 applied over five column volumes. The 2nd ion-exchange step
was performed similarly except that a smaller amount of protein (about 25 mg) was
applied. Also, prior to initiating the 2nd round of Source30Q chromatography the
column was reversed in orientation and cleaned successively with 2M NaCI (3 ml/min
30-60 min), 1 N NaOH (3 ml/min 1-2 hours) followed by 70% ethanol (1ml/min 1-2
hours). Following chromatography, proteins recovered in fractions (6 mL) were tested
for Nit activity with FMN. For this purpose, 90pl of each fraction was placed in 2 ml
sealed HPLC vial to which 10 mM fumaronitrile was added. Vials were incubated for
90 min to 2 h and analyzed for evidence of ammonia formation by visible inspection.
Those fractions exhibiting activity were collected and desalted with Na-K buffer (3
times original volume) (50 mM, pH 7.0) containing 1 mM dithiothreitol, and 1% glycerol16
Upcoming Pages
Here’s what’s next.
Search Inside
This thesis can be searched. Note: Results may vary based on the legibility of text within the document.
Tools / Downloads
Get a copy of this page or view the extracted text.
Citing and Sharing
Basic information for referencing this web page. We also provide extended guidance on usage rights, references, copying or embedding.
Reference the current page of this Thesis.
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/m1/26/: accessed May 6, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .