Extracellular Poly Research Articles

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X1-Ser-X2-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that 20Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.

Extracellular poly(alpha-L-guluronate)lyase from Corynebacterium sp.: purification, characteristics, and conformational properties.

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure-function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55 degrees C, respectively. Metal compounds such as MnCl2 and NiCl2 increased the enzyme activity. The enzyme was identified as the endolytic poly(alpha-L-guluronate)lyase, which was active on poly(alpha-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215 nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the beta-form of the enzyme molecule and resembled poly(beta-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(alpha-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The beta-sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.

Microbial degradation of polyesters: a review on extracellular poly(hydroxyalkanoic acid) depolymerases

Thermoplastic polyesters such as biologically produced poly[( R)-3-hydroxy-butyric acid], other polyhydroxyalkanoic acids and related chemosynthetic polyesters, have properties which make them valuable for many applications. In addition, these materials can be biodegraded to water and carbon dioxide. Many aerobic and anaerobic polyester-degrading microorganisms have been found in all moderate ecosystems. The microorganisms decompose the polymers by secretion of extracellular polyester depolymerases and utilize low molecular weight degradation products. This review summarizes the present knowledge on microbial extracellular polyester depolymerases.

Enzymatic degradation of poly(hydroxyalkanoate) by Corynebacterium aquaticum IM-1 isolated from activated sludge

Poly(4-hydroxybutyric acid)-degrading bacteria were isolated from activated sludge. One strain was selected, which was identified as Corynebacterium aquaticum. The strain C. aquaticum IM-1 excreted an extracellular poly(hydroxyalkanoate) [PHA]depolymerase and grew on 4-hydroxybutyric acid as the sole carbon source. The PHA depolymerase was purified from the cultured broth medium containing 4-hydroxybutyric acid as the sole carbon source by ultrafiltration, gel filtration and hydrophobic column chromatography. The molecular weight of the depolymerase was determined as approximately 33000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum activity of the depolymerase on the degradation of poly(4-hydroxybutyrate) (P(4HB)) was observed at pH 6.5 and 40 °C. Lipase activity of the depolymerase was not detected, and N-terminal sequences of the depolymerase were not applicable to the well-known enzymes. The enzymatic degradation of polyesters was studied by the erosion rate of solution-cast films of poly(3-hydroxybutyrate- co-4-hydroxybutyrate) and poly(ω-hydroxyalkanoate) of different chain lengths (C3–C6). The water-soluble products of poly(3-hydroxybutyrate co-97mol%4-hydroxybutyrate) film were revealed by 1H NMR and FAB-MS analysis, and the main product was the dimer of 4-hydroxybutyric acid.

Purification and characterization of extracellular poly(beta-D-1,4-mannuronide) lyase from Dendryphiella salina IFO 32139.

An extracellular endo poly(beta-D-1,4-mannuronide) lyase of Dendryphiella salina IF 32139 was purified to homogeneity by Q Sepharose FF and Sephacryl S-200 HR column chromatographies. The purified enzyme had a molecular weight of 35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.65 by isoelectric focusing. The optimum pH and temperature for enzyme activity were pH 5.0 and 45 degrees C, respectively. The enzyme was stable from pH 4 to 10 and at temperature below 40 degrees C. Some divalent cations, Ca2+, Mn2+, and Zn2+, increased the enzyme activity. Hg2+ and NBS strongly inhibited the activity. This enzyme susceptibly degraded poly-M, produced a wide range of 4,5-unsaturated oligomannuronic acids, and further degraded these unsaturated oligomannuronic acids to produce the unsaturated monomer and dimer as final products.

Characterization of a poly(3-hydroxybutyrate) depolymerase fromaureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified fromAureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11.N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-dl-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues byp-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds.A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films byA. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukaiet al. with poly(hydroxyalkanoates) depolymerases fromAlcaligenes faecalis, Pseudomonas pickettii, andComamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found withA. saperdae depolymerase.

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene.

An extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(-)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.

Enzymatic Degradation of Bacterial Poly(3-hydroxybutyrate) by a Depolymerase from Pseudomonas lemoignei

Extracellular poly(3-hydroxybutyrate)(PHB)-depolymerases A and B were purified from the culture medium of succinate-grown cells of Pseudomonas lemoignei by ammonium sulfate precipitation and chromatography on CM Sepharose CL-6B. The relative molecular weights of the sodium dodecyl sulfate (SDS)-treated enzymes were estimated to be 67 000 for both depolymerases. PHB-depolymerase A was homogeneous, as revealed by SDS−PAGE, and was used for degradation experiments on compression molded PHB films, exhibiting a spherulitic morphology. The extent of enzymatic degradation was found to increase with increasing depolymerase concentration (CE) and reach a plateau at CE ≥ 3 μg/mL. Slight morphological changes were induced on PHB films by different thermal treatments, in order to study their effect on degradation rate at a constant overall degree of crystallinity. It was found that the enzymatic degradation rate of PHB decreases as a consequence of an increase of the average size of the crystals, i.e. of crystalline ...

Enzymatic Degradation of Bacterial Poly(3-hydroxybutyrate) by a Depolymerase from Pseudomonas lemoignei

Extracellular poly(3-hydroxybutyrate)(PHB)-depolymerases A and B were purified from the culture medium of succinate-grown cells of Pseudomonas lemoignei by ammonium sulfate precipitation and chromatography on CM Sepharose CL-6B. The relative molecular weights of the sodium dodecyl sulfate (SDS)-treated enzymes were estimated to be 67 000 for both depolymerases. PHB-depolymerase A was homogeneous, as revealed by SDS−PAGE, and was used for degradation experiments on compression molded PHB films, exhibiting a spherulitic morphology. The extent of enzymatic degradation was found to increase with increasing depolymerase concentration (CE) and reach a plateau at CE ≥ 3 μg/mL. Slight morphological changes were induced on PHB films by different thermal treatments, in order to study their effect on degradation rate at a constant overall degree of crystallinity. It was found that the enzymatic degradation rate of PHB decreases as a consequence of an increase of the average size of the crystals, i.e. of crystalline ...

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.

Morphology and biodegradability of a binary blend of poly((R)-3-hydroxybutyric acid) and poly((R,S)-lactic acid).

The miscibility, morphology, and biodegradability of a binary blend of bacterial poly((R)-3-hydroxybutyric acid)(P((R)-3HB);Mn = 300,000) with atactic poly((R,S)-lactic acid)(P((R,S)-LA);Mn = 9,000) were studied by means of differential scanning calorimetry, optical microscopy, scanning electron microscopy, and hydrolysis with an without enzyme. Differential scanning calorimetry revealed that a P((R)-3HB)-P((R,S)-LA) blend had a single glass-transition temperature for all proportions of the components. The spherulites of P((R)-3HB) were volume filled in the blend films, indicating the inclusion of amorphous P((R,S)-LA) within the spherulites. The spherulitic growth rate decreased with an increase in the content of P((R,S)-LA). These results indicate that the P((R)-3HB)-P((R,S)-LA) blend is miscible in the melt and in the amorphous state. The enzymatic hydrolysis of P((R)-3HB)-P((R,S)-LA) blend films was carried out at 37 degrees C for 19 h in 0.1 M potassium phosphate buffer (pH 7.4) with an extracellular poly(hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. The rate of enzymatic surface erosion decreased with increasing P((R,S)-LA) content in the blend films. The simple hydrolysis of P((R)-3HB)-P((R,S)-LA) blend films without enzyme was also conducted at 37 degrees C in a 0.01 M potassium phosphate buffer (pH 7.4) for 150 days. The hydrolytic scission of P((R)-3HB) polymer chains was accelerated by blending with P((R,S)-LA). However, the rate of enzymatic hydrolysis was much faster than the rate of nonenzymatic hydrolysis.

Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13.

The isolation of poly(3-hydroxyoctanoic acid)- and poly(6-hydroxyhexanoic acid)-degrading bacteria yielded 28 strains with abilities to degrade various polymers. The most versatile strains hydrolyzed five different polyesters comprising short chain length and medium chain length poly(hydroxyalkanoates). The new isolates together with previously isolated poly(hydroxyalkanoate)-degrading bacteria were classified into 11 groups with respect to their polymer-degrading specificities. All PHA depolymerases studied so far have been characterized by the lipase consensus sequence Gly-X-Ser-X-Gly in their amino acid sequence, which is a known sequence for serine hydrolases. When we replaced the central residue, Ser-172, in the corresponding sequence Gly-Ile-Ser-Ser-Gly of the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, with alanine the enzyme lost its activity completely. This result of the mutational experiment indicates that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.

Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHA depolymerases

The kinetics and mechanism of enzymatic degradation on the surface of poly[(R)-3-hydroxybutyrate] (P[(R)-3HB]) film have been studied using three types of extracellular poly(hydroxyalkanoate) (PHA) depolymerases from Alcaligenes faecalis, Pseudomonas pickettii and Comamonas testosteroni. The monomer and dimer of 3-hydroxybutyric acid were produced during the course of the enzymatic degradation of P[(R)-3HB] film, and the rate of production was determined by monitoring the increase in absorbance at 210 nm on a spectrophotometer. The rate of enzymatic degradation increased to a maximum value with the concentration of PHA depolymerase, followed by a gradual decrease. The kinetic data were accounted for in terms of a heterogeneous enzymatic reaction, involving enzymatic degradation on the surface of P[(R)-3HB] film via two steps of adsorption and hydrolysis by a PHA depolymerase with binding and catalytic domains. The kinetic results suggest that the properties of the catalytic domains are very similar among the three PHA depolymerases, but that those of the binding domains are strongly dependent on the type of depolymerase.

Enzymatic degradation of poly(hydroxyalkanoates) by Pseudomonas pickettii

A bacterium capable of degrading poly(3-hydroxybutyrate) [P(3HB)] was isolated from laboratory media and identified as Pseudomonas pickettii. The strain P. pickettii excreted an extracellular poly(hydroxyalkanoate) (PHA) depolymerase and grew on P(3HB) as the sole carbon source. P. pickettii also grew on 3-hydroxybutyrate, glucose, fructose, citrate or succinate. However, only 3-hydroxybutyrate apart from P(3HB) induced the secretion of PHA depolymerase. The PHA depolymerase was purified from the culture medium containing 3-hydroxybutyrate at the sole carbon source by hydrophobic column chromatography and gel filtration, and its molecular weight was determined as about 40 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The optimum activity of degrading P(3HB) by the depolymerase was observed at pH 5.5 and 40°C. The enzymatic degradation of microbial copolyesters was studied by the weight loss (erosion) of solution-cast films of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). 1H-n.m.r. analysis of the water-soluble products after the hydrolysis of P(3HB) film by PHA depolymerase revealed that the main product was the monomer 3-hydroxybutyric acid.

Enzymatic degradation of poly([R,S] ?-hydroxybutyrate)

The synthetic analogue of a bacterially produced polyester, poly(β-hydroxybutyrate) (PHB) was synthesized from racemic β-butyrolactone using anin situ trimethyl aluminum-water catalyst. The polymer was fractionated into samples differing in molecular weight and isotactic diad content. The latter was closely related to degree of crystallinity. The biodegradation of these fractions were examined by monitoring mass loss over time in the presence of anAlcaligenes faecalis T1 extracellular bacterial poly(β-hydroxybutyrate) depolymerase. The fraction with high isotactic diad tacticity content showed little or no degradation over a 50 hour incubation period, whereas the fraction of intermediate isotactic diad content degraded in a continuous steady fashion at a rate that was less than that for bacterial PHB. The low isotactic diad fraction underwent a rapid initial degradation, followed by no further mass loss. The presence of stereoblocks in the polymer structure of the various fractions was an influence on the degree of susceptibility towards degradation and is related to sample crystallinity.

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.

Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: General characteristics and active site studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase has been isolated from Penicillium funiculosum cultural medium by a single hydrophobic column chromatography. The enzyme is a glycoprotein composed of a single polypeptide chain with a molecular mass of about 37,000 Da as analyzed by denatured sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by native gel filtration on Sephadex G-100. Its optimum activity occurs at pH 6.0. It has an isoelectric point of 5.8 and has a K m for PHB (average molecular weight = 45,000 Da) of 0.17 mg/ml. Various nonionic detergents competitively inhibit the enzyme with K i values of 0.56 and 0.014% for Tween 80 and Triton X-100, respectively. The enzyme is extremely sensitive to diisopropyl fluorophosphate, mercuric ion, and dithiothreitol (DTT). However, sulfhydryl reagents have little or no effect on its activity. The inactivation by mercuric ion and DTT is reversible by mercaptoethanol and hydrogen peroxide, respectively. These data suggest that the enzyme may be a serine esterase and may contain an important disulfide bond. The enzyme is also inactivated by diazoacetyl and epoxide compounds at low pH, which can be prevented by PHB, indicating the presence of a critical carboxyl group at the active site. These characteristics of the enzyme are compared to other extracellular polymerases isolated from bacterial culture media.

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.

Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity

The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis Tl, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of d(−)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mild trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.