Poly(aspartate) (PAA) is a bio-based, biocompatible, biodegradable alternative to polycarboxylate of poly(acrylate). Bacterial PAA-hydrolyzing enzyme can also perform synthesis of poly(alpha-ethyl beta-aspartate). This family is close to PHB depolymerase. The LpqC, poly(3-hydroxybutyrate) depolymerase from Bordetella parapertussis (CASP Target) (structure 3D0K) belongs to this family. Polyaspartic acid (PASA) is also a natural polymer as fragment of larger proteins with length up to 50 amino acids. Synthetic tPAA(thermally synthesized PAA) consists of beta-amide (70%) and alpha-amide,(30%) D- and L-aspartate units; the alpha- and beta-amide units are randomly distributed. PAA hydrolase-1 is an alpha beta hydrolase inhibited by PMSF and DFP. PAA hydrolase-2 is closer to metallo carboxypeptidase inhibited by DFP and PMSF
Thermally synthesized poly(aspartate) (tPAA) shows potential for use in a wide variety of products and applications as a biodegradable replacement for non-biodegradable polycarboxylates, such as poly(acrylate). The tPAA molecule has unnatural structures, and the relationship between its biodegradability and structures has been investigated. Two tPAA-degrading bacteria, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2, were isolated from river water; from them, two PAA-hydrolyzing enzymes, PAA hydrolases-1 and -2, were purified and biologically and genetically characterized. Interestingly, not only are PAA hydrolases-1 from those two strains novel in terms of structural genes and substrate specificities (they specifically cleave the amide bond between beta-aspartate units in tPAA), they also probably play a central role in tPAA biodegradation by both strains. In green polymer chemistry, one active area of research is the use of purified enzymes for the enzyme-catalyzed synthesis of polypeptides by taking advantage of their substrate specificities. Recently, beta-peptides have attracted academic and industrial interest as functional materials as they possess both functions of alpha-peptides and excellent metabolic stability. As one of the attractive applications of PAA hydrolases, we report here the enzyme-catalyzed synthesis of poly(alpha-ethyl beta-aspartate), which is composed of only beta-linkages and belongs to beta-peptides, using the unique substrate specificity of the enzyme from Pedobacter sp. KP-2.
Title: Cloning of poly(aspartic acid) (PAA) hydrolase-1 gene from Pedobacter sp. KP-2 and hydrolysis of thermally synthesized PAA by its gene product Hiraishi T, Masuda E, Kanayama N, Nagata M, Doi Y, Abe H, Maeda M Ref: Macromol Biosci, 9:10, 2009 : PubMed
Pedobacter sp. KP-2 can degrade and metabolize thermally synthesized alpha,beta-poly(D,L-aspartic acid) (tPAA), which contains 70% of unnatural beta-amide units, with high-molecular-weight. In this study, gene cloning and molecular characterization of PAA hydrolase-1 from KP-2 was carried out. Gene analysis reveals that deduced amino acid sequence of the enzyme shows a similarity to only that of PAA hydrolase-1 from Sphingomonas sp. KT-1. GPC and NMR analyses of the hydrolyzed products of tPAA by PAA hydrolase-1 of KP-2 indicate that this enzyme cleaves the beta-beta amide linkage via endo-mode to yield oligo(aspartic acid) from tPAA. Taking the composition of tPAA and the substrate specificity of PAA hydrolase-1 into consideration, the enzyme possibly plays a crucial role in tPAA biodegradation by KP-2.
Title: Genetic Analysis and Characterization of Poly(aspartic acid) Hydrolase-1 from Sphingomonas sp. KT-1 Hiraishi T, Kajiyama M, Tabata K, Yamato I, Doi Y Ref: Biomacromolecules, 4:80, 2003 : PubMed
Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.
