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High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiaeASP3 gene

Paper ID Volume ID Publish Year Pages File Format Full-Text
17615 42684 2010 6 PDF Available
Title
High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiaeASP3 gene
Abstract

The enzyme asparaginase is used for the treatment of haematopoietic diseases, such as acute lymphoblastic leukaemia and non-Hodgkin lymphomas. The extraction of the periplasmic asparaginase produced in high levels by a recombinant Pichia pastoris strain harbouring the Saccharomyces cerevisiaeASP3 gene was studied. We submitted the yeast cells to freeze–thaw cycles, ethanol treatment and alkaline extraction in the presence and absence of cysteine. The use of six freeze–thaw cycles, followed by extraction with 20 mM potassium phosphate buffer pH 7.0 for 20 h, resulted in 85% enzyme recovery whereas the alkaline extraction using 500 mM potassium phosphate at pH 11.5 in the presence of 10 mM cysteine allowed 100% enzyme recovery. The protein and asparaginase concentrations in the crude extract for the alkaline cysteine treatment (1220 mg L−1 protein; 19,134 U L−1 asparaginase) were higher than those observed for the freeze–thaw procedure (840 mg L−1; 13,274 U L−1). The activities of the two aforementioned asparaginase crude preparations were stable upon storage at −18 °C for several months. SDS-PAGE analysis of the two extracts displayed two major protein bands from each extraction protocol, that were both identified as asparaginase II from S. cerevisiae by mass spectrometric analyses.

Keywords
Pichia pastoris; Asparaginase production; Periplasmic asparaginase extraction; Yeast periplasmic enzyme; ASP3 gene
First Page Preview
High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiaeASP3 gene
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 47, Issue 3, 5 August 2010, Pages 71–76
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering