Characterization of a thermolabile poly(3-hydroxybutyrate) depolymerase from the marine bacterium Shewanella sp JKCM-AJ-6,1α

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) [P(3HB)] depolymerase was cloned from the genomic DNA of the marine bacterium Shewanella sp. strain JKCM-AJ-6,1 alpha (wild-type strain) from seawater. The gene was 2049 bp and encoded 683-amino acids protein with a molecular mass of 70,382 Da. A sequence homology revealed that the deduced protein contains a signal peptide, catalytic domain (CD), fibronectin type III linker domain (LD), and two substrate-binding domains (SBDs). Meanwhile, a P(3HB) depolymerase purified from wild-type strain (wPhaZ(She)) had a smaller molecular mass (47 kDa) because it lacked the SBDs, compared with calculated one based on the sequence. wPhaZ(She) was unstable above 15 degrees C and had relatively high enzymatic activity in the presence of 0.5 M NaCl, demonstrating that the enzyme is thermolabile and halotolerant. In addition, there was no decrease in the activity by self-inhibition. In contrast, a recombinant form of the enzyme (rbPhaZ(She)) produced in Escherichia coil BL21 had a stronger binding affinity for P(3HB) and exhibited self-inhibition. These results demonstrate that a truncated form P(3HB) depolymerase lacking SBDs (wPhaZ(She)) has an advantage for P(3HB) degradation in marine environments with relatively high NaCl concentrations owing to a less self-inhibitory effect. (C) 2016 Elsevier Ltd. All rights reserved.