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Partition separation and characterization of the polyhydroxyalkanoates synthaseproduced from recombinant Escherichia coli using an aqueous two-phase system

John Chi-Wei Lan,* Chun-Yi Yeh, Chih-Chi Wang, Yu-Hsuan Yang, and Ho-Shing Wu

Bio-renery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, 135 Yuan-Tung Road, Chung-li, Taoyuan 320,

Taiwan

Received 18 October 2012; accepted 7 April 2013

Available online 15 May 2013

Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either bynumerous of microorganismsin vivoor synthase in vitro. The synthesis of PHAs in vitro requires an efcient separationfor high yield of puried enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstoniaeutropha H16 was cultivated in the chemically dened medium for overexpression of synthase in the present work. Thepurication and characteristics of PHA synthase from claried feedstock by using aqueous two-phase systems (ATPS)was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4C. Theresults showed that the partition coefcient of enzyme activity and protein content are 6.07 and 0.22, respectively. Thespecic activity, selectivity, purication fold and recovery of phaCRe achieved 1.76 U mg

L1, 29.05, 16.23 and 95.32%,respectively. Several metal ions demonstrated a signicant effect on activity of puried enzyme. The puried enzymedisplayed maximum relative activity as operating condition at pH value of 7.5 and 37 C. As compared to conventionalpurication processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation oflarge quantity of PHA synthase on synthesis of P(3HB) in vitro.

2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Aqueous two-phase systems; Recombinant Escherichia coli; Recovery; Polyhydroxyalkanoates; PHA synthase]

Polyhydroxyalkanoates (PHAs) are known as the most fasci-

nating and largest group of biopolyesters, and are characterized

with dissimilar properties and functionalities (1). Many environ-

mental bacteria have been found to accumulate PHAs under natural

conditions(2). PHAs have drawn considerable attention from both

academic and industrial circles because of their superior charac-

teristics of biodegradability, bioabsorbability, and biocompatibility

(3). Poly(3-hydroxybutyrate) [P(3HB)] is a well-known polymer and

is the most widely used among the PHAs. P(3HB) can be synthe-

sized using the enzymatic method either in vivoor in vitro through

the polymerization of (R)-3HBCoA molecules by PHA synthases

(phaC)(4e7). The synthases themselves can be divided into fourclasses based on their size, subunit composition, and substrate

specicity(8). Several reports have been conducted on the in vitro

synthesis of P(3HB) to understand the mechanisms involved, as a

production method and as a technique to enhance the surface

properties of other materials for specialized and novel applications

(9). However, the difculty of obtaining a high-purity PHA synthase

derived from host strains by using multiple purication steps in

downstream processing and a high recovery cost have become

obstacles for further application of this enzyme.

Liebergesell et al. conducted two-step chromatography to sepa-

rate the phaCReprotein from recombinant Escherichia coli for the

synthesis of PHB in an in vitro study; however, the purication fold

and yield reached only 4.4 and 34.6%, respectively(10). Gerngross

etal. demonstratedthata 9.8purication foldwith 24%recovery was

achieved in the purication of the PHA synthase from Alcaligenes

eutrophus (11). A direct separation of PHA synthase by using afnity

Ni-tag was performed by Qi et al., but a 1% recovery yield was found

(4). Song et al. introduced a two-step operation of ammonium sul-

fate precipitation and the hydrophobic chromatography method to

achieve a 43% yield of high-purity synthetic enzymes (12). The

highest recovery yield, 44.1%, was achieved under sequential oper-ations of ion exchange and afnity chromatography(13). However,

they concluded that a low recovery of the phaCReenzyme obtained

from a time-consuming and complex process resulted in the prep-

aration restriction of high-purity, high-quantity PHA synthases for

its application such as in in vitro PHB polymerization(13e15).

Aqueous two-phase systems (ATPSs) consist of two liquid pha-

ses that are immiscible beyond a critical concentration (16). The

systems are formed by mixing two mutually incompatible poly-

mers or one polymer and an inorganic salt. ATPSs appeal as

excellent tools for the partitioning of targeted proteins from feed-

stock (17e19). Biomolecule partitioning in an ATPS is a complex

function of various factors, including the polymer molecular

weight, the concentration of polymer and salt, the pH values, the* Corresponding author. Tel.:886 34638800x3550; fax: 886 34559373.

E-mail address: [email protected](J.C.-W. Lan).

www.elsevier.com/locate/jbiosc

Journal of Bioscience and BioengineeringVOL. 116 No. 4, 499e505, 2013

1389-1723/$e see front matter 2013, The Society for Biotechnology, Japan. All rights reserved.http://dx.doi.org/10.1016/j.jbiosc.2013.04.010
mailto:[email protected]://www.elsevier.com/locate/jbioschttp://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://dx.doi.org/10.1016/j.jbiosc.2013.04.010http://www.elsevier.com/locate/jbioscmailto:[email protected]

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temperatures of the system, the net charge, and so on. This tech-

nique offers many advantages, including a short processing time,

low material cost, low energy consumption, good resolution, a high

yield, and a relatively high capacity(20,21).Moreover, the system

can be easily scaled up and applied in the downstream process. The

purpose of this study was to investigate the feasibility of using an

ATPS to separate PHA synthase from claried recombinant E. coli

feedstock. The partition behavior of PHA synthase was studied tooptimize recovery. Furthermore, the optimal activity of the puried

enzyme was estimated in respect to temperature, pH values, and

trace metal elements.

MATERIALS AND METHODS

Cultivation of recombinant E. coli and feedstock preparation The

microorganism and plasmids employed in this study are listed inTable 1.TheE. coli

strains JM109 and BL21(DE3) were used as the cloning host and expression host,

respectively. The chemically dened medium (glucose, 10 g L1; Na2SO4, 2 g L1;

(NH4)2SO4, 2.7 g L1; NH4Cl, 0.5 g L

1; K2HPO4, 12 g L1; NaH2PO4, 3.5 g L

1;

(NH4)2-H-citrate, 1.0 g L1; yeast extract, 1 g L1; thiamine, 0.01 g L1; 1 M

MgSO4, 2 m L L 1; and the trace element solution, 2 mL L1; pH 7) containing

ampicillin (Ap, 100mg mL1) was introduced for recombinant cell cultivation (22).

TheE. coli BL21(DE3) cell-harboring pET-15bT

phaCRe was cultivated to an op-ticaldensityof 600nm (OD600) equals0.6to 0.8 at37C,and 200rpm.Isopropyl-b-D-

()-thiogalactopyranoside (IPTG) was subsequently induced to a nal concentration

at 0.5 mM. The cells were further cultured at 15 C, at 200 rpm for 12 h for enzyme

overexpression. The harvested cells were resuspended with phosphate buffered

saline (PBS) at pH 7.5, and expressed synthase was released by introducing soni-

cation (XL2000, Microson, USA). The claried feedstock was collected from the su-

pernatant derived from a further centrifugation step, and was subjected to phaC Repurication in the ATPS.

Analysis of PHA synthase activity The PHA synthase activity was assayed

according to the method described by Satoh et al. (23).The PHA synthase activity

was measured by the CoA released during the polymerization reaction of 3HB-

CoA in a reaction buffer (100 mM sodium phosphate; pH 7.5). The assay mixture

(a total volume of 0.4 mL) contained 100 mM sodium phosphate (pH 7.5), 1 mM

3HBCoA, 5% (v/v) glycerol, and phaCRe. The reaction was initiated with the

addition of phaCRe, and then at dened time points the aliquots (45 mL) were

mixed with 90mL of 5% (w/v) trichloroacetic acid (TCA) to terminate the reaction.

After removal of the precipitated protein by centrifugation, 120 mL of th e

supernatant was added to 680 mL of a solution containing 0.1 mM 5,5 0-dithio-

bis(2-nitrobenzoic acid) (DTNB) in 0.5 M potassium phosphate (pH 7.5), and

incubated for 10 min at 30C. Subsequently, the absorbance was measured at

412 nm. The concentration of CoA was determined by measuring the absorbance

at 412 nm and correlating with a molar absorption coefcient of

13,600 M1 cm1. One unit (U) was dened as the amount of enzymes that

catalyzed the generation of 1mmol of CoA in 1 min.

Determination of protein concentrations and gel analysis of the protein

prole The total protein concentrations of the cell extracts were determined

according to the method described by Bradford using bovine serum albumin (BSA)

as a protein standard. The dye reagent (Bio-Rad,USA) wascomposed by diluting one

part concentrated dye with four parts deionized water. One milliliter of the diluted

dye reagent was added to the 1.5 mL test tube, and the 20 mL protein sample was

pipetted intoa 1.5 mL testtube. Proteinsolutions aregenerally mixed and assayed in

triplicate to yield a mean value. The percentage deviation was within an average

error of less than 5%. Absorbance at 595 nm was measured after 5 min of reaction

time. The results were expressed relative to a calibration plotderived fromthe assay

of the standard BSA protein(24). The purity of the enzyme solution was determined

by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

with a full-range rainbow molecular weight marker (GE Healthcare, USA). The

solution preparation and procedure were performed according to the methods

that have been described previously(25).

Aqueous two-phase systems: phase diagram and construction The

binodal curves were estimated using the cloud point method, as described by

Albertsson (16). The tie-line length (TLL) describes the compositions of the two

phases, which are in equilibrium, and was calculated as follows:

Tie line length ffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffiffiffiDWT DWB

2PEG DWT DWB

2Phosphate

q (1)

where (DW)PEG and (DW)Phosphate are the differences between the PEG and phos-

phate concentration, respectively, in the two phases. The concentration of PEG and

salts was analyzed using the refractive index and conductivity measurement,

respectively, according the method described by Hatti-Kaul(19).

The ATPSs were constructed by successively adding polyethylene glycol with

molecular masses of 6000 or 8000 g mol1 [Sigma; 50% (w/w) stock solution]

and dibasic/monobasic potassium phosphate [Sigma; 40% (w/w) stock solution]

to yield the appropriate weight percent system at the desired pH. The solution

was mixed according to the binodal partition diagrams. The phase systems

were prepared in 15 mL graduated centrifuge tubes by weighing the 80%

(w/w) stock solution and 20% (w/w) claried feedstock. Distilled water was then

added to each system to obtain a nal mass of 10 g. The centrifuge tubes were

mixed for 30 min using a laboratory mixer to achieve effective mixing between

the phase-forming chemicals and proteins. Each tube was then centrifuged at

3000 g for 3 min to accelerate phase separation. The volumes of the top and

bottom phases were measured, and samples were drawn from each phase and

suitably diluted before determining the phaCRe activity assay and total proteinconcentration.

Determination of the partition coefcient, purication factor, phase

volume, and recovery yield The partition coefcient is dened as the enzyme

activity or protein concentration in the top phase divided by the correspondent

value in the bottom phase, as shown in Eqs. 2 and 3.

KE ATAB

(2)

KP CTCB

(3)

where AT and AB are the enzyme activity (U) in the top and bottom phases,

respectively. TheCT andCBare the total protein concentrations (mg mL1) of the top

and bottom phases, respectively.Selectivity (S) wasdened as the ratioof the phaCReenzyme partition coefcient (KE) to the protein partition coefcient (KP) (as shown

in Eq.4).

S KEKP

(4)

Specic activity(SA) was denedas theratio betweenthe enzyme activity(U) in

the phase sample and the total protein concentration (mg) (Eq. 5).

SA

U mg1

Enzyme activityU

Proteinmg (5)

To evaluate the purication process, the enzyme SA (expressed in units

per milligram of protein), the purication factor (PF), and the enzyme recovery

yield inthe top phase (RT, %)andbottom phase (RB, %) were alsocalculated according

to the given equations (Eqs. 6e9), which have been well described by Porto

et al.(26).

PF SASAi

(6)

TABLE 1.Bacterial strains and plasmid used in this study.

Strain or plasmid Description Source or reference

Strain

E. coliJM109 recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1,

D(lac-proAB)/F-[traD36, proAB, lacI, lacZDM15]

Toyobo

E. coliBL21(DE3) F ompT hsdSB(rB mB

) gal dcm(DE3) Invitrogen

Plasmid

pET-15b N-terminus His-tagged fusion protein

expression vector, Ap

Novagen pET System Manual, 10th ed.

pBHR68 pBluescript SK-derivative, containing

R. eutrophaH16 PHB operon

Spiekermann et al. (22)

pET-15b:phaCRe pET-15b derivative; phaC fromR. eutrophaH16 This study

500 LAN ET AL. J. BIOSCI. BIOENG.,

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RT 100

1 1

VRKE

(7)

RB 100

1 VRKE(8)

VR VT

VB(9)

whereVRis the volume ratio, and VT andVBare the volumes of the top and bottom

phases, respectively.The enzyme SAcan be evaluated for bothphases by introducing

Eq.4with the corresponding enzyme activity and the protein concentration in the

selected phases. In Eq.5, SAi represents the SA for the initial sample (before puri-

cation). The partition experiments were conducted in triplicate, and the average

results are the values reported in this study.

Effects of pH and temperature on the activity of the puried PHA

synthase Enzyme activity was determined using 3HB-CoA as the substrate at

30C with value increments of 1 pH for the following buffer solutions: 100 mM of

sodium acetate (pH 5.0e6.0) and sodium phosphate (pH 7.0e9.0). The reaction was

initiated with the addition of the puried enzyme to the buffer solutions. The

mixture was incubated for 1 h at 30C. The residual activity of the enzyme was

subsequently determined with 0.3 mL of this mixture by using 3HB-CoA as the

substrate. The buffer solution contained the substrate without the addition of the

puried enzyme, which was introduced as the control.

A temperature gradient was employed to determine the optimal activity of the

enzyme. The puried enzyme and the equilibrium buffer were incubated over

temperatures ranging between 4C and 48C for 1 h attheobserved optimal pH. The

relative enzyme activity was determined spectrophotometrically at a specic tem-

perature as rapidly as possible.

Effect of metal ion on activity of the puried PHA synthase The effect of

metal ions on the enzyme activity was veried with the addition of the 1.0 mM

metal ion solution; Na and K for monovalent ions, Ca2, Zn2, Mg2, Mn2, Co2,

and Cu2 for divalent ions; and Fe3 for trivalent ions. Each metal ion solution was

mixed with the puried enzyme at a 1:1 ratio, and the mixture was then incubated

at an optimal temperature for 1 h. The relative enzyme activities were expressed in

percentages by comparing the enzyme activity with the standard assay mixture,

with no metal ion added at the optimal pH and temperature.

RESULTS AND DISCUSSION

The effect of system pH on the partition behavior ofphaCRe A series of phase systems at pH values from 5.5 to 8.7

was assessed to investigate the pH effect on the partition behavior

of the phaCRe protein. The inuence of pH values on the parti-

tioning of the phaCReprotein is shown inFig. 1.The results showed

a slightly increscent partition coefcient in the enzyme activity (KE)

and selectivity between pH 5.5 and pH 7. A dramatic increase on the

partition coefcient of the enzyme activity and selectivity was

observed from pH 7 to pH 8.7. However, the systems with the

protein concentration of the partition coefcient (KP) were

unaffected in a signicant manner by the change of pH values.

The average KP is 0.49 0.02. This indicates that the proteins

accumulate mainly in the bottom phase because the value of the

partition coefcient is lower than 1.0. As the pH of the system

changes, phaCReis partitioned according to the net charge of the

protein and surface properties other than the charge. The phaCReenzyme is characterized with highly hydrophobic activation sites

having an isoelectric point (pI) of 6.09 and a molecular weight of

67,000 Da(27). The synthase is negatively charged at above pH 7,and the PEG polymer tends to act as a positive charge (28e30),

which can attract the target enzyme by ionic interaction. This

interaction improved the partitioning of phaCRe because the

highest selectivity (S 2.50) was achieved at pH 8.7. Therefore, a

pH value of 8.7 was selected for further study.

Effect of the PEG molecular weight and TLL on the partition

behavior of phaCRe Fig. 2A and B shows the phase diagram for

the PEG-phosphate system with a comparable TLL. These systems

were formed using different molecular masses of PEG with an

increasing TLL while maintaining VRand the pH value at 1.0 and

8.7, respectively. To examine the effect of differing TLLs for the

PEG 6000 and PEG 8000 systems separately, a series of solutions

containing 29.6e44.4% (w/w) of the TLL were performed. The

effects of the TLL on different PEG systems on phaC Repartitioning

are shown in Fig. 3. For the PEG 6000 system, it displayed a

remarkable increase in the enzymatic activity of the partition

coefcient between 35.4% and 44.2% (w/w). For the PEG 8000

pH

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

Partitioncoefficient(K)

0.4

0.6

0.8

1.0

1.2

KEnzyme activity

KProtein concentration

FIG. 1. The pH values effect on partition coefcients.

Potassium phosphate pH 8.7 (%, w/w)

0 5 10 15 20 25 30

0 5 10 15 20 25 30

PEG6000

(%,w/w)

0

10

20

30

40

50

Potassium phosphate pH 8.7 (%, w/w)

PEG8000(%,w/w)

0

10

20

30

40

50

A

B

FIG. 2. The binodal curve and tie-line length. (A) Used for PEG 6000e

potassiumphosphate system at pH 8.7. (B) Used for PEG 8000 e potassium phosphate system at

pH 8.7.

VOL. 116, 2013 PARTITION SEPARATION OF PHA SYNTHASE USING ATPS 501

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system, a gradual increase in the enzyme activity of the partition

coefcient was observed between 30.9% and 44.4% (w/w).

However, the KP values were raised with the increase in weight

percentages of the TLL for both the PEG 6000 and 8000 systems

(see Fig. 3B). The PEG-8000/phosphate system demonstrated a

lower value of KP and recovery yield compared to PEG 6000/

phosphate under the same TLL percentage. The partition

coefcient of the protein was indicated to usually decrease as the

PEG chain length increases (30). The exclusion of the polymer

from the proteins with increasing PEG molecular mass reduces

the interaction between PEG and proteins. The PEG solution ofthe high concentration percentage allowed the hydrophobic PEG

molecules to interact with the hydrophobic region of the

proteins. The impact of the PEG molecule upon separation of the

target protein can be attributed to an adequate balance between

PEG exclusion and PEG-protein binding through the hydrophobic

area(30). Consequently, PEG-6000 was selected for further study.

Partition behavior of phaCRein the presence of the addition

of NaCl A series of the PEG 6000-phosphate system containing

0e5% (w/w) of additional NaCl was employed for the TLL (44.2%) at

pH 8.7 at room temperature, and the result is shown in Fig. 4. The

concentration and variation of salts result in an electrical potential

difference between the two phases, which are caused by unequal

ion distribution (31). Such a phenomenon strongly affects the

partitioning of charged enzymes. Numerous studies have

reported that the addition of NaCl or other neutral salts in the

ATPS would affect the water structure and hydrophobic

interactions between the hydrophobic chain (ethylene group) ofPEG and the hydrophobic surface area of the partitioning enzyme,

in which the recovery yield can be improved (18,21,32). In this

study, the system containing a higher NaCl content displayed

stronger repression in respect to the recovery, selectivity, and

purication factor. The KE values are approximately to 1.0, and

the majority of proteins remained in the bottom phase (i.e., KPvalues below 0.2). A similar result was reported in the recovery of

porcine pancreatic lipase using the PEG/phosphate system (32).

This can be due to unequal partition behavior between two

phases that occurred because the chemical potential of a solute is

strongly affected by inducing salt in the ATPS (33). Because of

these results, the systems without NaCl addition were suggested

in subsequent experiments.

Effects of VR and operating temperature on phaCRepartitioning The differential partition behaviors of phaCRe for

TLL 44.2% (w/w) at different phase volumeratios (from 0.31 to 3.25)

and operating temperatures (from 4C to 26C) are shown in Fig. 5A

and B. We found that the partition coefcient of the phaCRe enzyme

increased with VR. The highest values ofKE can be achieved with

6.07, with a volume ratio of 3.25 and 4C. Moreover, the

decreasing trend of KP values corresponding to the increasing VRimproved the selectivity of the target protein within the system.

Theoretically, the partition behavior of proteins is not

signicantly inuenced by altering VRof the biphasic system(33).

Usually, a natural product from the E. coli homogenate includes

several hydrophobic proteins, cell debris, and some electrolytes

and peptides. The hydrophobic proteins may precipitate in an

irreversible manner when the crude homogenate is added to the

biphasic system by interacting with its components. The presence

of protein precipitation was observed at the interface as the top

phase volume was reduced. An increasing volume in the top

phase may extend the hydrophobic interaction between PEG and

phaCRe. The temperature could be an important factor in protein

partitioning. The temperature effect on protein partitioning varies

for different phase systems depending on the type of polymer

used. Table 2 presents a summary of the evaluation, and shows

that the decrease in the system temperature promoted the

purication of the phaCRe protein from claried feedstock. This

may be attributed to an extended structure of PEG at a higher

temperature, in which its preferential interaction with the protein

decreases, thereby reducing the partition coefcient (34). The

recovery properties of phaCRe are summarized in Table 2. The

selectivity, purication factor, and recovery yield of the top phase

of phaCRe recovery in the ATPS at 4C were 29.05, 16.23, and

KEnzym

eactivity

Tie-line length (%, w/w)

28 30 32 34 36 38 40 42 44 46

1.0

1.5

2.0

2.5

3.0

3.5

4.0

PEG 6000

PEG 8000

Tie-line length (%, w/w)

28 30 32 34 36 38 40 42 44 46

KProteinconcentration

0.20

0.25

0.30

0.35

0.40PEG 6000

PEG 8000

A

B

FIG. 3. Inuence of molecular weight of PEG with different tie-line length. (A) Enzyme

activity of partition coefcient for PEG 6000 and PEG 8000. (B) Protein concentrationof partition coefcient for PEG 6000 and PEG 8000

NaCl (%, w/w)

0 1 2 3 4 5 6

KEnz

ymeactivity

1

2

3

4


0.1

0.2

0.3

0.4

0.5KEnzyme activity

KProtein concentration

FIG. 4. Inuence of the addition of NaCl.


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95.32, respectively. The consequence suggested that a lower

temperature would be favorable for phaCReseparation in the PEG/

phosphate system.

Characterizations of the puried PHA synthase Fig. 6

displays the prole of phaCRe partitioning from claried

feedstock at 4C and 26C, respectively. The gure also shows

that a high-purity PHA synthase can be recovered by using the

ATPS. The puried enzyme was subjected to optimization activity

for the pH and temperature. The results demonstrated that the

enzyme activity increased with the temperature from 4C to37C, but decreased during operation at 48C. The highest activity

of most enzymes occurs under a moderate operating

temperature. A high temperature may cause a denatured effect

on enzyme activity. The optimal temperature was 37C, as

displayed in Fig. 7A. The gure shows that the highest enzyme

activities were found at a pH value of 8 at several operating

temperatures. However, Zhang et al. showed an optimal pH at 7.0

for phaCRe polymerase, which was puried by several

chromatography steps(35). Therefore, further determination was

performed in the pH range of 7.0e9.0 (seeFig. 7B). The optimal

pH value of 7.5 for the expression of phaCRe activity under

different operating temperatures was observed.

Jossek and Steinbchel demonstrated dramatic effects of several

metal-salts at a concentration range of 10e100 mM upon the yieldand activities of PHA synthase from Chromatium vinosum (7).

Because those metal ions were employed in the culture medium

and may remain in the aqueous two-phase system, it is necessary to

investigate the effect of metal ions on puried enzymes. Table 3

presents a summary of the impact of metal ions on phaCReactiv-

ity. It indicates that Cu2, Co2, Mg2, Mn2, and Fe3 displayed a

signicant inhibition on enzyme activity. The relative activities

were reduced to the range of 70.45e32.27%. This might be attrib-

uted to the stronger afnity between certain divalent/trivalent ions

and synthase. However, how these components interact with the

PHA synthase remains unclear.

The enzymes produced from recombinant E. coliare generally a

complex of various contaminants. Employing an aqueous two-

phase system for direct recovery of phaCResynthase from clariedfeedstock was found to be highly attractive and efcient. The

optimized system was formed with 30% w/w of the PEG 6000

polymer and 8% w/w of potassium phosphate at pH 8.7 and 4C. A

low volume ratio and the addition of NaCl in this biphasic system

showed a negative effect on the partitioning of the phaC Reprotein.

A system temperature of over 15C reduced the recovery efciency

TABLE 2. Purication summary of PHA synthase from claried feedstock.

Samples Temperature (C) Protein content (mg) Total activity (U) Specic activity (U mg1) Sel ecti vi ty Purication fold RT(%)

Claried feedstock 26 40.59 4.41 0.11 e 1.00 100

Purication

ATPS pH 5.5 26 15.73 4.65 0.30 1.02 2.72 67.75

pH 8.7 4 7.19 12.68 1.76 29.05 16.23 95.32

pH 8.7 15 7.11 11.84 1.66 28.35 15.31 95.11pH 8.7 26 7.06 11.00 1.56 27.34 14.33 94.93

FIG. 6. SDS-PAGE analysis of purication step in ATPS. Lane M, Markers; lane 1, crude

protein prole; lane 2, claried feedstock; lane 3, sample from top phase after puri-

cation by ATPS at 4C; lane 4, sample from bottom phase after purication by ATPS at

4C; lane 5, sample from top phase after purication by ATPS at 26C; lane 6, sample

from bottom phase after purication by ATPS at 26C.

Volume ratio (VR)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

KEnzymeactivity

2

3

4

5

64 oC

15 oC

26 oC

Volume ratio (VR)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5


0.2

0.3

0.4

0.5

0.64 oC

15 oC

26 oC

A

B

FIG. 5. Inuence of system temperature with different volume ratio. (A) Enzyme ac-tivity of partition coefcient for 4C, 15C and 26C, respectively. (B) Protein concen-

tration of partition coefcient for 4C, 15C and 26C, respectively


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and purication factor of the enzyme. The optimal pH and tem-

perature conditions for enzyme activity were 7.5 and 37C,

respectively. Several divalent ions and trivalent ions played an

inhibitory role on the phaCRe activity. The potential of using the

ATPS could be an alternative process for phaCRe recovery with

respect to high-purity, a high recovery yield, and a low cost.

ACKNOWLEDGMENT

This study is nancial supported by National Science Council

(Taiwan) with project number of NSC 101-2632-E-155-001-MY3.

References

1. Hazer, B. and Steinbchel, A.: Increased diversication of poly-

hydroxyalkanoates by modication reactions for industrial and medical ap-

plications, Appl. Microbiol. Biotechnol., 74, 1e12 (2007).

2. Anderson, A. J. and Dawes, E. A.:Occurrence, metabolism, metabolic role, and

industrial uses of bacterial polyhydroxyalkanoates, Microbiol. Mol. Biol. Rev.,

54, 450e472 (1990).

3. Sudesh, K., Abe, H., and Doi, Y.: Synthesis, structure and properties of poly-hydroxyalkanoates: biological polyesters, Prog. Polym. Sci., 25, 1503e1555

(2000).

4. Qi, Q., Steinbchel, A., and Rehm, B. H. A.: In vitro synthesis of poly(3-

hydroxydecanoate): purication and enzymatic characterization of type II

polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aerugi-

nosa, Appl. Microbiol. Biotechnol., 54, 37e43 (2000).

5. Rehm, B. H. A., Qi, Q., Beermann, B. B., Hinz, H. J., and Steinbuchel, A.:Ma-

trix-assisted in vitro refolding of Pseudomonas aeruginosa class II poly-

hydroxyalkanoate synthase from inclusion bodies produced in recombinant

Escherichia coli, Biol. Chem., 358, 263e268 (2001).

6. Kichise, T., Fukui, T., Yoshida, Y., and Doi, Y.: Biosynthesis of poly-

hydroxyalkanoates (PHA) by recombinantRalstonia eutrophaand effects of PHA

synthase activity onin vivoPHA biosynthesis, Int. J. Biol. Macromol., 25, 69e77

(1999).

7. Jossek, R., Reichelt, R., and Steinbuchel, A.: In vitro biosynthesis of poly(3-

hydroxybutyric acid) by using puried poly(hydroxyalkanoic acid) synthase of

Chromatium vinosum, Appl. Microbiol. Biotechnol., 49, 258e266 (1998).

8. Rehm, B. H. A.:Polyester synthases: natural catalysts for plastics, Biol. Chem.,

376, 15e33 (2003).

9. Thomson, N., Roy, I., Summers, D., and Sivaniah, E.: In vitro production of

polyhydroxyalkanoates: achievements and applications, J. Chem. Technol.

Biotechnol., 85, 760e767 (2010).

10. Liebergesell, M., Sonomoto, K., Madkour, M., Mayer, F., and Steinbuchel, A.:

purication and characterization of the poly(hydroxyalkanoic acid) synthase

from Chromatium vinosum and localization of the enzyme at the surface of

poly(hydroxyalkanoic acid) granules, FEBS J., 226, 71e80 (1994).

11. Gerngross, T. U., Snell, K. D., Peoples, O. P., Sinskey, A. J., Csuhai, E.,

Masamune, S., and Stubbe, J.: Overexpression and purication of the soluble

polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a

required posttranslational modication for catalytic activity, Biochemistry, 33,

9311e9320 (1994).

12. Song, J. J., Zhang, S., Lenz, R. W., and Goodwin, S.:In vitropolymerization and

copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from

Ralstonia eutropha, Biomacromolecules,1, 433e439 (2000).

13. Zhang, S., Kolvek, S., Goodwin, S., and Lenz, R. W.:Poly(hydroxyalkanoic acid)biosynthesis in Ectothiorhodospira shaposhnikovii: characterization and reac-

tivity of a type III PHA synthase, Biomacromolecules, 5, 40e48 (2004).

14. Tajima, K., Tannai, H., Satoh, Y., and Munekata, M.: Synthesis of poly(3-

hydroxybutyrate) by immobilized poly(3-hydroxybutyrate) synthase, Polym. J.,

35, 407e410 (2003).

15. Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and

Doi, Y.:Molecular cloning of two (R)-specic enoyl-CoA hydratase genes from

Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis,

FEMS Microbiol. Lett., 184, 193e198 (2000).

16. Albertsson, P. A.:partition of cell particles and macromolecules, 3rd ed. Wiley,

New York (1986).

17. Ooi, C. W., Tey, B. T., Hii, S. L., Kamal, S. M. M., Lan, J. C. W., Ariff, A., and

Ling, T. C.: Purication of lipase derived from Burkholderia pseudomallei with

alcohol/salt-based aqueous two-phase systems, Process Biochem., 44,

1083e1087 (2009).

18. Ooi, C. W., Hii, S. L., Kamal, S. M. M., Ariff, A., and Ling, T. C.: Extractive

fermentation using aqueous two-phase systems for integrated production andpurication of extracellular lipase derived from Burkholderia pseudomallei,

Process Biochem., 46, 68e73 (2011).

19. Hatti-Kaul, R.: Aqueous two-phase systems, methods in biotechnology.

Humana Press Inc., Totowa, New York (2000).

20. Rito-Palomares, M.: Practical application of aqueous two-phase partition to

process development for the recovery of biological products, J. Chromatogr. B,

807, 3e11 (2004).

21. Hiraishi, T., Kikkawa, Y., Fujita, M., Normi, Y. M., Kanesato, M., Tsuge, T.,

Sudesh, K., Maeda, M., and Doi, Y.: Atomic force microscopic observation

of in vitro polymerized poly[(R)-3-hydroxybutyrate]: insight into possible

mechanism of granule formation, Biomacromolecules, 6, 2671e2677

(2005).

22. Spiekermann, P., Rehm, B. H. A., Kalscheuer, R., Baumeister, D., and

Steinbchel, A.:A sensitive, viable-colony staining method using Nile red for

direct screening of bacteria that accumulate polyhydroxyalkanoic acids and

other lipid storage compounds, Arch. Microbiol., 171, 73e80 (1999).

23. Satoh, Y., Tajima, K., Tannai, H., and Munekata, M.: Enzyme-catalyzed

poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and

NADPH regeneration in vitro, J. Biosci. Bioeng., 95, 335e341 (2003).

A

B

FIG. 7. Optimization of phaCReactivity by different temperature and pH conditions. The

identical amount of puried phaCRe was loaded to the substrate solution under several

of operating pH and temperature conditions. (A) pH range from 5 to 9; (B) pH range

from 7 to 9.

TABLE 3. Inuence of different metal elements on the activity of phaCRe.

Composition Final activity (U) Relative activity (%)

Control 0.261 100

Na2EDTA 0.260 99.56

CaCl2 0.251 95.91

Na2HPO4 0.247 94.47

ZnCl2 0.245 93.47

MnCl2 0.186 70.45

FeCl3 0.168 63.15

MgSO4 0.139 51.53CoCl2 0.119 43.78

CuSO4 0.090 32.27


8/13/2019 1-s2.0-S138917231300131X-main

7/7

24. Bradford, M. M.: A rapid and sensitive method for the quantitation of micro-

gram quantities of protein utilizing the principle of protein-dye binding, Anal.

Biochem., 72, 248e254 (1976).

25. Lan, C. W., Ling, T. C., OSullivan, D. A., and Lyddiatt, A.: Comparison of two

matrices for selective recovery of C595 diabody fragment (dbFv) from Escher-

ichia coli lysates, Process Biochem., 42, 335e343 (2007).

26. Porto, T. S., Medeiros-Silva, G. M., Porto, C. S., Cavalcanti, M. T. H., Neto, B. B.,

Lima-Filho, J. L., Converti, A., Porto, A. L. F., and Pessoa, A.: Liquid-liquid

extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system, Chem. Eng. Process., 47, 716e721 (2008).

27. Antonio, R. V., Steinbchel, A., and Rehm, B. H. A.:Analysis ofin vivosubstrate

specicityof PHAsynthasefrom Ralstoniaeutropha: formationof novel copolyesters

in recombinant Escherichia coli, FEMS Microbiol. Lett., 182, 111e117 (2000).

28. Huddleston, J. G., Ottomar, K. W., Ngonyani, D. M., and Lyddiatt, A.:Inuence

of system and molecular parameters upon fractionation of intracellular pro-

teins from Saccharomyces by aqueous two-phase partition, Enzyme Microb.

Technol., 13, 24e32 (1991).

29. Tanuja, S., Srinivas, N. D., Raghava Rao, K. S. M. S., and Gowthaman, M. K.:

Aqueous two-phase extraction for downstream processing of amyloglucosi-

dase, Process Biochem., 32, 635e641 (1997).

30. Lima, A. S., Alegre, R. M., and Meirelles, A. J. A.:Partitioning of pectinolytic

enzymes in polyethylene glycol/potassium phosphate aqueous two-phase

systems, Carbohydr. Polym., 50, 63e68 (2002).

31. Johansson, G.: Partition of salts and their effects on partition of proteins in a

dextran-poly(ethylene glycol)-water two-phase system, Biochim. Biophys.

Acta, 221, 387e390 (1970).

32. De Sourza, R. L., Barbosa, J. M. P., Zanin , G. M., Lobo, M. W., Soares, C. M. F.,

and Lima, A. S.:Partitioning of porcine pancreatic lipase in a two-phase sys-

tems of polyethylene glycol/potassium phosphate aqueous, Appl. Biochem.Biotechnol., 161, 288e300 (2010).

33. Forciniti, D.: Studying the inuence of salts on partitioning of proteins, pp.

23e33,in: Kaul, R. H. (Ed.),Methods in biotechnology, vol. 11.Aqueous twophase

system: methods and protocols. Humana Press Inc., Totowa, NJ, USA (2000).

34. Nucci,H. D., Nerli, B., and Pico, G.: Comparison between the thermodynamic

features ofa 1-antitrypsin and human albumin partitioning in aqueous two-

phase systems of polyethyleneglycoledextran, Biophys. Chem., 89, 219e229

(2001).

35. Zhang, S., Yauso, T., Lenz, R. W., and Goodwin, S.: Kinetic and mechanistic

characterization of the polyhydroxybutyrate synthase f romRalstonia eutropha,

Biomacromolecules, 1, 244e251 (2000).


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