A New Type of Thermoalkalophilic Hydrolase of Paucimonas lemoignei with High Specificity for Amorphous Polyesters of Short Chain-length Hydroxyalkanoic Acids

Abstract

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholatecoated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-␣-benzoyl-L-arginine-4-nitranilide, or starch. The purified enzyme (M r 36,209) was remarkably stable and active at high temperature (60°C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.Poly((R)-3-hydroxyalkanoic acids) (PHAs) 1 are a class of storage compounds that are synthesized during unbalanced growth by many bacteria. PHAs are deposited intracellularly in the form of inclusion bodies ("granules") to levels up to 90% of the cellular dry weight. The subject was reviewed recently (1). Poly((R)-3-hydroxybutyric acid) (PHB) is the most abundant polyester in bacteria. Bacterial copolymers containing randomly distributed (R)-3-hydroxybutyric and (R)-3-hydroxyvaleric units (poly(3HB-co-3HV)) have been commercialized for over a decade under the trade name Biopol ® . Any research on the biodegradation of PHA should clearly distinguish between (i) extracellular PHA degradation and (ii) intracellular PHA degradation. (i) Extracellular degradation is the utilization of an exogenous carbon/energy source by a notnecessarily-accumulating microorganism. The source of this extracellular polymer is PHA-released by accumulating cells after death. The ability to degrade PHA is widely distributed among bacteria and depends on the secretion of specific PHA depolymerases that are carboxyesterases (EC 3.1.1) a...

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

“…Recently, amorphous PHB-specific extracellular PHB depolymerase was found from Paucimonas lemoignei (PhaZ7 ple ) (9). Although PhaZ7 ple show a high PHB-hydrolyzing specific activity, the amino acid sequence was not similar to either the type I or type II catalytic domain.…”

Cloning and Sequencing of a Poly( dl -Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli

“…The deduced amino acid sequence showed 45 to 50% sequence identity with mesophilic Bacillus family I-4 lipases (3,18,21,34), ϳ20% sequence identity with thermophilic Bacillus family I-5 lipases (2,11,29), but no identity with PHA depolymerase from P. lemoignei, except for the conserved pentapeptide (6). Functional expression and purification of the recombinant enzyme in E. coli.…”

“…The PHA depolymerase from P. lemoignei, PhaZ7, is included in the lipases possessing an Ala-His-SerMet-Gly pentapeptide. Because of its unique sequence, Handrick et al suggested that PhaZ7 should be classified into a new family of carboxylesterases (6). The purified recombinant PLA depolymerase could not degrade P(HB-co-HV) ( Table 2), and the entire amino acid sequences of PlaA and PhaZ7 were different, except for the catalytic pentapeptide.…”

Unraveling the Function of the Rhodospirillum rubrum Activator of Polyhydroxybutyrate (PHB) Degradation: the Activator Is a PHB-Granule-Bound Protein (Phasin)

“…Semicrystalline dPHB was isolated from accumulating cells of R. eutropha H16 by sodium hypochlorite digestion and subsequent solvent extraction of the dried polymer with acetone-diethyl ether (2:1). nPHB granules with an intact surface layer were prepared from crude extracts (French press) of PHB-rich cells of R. eutropha H16 or from recombinant E. coli harboring the PHB biosynthetic genes phaCBA of R. eutropha with or without phaP (17) by two sodium phosphate-buffered glycerol density gradient centrifugation steps as described previously (10). nPHB granules were isolated from PHB-accumulating cells of Chromobacterium violaceum, Bacillus megaterium, Bacillus cereus, and R. rubum by the same procedure.…”