Volume 46, No. 03, Month MAY, Year 2019, Pages 417 - 430
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
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.