Abstract
We previously reported that poly(Asp) hydrolase-1 (PahZ1KP-2) from Pedobacter sp. KP-2 selectively, but not completely, cleaved the amide bonds between β-Asp units in thermally synthesized poly(Asp) (tPAA). In the present study, the enzymatic hydrolysis of stereoisomeric β-tri(Asp)s by PahZ1KP-2 was investigated to clarify the substrate stereoselectivity of PahZ1KP-2 in the hydrolysis of tPAA. The results suggest the following structural features of PahZ1KP-2 at its substrate binding site: (1) the active site contains four subsites (2, 1, −1, and −2), three of which need to be occupied by Asp units for cleavage to occur; (2) for the hydrolysis to proceed, subsite 1 should be occupied by an l-Asp unit, whereas the other three subsites may accept both l- and d-Asp units; (3) for the two central subsites between which cleavage occurs, the (l-Asp)-(d-Asp) sequence is the most favorable for cleavage.
Highlights
Introduction βPeptides, in which monomer units are connected at the β-position, fold into a conformationally ordered state in solution to exert their unique properties and potentially show their enzymatic and metabolic stabilities, which are consistent with the advantages of α-peptides such as interactions with proteins, DNA/RNA, and cell membranes (Seebach et al 2004; Seebach and Gardiner 2008)
Enzymatic hydrolysis of (α‐l‐Asp)‐(α‐l‐Asp)‐(l‐Asp) and (β‐l‐Asp)‐(β‐l‐Asp)‐(l‐Asp) by PahZ1KP‐2 To examine the effects of α- and β-amide linkages on the hydrolysis by PahZ1KP-2, the products of the enzymatic hydrolysis of (α-l-Asp unit in stereoisomeric β-tri(Asp)s to generate (l-Asp))-(α-l-Asp)-(l-Asp) and (β-l-Asp)(β-l-Asp)-(l-Asp) (LLL) were analyzed by Gel permeation chromatography (GPC)
GPC analysis of products on hydrolysis of β‐tri(Asp)s by PahZ1KP‐2 To elucidate the importance of the sequences of l- and d-Asp units in β-tri(Asp)s in enzymatic cleavage, eight β-tri(Asp)s with all possible combinations of l- and dAsp units were designed and enzymatically hydrolyzed by 1 μM PahZ1KP-2
Summary
Introduction βPeptides, in which monomer units are connected at the β-position, fold into a conformationally ordered state in solution to exert their unique properties and potentially show their enzymatic and metabolic stabilities, which are consistent with the advantages of α-peptides such as interactions with proteins, DNA/RNA, and cell membranes (Seebach et al 2004; Seebach and Gardiner 2008). Β-peptides have recently attracted a wide variety of interests as functional materials. Poly(Asp) (PAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that shows high polyanionic properties because of its high content of carboxyl groups relative to its molecular weight (Freeman et al 1996; Kim et al 1996; Tang and Wheeler 2001; Joentgen et al 2003; Thombre and Sarwade 2005). Owing to the unique structural and functional characteristics of tPAA, tPAA can be used as an environmentally benign material for dispersing agents and scale inhibitors. In addition to its application as a homopolymer, tPAA derivatives, including block copolymers with poly(ethylene glycol) and hydrogel cross-linked via diamine, have attracted attention owing to their potential applications in the medical and industrial fields as drug delivery systems. It is considered that tPAA biodegradability should be carefully taken into consideration in its applications for practical use
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