Poly(L-lactide)-Degrading Enzyme from Laceyella sacchari LP175: Cloning, Sequencing, Expression, Characterization and Its Hydrolysis of Poly(L-lactide) Polymer
Thanasak Lomthong, Marie Guicherd, Gianluca Cioci, Sophie Duquesne, Alain Marty, Saisamorn Lumyong and Vichien Kitpreechavanich* Author for corresponding; e-mail address: fsciwck@ku.ac.th
Volume :Vol.46 No.3 (May 2019)
Research Article
DOI:
Received: 3 June 2018, Revised: -, Accepted: 13 November 2018, Published: -
Citation: Lomthong T., Guicherd M., Cioci G., Duquesne S., Marty A., Lumyong S., et al., Poly(L-lactide)-Degrading Enzyme from Laceyella sacchari LP175: Cloning, Sequencing, Expression, Characterization and Its Hydrolysis of Poly(L-lactide) Polymer, Chiang Mai Journal of Science, 2019; 46(3): 417-430.
Abstract
In this study, the poly-(L-lactide) (PLLA)-degrading enzyme from the thermophilic filamentous bacterium, Laceyella sacchari LP175 was characterized for application in biological recycling process. The gene encoding the PLLA-degrading gene (plla_lp175) was cloned and expressed in Escherichia coli. The native protein comprises 383 amino acids with a molecular mass and pI of 39.45 kDa and 8.26, respectively. The recombinant protein was purified by one-step purification, and was found to have a molecular weight of 28 kDa. Functional expression of PLLA_LP175 in E. coli with a pMAL-c5X vector enhanced the expression of PLLA-degrading enzyme up to 756 U/mg of protein, which is a 2.3-fold increase compared to the native strain. The purified recombinant protein is active in the range of pH 7.0–9.0 and 45- 60 °C, with optimum activity at pH 9.0 and 60 °C. The recombinant enzyme could hydrolyse PLLA objects to a different extent depending on PLLA composition, at pH 9.0 and 50 °C within 24 h. Monomeric lactic acid was detected as the product from enzymatic degradation of PLLA objects. A scanning electron micrograph, showing a rough surface with holes of all PLLA objects after treatments with the recombinant enzyme, confirmed the ability of this enzyme to degrade PLLA objects.