Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli

Abstract

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

FIG. 1.

Clear-zone formation obtained with recombinant E. coli XL10-Gold on agar plates containing emulsified PLA05 (A) and PBSA3020 (B). The plates were incubated at 37°C for 48 h.

FIG. 2.

Restriction map of the 2.9-kbp HindIII fragment from pLA29, showing the positions of the HindIII (Hin), SphI (Sph), SmaI (Sma), EcoRI (Eco), and PstI (Pst) restriction sites. The location of the plaA gene is indicated by an arrow. E. coli XL10-Gold transformed with each plasmid was grown on an agar plate containing emulsified PLA05 or PBSA3020 at 37°C for 24 h.

FIG. 3.

Nucleotide sequence of the PLA depolymerase gene. The deduced amino acid sequence is shown below the nucleotide sequence. A putative promoter region (−10 and −35), a Shine-Dalgarno (S.D.) sequence, and a ρ-independent terminator (horizontal arrows) are indicated. A sequence that exhibits the characteristics of a signal peptide is underlined, and the putative active site is doubly underlined. SphI and SmaI restriction sites at the beginning and end of the sequence, respectively, are underlined.

FIG. 4.

PLA and PBSA degradation by the purified recombinant PLA depolymerase. Emulsions of PLA05 (open square), PLA10 (open diamond), PLA15 (open circle), PLA20 (open triangle), PBSA3020 (closed square) PBSA3001 (closed diamond), PBS1020 (closed circle), and PBS1001 (closed triangle) were used as substrates. The decrease in the turbidity at a wavelength of 580 nm (left) and the TOC concentrations (right) were measured as described in Materials and Methods.

FIG. 5.

Dendrogram of the PLA depolymerase from P. amylolyticus strain TB-13 (PlaA) and related lipases. On the basis of the amino acid alignment, the dendrogram was constructed as described in Materials and Methods. The numbers at the nodes indicate the percent recovery in 100 bootstrap resamplings. The accession numbers for the amino acid sequences are indicated in Table 1.