Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1.
We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.
Matsuda
Emiko
E
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan.
Abe
Naoki
N
Tamakawa
Hideyuki
H
Kaneko
Jun
J
Kamio
Yoshiyuki
Y
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002208:Caproates / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019344:Lactic Acid / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010196:Pancreatic Elastase; D011091:Polyesters / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics; D048170:Streptomyces lividans / Q000235:genetics
D001426:Bacterial Proteins; D002208:Caproates; D004269:DNA, Bacterial; D007783:Lactones; D011091:Polyesters; D011108:Polymers; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019344:Lactic Acid; C033616:poly(lactide); C121056:caprolactone; D002265:Carboxylic Ester Hydrolases; C425772:poly(lactic acid) depolymerase; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D020155:3-Hydroxybutyric Acid