The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst ) of Pseudomonas stutzeriwas cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B fromClostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S -transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

The mechanism of enzymatic hydrolysis for (R)-3-hydroxybutyrate (3HB) oligomers with poly[(R)-3-hydroxybutyrate] [P(3HB)] depolymerase (PhaZpst) from Pseudomonas stutzeri was investigated by two deletion mutants lacking the substrate-binding domain and linker region, PhaZpst delta sbd and PhaZpstcore. The two deletion mutants had no ability for hydrolysis of water-insoluble P(3HB), while the hydrolysis activities of two deletion mutants for water-soluble 3HB oligomer and its derivatives (dimer, trimer, and tetramer) were identical with those of the wild type, indicating that the function of catalytic domain is independent of its substrate-binding domain and linker region. The hydrolyzed products analysis of 3HB oligomers by HPLC showed that the active site of catalytic domain recognizes at least two 3HB units for hydrolysis. The initial rates of hydrolysis of dimer derivative were lower by 2 orders of magnitude than those of trimer and tetramer derivatives, suggesting that 3HB oligomer derivatives larger than trimer are favorite substrates for PhaZpst.

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The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate (4HB) with high mole fractions of 4HB units by a wild‐type strain of Ralstonia eutropha H16 was investigated in culture solutions containing 4‐hydroxybutyric acid (4HBA) and various carbon substrates in the presence of a nitrogen source such as ammonium sulfate. The addition of glucose or acetic acid to the culture solution containing 4HBA in the presence of ammonium sulfate resulted in the production of random copolymers of P(3HB‐co‐4HB) with compositions of up to 82 mol% 4HB, but the yield of copolymers was less than 7 wt% of dried cell weights. In contrast, when n‐alkanoic acids such as propionic acid, butyric acid, valeric acid and hexanoic acid, being subject to β‐oxidation metabolism in the cell, were used as the co‐substrates of 4HBA in the presence of ammonium sulfate, a mixture of copolymers with two different 4HB compositions was produced, and copolyesters with compositions of 93–100 mol% 4HB were isolated from chloroform–n‐hexane insoluble fractions in the mixture of copolymers. Especially, when this wild‐type Ralstonia eutropha H16 was cultivated in a medium containing 4HBA (15 g litre−1), propionic acid (5 g litre−1) and ammonium sulfate (5 g litre−1), namely C/N (mol/mol) = 10, the P(4HB) homopolymer was produced at maximally 34 wt% of dry cell weight (7.8 g litre−1), and the conversion yield of 4HBA to P(4HB) homopolymer resulted in values as high as 21 mol%. © 1999 Society of Chemical Industry

Poly(3-hydroxybutyrate) [P(3HB)] was prepared by the ring-opening polymerization of (R,S)-β-butyrolactone (BL) using two types of PHB depolymerase with or without substrate-binding domains (SBD) as the catalyst. The SBD lacking PHB depolymerase exhibited better catalytic activities for the ring-opening polymerization of BL.