Polymer Degradation and Stabiltiy 58 (1997) 275-281

0 1997 Elsevier Science Limited

Printed in Northern Ireland. All rights reserved
ELSEVIER PII: SOl41-3910(97)00058-X 0141.3910/97/$17.M)

Enzymatic degradation of poly[ (R )-

3-hydroxybutyrate]: secretion and properties of
PHB depolymerase from Pseudomonas
stutzeri

Megumi Uefuji, Ken-ichi Kasuya & Yoshiharu Doi*

Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-l Hirosawa, Wako-shi,
Saitama 351-01, Japan

(Received 24 January 1997; accepted 10 February 1997)

The secretion mechanism and properties of an extracellular polyhydroxybuty-

rate (PHB) depolymerase of Pseudomonas stutzeri YM1006, which had been
isolated from sea water, were studied in the culture containing poly[(R)-
3-hydroxybutyrate] [P(3HB)], (R)-3-hydroxybutyrate [(R)-3HB] or (S)-
3-hydroxybutyrate [(S)-3HB] as sole carbon source. R stutzeri YM1006 was
able to grow on P(3HB), (R)3HB and (S)3HB. The bacterium secreted a
PHB depolymerase into the culture supernatant during growth on P(3HB) and
(R)-3HB, while it did not secrete the enzyme on (S)3HB. The PHB
depolymerase was purified to electrophoretic homogeneity from the culture
medium of I? stutzeri by hydrophobic interaction chromatography, and its
molecular weight was determined as 60 kDa by electrophoresis on polyacryl-
amide gel in the presence of sodium dodecyl sulfate. The enzyme was stable at
temperatures up to 50C in aqueous solution at pH values of 6 to 12. The
optimum activity of degrading P(3HB) by the enzyme was observed at pH 7.0.
A kinetic study of enzymatic hydrolysis of P(3HB) film was carried out at
temperatures from 30 to 45C at pH 7.4 in 0.1 M potassium phosphate buffer.
The enzymatic degradation product contained monomer and dimer of
3-hydroxybutyric acid, and the monomer was a major product over the whole
range of enzyme concentration. 0 1997 Elsevier Science Limited

1 INTRODUCTION Several PHB depolymerases have been purified

from some microorganisms, and their properties
Poly[(R)-3-hydroxybutyrate] [P(3HB)] and its have been characterized.5m12 The structural genes
copolyesters are produced by a wide variety of of PHB depolymerases of Alcaligenes fuecaZis,13
bacteria and have some attractive physical Pseudomonas lernoignei,66 Pseudomonas pick-
properties comparable to those of conventional ettii and Cornamonas sp. have been cloned and
plastics. A remarkable characteristic of P(3HB) sequenced. Analysis of the primary structure has
is its biodegradability in the environment.2~3 revealed that the enzymes of 393438 amino acids
Aerobic and anaerobic P(3HB)-degrading bacte- are composed of two functional domains and a
ria and fungi have been isolated from various region linking the two domains. One is a catalytic
environments such as soi1,4,5 sludge,6 fresh water domain containing the catalytic triad of serine,
and sea water. The microorganisms excrete aspartate and histidine, while the other is a
extracellular PHB depolymerases to degrade substrate-binding domain which adsorbs on the
P(3HB) and utilize the decomposed compounds surface of partially crystalline P(3HB) and orients
as nutrients. the catalytic domain towards the substrate. In
previous papers19.0 we studied the kinetics of
* To whom correspondence should be addressed. surface hydrolysis of P(3HB) film in the presence
275
276 M. Uefuji et al.

of PHB depolymerases of A. faecalis and R shaker in 10 flasks containing 200 ml of the

pickettii, and demonstrated that the surface medium. The cultivation was performed for 24 h.
hydrolysis of P(3HB) film took place via two The resulting culture was centrifuged at
steps of adsorption and hydrolysis by the enzyme 10 000 X g for 30 min to remove the bacterial
with two functions of binding and catalytic cells, and the supernatant (2000 ml) was used for
domains. enzyme preparation. All purification procedures
In a previous paper we isolated two types of of enzyme were carried out at 0-4C.
P(3HB)-degrading bacteria, Comamonas testster- The supernatant was brought to the final
oni YM1004 and Pseudomonas stutzeri YM1006, concentration of 0.3 M with solid ammonium
from sea water, and studied the properties of sulfate, and allowed to stand for 1 h. The
PHB depolymerase purified from C. testosteroni. precipitate was discarded by centrifugation at
In the present paper, we report the secretion and 10 000 X g for 10 min. The supernatant was
properties of an extracellular PHB depolymerase applied to a Butyl-Toyopearl column (3 cm x 30
from the other marine bacterium, R stutzeri. cm) equilibrated with 10 mM phosphate buffer
(pH 7.0) containing 0.3 M ammonium sulfate.
The hydrophobic interaction chromatography
2 EXPERIMENTAL column was washed with two bed volumes of the
same salt solution, and the adsorbed enzyme was
2.1 Materials eluted with a linear gradient of ammonium
sulfate from 0.3 M to 0 M, and then with a linear
P(3HB) homopolymer was produced from gradient of ethanol from 0 to 40%. Fractions with
butyric acid by A. eutrophus. The film of a high activity were pooled and concentrated by
P(3HB) was prepared by solvent-casting tech- Sumikagel (Sumitomo Chemical Industrial). The
niques from a chloroform solution of P(3HB) concentrated enzyme solution was dialyzed
using a glass Petri dish as casting surface. The against 10 mM Tris-HCl buffer (pH 7.5) and
solution-cast films were aged for 3 weeks to reach stored at 4C.
equilibrium crystallinity prior to analysis.
P(3HB) granules were isolated from cells of A. 2.4 Enzyme assay
eutrophus by sonic oscillation and treatment with
Assays of the enzymatic hydrolysis of P(3HB)
alkaline hypochlorite. The partially crystalline
granules were conducted as previously
P(3HB) granules were used for the enzymatic
described.
assays.
2.5 Enzymatic hydrolysis of P(3HB) films
2.2 Culture medium
The enzymatic hydrolysis of P(3HB) films by the
The basal liquid mineral medium consisted of purified PHB depolymerase was carried out at
0.23 % KH2P04, 0.58 % Na,HP0,.12H,O, 0.3 % temperatures from 30 to 45C in 0.1 M potassium
NaCl, 0.05% MgSO,.7H,O, 0.1% (NH,),SO, and phosphate buffer (pH 7.4). The enzyme solution
a microelement solution as described previously. (10 ~1) of different concentrations was added to
The pH was adjusted to 6.8. Mineral agar plates the reaction cuvette containing 1 ml of 0.1 M
were prepared by the addition of 1.5% agar to potassium phosphate buffer. The cuvette was
the above liquid culture. maintained at the reaction temperature. The
reaction was started by the addition of P(3HB)
2.3 Purification of extracellular PHB film (initial weight, about 8-O mg; initial film
depolymerase dimensions, 10 mm X 10 mm X 0.07 mm). The
amount of 3-hydroxybutyrate (3HB) units lib-
A strain YM1006 isolated from the sea water was erated from the P(3HB) film during enzymatic
precultured in a basal mineral medium containing hydrolysis was determined by the ultraviolet
0.4% sodium succinate. Aliquots (4 ml) of the (UV) method described in previous papers..20
culture were transferred to the basal mineral The water-soluble products after enzymatic
medium containing 0.2% P(3HB) granules as sole degradation of P(3HB) film were examined by
carbon source, and the strain was cultivated high-performance liquid chromatography
under aerobic conditions at 30C on a reciprocal (HPLC) analysis of the reaction solution.
 Enzymatic degradation of P(3HB) 277

2.6 Analytical procedures of enzyme from the activities of the culture supernatant for
the hydrolysis of P(3HB) granules. When P(3HB)
Polyacrylamide gel electrophoresis of enzyme in and its enzymatic degradation product, (R)-
the presence of sodium dodecyl sulfate (SDS) was 3_hydroxybutyrate, were used as a carbon source,
carried out on a 12.5% gel according to the PHB depolymerase activity was observed in the
method of Laemmli,24 using a molecular weight culture supernatant. However, no activity was
calibration kit (Pharmacia Biotech). Protein detected in the supernatant of glucose, succinate,
concentrations were determined by the method citrate and (S)-3-hydroxybutyrate grown cells.
of Bradford,25 using the Bio-Rad Protein Assay The influence of (R)- and (S)-3-hydroxybutyr-
Kit II with bovine serum albumin as the standard. ate upon the secretion of PHB depolymerase was
Proteins were silver-stained. investigated. Figure 1 shows the growth curves
and the depolymerase activities in the super-
3 RESULTS AND DISCUSSION natants of E stutzeri grown on (R)- or (S)-
3-hydroxybutyrate. When (R)-3-hydroxybutyrate
3.1 Secretion of PHB depolymerase from p
was used as the sole carbon source, the activity of
stutzeri PHB depolymerase in the supernatant increased
Two independent P(3HB)-degrading bacteria with the growth of cells. In contrast, no activity
were isolated from sea water (Kanagawa, Japan), was detected at all in the supernatant of cells
as reported in a previous paper. One of them grown in the presence of (S)-3-hydroxybutyrate.
(strain YM1004) was Comamonas teststeroni, and It may be concluded that (R)-3_hydroxybutyrate,
the other (strain YM1006) was used in the as a product of enzymatic hydrolysis of P(3HB),
present study. The strain YM1006 is a gram- induces the secretion of PHB depolymerase from
negative rod and oxidase positive, and it utilizes I? stutzeri cells.
glucose, D-mannitol, maltose, malate, succinate
and citrate as carbon sources. On the basis of 3.2 Properties of PHB depolymerase
further characterization, strain YM1006 was
The highest activity of degrading P(3HB)
identified as Pseudomonas stutzeri.
Table 1 shows the effects of carbon sources on granules was observed in the supernatant of R
stutzeri grown on P(3HB). Therefore, P(3HB)
both the growth of ? stutzeri and the secretion of
PHB depolymerase. The secretion of PHB granules were used for the preparation of PHB
depolymerase from bacterial cells was evaluated depolymerase. The activity of PHB depolymerase
was maximum at the stage from the end of the
Table 1. PHB depolymerase activities in the culture super-
1.2 0.3
natants of E! stutzeri YM1006 grown on various carbon
sources
1.0
Carbon source Growth Activity +_
$
(units ml-) +
+ 0.8 0.2 =
Poly[(R)-3-hydroxybutyrate] ++ 0.45 -z
Sodium (R)-3-hydroxybutyrate +++ 0.24 8 0.6 s
Sodium (S)-3-hydroxybutyrate ++ 0 2 3
Glucose + 0 2 0.4 0.1 .g
Fructose + 0 d .$
Sodium malate + 0 Y
Sodium succinate +++ 0 0.2
Sodium citrate ++ 0
Sodium 3-hydroxyoctanoate + 0 0.0 0.0
Sodium butyrate + 0.02 0 6 12 18 24
Olive oil ++ 0
Tween 80 ++ 0 Time (b)
Sodium lactate + 0 Fig. 1. Growth curves of 19 stutzeri YM1006 and the
L-Glutamate + 0 activities of PHB depolymerase. The bacterium was grown at
3-Hydroxypropionate - 30C in the liquid culture medium containing 0.2% sodium
Sodium 2-hydroxybutyrate - 3-hydroxybutyrate as sole carbon source. Cell growth was
monitored by measuring the turbidity at 6.50 nm (closed
n - , No growth, +, poor growth; ++, medium growth; symbols), and PHB depolymerase activity (open symbols)
+ + + , high growth. was determined. 0.2% (R)3HB (@,O) or 0.2% (S)-3HB
The maximum enzyme activity in the culture supernatant. (A,n) was used as the sole carbon source.
278 M. Uefkji et al.

Table 2. Purification of PHB depolymerase from E stutzeri Table 2, and the elution profile by hydrophobic
YM1006
interaction chromatography is shown in Fig. 2.
step Total Total Specific Yield (%) After the procedure, the purified enzyme was
activity protein activity homogeneous as judged by polyacrylamide gel
(units) (mg) (units mg )
electrophoresis in the presence of sodium dodecyl
1. Supernatant 1480 49.3 30 100 sulfate (SDS-PAGE). The molecular weight of
2. Butyl-Toyopearl 360 9.6 3x 24 PHB depolymerase from R stutzeri was about
60 kDa as estimated by SDS-PAGE. The enzyme
had an isoelectric point of about 7.3 by isoelectric
logarithmic growth phase to the stationary phase.
focusing.
The purification of PHB depolymerase was
Figure 3 shows the pH dependence of the
performed by hydrophobic interaction (Butyl-
activity of P(3HB) degradation by the purified
Toyopearl) column chromatography. The purifica-
enzyme as determined by the turbimetric method
tion steps of the enzyme are summarized in
with P(3HB) granules. The optimum pH for the
20
100
90
+
15 g 80
; 70
E Y
-3 .; 60
J 50
z
s 40
Z
d 30
20

0 0 2 4 6 8 10 12 14
20 40 60 80 loo
PH
Fraction Number

1
Fig. 2. Elution profiles of PHB depolymcrase from I-I
stutzeri YM1006. The supernatant was applied to a
hydrophobic (Butyl-Toyopearl) column. and the enzyme was
eluted with linear gradients of 0.3-O M ammonium sulfate
and O-40% ethanol.

0 20 40 60 80

Temperature(C)
Fig. 4. Effects of pH and temperature on the stability of
PHB depolymerase from J? stutzeri YMl006. (A) The
enzyme was incubated in O-1 M buffers of diffcrcnt pH
values at 37C for 5 h. and the residual activity was
measured at pH 7.4 and 37C by the turbidimetric method.
2 4 6 8 10 12 14 The activity was determined in 0.1 M sodium acetate buffer
(X). sodium phosphate buffer (0). Tris-HCI buffer (A).
PH borate-NaOH buffer (+), glycine-NaOH buffer (0).
Fig. 3. The pH profile of PHB depolymerasc activity. The NaHCO, buffer (0) and KCI-NaOH buffer (A). (B) The
activity was determined in 0.1 M sodium acetate buffer (0), enzyme was incubated in 50 mM Tris-HCI buffer (pH 7.5)
sodium phosphate buffer (O), Tris-HCl buffer (0) and for 30 min. and the residual activity was measured at pH 7-4
glycine-NaOH buffer (m). and 37C by the turbidimctric method.
 Enzymatic degradation of P(3HB) 279

hydrolysis of P(3HB) granules was the neutral of monomer and dimer increased with the
region between 7.0 and 7.5. reaction time. The rate of the formation of
Figure 4(A) shows the effect of pH on the monomer and dimer was a maximum at enzyme
stability of PHB depolymerase. The enzyme was concentrations of 1 to 3 ,ug ml-. At a high
incubated at 37C for 5 h at different pH values, enzyme concentration of 20 pug ml-, only
and the remaining activity was measured at pH monomer of 3-hydroxybutyric acid was detected,
7.4 and 37C. The PHB depolymerase was stable and the rate of formation was relatively low.
at pH values from 6 to 12. The effect of
3.3 Kinetic analysis of enzymatic hydrolysis
temperature on the stability of the enzyme is
shown in Fig. 4(B). The PHB depolymerase was As reported in previous papers,9,2 the water-
stable at temperatures up to 50C at pH 7.4. soluble products can be detected quantitatively
The composition of water-soluble product was by monitoring the adsorption at 210 nm due to
measured by HPLC analysis during the enzymatic the carbonyl groups of monomer and dimer.
hydrolysis of P(3HB) film at 37C in 0.1 M Figure 6 shows the time-dependent changes in the
potassium phosphate buffer (pH 7.4) with amounts of 3-hydroxybutyrate units liberated as
different concentrations of enzyme: 0.2, 1.0, 3.0 water-soluble product during the course of
and 20 t_Lg mll. In this experiment the reaction enzymatic hydrolysis of P(3HB) film at 37C in
solutions were collected after the enzymatic O-1 M phosphate solutions (pH 7.4) containing
hydrolysis and analyzed by HPLC. This analysis different amounts of PHB depolymerase. An
revealed that monomer and dimer of 3-hydroxy- induction period was observed for about 60 min
butyric acid were produced during the enzymatic at the initial stage of enzymatic hydrolysis, and
hydrolysis of P(3HB) film. Figure 5 shows the then the amount of water-soluble products
time-dependent changes in relative amounts increased with time. The rate of enzymatic
(HPLC peak areas) of 3HB units generated as hydrolysis was determined from the linear slopes
monomer and dimer of 3-hydroxybutyric acid. of the curves against time. The rate of enzymatic
Over the whole range of enzyme concentrations hydrolysis was strongly dependent on the concen-
monomer was a major product. The total amount tration of enzyme.

(A) g [E]=l.Opg/ml (B)

I 7
6 I

0 5 10 15 20 25 0 5 10 15 20 25

9
g [E]=3.0pg/ml (0

0 5 10 15 20 25 0 5 10 15 20 25
Time(h) Time(h)
Fig. 5. The relative HPLC peak areas of monomer and dimer of 3-hydroxybutyric acid generated as water-soluble products
during the enzymatic degradation of P(3HB) film at 37C and pH 7.4 with different concentrations of PHB depolymerase from
P stutzeri YM1006. W, monomer peak area; 0, dimer peak area.
280 M. Uefuji et al.
6
Concentration of enzyme
W/ml)
+ 0.1

-f 0.5

--k-l

+2

-0-4

u8

* 10
0 1 2 3 4 5

Time (h)
Fig. 6. The amounts of 3HB units liberated as water-soluble products during the enzymatic degradation of P(3HB) film in
aqueous solutions (pH 7.4 and 37C) containing different amounts of PHB depolymerase from P stutzeri.

Figure 7 shows the effect of temperature on the value, followed by a gradual decrease with
rate of enzymatic hydrolysis of P(3HB) film, as further increase in enzyme concentration.
determined from the generation rate of water- The same dependence of enzyme concentration
soluble products. The rate of enzymatic hydro- on the rate of enzymatic hydrolysis of P(3HB)
lysis at a given concentration of enzyme increased film was reported with other PHB depolymerases
with temperature from 30 to 45C and maximum from I? pickettii, C. testosteroni. and A.
rates at different temperatures were observed at faecalis. The rate, R, of enzymatic hydrolysis of
an enzyme concentration of 2-5 pug ml-. The P(3HB) film could be expressed by,20
rate of enzymatic hydrolysis increased with the
concentration of enzyme to attain a maximum R = k,K[ El/( I + K[ El) (1)
4-
(A)
30C 34C
N^ 3

$ 2_
z
s
g 1-o
0
,

0 I
0 5 10 15 20

4
U-V
0
45C

0 5 10 15 20 0 5 10 15 20
Wl(~mo~ml) fEl(umo~ml)
Fig. 7. Effects of enzyme concentration, [El, on the rate, R, of 3HB unit liberation at different temperatures during the
enzymatic degradation of P(3HB) film in the aqueous solution (pH 7.4) of PHB depolymerase.
 Enzymatic degradation of P(3HB) 281

Table 3. The adsorption equilibrium constants (K) and REFERENCES

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