ORIGINAL PAPER

A Novel Affinity Chromatographic Material for the Purificationof Extracellular Polyhydroxybutyrate Depolymerases

Christos P. Papaneophytou • Anastasia A. Pantazaki

Published online: 20 August 2011

� Springer Science+Business Media, LLC 2011

Abstract A novel affinity chromatographic material,

which is composed of silica matrix, coated with poly-

hydroxybutyrate (PHB) powder, suitable for the purifica-

tion of PHB depolymerases, was developed. The surface

morphology of the PHB-silica coated particles (silica-PHB

composite particles) was examined by scanning electron

microscopy and revealed a successful uniform coating of

silica particles with PHB. Moreover, the complex of these

materials retained its homogeneity even after incubation at

80 �C for 6 h, whereas the strong binding of PHB on silica

surface was further verified by thermal gravimetric analysis

and by PHB extraction- from silica surface- experiments.

This novel material was demonstrated to be suitable for

both, the one-step on-batch and on-column purification of

Thermus thermophilus extracellular PHB depolymerase.

The enzyme exhibited higher affinity against the composite

of silica-PHB particles than PHB powder, since the one-

step purification-fold and the overall recovery of the

enzyme were 2.8 and 4 times higher respectively, in the

first case. Reusability of the silica-PHB composites parti-

cles was examined by determining the recoveries of PHB

depolymerase. The enzyme recoveries were ranged from

30 to 35% for the first five uses, whereas for further uses

recoveries gradually dropped to 15–18% indicating that the

particles could be used repeatedly for five times. This

material could be also a suitable support for lipases or other

proteins that exhibit strong affinity to hydrophobic

materials.

Keywords Affinity chromatography � Silica-PHB

composite particles � Extracellular PHB-depolymerase �Thermus thermophilus HB8

Introduction

Polyhydroxyalkanoates (PHAs) are storage compounds of

carbon and energy accumulated by many microorganisms.

PHAs have similar physical properties as chemically pro-

duced polymers such as polyethylene or polypropylene and

are synthesized from renewable resources. In addition,

PHAs are biodegradable to water and carbon dioxide and

thus are considered as environmental-friendly alternatives

to petrol-based polymers [1, 2]. Amongst various bacteri-

ally synthesized PHA polymers, polyhydroxybutyrate

(PHB) is the most thoroughly characterized biopolymer

due to its early discovery in 1926 by Lemoigne [3].

The ability to degrade extracellular PHAs is widely

distributed among bacteria and depends on the secretion of

specific polyester depolymerases, which hydrolyze the

water-insoluble polyester to water-soluble monomers or

oligomers [4–6]. Many studies have been done on the

degradation of PHAs in soil [7], the ocean [8] and compost

[9]. In addition, many extracellular PHA depolymerases

have been purified from various microorganisms and

characterized. However, most purified depolymerases are

specific mainly for PHB. The PHB depolymerases of sev-

eral PHB-degrading bacteria, such as Alcaligenes faecalis

AE122 [10], Comamonas sp. [11], Pseudomonas lemoignei

[12, 13], Pseudomonas putida [14] and Agrobacterium sp.

K-03 [15] have been extensively studied. Some eukaryotes

such as Aspergillus fumigatus [9], Paecilomyces lilacinus

D218 [16], Penicillium funiculosum ATCC 9644 [17]

Penicillium simplicissimum LAR13 [18] and Emericellopsis

C. P. Papaneophytou � A. A. Pantazaki (&)

Department of Chemistry, Laboratory of Biochemistry, Aristotle

University of Thessaloniki, Thessaloniki 54124, Greece

e-mail: natasa@chem.auth.gr

123

J Polym Environ (2011) 19:876–886

DOI 10.1007/s10924-011-0345-x

minima W2 [19] are also known to be capable of PHB

degradation.

In general, extracellular PHB depolymerases share

several characteristics: (1) the Mr is relatively small (below

100 kDa; for many PHA depolymerases between 40 and

50 kDa); (2) PHA depolymerases do not bind to anion

exchangers such as DEAE but have a pronounced affinity

to hydrophobic materials; (3) the pH optimum is in the

alkaline range (7.5–9.8). Only the depolymerases of

R. pickettii and of Penicillium jiiniculosum have pH optima

at 5.5 or 6.0, respectively; (4) most PHA depolymerases are

inhibited by serine esterase inhibitors such as diisopropyl-

fluorylphosphate (DFP) or acylsulfonyl compounds, which

have been shown to bind covalently to the active site serine

of serine hydrolases [20]. The optimum temperatures of

these well-studied PHB depolymerases are less than 55 �C.

Nevertheless, a few publications reports for thermostable

PHB depolymerases have been reported [21–23]. Recently,

our group demonstrated that the thermophilic bacterium

Thermus thermophilus HB8 is a PHB degrading microor-

ganism [24]. T. thermophilus extracellular PHB depoly-

merase has an apparent molecular mass of 42 kDa and

exhibits great thermostability.

The literature describes several methods for the purifi-

cation of extracellular PHB depolymerases. Amongst them,

there are some one-step affinity chromatography methods

using PHB [25, 26] or multiple purification steps including

various combinations of ammonium sulfate precipitation,

anion exchange chromatograph, hydrophobic interaction

chromatography and gel filtration chromatography [10, 12,

16, 18, 19, 22, 27–36]. Nevertheless, protein purification

technologies remain a hot topic of the today’s separation

science.

The synthesis of core–shell nano-objects comprising of

an inorganic core and a polymer shell has recently gathered

great scientific interest with a view towards new opportu-

nities for constructing functional nano-structured materials

[37, 38]. Particles coating to change the surface properties

and/or functionality of powders appear as a very important

process for many industries. Typical applications include

modification of flowability, wettability (hydrophobic/

hydrophilic properties), solubility, dispensability, flavor,

particle properties. In general particle coating processes,

materials with relatively large particle size (host particles;

1–500 lm) are coated with fine particles (guest particles;

0.1–50 lm) in order to create new functionality or to

improve their initial characteristics [39]. Since the size of

the guest particles is so small, van der Waals interactions

are strong enough to keep them firmly attached to the host

particles. Thus, either a discrete or continuous coating of

guest particles can be achieved depending on a variety of

operating conditions including processing time, rotation

speed, weight fraction of guest to host particles and particle

properties [40].

Until now, studies of biodegradable polymer/inorganic

hybrid composites have traditionally focused mainly on

resulting physical properties of these complexes. In this

study a novel affinity chromatographic material, composed

of silica particles coated with PHB specific and suitable for

the purification of PHB depolymerases has been developed

and tested with T. thermophilus extracellular PHB

depolymerase.

Materials and Methods

Materials

PHB and crotonic acid standards were obtained from Fluka

Chemica GmbH (Steinheim, Germany). All other chemi-

cals were purchased from Sigma Chem. Co. (St. Louis,

MO, USA).

Bacteria Strain and Growth Conditions

Thermus thermophilus HB8 (DSM 579) was used in this

study. For the over expression of extracellular PHB

depolymerase, T. thermophilus was grown in mineral salt

medium (MSM) containing PHB as sole carbon source as

previously described [24]. Cell-free supernatant was con-

centrated approximately 100-fold by passage through an

ultra-filtration nitrocellulose membrane (YM 30; Amicon,

Beverly, MA) and subsequently was diluted with 50 mM

Tris–HCl buffer, pH 8. The concentrated enzyme solution

was used in all experiments unless is indicated in the text.

Coating of Silica Particles with PHB

The affinity support was synthesized by adsorbing PHB

(average molecular mass 540,000 g/mol) from a solution in

chloroform onto silica particles. Thirty grams of silica

(Kiesegel 60, 0.063–0.2 mm) were dried under reduced

pressure at 150 �C for 12 h, while 3 g of PHB were dis-

solved in 25 mL chloroform and filtered. Silica particles

were mixed with the PHB solution, and 20 min of soni-

cation (20 kHz) was allowed for their mixing. The mixture

was then left at room temperature for 24 h under gentle

agitation in a rotor mixer and subsequently was filtered

under nitrogen pressure. The PHB-coated silica particles

(PHB-silica composite particles) were washed with 50 mL

of chloroform and subsequently with equal volume of

methanol. The washing procedure was repeated three

times. After washings the silica-PHB composites were air-

dried [41, 42].

J Polym Environ (2011) 19:876–886 877

123

Determination of PHB Adsorbed on Silica Particles

The effectiveness of the PHB coating as well as the amount

of the PHB adsorbed onto the silica surface was quantified

indirectly by HPLC using a variation of the method of Karr

et al. [43] based on the acid-catalyzed b-elimination of PHB

to crotonic acid, as previously described [44]. The procedure

was followed as described [44] with the difference that ali-

quots (0.2 g) either of silica particles or silica particles

coated with PHB were incubated with concentrated H2SO4 at

92 �C for 8 h to transform PHB to crotonic acid. The final

filtrate was analyzed by HPLC on an Aminex HPX-87H

ion exclusion organic acid analysis column (360 mm 9

7.8 mm; Biorad, Espoo, Finland) applying standard condi-

tions (Huang and Reusch [44]) [39], and using a Shimadzu

(Tokyo, Japan) HPLC Chromatography System. Quantita-

tion was performed following comparison of peak absor-

bance with those of crotonic acid standards. The millimolar

extinction coefficient was A208 = 14.12 L�mol-1�cm-1.

In addition, the surface homogeneity and the morphol-

ogy of the silica-PHB composites as well as the surface

morphology of intact silica particles and PHB were mon-

itored using scanning electron microscopy (SEM) as is

following described.

Scanning Electron Microscopy

The structure and the surface morphology of the starting

materials of silica particles, of PHB and of silica coated

with PHB were monitored by a JSM-6300, Jeol SEM. All

materials were coated with gold using an ion coater.

Stability of PHB-Coated Silica Particles

To investigate the stability of PHB-silica composites, a

20 mL suspension (5% w/v) of this material in 50 mM

Tris–HCl buffer pH 8.0 was prepared, and incubated for

6 h at 80 �C. Samples of 2 mL at various time intervals

were taken and filtrated by passing through a Whatman

filter paper No. 5 and the silica particles coated with PHB

were recovered. Subsequently, their morphology was

examined by SEM, before and after the heat treatment to

monitor the influence of prolonged heating in the coating

integrity and stability. In addition, the resulted supernatants

were concentrated to dryness in a using rotary evaporator

and the remained pellets were in a GC chromatographer as

following described in order to detect the amount of PHB

realising from silica surface.

Gas Chromatography Analysis

The amount of PHB in various samples, as indicating in the

text, was determined by gas chromatography (GC) [45],

while samples were previously subjected to methanolysis

as before described [45]. Gas chromatography was per-

formed with an Agilent gas chromatograph (5975c Series

GC/MSD, California, USA) equipped with an HP-Innowax

capillary column (30 m 9 0.5 lm; J&W Scientific, Cali-

fornia, USA) and a flame ionization detector. Two micro-

liters of the organic phase was injected in the column.

Nitrogen (0.9 mL/min) was used as the carrier gas. The

temperature of the injector and detector were 220 and

250 �C, respectively. A temperature program was used for

efficient separation of the esters (50 �C for 3 min, tem-

perature increase 9 �C/min until 250 �C, (250 �C for

1 min)). Under these conditions, the retention time (in

minutes) of the 3-hydroxybutyric acid methyl ester stan-

dard was 11.27 min.

Thermal Gravimetric Analysis

Thermal gravimetric analysis (TGA) data of silica parti-

cles, PHB powder and PHB-coated silica particles were

taken on a Perkin Elmer (Pyris 1) instrument in a nitrogen

atmosphere at a rate of 10 �C/min. The microwave

apparatus used for the polycondensation was a Samsung

(South Korea) microwave oven (2,450 MHz, 900 W).

Affinity of Extracellular PHB Depolymerase for Silica

Particles, PHB and PHB- Coated Silica Particles

In order to investigate the affinity of extracellular PHB

depolymerase for silica particles, PHB and silica-PHB

composite particles, various concentrations of each mate-

rial ranging from 20 to 200 lg/mL were added in 1 mL of

enzyme preparation from concentrated cell-free superna-

tant. All assay mixtures were incubated for 1 h at room

temperature and subsequently the supernatants were sepa-

rate from the solid pellets by passing through a Whatman

No. 5 filter paper. After the adsorption step of the enzyme

to the various materials used, remaining PHB depolymer-

ase activities in all resulted supernatants, were determined.

Determination of PHB Depolymerare Activity

Polyhydroxybutyrate depolymerase activity was determi-

nate using either PHB of para-nitrophenylbutyrate (pNPB)

as substrates as previously described [24].

Purification of the T. thermophilus PHB Depolymerase

The extracellular PHB depolymerase was isolated from the

cell-free supernatant of a T. thermophilus culture, grown

for 48 h at 70 �C in MSM, containing 0.1% (w/v) PHB as

sole carbon source. After centrifugation at 10,000 9g for

15 min the resulted cell-free supernatant was concentrated

878 J Polym Environ (2011) 19:876–886

123

100-fold by passage through an ultra-filtration nitrocellu-

lose membrane (YM 30; Amicon, Beverly, MA). The

concentrated protein solution was dialyzed with 50 mM

Tris–HCl buffer, pH 8 at 4 �C and used for extracellular

PHB depolymerise purification.

Purification of T. thermophilus Extracellular PHB

Depolymerase on Batch Using the PHB-Coated Silica

Particles or PHB

For on batch-purification of extracellular PHB depoly-

merase, the concentrated cell-free supernatant was first

heated at 80 �C for 30 min and the precipitated proteins

were removed by centrifugation at 20,000 9g for 15 min.

Subsequently, a 0.1% (w/v) suspension of silica-PHB

composites was added to the enzyme solution for selective

binding of the PHB depolymerase activity at 70 �C under

continuous stirring for 30 min. The resulting mixture was

filtered through a Whatman No. 5 filter paper, and the solid

material washed twice with 50 mM Tris–HCl buffer pH

8.0. Subsequently the PHB depolymerase was eluted with

1 M NaCl in the same buffer and the eluant was then

dialyzed against 50 mM Tris–HCl buffer pH 8.0. For sil-

ica-PHB composites regeneration, the material was initially

washed with 0.25 M NaCl in buffer 50 mM Tris–HCl pH 8

and subsequently with the same buffer before the next use.

In addition, extracellular PHB depolymerase was puri-

fied using a suspension of PHB as previously described

[24], instead of silica-PHB composite particles.

Purification on a PHB-Coated Silica Particles Column

Thermus thermophilus extracellular PHB depolymerase

was also purified using a column filled with PHB-coated

silica particles. The procedure was took place at room

temperature unless is indicated in the text. The concen-

trated and dialyzed with 50 mM Tris–HCl buffer, pH 8 at

4 �C protein, was subsequently loaded on a column

(2.6 9 25 cm) filled with PHB-coated silica particles,

previously equilibrated with the same buffer. The column

was washed 50 mM Tris–HCl buffer, pH 8.0. Elution of

PHB depolymerase was performed using an increasing

gradient (0–1 M of NaCl in 50 mM Tris–HCl, pH 8.0.

Fractions exhibiting PHB depolymerase activity were

pooled, dialysed against 50 mM Tris–HCl buffer, pH 8 and

concentrated with an Amicon concentrator with a PM-10

membrane at 4 �C.

Protein Determination and Gel Electrophoresis

The concentration of protein in the samples was deter-

mined by the Bradford method [46] and bovine albumin

was used as standard. Proteins were separated by

electrophoresis in 10% (w/v) SDS–polyacrylamide gel

electrophoresis (SDS–PAGE) as previously described [47].

Results

Coating of Silica Particles with PHB, Surface

Morphology and Stability of PHB-Coated Silica

Particles

The formation of a new and selective composite material,

for the purification of extracellular PHB depolymerases,

has been developed by coating silica beads with PHB, the

natural substrate of PHB depolymerases, as illustrated in

Fig. 1.

The determination of the PHB amount, which was

absorbed on the surface of the silica particles, was based on

the acid-catalyzed b-elimination of PHB to crotonic acid,

followed by HPLC analysis (see Methods). The results

revealed that a high amount of PHB was adsorbed on the

surface of silica particles, since a large peak of crotonic

acid was detected (data not shown). Based on the standard

curve, it was estimated that approximately 72% of the PHB

was absorbed on silica matrix. The amount of PHB

adsorbed (attached) on the silica was determined to be

0.126 lmol/g. In contrast, crotonic acid did not detect in

the starting materials of intact silica particles (data not

shown), as it was expected.

Fig. 1 Schematic representation of formation of composite of silica-

PHB particles. Silica particles were dried at 150 �C for 12 h and

subsequently mixed with a PHB solution in chloroform. The mixture

was sonicated for 20 min and left at room temperature for 24 h under

gentle agitation. The suspension was filtered and the resulted PHB-

coated silica particles were washed with chloroform and methanol

J Polym Environ (2011) 19:876–886 879

123

The uncoated and coated silica particles were examined

by means of a SEM to study the surface morphology and

the particles shape before and after coating. SEM images

indicate that the silica particles are irregularly shaped and

the surface is rough as illustrated in Fig. 2a. In addition,

Fig. 2b shows the structure of PHB powder, while the

structure of a silica particle coated with PHB is illustrated

in Fig. 2c, and as it can been seen the surface of silica

particle was uniformly covered with PHB. The stability of

the formed silica-PHB composite particles was initially

verified by incubating this material in 50 mM Tris–HCl

buffer, pH 8.0, at 80 �C for 6 h. The silica-PHB composite

particles were recovered by filtering the mixture through a

Whatman filter paper No. 5 in order to examine their

morphology by SEM while the resulted supernatant were

analysed by GC to verify the amount of PHB released after

incubation at 80 �C (see ‘‘Materials and Methods’’). As

shown in Fig. 2d, the PHB-coated silica matrix is very

stable since it retained its homogeneity even after incuba-

tion at 80 �C for 6 h. In addition, in the resulted

supernatants only traces of PHB were detected by GC

analysis (data not shown). These results revealed that the

absorption of PHB on silica beads is very strong and the

strength of interaction between PHB and silica did not

break at 80 �C even after 6 h of incubation.

Thermal gravimetric analysis was performed for PHB,

silica particles and for PHB-coated silica particles, where

the weight loss due to the volatilization of the degradation

products was monitored as a function of temperature as

shown in Fig. 3a. As it can be seen silica particles, prior

coating with PHB, exhibited high thermostability and the

weight loss percentage is only 5% after heating to 500 �C

in nitrogen, while intact PHB lost weight precipitously at

220–340 �C. In addition after the coating of silica particles

with PHB the weight loss percentage is 15% after heading

to 500 �C indicating that the adsorption of PHB on silica

surfaces is very strong.

To confirm the strong adsorption of PHB on silica sur-

face, the PHB-coated silica particles (50 mg) were incu-

bated in boiling chloroform (1 mL) in screw cup tubes for

Fig. 2 Scanning electron images of the starting constituents and of

the affinity support surface used for the purification of T. thermophi-lus extracellular PHB depolymerase. Silica particle (a), PHB (b),

homogenous coating of silica particles with PHB before (c) and after

incubation at 80 �C for 6 h (d)

880 J Polym Environ (2011) 19:876–886

123

various times intervals during 1–12 h. All samples were

then rapidly cooled in ice and subsequently centrifuged at

5,000 9g for 5 min to recover the tied loosely PHB. The

layer of chloroform containing the released PHB from

silica surface was concentrated to dryness in a rotary

evaporator and subjected in GC analysis (as is described in

the material section) to quantify PHB amount. As shown in

Fig. 3b the PHB released from the silica surface was

increasing almost linearly until 6 h of extraction. In pro-

longed incubation, only a slight increase of the amount of

PHB released from silica surface was observed. Never-

theless, it is notable that the amount of PHB released from

silica surface was significant low, approximately 4% of the

initial amount of PHB, which was adsorbed on the silica

surface, confirming the high stability of the resulting

material.

Affinity of Extracellular PHB Depolymerase for Silica

Particles, PHB and Silica-PHB Composite Particles

and Effectiveness of the Materials

Polyhydroxybutyrate depolymerase revealed negligible

affinity for uncoated silica particles as almost the total of

enzyme activity of the concentrated cell-free supernatants

remained in samples after adsorption onto silica particles,

even when 200 lg/mL of silica was used, as it shown in

Fig. 4a.

In contrast, extracellular PHB depolymerase exhibited

high affinity for PHB while the enzyme exhibited the

highest affinity for the PHB-coated silica particles. The

remaining PHB depolymerase activity decreased gradually

in the resulted supernatants after the addition and removal

of PHB or PHB -coated silica particles. Moreover, the

enzyme activity in the resulted supernatants was reduced

almost linearly as the concentration of PHB-coated silica

beads was increased from 0 to 100 lg/mL, while at higher

concentrations further decreased of enzyme activity did not

observed. Moreover, the enzyme activity in the resulted

supernatants was reduced almost linearly as the concen-

tration of silica-PHB composites was increased from 0 to

100 lg/mL reaching a plateau, while addition of higher

concentrations of materials did not result in further

adsorption of enzyme (Fig. 4a).

To compare the effectiveness of the silica-PHB com-

posites against PHB, for adsorption and purification of the

extracellular PHB depolymerase, both materials were used

to absorb the enzyme from a concentrated cell–free

supernatant of T. thermophilus culture containing PHB as

sole carbon source. As shown in Fig. 4b, the silica-PHB

composite particles were more efficient for the enzyme

purification compared to PHB in terms of enzyme purifi-

cation -fold and recovery. The usage of PHB for the

extracellular PHB depolymerase purification resulted in a

28.24-fold purification of enzyme with an overall recovery

of 8.36%. In contrast, when a suspension of silica-PHB

composites was used, the purification -fold of the enzyme

was increased 2.8 times (reaching 81.26-fold), while the

overall recovery was 4 times higher (33.8%) than that when

PHB was used.

Fig. 3 (a) TGA curves for silica particles, PHB and silica-PHB

composite particles. (b) Effect of reaction time on silica-PHB

composite particles stability. The silica-PHB composite particles

were incubated in boiling chloroform for various time intervals and

the particles were removed by centrifugation at 5,000 9g for 5 min.

The resulted supernatants were subjected in GC analysis in order to

determine the amount of PHB released form silica surface. The

concentration of releasing PHB was estimated based on the initial

amount of polymer which was adsorbed on silica surface

J Polym Environ (2011) 19:876–886 881

123

Purification of Extracellular T. thermophilus PHB

Depolymerase Using the Novel Silica-PHB Composites

On-Batch Purification and Reusability of Silica-PHB

Particles

A one-step substrate-affinity and hydrophobic interaction

chromatographic method was developed for the purifica-

tion of an extracellular PHB depolymerase based on the

coating of silica particles with PHB. The enzyme was

purified 83-fold with an overall recovery of 34.2%

(Table 1). SDS–PAGE analysis of the purified enzyme

preparation revealed a single band of molecular mass of

approximately 42 kDa as shown in Fig. 5.

Reusability of the silica-PHB particles was investigated

after regeneration of the particles by assessing the recovery

of the PHB depolymerase for 7 successive times. Recovery

of the enzyme from the first use of the particles was

approximately 35% and recoveries from the second and the

third uses (after the first and second regeneration) were

31–33% and the recoveries from the forth and fifth uses

(after the 3–4 regenerations) were 30–32%. However,

recoveries from the sixth-seventh uses gradually dropped to

15–18%. These results indicated that the newly prepared

silica-PHB composite particles could be used repeatedly

for 5 times after regeneration. The causes of gradually

decrease of PHB depolymerase recovery is probably due to

the deterioration of material after extensive use. Never-

theless, the causes of gradually decrease of PHB depoly-

merase recovery are under investigation.

On-Column Purification

The chromatogram and the elution pattern of extracellular

PHB depolymerase on a silica-PHB composite particles

column is illustrated in Fig. 6. The enzyme was purified

approximately 57-fold with an overall recovery of 33.03%

as illustrated in Table 2. SDS–PAGE analysis of the puri-

fied enzyme preparation revealed the same single band at

42 kDa (data not shown). These results revealed the silica-

PHB composite particles are suitable for the on-column

purification of extracellular PHB depolymerase. In addi-

tion, the on-column and batch purifications procedures of

T. thermophilus PHB depolymerase using the PHB-coated

silica particles are comparable.

Discussion

In the last few decades, organic–inorganic hybrid materials

have attracted much interest because of their extensive

potential applications in various fields of material science,

ranging from paints, magnetic fluids, and high-quality paper

coating to microelectronics and biotechnology [48]. These

materials represent a new class of polymeric materials which

combine the properties of the inorganic particles, such as

mechanical strength and thermal stability, with the process

ability and the flexibility of the organic polymer matrix. Such

materials could be obtained by simply mixing the organic

and inorganic components [49]. These composites combine

the favored properties of the inorganic material (e.g.,

increased strength and modulus, high thermal stability and

better gas barrier property) and the organic polymer matrix

(e.g., flexibility, low density and processability) [50–52].

It is well known that (bio) macromolecules have a ten-

dency of adsorbing onto silica particles, and this property

has often been utilized for the preparation of ligand-

Fig. 4 (a) Affinity of extracellular PHB depolymerase against silica

particles (filled triangle), PHB (filled circle) and silica-PHB compos-

ite particles (filled square). (b) Comparison of the affinity materials

PHB beads and silica-PHB composites for the extracellular PHB

depolymerase purification. White columns: Recovery of extracellular

PHB depolymerase (%); Line-drawing columns: PHB depolymerase

purification -fold

882 J Polym Environ (2011) 19:876–886

123

modified stationary phases. However, most studies for

organic/inorganic hybrids mainly focus on the physico-

chemical properties of these complexes and not on their

potential applications. For example, Lim et al. [53] studied

the effect of hydrogen bonding in the crystallization

behavior of biodegradable poly(3-hydroxybutyrate-co-3-

hydroxyhexanoate)/silica composites. Lee et al. [54] pre-

pared hydrophobic silica particles either by chemical graft

of alkyl chains or by physical adsorption of cationic sur-

factants alkyl-tri-methyl ammonium bromide and the

effects of the two modification methods on the monolayer

behavior of silica particles at the air/water interface were

studied. In addition, Kawano et al. [55] prepared sparsely-

distributed silica/poly(methyl methacrylate) composite

particles with potential applications in chromatographic

analysis, separation and bio-compatible materials.

In this paper we employ the tendency of PHB to be

absorbed on silica surface in order to develop a novel

material for PHB depolymerase purification (Figs. 1 and

2). To our knowledge this is the first attempt to develop a

hybrid composite which would be used for enzymes puri-

fication. The silica-PHB composites exhibited high ther-

mostability as resulted by TGA analysis and PHB

extraction –from silica surface- experiments (Fig. 3)

revealing that the adsorption of PHB onto silica is very

strong.

Our results demonstrated that T. thermophilus extracel-

lular PHB depolymerase revealed higher affinity to the

novel silica-PHB composite particles than PHB powder

(Fig. 4). The advantage of this support is that the PHB-

coated particles probably attributing a high specific surface

Table 1 Purification of

T. thermophilus PHB

depolymerase using the

PHB-coated silica particles

Purification step Total

protein

(mg)

Total

activity

(U)

Specific

activity

(U/mg)

Purification

fold

Recovery

(%)

Concentrated supernatant 102 208 2.04 1 100

Heat treatment at 80 �C 58.65 142.56 2.43 1.19 68.54

Affinity binding to PHB

coated-silica beads

0.42 71.12 169.3 83.0 34.2

Fig. 5 SDS-PAGE analysis of the purified PHB depolymerase.

Purified enzyme (5 lg) was subjected to SDS–polyacrylamide gel

in 10% (w/v) polyacrylamide. The gel was stained with silver nitrate.Lane 1: cell free supernatant, lane 2: purified PHB depolymerase,

lane 3: molecular weight markers of the indicate masses

Fig. 6 Chromatogram of

extracellular PHB depolymerase

from T. thermophilus HB8. The

concentrate cell-free

supernatant was applied to a

column (2.6 9 25 cm) filled

with PHB-coated silica particles

and previously equilibrated with

50 mM Tris–HCl buffer, pH

8.0. After washing, the column

was developed with an

increasing linear salt gradient

ranging from 0 to 1 M NaCl.

Continues line: protein (A280

nm); filled square: PHB

depolymerase activity

J Polym Environ (2011) 19:876–886 883

123

to this support compared with PHB powder which was

previously used [24–26].

This material was used for the purification of extracel-

lular PHB depolymerase of T. thermophilus. This adsorbent

was demonstrated to be highly suitable for the PHB

depolymerase purification from the extracellular crude

extract using on batch and on column utilization (Fig. 6)

The novel single purification step procedure resulted in a

highly purified and homogeneous enzyme preparation

(Fig. 5) while newly prepared silica-PHB composite par-

ticles could be used repeatedly for 5 times after regenera-

tion. The usage of this material for the purification of

extracellular PHB depolymerases of other strains is cur-

rently in progress in our laboratory.

Until now, all the one-step purification protocols for the

purification of extracellular PHB depolymerases have been

performed using PHB powder as affinity material. Using

PHB powder as substrate the extracellular PHB depoly-

merase of T. thermophilus was purified with an overall

recovery of 8.2% and 28.2-fold purification [24] while the

a 1.9-fold purification and a recovery yield of 10.3% has

been reported for the extracellular PHB depolymerase of

S. thermodepolymerans [26]. In addition, using the PHB

powder as affinity substrate the extracellular PHB depol-

ymerase of A. fumigatus has been purified 33-fold with an

overall recovery of 86% [25]. Thus, the recovery yield

(83%) and purification (34.3-fold) achieved by this novel

affinity chromatography method is better or comparable

with other one-step purification methods reported to date.

On the other hand various multistep purification strate-

gies that have been previously employed for the enzyme

purification. Most of these purification procedures were

based on a combination of several non- specific techniques,

such as ammonium sulfate precipitation, and ultra-filtra-

tion, gel filtration and ion-exchange chromatography as

demonstrated in Table 3. The maximum purification fold

reported so far is 562.2-fold from Arthrobacter sp. stain

W6 [28], using a three-step purification. A high purification

fold, 317.8 times, and a recovery yield of 22% have been

reported for an extracellular PHB depolymerase from Le-

ptotrix sp. using ammonium sulfate precipitation and

chromatography on Toyopearl HW-55F [32]. However, the

obtained overall recovery and purification of T. thermo-

philus PHB depolymerase, using this novel material is

better than most of the multistep purification strategies

which are summarized in Table 3 and moreover is less

time-consuming.

Purification of the PHB depolymerases is essential to

understanding and differentiation between the functions of

the different depolymerases and the modes of substrate

hydrolysis and distinguishing endo- and exo-type hydro-

lases. Purification of PHB depolymerases in one-step

substrate-affinity and hydrophobic interaction chromato-

graphic method could be proposed as useful in a wide

range of applications such as for producing pure monomers

of PHA and useful chemicals, including D-3-hydroxy-

carboxylic acids such as pure D-3-hydroxybutyric acid,

which is the main component of the degradation products

obtained by enzymatic degradation of PHB [22]. A pure

monomer of PHB, D-3-hydroxybutyric acid, is also an

important precursor of 4-acetoxyazetidinone, which is used

in making carbapenem antibiotics [56].

Extracellular PHB depolymerases are secreted from

various microorganisms and several have been isolated and

purified, are ubiquitous and play an important role in the

metabolism of PHB in the environment. Analysis of their

primary structures revealed that the enzymes are composed

of substrate-binding domain, catalytic domain, and a linker

region connecting the two domains. The substrate-binding

domain plays a role in binding to the solid PHB [57]. PHB

depolymerases belong to a group of specific hydrolases,

namely the serine hydrolases which are also characterized

as polyester cleaving enzymes and which are able to

catalyze the hydrolytic cleavage of the C–O and C–N–

bonds[58]. This group of enzymes also includes lipases,

esterases, and serine endopeptidases [58]. The active site

of the enzymes belonged to the serine hydrolase super-

family is composed of a catalytic triad Ser-Asp(Glu)-His.

Serine is a part of a pentapeptide sequence (GX1SX2G)

which is known as ‘‘lipase box’’ [20]. The catalytic cat-

alytic triad (Ser-His-Asp) has also been found in all

reported so far extracellular PHB depolymerases, and the

serine is part of the lipase box. [59]. Conclusively, a

similar substrate-binding domain might probably be

involved and play also similar role in the binding of PHB

depolymerase to the PHB-coated silica support of this

work, since uncoated silica particles did not adsorb the

enzyme. Moreover, the PHB depolymerase catalytic

domain seems to be unaffected by the adsorption proce-

dure of the enzyme to the PHB-coated silica support

Table 2 Purification of

T. thermophilus extracellular

PHB-depolymerase using a

chromatographic column filled

with PHB-coated silica particles

Purification step Total

protein

(mg)

Total

activity

(U)

Specific

activity

(U/mg)

Purification

fold

Recovery

(%)

Concentrated supernatant 117 218 1.86 1.0 100

Chromatography on PHB-coated

silica particles

0.68 72 105.88 56.92 33.03

884 J Polym Environ (2011) 19:876–886

123

forasmuch as the enzyme retained catalytic activity after

elution.

In addition, lipases have a common amino acid sequence

around the active site, Gly-X1-Ser-X2-Gly, similar to PHA

depolymerases, while the X1 residue is a leucine residue in

PHA depolymerases instead of a histidine in bacterial

lipases suggesting that lipases and PHA depolymerases

may share a similar mechanism of substrate hydrolysis

[60]. Thus, the novel silica-PHB composite particles, from

another point of view, might be used as a support for

lipases or other proteins that have strong affinity to

hydrophobic materials. The usage of this material for the

purification of other proteins is currently in progress in our

laboratory.

Based on this idea, the coating with PHB of the inner

wall surface of fused-silica capillaries was successfully

attempted (unpublished data), and the development of

applications on the field of proteins separations and of

affinity capillary electrophoresis constituted a future pros-

pect of our studies which are in progress.

References

1. Anderson AJ, Dawes EA (1990) Microbiol Rev 54:450

2. Madison LL, Huisman GW (1999) Microbiol Mol Biol Rev 63:21

3. Lemoigne M (1926) Bull Soc Chim Biol 8:770

4. Jendrossek D (2002) Extracellular polyhydroxyalkanoate dep-

olymerases: the key enzymes of PHA degradation. In: Doi Y,

Steinbuchel A (eds) Biopolymers, vol. 3b. Polyesters II. Wiley-

VCH, Hoboken, pp 41–83

5. Jendrossek D, Handrick R (2002) Annu Rev Microbiol 56:403

6. Jendrossek D, Schirmer A, Schlegel HG (1996) Appl Microbiol

Biotechnol 46:451

7. Mergaert J, Webb A, Anderson C, Wouters A, Swings (1993)

Appl Environ Microbiol 59:3233

8. Mergaert J, Wouters A, Anderson C, Swings J (1995) Can J

Microbiol 41 (suppl 1):154

Table 3 Comparison of methods for the purification of extracellular PHB depolymerase of various PHB-degrading microorganisms

Strain study Method Purification

fold

Recovery

(%)

Reference

T. thermophilus HB8 Affinity binding to a novel material composed of silica particles

coated with PHB

83.0 34.2 This study

T. thermophilus HB8 Affinity binding to PHB 28.2 8.2 [24]

S. thermodepolymerans Affinity binding to PHB 1.9 10.3 [26]

Aspergilus fumigatus Affinity binding to PHB 33 86 [25]

Streptomyces sp. Chromatography on HiPrep 16/10 butyl and on Superdex 75 32 19 [22]

P. simplicissimum LAR 13 Chromatography on Sepharose CL-6B column 2.4 61 [17]

Bacillus subtilis WB800 Ammonium sulfate precipitation and chromatography

on CM-Sepharose

8.0 8 [27]

Emericellopsis minima W2 Chromatography on octyl Sepharose CL-4B and Sephaden G-100 20.0 51.1 [19]

Arthrobacter sp. Strain W6 Holow fiber and chromatography on DEAE-Toyopearl

and Butyl-Toyopearl

562.2 8 [28]

Paecilomyces lilacinus D218 Chromatography on CM-Toyopearl 650 M and Hyroxylapatite 94.7 46.7 [16]

Comamonas acidovorans Chromatography on Butyl-Toyopearl and Sephadex G-200 9.1 8 [29]

Pseudomonas Stutzeri Chromatography on Phenyl Sepharose, Q Sepharose and Mono Q 8 4 [30]

Aspergillus fumigatus Spiral Cartridge, Stirred Cell and chromatography

on Butyl-Sepharose

14.4 66 [31]

Leptothrix sp. Ammonium sulfate precipitation and chromatography

on Toyopearl HW-55F

317.8 22 [32]

Alcaligenes faecalis AE122 Chromatography on DEAE-cellulose (DE52),

Phenyl-Sepharose CL-4B and

DEAE-Toyopearl

37 15 [10]

Algaligenes faecalis T1 Chromatography on Butyl-Toyopearl N.D 47 [33]

Comomonas testosterone Ammonium sulfate precipitation and chromatography

on Butyl-Toyopearl

27.3 21 [33]

Comamonas sp. Ammonium sulfate precipitation and chromatography

on DEAE Sephacel and Butyl-Sepharose (4B)

13 37 [12]

Algaligenes faecalis T1 Ammonium sulfate precipitation and chromatography

on SEAE-cellulose and butyl Toyoperal

6.7 59 [35]

Pseudomonas lemoignei (PHBdepolymerase Isozyme A1)

Ammonium sulfate precipitation and chromatography

on DEAE-Sephorose CL-6B and CM-Sepharose CL-6B

5.9 19 [36]

J Polym Environ (2011) 19:876–886 885

123

9. Scherer TM (1996) Biological and enzymatic mechanisms of

polyester biodegradation by fungi. PSh. D. Thesis, University of

Massachusetts, Amherst

10. Kita K, Ishimaru K, Teraoka M, Yanase H, Kato N (1995)

Environ Microbiol 61:1727

11. Jendrossek D, Backhaus M, Andermann M (1995) Can J

Microbiol 41(suppl 1):160

12. Jendrossek D, Muller B, Schlegel HG (1993) Eur J Biochem

218:701

13. Jendrossek D, Frisse A, Behrends A, Andermann M, Kratzin HD,

Stanislawski T (1995) J Bacteriol 177:596

14. Ohura T, Kasuya KI, Doi Y (1999) Appl Environ Microbiol

65:189

15. Nojima S, Mineki S, Iida M (1996) J Ferment Bioeng 81:72

16. Oda Y, Osaka H, Urakami T, Tonomura K (1997) Curr Microbiol

34:230

17. Brucato CL, Wong SS (1991) Arch Biochem Biophys 290:497

18. Han JS, Kim MN (2002) J Microbiol 40:20

19. Kim DY, Yun JH, Kim WK, Bae KS, Rhee YH (2002) J

Microbiol 40:129

20. Jendrossek D (1998) J Polym Degrad 59:317

21. Kasuya K, Doi Y, Yao T (1994) Polym Degrad Stab 45:375

22. Calabia BP, Tokiwa Y (2006) Biotechnol Lett 28:383

23. Romen F, Reinhardt S, Jendrossek D (2004) Arch Microbiol

182:157

24. Papaneophytou CP, Pantazaki AA, Kyriakidis DA (2009) Appl

Microbiol Biotechnol 83:659

25. Iyer S, Shah R, Sharma A, Jendrossek D, Desai A (2000) J Polym

Environ 8:197

26. Elbanna K, Lutke-Eversloh T, Jendrossek D, Luftmann H,

Steinbuchel A (2004) Arch Microbiol 182:212

27. Braaz R, Wong SL, Jendrossek D (2002) FEMS Microbiol Lett

209:237

28. Asano Y, Watanabe S (2001) Biosci Biotechnol Biochem

65:1191

29. Kasuya K, Inoue Y, Tanaka T, Akehata T, Iwata T, Fukui T et al

(1997) Appl Environ Microbiol 63:4844

30. Hiraishi T, Ohura T, Ito S, Kasuya K, Doi Y (2000) Biomacro-

molecules 1:320

31. Scherer TM, Fuller RC, Lenz RW, Goodwin S (1999) J Environ

Polym Degrad 7:117

32. Takeda M, Koizumi J-I, Yabe K, Adachi K (1998) J Ferment

Bioeng 85:375

33. Saito T, Iwata A, Watanabe T (1993) J Environ Pol Degrad 1:99

34. Mukai K, Yamada K, Doi Y (1993) Int J Biol Macromol 15:361

35. Shirakura Y, Fukui T, Saito T, Okamoto Y, Narikawa T, Koide

K, et al. (1986) Biochim Biophys Acta 880:46

36. Nakayama K, Saito T, Fukui T, Shirakura Y, Tomita K (1985)

Biochim Biophys Acta 827:63

37. Caruso F (2001) Adv Mater 13:11

38. Golden JH, Deng H, DiSalvo FJ, Frechet JMJ, Thompson PM

(1995) Science 268:1463

39. Yoshihara I, Pieper W (1999) Swiss Pharma 6:21

40. Pfeffer R, Dave RN, Dongguang W, Ramlakhan M (2001)

Powder Technol 117:40

41. Millot M-C, Debranche T, Pantazaki A, Gherghi I, Sebille B,

Vidal-Madjar C (2003) Chromatographia 58:365

42. Loos-Neskovic C, Vidal-Madjar Cl, Jimenez B, Pantazaki A,

Tamburini A, Fedoroff M, et al. (1999) Radiochim Acta 85:143

43. Karr DB, Waters JK, Emerich DW (1983) Appl Environ

Microbiol 46:1339

44. Huang R, Reusch RN (1996) J Biol Chem 271:22196

45. Pantazaki AA, Tambaka MG, Langlois V, Guerin P, Kyriakidis

DA (2003) Mol Cell Biochem 254:173

46. Bradford MM (1976) Anal Biochem 72:248

47. Laemmli UK (1970) Nature 227:680

48. Ni KF, Sheibat-Othman N, Shan GR, Fevotte G, Bourgeat-Lami

E (2005) Macromolecules 38:9100

49. Qu A, Wen X, Pi P, Cheng J, Yang Z (2008) J Colloid Interface

Sci 317:62

50. Tian D, Dubois PH, Jerome R (1996) Polymer 37:3983

51. Tian D, Duboisa P, Blacher S (1997) Polymer 39:855

52. Sinha Ray S, Maiti P, Okamoto M, Yamata K, Ueda K (2002)

Macromolecules 35:3104

53. Lim JS, Noda I, Im SS (2007) Polymer 48:2745

54. Lee YL, Du ZC, Lin WX, Yang YM (2006) J Colloid Interface

Sci 296:233

55. Kawano S, Sei A, Kunitake M (2010) J Colloid Interface Sci

352:348

56. Chiba T, Nakai T (1985) Chem Lett 14:651

57. Tokiwa Y, Calabia BP (2004) Biotechnol Lett 26:1181

58. Webb EC (1992) Enzyme nomencluture. Academic Press, New

York

59. Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M,

Misset O (1994) FEMS Microbiol Rev 15:29

60. Jaeger KE, Steinbuchel A, Jendrossek D (1995) Appl Environ

Microbiol 61:3113

886 J Polym Environ (2011) 19:876–886

123