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Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1
Ken-ichi Kasuya1, Tsutomu Takano, Yoko Tezuka
1Material Science Laboratory, Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu-shi, Gunma 376-8515, Japan. kkasuya@bce.gunma-u.ac.jp
International Journal of Biological Macromolecules
|November 11, 2003
Summary
Marinobacter sp. poly(3-hydroxybutyrate) (P(3HB)) depolymerase gene was cloned and analyzed. Substrate-binding domains are crucial for hydrolyzing solid P(3HB).
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Poly(3-hydroxybutyrate) (P(3HB)) is a biodegradable polymer.
- Microbial enzymes are key to P(3HB) degradation.
- Marinobacter sp. possesses a P(3HB) depolymerase gene.
Purpose of the Study:
- To clone and characterize the P(3HB) depolymerase gene from Marinobacter sp.
- To elucidate the functional roles of different protein domains in P(3HB) hydrolysis.
Main Methods:
- Genomic DNA cloning and sequencing of the P(3HB) depolymerase gene.
- Expression of fusion proteins (GST-CD, GST-CD-SBD).
- Enzyme activity assays and substrate-binding tests.
Main Results:
- The gene encodes a 578-amino acid protein with signal peptide, catalytic domain (CD), linker domain (LD), and two substrate-binding domains (SBD).
- Fusion proteins with CD exhibited hydrolytic activity.
- Proteins lacking SBDs showed significantly reduced activity on denatured P(3HB) (dP(3HB)).
- SBDs specifically bound to dP(3HB), not emulsified P(3HB) (eP(3HB)).
Conclusions:
- The substrate-binding domains (SBDs) are essential for the enzymatic hydrolysis of solid P(3HB) (dP(3HB)).
- The findings provide insights into the mechanism of P(3HB) degradation by Marinobacter sp. P(3HB) depolymerase.