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Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae

Paper ID Volume ID Publish Year Pages File Format Full-Text
20306 43168 2015 5 PDF Available
Title
Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae
Abstract

•Saccharomyces cerevisiae strain overexpressing polyphosphatase PPN1 was constructed.•Recombinant PPN1 was purified and characterized.•Amino acid sequence of recombinant PPN1 was the same as in mature wild type PPN1.•The recombinant PPN1 was localized in the cytoplasm and vacuoles.•PPN1 polyphosphatase overexpression decreases short-chain polyphosphate level.

Inorganic polyphosphate performs many regulatory functions in living cells. The yeast exopolyphosphatase PPN1 is an enzyme with multiple cellular localization and probably variable functions. The Saccharomyces cerevisiae strain with overexpressed PPN1 was constructed for large-scale production of the enzyme and for studying the effect of overproduction on polyphosphate metabolism. The ΔPPN1 strain was transformed by the vector containing this gene under a strong constitutive promoter of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae. Exopolyphosphatase activity in the transformant increased 28- and 11-fold compared to the ΔPPN1 and parent strains, respectively. The content of acid-soluble polyphosphate decreased ∼6-fold and the content of acid-insoluble polyphosphate decreased ∼2.5-fold in the cells of the transformant compared to the ΔPPN1 strain. The recombinant enzyme was purified. The substrate specificity, cation requirement, and inhibition by heparin were found to be similar to native PPN1. The molecular mass of a subunit (∼33 kD) and the amino acid sequence of the recombinant enzyme were the same as in mature PPN1. The recombinant enzyme was localized mainly in the cytoplasm (40%) and vacuoles (20%). The overproducer strain had no growths defects under phosphate deficiency or phosphate excess. In contrast to the parent strains accumulating polyphosphate, the transformant accumulated orthophosphate under phosphate surplus.

Keywords
Exopolyphosphatase; Polyphosphate; Phosphate surplus; Saccharomyces cerevisiae; PPN1 gene; Constitutive promoter
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Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 119, Issue 1, January 2015, Pages 52–56
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us