Matsuda Emiko, Abe Naoki, Tamakawa Hideyuki, Kaneko Jun, Kamio Yoshiyuki
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.
J Bacteriol. 2005 Nov;187(21):7333-40. doi: 10.1128/JB.187.21.7333-7340.2005.
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.
我们分离出了一种聚丙交酯或聚（L-乳酸）（PLA）降解细菌——拟无枝酸菌属菌株K104-1，并从该细菌的培养液中纯化出了PLA解聚酶（PLD）。在此，我们在变铅青链霉菌1326中克隆并表达了编码PLD的pld基因，并对重组PLD（rPLD）制剂进行了表征。我们还描述了从新生PLD到成熟PLD的加工机制。pld基因编码的PLD是一种由238个氨基酸组成的24,225道尔顿的多肽。对PLD及其前体的生化分析和蛋白质免疫印迹分析表明，PLD以前体（前原类型）形式合成，需要对包括26个氨基酸信号序列和9个氨基酸前导序列的N端35个氨基酸延伸进行蛋白水解切割，以产生20,904道尔顿的成熟酶I。发现前导序列的切割是自催化的。PLD与许多真核丝氨酸蛋白酶具有约45%的相似性。此外，在真核丝氨酸蛋白酶中参与形成切割底物酰胺键所需催化三联体的三个氨基酸残基H57、D102和S195（胰凝乳蛋白酶编号）在PLD中作为H74、D111和S197残基保守存在。在PLD中，与S195残基形成氧阴离子洞并与可裂解肽键的羰基形成重要氢键的G193残基（胰凝乳蛋白酶编号）也保守存在。对PLD突变体H74A、D111A和S197A的功能分析表明，H74、D111和S197残基对PLD的解聚酶活性和酪蛋白分解活性以及从前体类型PLD切割前导序列以形成成熟PLD很重要。PLD制剂具有弹性蛋白酶活性，1 mM弹性蛋白酶抑制剂不能抑制该活性，该浓度比完全抑制猪胰弹性蛋白酶所需浓度高10倍。rPLD制剂将平均分子量为220 kDa的PLA通过乳酸低聚物降解为乳酸二聚体，最终降解为乳酸。PLD制剂与3-羟基丁酸、ε-己内酯和丁二酸丁二醇酯的高聚物以及PLA结合，但它只降解PLA。
