41
3
Extracellular
Polyhydroxyalkanoate
Depolymerases: The Key
Enzymes of PHA
Degradation
Priv.-Doz. Dr. rer. nat. Dieter Jendrossek
Institut f¸r Mikrobiologie der Universit‰t Stuttgart, Allmandring 31,
D-70569 Stuttgart, Germany; Tel.: ‡ 49-711-685-5483; Fax: ‡ 49-711-685-5725;
E-mail: [email protected]
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
2
Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
3
Identification and Isolation of Extracellular d-Poly(HA)-Degrading Microorganisms
50
4
Characterization of Poly(HA )-Degrading Microorganisms . . . . . . . . . . . .
51
5
Biochemical Properties of Extracellular d-Poly(HA ) Depolymerases . . . . . .
54
6
Molecular Biology and Functional Analysis of d-Poly(3HASCL ) Depolymerases.
56
7
PhaZ7, a new Type of Thermoalkalophilic Hydrolase of P. lemoignei with high
Specificity for Amorphous Poly(HASCL ) . . . . . . . . . . . . . . . . . . . . . . .
62
8
Molecular Biology and Functional Analysis of d-Poly(HAMCL ) Depolymerases .
65
9
Enantioselectivity and Hydrolysis Products of Poly(HA ) Depolymerases . . .
66
10
Regulation of Poly(HA ) Depolymerase Synthesis . . . . . . . . . . . . . . . . .
69
11
Influence of Physico-chemical Properties of the Polymer on its Biodegradability
72
12
Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
74
13
Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
74
14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
42
3 Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation
3HB
3HV
d-poly(3HB )
d-poly(HA )
MCL
n-poly(3HB )
n-poly(HA )
poly(3HB )
poly(3HO )
poly(3HV )
poly(HA )
SCL
SEM
3-hydroxybutyric acid
3-hydroxyvaleric acid
denatured poly(3-hydroxybutyric acid)
denatured poly(hydroxyalkanoic acids)
medium-chain-length
native poly(3-hydroxybutyric acid)
native poly(hydroxyalkanoic acids)
poly(3-hydroxybutyric acid) ˆ poly(3-hydroxybutanoic acid) ˆ poly(3-hydroxybutyrate)
poly(3-hydroxyoctanoic acid) ˆ poly(3-hydroxyoctanoate)
poly(3-hydroxyvaleric acid) ˆ poly(3-hydroxypentanoic acid) ˆ
poly(3-hydroxyvalerate)
poly(hydroxyalkanoic acids) ˆ poly(hydroxyalkanoates)
short-chain-length
scanning electron microscopy
1
Introduction
Poly((R )-3-hydroxyalkanoic acids) (poly(HA )) are a class of bacterial storage compounds that are synthesized during unbalanced growth by many Gram-negative and
Gram-positive bacteria. Poly(HA ) are deposited intracellularly in the form of inclusion
bodies (™granules∫) to levels up to 90% of the
cellular dry weight (for reviews, see Anderson and Dawes (1990), Doi (1990), Steinb¸chel (1991), M¸ller and Seebach (1993),
Sharma and Ray (1995), Marchessault
(1996), Sasikala and Ramana (1996), Steinb¸chel and F¸rchtenbusch (1998), Braunnegg et al. (1998), Madison and Huisman
(1999), Babel et al. (2001), Kessler and
Witholt (2001), Steinb¸chel and Hein
(2001) and Chapter 2 and 6 of this book.
Poly[(R )-3-hydroxybutyric acid], poly(3HB ),
was the first of the poly(HA ) discovered by
Lemoigne (1925), and is the most abundant
polyester found in bacteria. Bacterial copolymers containing randomly distributed (R )-3hydroxybutyric acid (3HB ) and (R )-3hydroxyvaleric acid (3HV ) units [poly(3HBco-3HV )] have been sold commercially for
over a decade under the tradename Biopol¾.
About 150 hydroxyalkanoic acids other than
3HB have been identified as constituents of
poly(HA ) during the past three decades
[many are summarized in Steinb¸chel and
Hein (2001), Steinb¸chel and Valentin
(1995), and Kim and Lenz (2001)]. The
monomeric composition of poly(HA ) is
highly variable, and determines the molecule's physical and chemical characteristics.
Such composition depends on: (1) the
biochemical properties of the polymerizing
enzyme, the poly(HA ) synthase (see Chapter 7); (2) the metabolic capabilities of the
bacterial strain; and (3) the carbon source
applied during accumulation of the polymer
(see Chapters 6, 8, 9, 10).
Any research on the biodegradation of
poly(HA ) should clearly distinguish between extracellular poly(HA ) degradation
and intracellular poly(HA ) degradation.
1) Extracellular degradation is the utilization
of an exogenous carbon/energy source by
a not necessarily accumulating microorganism. The source of this extracellular
polymer is poly(HA ) released by accumulating cells after death. The ability to
1 Introduction
degrade poly(HA ) is widely distributed
among bacteria, and depends on the
secretion of specific poly(HA ) depolymerases which are carboxyesterases (EC
3.1.1) and hydrolyze the water-insoluble
polymer to water-soluble monomers and/
or oligomers.
2) Intracellular degradation is the active
mobilization (hydrolysis) of an endogenous carbon/energy storage reservoir
by the accumulating bacterium itself.
The differentiation between extra- and
intracellular degradation is necessary because poly(HA ) in vivo and outside of the
bacteria is present in two different biophysical states. In intracellular (native) poly(HA )
granules, the high molecular-weight polymer (MW 105±106 Daltons) is in the amorphous ™rubbery∫ state (highly mobile chains
in disordered conformation), and the gran-
ule surface layer consists of proteins and
phospholipids (Figure 1) (Lundgren et al.,
1965; Amor et al., 1991; Steinb¸chel et al.,
1995; Mayer et al., 1996). Upon extraction
from the cell, the granule surface layer is
either damaged or lost (Merrick et al., 1965;
Griebel et al., 1968; Griebel and Merrick,
1971), and the polyester chains tend to adopt
ordered helical conformations and to develop a crystalline phase (Figure 1) (Cornibert
and Marchessault, 1975; de Koning and
Lemstra, 1992). Extracellular poly(3HB ), for
example, is a partially crystalline polymer
(typical degree of crystallinity 50 ± 60%)
(Scandola et al., 1988) with an amorphous
fraction characterized by the same glass
transition temperature (Tg ) as native poly(3HB ) (Tg ~ 0 8C ) and a crystalline fraction
that melts in the range 170 ± 180 8C (Scandola et al., 1988). For the sake of clarity, in
Fig. 1 Physical state of poly(3HA). Left: schematic view of a section through a native poly(3HB ) granule;
structural and functional proteins and phospholipids constituting the surface layer are indicated (for details
see Chapter 8). Center: electron micrograph of a poly(3HB )-accumulating bacterium. Right: schematic view
of isolated denatured, partially crystalline poly(3HB ). CM, cytoplasmic membrane; DL, dense layer; OM,
outer membrane.
43
44
3 Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation
this chapter poly(HA ) in the native state
(i.e., in the intracellular granules with intact
surface layer) are indicated as n-poly(HA ),
whereas the same polyesters in the partially
crystalline form are denoted as denatured dpoly(HA ) according to the nomenclature
introduced by Merrick and Doudoroff
(1964). The same notation is used to differentiate poly(HA ) depolymerases according
to their ability to hydrolyze n-poly(HA ) [npoly(HA ) depolymerases] or d-poly(HA ) [dpoly(HA ) depolymerases].
Poly(HA ) can be biodegraded to water and
carbon dioxide or methane by a large variety
of ubiquitous microorganisms present in
many ecosystems (Figure 2). This fairly easy
biodegradability came as a surprise given the
inertness of the water-insoluble, hydrophobic and (partially) crystalline polymers. The
key enzymes of poly(HA ) degradation are
poly(HA ) depolymerases, and this chapter
focuses on the biochemical and molecular
properties of these intriguing enzymes. The
mechanism of d-poly(HA ) degradation has
been studied extensively during the past
decade [ for reviews on poly(HA ) degradation see Brandl et al. (1995b), Jendrossek
et al. (1996), and Jendrossek (1998, 2001a)].
Fig. 2 Biodegradation of poly(HA ). (A ) Degradation of Biopol¾ bottles. Bottles were incubated for 0, 2, 4, 6 and 8 weeks in
aerobic sewage sludge. (B ) Growth
of a poly(HASCL )-degrading bacterium on opaque agar with poly(3HB ) as the sole carbon and
energy source. Clearing zone formation (halos) around the colonies
indicates extracellular hydrolysis of
the water-insoluble polymer to water-soluble products.
2 Historical Outline
This chapter will be an update of these
contributions, but will focus only on the
extracellular degradation of poly(HA ) by
extracellular depolymerases. Related enzymes, such as extracellular cutinases or
suberinases are not included in this review
but have been reviewed elsewhere (Kolattukuday, 2001) and are subject of Chapters 1
and 2.
The study of intracellular n-poly(HA )
degradation and mobilization of the storage
polymer, after being the subject of detailed
physiological studies during the 1960s, has
only very recently become the center of
attention again, and accordingly its molecular mechanism is only poorly understood.
The intracellular n-poly(HA ) mobilization
by intracellular n-poly(HA ) depolymerases
has been described elsewhere (Hippe, 1967;
Hippe and Schlegel, 1967; Saito et al., 1992,
1995; Handrick and Jendrossek, 1998; Yoon
and Choi, 1999; Handrick et al., 2000;
Jendrossek, 2001a; Ruiz et al., 2001; Saegusa
et al., 2001) and is reviewed in Chapter 9.
2
Historical Outline
Since the discovery of poly(3HB ) as a
constituent of B. megaterium in 1925 by
Maurice Lemoigne, and the finding that
3HB appeared after incubation of poly(3HB )-rich cells of B. megaterium in water
(Lemoigne, 1925, 1926), it was almost 40
years until the first (published) study on the
(extracellular) degradation of poly(HA ) was
performed. In these studies, poly(3HB )degrading bacteria were isolated by selection
for microorganisms able to utilize poly(3HB ) as the sole source of carbon and
energy (Chowdhury, 1963). Such organisms
produced clearing zones on opaque agar. In
this early study the ability of poly(3HB )
degradation in Gram-negative bacteria (Pseu-
domonas sp. P1) and in Gram-positive bacteria (Bacillus sp.), as well as in actinomycetes (Streptomyces sp.), was documented.
Unfortunately, these early isolates of poly(3HB )-degrading bacteria have not been
preserved, but based on the physiological
and biochemical properties of the isolated
strain and its characterized depolymerase
(e.g. pH optimum 9.5 ± 10, monomeric 3HB
as main end-product of enzymatic poly(3HB ) hydrolysis), it is likely that Pseudomonas sp. P1 is related to Comamonas sp. and
Comamonas acidovorans (see below and
Tables 1 and 2). Spore-forming poly(3HB )degrading bacteria that were isolated from
pasteurized soil samples on poly(3HB ) agar
by Chowdhury were not analyzed in detail
because of the small size of the clearing
zones. Presumably, they represent B. megaterium strains because many such strains (if
not all) produce only very small halos on
poly(3HB ) agar (D. Jendrossek, unpublished
observation). This first study on extracellular
poly(3HB ) degradation, which often is overlooked (probably because it has been published in the German language) was performed in the department of H. G. Schlegel
at the University of Gˆttingen, Germany. It
was also Schlegel's department in which the
most important and best-studied poly(HA )accumulating bacterium (Hydrogenomonas
eutropha H16, reclassified as Alcaligenes
eutrophus, reclassified as Ralstonia eutropha;
Yabuchii et al., 1995) was isolated ( Wilde,
1962) and in which many studies on the
synthesis and degradation of poly(HA ) have
been initiated by A. Steinb¸chel (in 1986)
and by the present author in 1989. The
second report on extracellular poly(3HB )
degradation was published in 1965 by Delafield, working with M. Doudoroff (Delafield
et al., 1965b). In this publication a large
variety of mainly Gram-negative poly(3HB )degrading bacteria were described. Some of
the isolates appeared to be highly adapted for
45
46
Overview on poly(HA )-degrading microorganisms and biochemical characterization of purified poly(HA )-depolymerases
Strain analyzed
Depol. purified
Binding Mr
to DEAE (SDSPAGE )
d-Poly(HASCL ) depolymerases of Gram-negative aerobic bacteria
Acidovorax sp.
Poly(3HB ) depol.
Depen- Topt (8C ) pHopt
dence
on Ca2‡
< 40
50
Poly(3HB ) depol.
Poly(3HB ) depol
Poly(3HB ) depol.
A. faecalis T1
Recombinant hybrid poly(3HB ) depol. domain mutants
A. faecalis T1
Poly(3HB ) depol.
A. faecalis AE122
Poly(3HB ) depol.
A. faecalis AE122
Aureobacterium saperdae
Comamonas sp.
PhaZ Afa122-reca
Poly(3HB ) depol.
Poly(3HB ) depol.
C. acidovorans YM1609
Poly(3HB ) depol.
C. testosteroni ATSU
Poly(3HB ) depol.
C. testosteroni YM1004
C. testosteroni YM1004
Marinobacter sp. NK-1
Paucimonas lemoignei
P. lemoignei
P. lemoignei
Poly(3HB ) depol.
50
9.5-10
Glutathione-S-transferase- poly(3HB ) depol. binding domain fusion
Poly(3HB ) depol.
yes
> 37
8.0
partial
Poly(3HB ) depol A, B
no
no
P. lemoignei
A1, A2
B1, B2
PhaZ1Ple-reca
Poly(3HB ) depol. A
no
no
no
no
58
44
59
54
yes
yes
yes
65
Poly(3HB ) depol. B
Poly(3HV) depol.
no
no
67
54
yes
yes
55
Poly(3HB ) depol. A
Poly(3HB ) depol. B
Poly(3HB ) depol. C
no
no
no
55
67
P. lemoignei
no
62.5
43
44
nob
8.5
Alcaligenes faecalis T1
A. faecalis T1
A. faecalis T1
P. lemoignei
P. lemoignei
50
no
no
Carbo- Remarks
hydrate
content
8.6
45
29±35
8.0
9.4
45 (48.6) no
37
9.0
49
70
8.5
no
no
yes
yes
yes
8.0
References
Purified depolymerase hydrolyzes polypropiolactone Kobayashi et al. (1999)
in addition to poly(3HB )
Tanio et al. (1982)
Indication for substrate-binding site
Shirakura et al. (1986)
Identification of the catalytic site serine by siteShinohe et al. (1996)
directed mutagenesis
Type and number of linker domains do not affect
Nojiri and Saito (1997)
activity
Detailed mechanistic study using defined 3HB
Bachmann and Seebach
oligomers
(1999)
Isolate from seawater, depolymerase with unusual Kita et al. (1995)
high apparent Mr
Two poly(3HB ) binding domains
Kita et al. (1997)
Soil isolate
Sadocco et al. (1997)
Monomers are the only hydrolysis end-products
Jendrossek et al. (1993a,
1995a)
Monomers are the end-product of hydrolysis at high Kasuya et al. (1997)
enzyme concentration
Depolymerase with the highest temperature opt.,
Kasuya et al. (1994)
however unstable at 70 8C
Isolate from seawater
Mukai et al. (1993b)
Function of C-terminal domain
Shinomiya et al. (1997)
Purified depolymerase active in 0.5 M NaCl;
Kasuya et al. (2000)
Reclassification of Pseudomonas lemoignei
Jendrossek, (2001b)
Detailed microbiological and physiological study
Delafield et al. (1965b)
Thermal inactivation at 60 8C
Lusty and Doudoroff
(1966)
Nakayama et al. (1985)
8.0
Jendrossek et al. (1993)
M¸ller and Jendrossek
(1993)
8.0
yes
yes
Poly(HA )SCL depolymerase with high poly(3HV)
depolymerase activity
Survey of poly(HA ) depolymerases
Briese et al. (1994b)
3 Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation
Tab. 1
Tab. 1
(cont.)
Strain analyzed
Depol. purified
Binding Mr
to DEAE (SDSPAGE )
Depen- Topt (8C ) pHopt
dence
on Ca2‡
d-Poly(HASCL ) depolymerases of Gram-negative aerobic bacteria continued
Poly(3HV) depol.
no
P. lemoignei
Poly(3HB ) depol. A
P. lemoignei
no
P. lemoignei A62
P. lemoignei
no
Poly(3HB ) depol.
P. lemoignei
Poly(3HB ) depol. A
P. lemoignei
no
P. lemoignei
no
P. lemoignei
Poly(3HV) depol. PhaZ6
Pseudomonas P1
P. pickettii
P. pickettii
isolate Z925
isolate T107
isolate S2
isolate A1
P. stutzeri YM1414
P. stutzeri YM1006
P. stutzeri YM1006
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
Poly(3HB ) depol.
PhaZPst-reca
6 Pseudomonas sp.
no
no
no
no
no
44
46.5
65.5
49
51
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
61.5
54.5
50
51
yes
no
yes
40
49
45
45
43
49
48
60
57.5
45
40
yes
55
9.8
5.5
6.0
9.5
9.5
9.5
9.5
9.5
7-7.5
References
Contains glucose and N-acetylglucosamine, Survey Jendrossek et al. (1995b)
of P. lemoignei poly(3HB ) depolymerases
Contains glucose and N-acetylglucosamine
Contains glucose and N-acetylglucosamine
Contains glucose and N-acetylglucosamine
Glycosylation not essential for activity
Glycosylation not essential for activity
Glycosylation not essential for activity
Glycosylation not essential for activity
Identification of a C-terminal polyester-binding
Behrends et al. (1996)
domain
Specific isolation procedure for P. lemoignei, no
Mergaert et al. (1996 b)
plasmid in type strain;
Strain A62 contains 200 kbp megaplasmid
Mergaert et al. (1996)
Identification of the catalytic site serine by siteShinohe et al. (1996)
directed mutagenesis
Simplified purification procedure for poly(3HB )
Tomasi et al. (1996)
depolymerase A
Relationship between poly(3HB ) depolymerase
Terpe et al. (1999)
synthesis and uptake of succinate
Analysis of artificial and n-poly(3HB) hydrolysis by Merrick et al. (1999)
cell-free culture fluid
PhaZ6 and not PhaZ4 is the true poly(3HV )
Schˆber et al. (2000)
depolymerase of P. lemoignei
Strains are not preserved
Chowdhury (1963)
Depolymerase with acidic pH optimum
Yamada et al. (1993)
Shiraki et al. (1995)
Poly(3HB ) depolymerase from a marine isolate
Poly(3HB ) depolymerase with cadherin-like linker
domain and two poly(3HB ) binding domains
Development of a method for preparing stable
Poly(HAMCL ) emulsions
Mukai et al. (1994)
Uefuji et al. (1997)
Ohura et al. (1999b)
Ramsay et al. (1994)
2 Historical Outline
P. lemoignei
Poly(3HB ) depol. B
Poly(3HB ) depol. C
Poly(3HV) depol.
PhaZ1Ple-reca
PhaZ2Ple-reca
PhaZ4Ple-reca
PhaZ5Ple-reca
PhaZ4Ple-reca (truncated)
Carbo- Remarks
hydrate
content
47
48
(cont.)
Strain analyzed
Depol. purified
Binding Mr
to DEAE (SDSPAGE )
several species
6 lipases and 9 poly(HA ) depolymerases
Depen- Topt (8C ) pHopt
dence
on Ca2‡
Carbo- Remarks
hydrate
content
Lipases hydrolyze polymers of w-hydroxyalkanoic
acids
Ecological study
Taxonomical studies
no
no
d-Poly(HASCL ) depolymerases of Gram-positive aerobic bacteria
Streptomyces exfoliatus K10 poly(3HB ) depol.
49
no
40
8.5±9.0
d-Poly(HASCL ) depolymerases of anaerobic bacteria
Ilyobacter delafieldii
no
anaerobic consortium
Clostridium-like isolates
48
37
40
d-Poly(HAMCL ) depolymerases of Gram-negative aerobic bacteria
P. fluorescens GK13
Poly(3HO ) depol.
25
P. fluorescens GK13
45
45±60
no
45
Anaerobic poly(3HB ) degrading bacterium that
ferments poly(3HB )
to acetate, butyrate and H2
Janssen and Harfoot
(1990)
Janssen and Schink (1993)
Budwil et al. (1992)
Mergaert et al. (1996a)
P. fluorescens GK13
PhaZPfl-rec
a
no
28
P. fluorescens GK13
PhaZPfl-reca
no
28
P. maculicola
Comamonas sp. P37C
Xanthomonas sp. JS02
no
Oda et al. (1997b)
6.0
yes
8.5
yes
Poly(3HO ) depol.
no
Contains mannose, galactose and glucose
Exo- and endo-type hydrolysis of poly(3HB )
Purification via PHB affinity chromatography, enzyme tends to aggregate
Direct activity staining in poly(3HB )-containing
SDS-polyacrylamide gels
Brucato and Wong (1991)
Scherer et al. (2000)
Iyer et al. (2001)
Poly(3HO ) depolymerase that does not hydrolyze
poly(3HB )
Degradation of unsaturated and cross-linked poly(3HO ) derivatives
In rec. E. coli processing of poly(3HO ) depolymerase
differs from wild-type
Site-directed mutagenesis of catalytic triad serine
Isolation of bacteria able to hydrolyse poly(HA )SCL
and poly(HA )MCL
Schirmer et al. (1993)
Poly(HAMCL ) depol.
41.7
60
40
70-73
8.5
Iyer et al. (1997)
de Koning et al. (1994)
Schirmer and Jendrossek
(1994)
Schirmer et al. (1995)
Hydrolyzes poly(HAMCL ) with aromatic side chains
Foster et al. (1995)
Quinteros et al. (1999)
Kim et al. (2000a)
3HB-oligomer hydrolyzed with no activity towards
Oda et al. (1997)
Zhang et al. (1997)
no
Other microbial esterases related to poly(HA ) depolymerases
A. faecalis
PCL depolymerase/lipase
Pseudomonas sp. A1
Oligomer-hydrolase
Briese et al. (1994a)
Mergaert et al. (1993,
1994, 1995, 1996b)
Klingbeil et al. (1996)
7.0
8.5
Jaeger et al. (1995)
K10 hydrolyzes poly(3HB ) and poly(3HO )
no
d-Poly(HASCL ) depolymerases of fungi
Paecilomyces lilacinus
Poly(3HB ) depol.
D218
Penicillium funiculosum
Poly(3HB ) depol.
Aspergillus fumigatus
Poly(3HB ) depol.
Aspergillus fumigatus
Poly(3HB ) depol.
References
7.0
3 Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation
Tab. 1
Tab. 1
(cont.)
Strain analyzed
Depol. purified
Binding Mr
to DEAE (SDSPAGE )
Depen- Topt (8C ) pHopt
dence
on Ca2‡
Carbo- Remarks
hydrate
content
OhaZPsp-rec
Physarum polycephalum
PMA hydrolase
68
Comamonas acidovorans
PMA hydrolase
43
Comamonas acidovorans
strain TB-35
several species
Polyester-polyurethane
62
hydrolase
6 lipases and 9 poly(HA ) depolymerases
Amycolatopsis sp.
n-Poly(HASCL ) depolymerases
P. lemoignei
PhaZ7Ple
no
3.5
yes
40
8.1
yes
45
6.5
65
9.5-10 no
poly(3HB )
No true lipase box within the deduced amino acid
sequence
Extracellular, exo-type poly(b-l-malic acid) hydrolase Koherr et al. (1995); Liu
and Steinb¸chel (1996)
Membrane-bound oxo-type poly(b-l-malic acid) hy- Gˆdde et al. (1999)
drolase
Cell-surface bound hydrolase
Akutsu et al. (1998)
Nomura et al. (1998)
Lipases hydrolyze polymers of w-hydroxyalkanoic
Jaeger et al. (1995)
acids
Poly(l-lactide)-degrading microorganism
no
36
partial
References
Pranamuta et al. (1997)
Currently the only known extracellular n-poly(3HB ) Handrick et al. (2001)
depolymerase, no classical poly(3HB ) binding domain, new type of serine hydrolase, thermoalkalophilic enzyme
Enzyme purified from recombinant E. coli, bAddition of Ca2‡ or Mg2‡ not required for activity but influence of EDTA unknown; Abbreviations: depolymerase. An empty space in a column indicates that
the value has not been determined
a
2 Historical Outline
49
50
3 Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation
poly(3HB ) utilization because they did not
grow on common soluble substrates such as
sugars, amino acids and many other carbon
sources. These isolates were identified to
represent a new species which was named
Pseudomonas lemoignei in honor of Maurice
Lemoigne who discovered poly(3HB ) (see
Section 1). This species ± which was recently
reclassified as Paucimonas lemoignei (Jendrossek, 2001b) ± has been studied almost
continuously since its isolation (e.g., Lusty
and Doudoroff, 1966; Stinson and Merrick,
1974; Nakayama et al., 1985; M¸ller and
Jendrossek, 1993; Jendrossek et al., 1995b).
It transpired that P. lemoignei has at least
seven isoenzymes of extracellular poly(3HB )
depolymerases, and is the most interesting
poly(HA )-degrading bacterium (see below).
3
Identification and Isolation of Extracellular
d-Poly(HA )-Degrading Microorganisms
Poly(HA )-degrading microorganisms can
be enriched from soil or liquid samples
collected from various ecosystems after
inoculation of mineral salt solutions which
contain d-poly(HA ) as a sole source of
carbon and energy. This procedure generally
results in enrichment of only one or a few
microbial species which exhibit the fastest
growth under the chosen laboratory conditions. These organisms are not necessarily
the most efficient poly(HA )-degrading
strains. Contaminating bacteria, which do
not degrade d-poly(HA ) but utilize the
primary degradation products (oligomers
of poly(HA )) might grow even faster and
may be difficult to remove. A more suitable
method to assess the distribution of microbial poly(HA ) degraders in a particular
sample is to plate (soil) suspensions of the
desired ecosystem on solid agar media which
contain the polymer as a sole source of
carbon in an opaque overlay prepared from
d-poly(HA ) granules. Only true poly(HA )degrading microorganisms secrete specific
poly(HA ) depolymerases, which hydrolyze
the polymer extracellularly to water-soluble
products, and produce transparent clearing
zones around the depolymerase-secreting
colonies (clear zone technique, Figure 2).
This technique can be performed aerobically,
anaerobically or in agar shake tubes for the
selection of aerobic, anaerobic or microaerophilic microorganisms, respectively.
Unfortunately, only a few short-chainlength d-poly(HA ) [d-poly(HASCL )], namely
poly(3HB ), its copolymers with 3HV and
poly(3-hydroxyvaleric acid), poly(3HV ), can
be prepared as a milky suspension of
denatured granules. Most other poly(HA ),
including poly(4-hydroxybutyrate) and all
medium-chain-length poly(HA ) [poly(HAMCL ], form large rubber-like aggregations which cannot be used directly for the
clear-zone technique. Mineral agar plates
containing ultrathin solution-cast films of
such, which have been stained with a dye
(e.g., Sudan red, Nile red) have been successfully applied for the isolation of poly(HAMCL )-degrading bacteria (Schirmer
et al., 1993; see also Gorenflo et al., 1999;
Spiekermann et al., 1999 for in vivo Nile redstaining of poly(3HB )). However, microorganisms with only a low activity of poly(HA )degrading enzymes may be missed when
using this method. A significant improvement was achieved by the development of
poly(HA ) emulsions (latices) (Ramsay et al.,
1994; Marchessault et al., 1995). These are
stable and heat-resistant emulsions which
are prepared by adding acetone-dissolved
poly(HA ) to cold water and evaporating the
solvent afterwards. The resulting emulsions
(named artificial poly(HA ) granules) have a
milky appearance and can be used for the
clear-zone method. This technique, with
slight modifications for different d-poly-
4 Characterization of Poly(HA )-Degrading Microorganisms
(HA ), was successfully applied to the isolation of a large variety of poly(HAMCL )degrading bacteria (Schirmer et al., 1995).
The growth of Pseudomonas fluorescens GK13
on a poly(3HO )-latex emulsified in the agar
is shown in Figure 3. The different diameters of the clearing zones and different
degrees of opacity are due to mutations in
the poly(3HO )-depolymerase gene. (For details, see Section 7 and legend to Figures 3
and 10.) Another similar method for latex
preparation was developed by Horowitz and
Sanders (1994, 1995), who prepared emulsions of poly(HA ) by dissolving the polymer
in chloroform, adding a surfactant and
emulsifying with water by sonication. The
solvent was removed by dialysis or evaporation, and an opaque, stable suspension of
artificial granules coated by the surfactant
was obtained. Since poly(HA ) generally are
Fig. 3 Growth of recombinant E. coli harboring the
poly(3HO) depolymerase gene of P. fluorescens on
solid medium with opaque latex of poly(3HO) as a
sole source of carbon and energy. Note large
diameter of clearing zones and complete clearing
of wild-type colonies (top) and different diameters
and variable degree of clearing zone formation in
strains harboring mutations in the poly(3HO )
depolymerase gene (right, bottom, left); for details
see text and Figure 10.
soluble in chloroform this method can be
used to prepare artificial granules from any
kind of poly(HA ). However, the surfactant
might inhibit bacterial growth and poly(HA )
depolymerase activity. In addition, artificial
poly(HA ) granules remain amorphous and
thus resemble n-poly(HA ). This might
prevent the isolation of degrading bacteria
with poly(HA ) depolymerases specific for dpoly(HA ). Recently, an alternative method
for preparing poly(3HB ) latices has been
developed by heating aqueous suspensions
of crystalline d-poly(3HB ) granules above
the melting temperature ( ~ 180 8C ) and
subsequent rapid cooling to room temperature. The melted poly(3HB ) granules remained in a metastable amorphous state for
several weeks (Horowitz et al., 1999). It is
likely that this method can be also applied to
poly(HA ) other than poly(3HB ).
Using the methods described above, poly(HA )-degrading microorganisms can be
easily identified and isolated. Bacteria with
rapid growth and high poly(HA ) hydrolysis
rates can be distinguished from those with
only low polymer-hydrolyzing abilities by
measuring the diameter of the colonies and
of the clearing zones. However, isolates with
large clearing zones do not necessarily
represent those strains which are most
important for the degradation of poly(HA )
in situ. Such organisms can be enriched by
incubation of (thin) pieces of the polymer or
polymer films in the ecosystem to be
analyzed. After incubation for several weeks
to months the organisms attached to the
polymer can be investigated.
4
Characterization of Poly(HA )-Degrading
Microorganisms
The ability to degrade extracellular poly(HA )
is widely distributed among bacteria and
51