Enzymatic Hydrolysis of Thioester Linkages in Bacterial Poly(3-hydroxybutyrate-co-3-mercaptopropionate)s by Poly(3-hydroxybutyrate) Depolymerase Isolated from Ralstonia pickettii T1

Abstract

KEY WORDSPoly(3-hydroxybutyrate-co-3-mercaptopropionate) / Enzymatic Degradation / Thioester / [DOI 10.1295/polymj.37.711] One of major approaches to modify the bacterial poly(3-hydroxybutyrate) (PHB) while reserving its biodegradability has involved the copolymerization of its monomer with some hydroxyalkanoates (HA) via bacterial fermentation, 1 comprising 3-, 4-, 5-and 6-hydroxyalkanoates with various length of the mainchain or various substituents at different positions, such as 3-hydroxypropionate (3HP), 2-5 3-hydroxyvalerate (3HV) 6,7 and so forth. Recently, Steinbüchel's group [8][9][10][11][12][13][14] has succeeded to isolate a novel class of biopolymers from the poly-(hydroxyalkanoate) (PHA)-accumulating bacterium, comprising one kind of completely different backbone constituents, that is, thioesters, such as poly(3-mercaptopropionate), poly(3-mercaptobutyrate), poly(3-mercaptovalerate), poly(3-hydroxybutyrate-co-3-mercaptopropionate) [P(3HB-co-3MP)] and poly(3-hydroxybutyrate-co-3-mercaptobutyrate).Physical properties, such as solubility, crystallinity and thermal stability, of the biodegradable polymers are modified with the introduction of thioester linkages. 8,15 However, few research have been devoted to the enzymatic hydrolysis of thioester linkages in P(3HB-co-3MP) except for the work most recently published. 16 It is found that the PHA depolymerase isolated from Schlegelella thermodepolymerans is specific for the oxoester linkages and that thioester linkages can not be cleaved by the enzyme. PHA depolymerases are known to be quite specific to the chemical structure and physical state of polyesters. It seems necessary to investigate further the enzymatic hydrolyzability of the thioester linkages with considering the possible substrate specificity of depolymerase. PHA depolymerase isolated from Ralstonia pickettii T1 (PhaZRpi) has been well purified and characterized, and thus is one ideal enzyme to study the degradation of P(3HB-co-3MP)s.In this communication, the enzymatic degradability of thioester linkages in the comonomer compositionally well-fractionated P(3HB-co-3MP)s by PHA depolymerase PhaZRpi is evidenced by analyzing degradation products. It is the first observation of the degradation of thioester linkages by PHA depolymerase. Moreover, in comparison with P(3HB-co-3HP)s, the comonomer compositional dependence of the enzymatic degradability will be established. EXPERIMENTAL Materials and Preparation of SamplesP(3HB) 17 and P(3HB-co-3MP)s 10-13,15 were synthesized by using Ralstonia eutropha H16. Films for the enzymatic degradation were compression-molded at 195 C for 5 min under a pressure of 5 MPa, using Toyoseiki Mini Test Press-10, and then quenched to 30 C for crystallization of at least 4 weeks. The enzyme was isolated from Ralstonia pickettii T1. 19 Analytical ProceduresWide-angle X-ray diffraction (WAXD) measurements were carried out on a Rigaku RINT-2000

Biosynthesis and Biodegradability of Copolythioesters from 3,3′‐Thiodipropionic Acid and Plant Oils by Cupriviadus necator

“…This order of weight loss values is similar to the order of enzymatic degradability. [20] From these results, it is clear that the weight loss of the copolymers in the activated sludge supernatant is achieved through microbial action. The results indicate a non-linear relationship between the 3MP unit fraction and weight loss behavior.…”

“…[19] Zhu et al investigated the enzymatic degradation of poly(3HB-co-3MP) by extracellular PHB depolymerase and reported that the PHB depolymerase hydrolyzed 3HB-3MP linkages but not 3MP-3MP ones. [20] However, no detail on the microbial degradation behavior of poly(3HB-co-3MP) has been reported. To examine a general feature of the microbial degradability of poly(3HB-co-3MP), the degradation behavior of the prepared copolymers was investigated in an activated sludge supernatant at 25 8C at a pH of 7.0.…”

Mechanical Properties of Comonomer‐Compositionally Fractionated Poly[(3‐hydroxybutyrate)‐co‐(3‐mercaptopropionate)] with Low 3‐Mercaptopropionate Unit Content

“…It can be seen that, similar to P(3HB-co-3HP), the T g value of P(3HB-co-3MP) decreases with increasing 3MP unit content within the comonomer range investigated here, which is in accordance with our previous study. [25] Therefore, from the analysis of the thermal properties it is reasonable to suggest that besides the second comonomer unit content, the sequence distribution is also an important factor that affects the thermal properties, probably by influencing the crystal morphology of the copolymer.…”

“…So far, research about the biosynthesis, [14,[19][20][21] thermal properties, [22,23] biodegradation behavior, [24][25][26] etc. of P(3HB-co-3MP) has been intensive.…”

“…[23,28] Furthermore, the thermal properties and the enzymatic hydrolysis rate of P(3HB-co-3MP) were found to show a strong dependence on the comonomer unit content. [23,25,28] Thus, bacterially as-produced P(3HB-co-3MP) samples should be comonomer-unit compositionally well fractionated in order to study the comonomer-unit composition or the dependence of the physical properties and biodegradability of P(3HB-co-3MP)s.…”