Immobilization of chloroperoxida onto highly hydrophilic polyethylene chains via bio-conjugation:Catalytic properties and stabilities
Gulay Bayramoglu a ,⇑,Begum Altintas b ,Meltem Yilmaz b ,M.Yakup Arica a
a Biochemical Processing and Biomaterial Rearch Laboratory,Faculty of Arts and Sciences,Gazi University,06500Teknikokullar,Ankara,Turkey b
Gazi University,Institute of Science and Technology,Department of Environmental Sciences,06570Ankara,Turkey
a r t i c l e i n f o Article history:
Received 6May 2010
Received in revid form 12August 2010Accepted 13August 2010
Available online 24August 2010Keywords:
Hydrophilic supports Enzyme immobilization Chloroperoxida Kinetic parameters Thermal stability
a b s t r a c t
Chloroperoxida (CPO)was covalently immobilized on poly(hydroxypropyl methacrylate-co-polyethyl-eneglycole-methacrylate)membranes,which were characterized,by swelling test,FT-IR spectroscopy,scanning electron microscopy,and contact angle measurement.The K m and V max values for free and immobilized CPO were found to be 34.6and 47.2l M,and 287.5and 245.2U/mg protein,respectively.The optimum pH for both the free and immobilized enzyme was obrved at 3.0.The immobilized enzyme showed wide pH and temperature profiles.Most importantly,the incread thermal,storage and operational stability of immobilized CPO should depend on the creation of a comfortable strong hydrophilic microenvironment on the designed support to the host e
nzyme molecule.
Ó2010Elvier Ltd.All rights rerved.
by and large1.Introduction
Immobilization of enzyme polymeric supports can occur via a variety of interactions including covalent immobilization,physical adsorption,entrapment and binding to metal-ion complexes (Arica and Bayramoglu,2004;Bayramoglu et al.,2010a;2010b Rekuc et al.,2010).Covalent immobilization involves the reaction be-tween reactive groups of supports and the functional groups in the enzymes.In addition,the covalent binding of enzymes on functional supports is required to be carried out under very mild experimental conditions to avoid inactivation of the enzymes (Pet-ri et al.,2004;Bai et al.,2006;Mateo et al.,2007).A wide range of natural and synthetic polymeric supports such as silica,polyethyl-ene,poly(vinylalcohol),polyacrylonitrile-g-polyaniline composite film,poly(methylmethacrylate-glycidylmethacrylate)bad mag-netic beads,poly(4-vinylpyridine-hydroxyethyl methacrylate)hydrogels,alginic acid and cellulo bad materials were modified to develop functional support for enzyme immobilization (Arica,2000;Huang et al.,2008;Neri et al.,2009).In some cas,immobi-lization of enzymes on hydrophilic supports leads to a pronounced l
oss of enzymatic activity.This decrea in the enzymatic can be due to a partial unfolding of the protein resulting from the adsorp-tion of proteins on solid surfaces (Arica and Bayramoglu,2006;Lahari et al.,2010).
The homo-polymer and copolymer of hydrogels such as 2-hydroxyethyl methacrylate (HEMA)and hydroxypropyl methac-rylate (HPMA)have found extensive applications in the field of enzyme technology becau of their good chemical stability and biocompatibility (Bayramoglu et al.,2005;Tyagi et al.,2009).The hydrogel polymers have also sufficient mechanical strength and hydrophilicity to immobilize enzymes and are not susceptible to microbial attack (Arica et al.,2009;Bayramoglu et al.,2004).The incorporation of polyethylene glycol to above hydrogels structures provides additional resistance to non-specific interactions between protein and support surfaces.Poly(ethylene glycol)(PEG)is a water-soluble,nontoxic,and a smart polymer.Surfaces containing PEG are interesting biomaterials becau they exhibit low degrees of protein interactions (Bayramoglu et al.,2009;Bergstrom et al.,1992;Bajpai and Bhanu,2003).In this concept,PEG not only pre-vents non-specific protein interaction with support surface but also act as a spacer arm to move the enzyme from the support sur-face.In solution,PEG is a highly hydrated polymer,where each monomer (ethylene oxide unit)can bind three molecules of water (Hinds and Kim,2002),and can prov
ide an aqueous comfortable microenvironment to the host enzyme molecule like in cells.Sev-eral PEG–enzyme conjugates described in the literature with in-cread enzymes activities and stabilities such as catala,urica,trypsin,alkaline phosphata,asparagina,superoxide dismuta,ribonuclea A,and insulin (Hinds and Kim,2002;Treetharnmathurot et al.,2008;Lim and Herron,1992).
Chloroperoxida (E.C.1.11.1.10;from Caldariomyces fumago )is a heme-containing enzyme that exhibits catala,peroxida and
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Corresponding author.Tel.:+903122135670;fax:+903122122279.
E-mail address:g_ (G.Bayramoglu).
cytochrome P450activities besides the halogenation reaction (Seelbach et al.,1996;van Deurzen et al.,1996).CPO has321ami-no acids with predominantly acidic residues and a pI in the range of3.2–4.0.Like most peroxidas,CPO can be ud infields such as analytical diagnosis,pharmaceuticals a
nd removal of toxic wastes(Kadima and Pickard,1990;Aburto et al.,2005).Immobili-zation of a variety of peroxidas have been reported onto syn-thetic and natural supports materials,but little work has been carried out on CPO immobilization(Wang et al.,2009;Bayramoglu et al.,2008;Seelbach et al.,1996;van Deurzen et al.,1996;Aburto et al.,2005;van de Velde et al.,2000).For example,hydrophilic polymer coated magnetic nano-particles was ud for covalent immobilization of CPO,and the immobilized enzyme was ud for enantiolective sulfoxidation reaction(Wang et al.,2009).
In this study,a ries of poly(hydroxypropyl methacrylate-co-poly(ethylene glycol)-methacrylate,(p(HPMA-co-PEG-MA)) hydrogels in the membrane form with different pHPMA/PEGMA ratios were prepared via redox polymerization technique.The sup-ports were characterized with FT-IR spectra,scanning electron microscopy,and contact angle studies.The hydroxyl groups of the p(HPMA-co-PEG-MA)were converted into epoxy groups by reacting with epiclorohydrin under alkaline condition.The epoxy groups of the hydrogels were ud for covalent bio-conjugation of CPO under mild experimental conditions.The activities and sta-bilities of(p(HPMA-co-PEG-MA)-1-3-CPO)conjugates were stud-ied using p(HPMA)-CPO as control system.
2.Methods
2.1.Materials
Chloroperoxida(CPO;EC 1.11.1.10;from Caldariomyces fumago:3000units/mL;the enzyme solution contained10.7mg/ mL protein;280.4units/mg protein),monochlorodimedone (MCD),were purchad from Sigma–Aldrich Chem.Co.(St.Louis, USA)and were ud without further purification.Hydroxypropyl methacrylate(HPMA)polyethyleneglycole-methacrylate(PEG-MA;M n$360),N,N0-methylenebisacrylamide(BISAA),ammonium persulfate(APS),and N,N,N0,N0-tetramethylethylenediamine (TEMED)were obtained from Fluka AG(Switzerland)and ud without further purifications.All other chemicals were of analyti-cal grade and were purchad from Merck AG(Darmstadt, Germany).
2.2.Preparation of p(HPMA)and p(HPMA-co-PEG-MA)membranes
The p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes were prepared by redox polymerization.The chemical structure of the unit copolymer membranes is prented in Supplementary Fig.S1.To check the effect of the monomer ratio on the membranes properties and recovered CPO-activity in the initial polymerization mixture,three different HPMA/PEG-MA ratios were ud[(1) 2.5:0.5(v/v);(2)2.0:1.0(v/v);and(3)1.5:1.5(v/v)],and they were referred as p(HPMA-co-PEG-MA)-1,p(HPMA-co-PE
G-MA)-2and p(HPMA-co-PEG-MA)-3,respectively.HPMA was prepared as a control system without addition PEG-MA macro-monomer in the polymerization mixture.For synthesis of hydrogels,above macro-monomer and monomer were transferred in3mL purified water in the existence of cross-linker BISAA(10mg).APS(10mg)and TEMED(20l L from10%solution w/v)are ud as redox initiators. The polymerization mixture was transferred into molds,and after aling,a nitrogen atmospheres was created in the mold.Polymer-ization was carried out at room temperature for30min and then the hydrogels obtained were cut into discs by means of a perforator (1.0cm in diameter and650l m in thickness).2.3.Characterization of p(HPMA-co-PEG-MA)membranes
After activation with epichlorohydrin,the amount of available reactive epoxy groups content of the p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes was determined by pyridine–HCl meth-od.The p(HPMA-co-PEG-MA)-3membrane was coated with a thin layer of gold under reduced pressure and their scanning electron micrographs were obtained using a JEOL(JSM5600)scanning elec-tron microscope.The morphology and surface structure of the membrane was obtained at the required magnification at room temperature.The FT-IR spectra of the p(HPMA-co-PEG-MA)-3 and CPO immobilized membranes were obtained by using a FT-IR spectrophotometer(FT-IR8000Series,Shimadzu,Japan).The dry sample(about0.01g)mixed with KBr(0.1g)and presd into a
tablet form.The FT-IR spectrum was then recorded.The water con-tent of the p(HPMA/PEGMA)-1-3membranes was determined at room temperature in phosphate buffer solution(50mM,pH7.0) with a gravimetric method.The pre-weighed dry samples were im-merd in buffer solution;the swollen membranes were removed after the excess surface–water was removed byfilter paper,and they were weighed on a nsitive balance.To ensure complete equilibration,the membrane samples were allowed to swell for 24h.The thickness of the hydrogel membranes were estimated with a micrometer thickness gauge.
Contact angles to water,glycerol and diiodomethane of the p(HPMA),p(HPMA-co-PEG-MA)-1-3and CPO immobilized p(HPMA-co-PEG-MA)-3membranes were measured by ssile drop method at ambient temperature by using a digital optical contact angle meter Phoneix150(Surface Electro Optics,Korea).The ssile drop was formed by depositing the liquid from the above using a manual micro-syringe on the membrane surfaces.Both the left and right contact angles and drop dimension parameters were automatically calculated from the digitalized image.The measure-ments were the average offive contact angles at least operated on three membrane samples.The free surface energy parameters of the p(HPMA),p(HPMA-co-PEG-MA)-1-3and CPO immobilized p(HPMA-co-PEG-MA)-3membranes were calculated using the con-tact angle data of the probe liquids.The result
s are analyzed accord-ing to acid–ba method(van Oss et al.,1986).In this method,the contact angles against at least three liquids with known values of c LW,c+and cÀare measured and the superscripts(LW),(+)and (À)refers to dispersive,Lewis acid and ba components,respec-tively.The(s)and(l)refers to solid and liquid phas,respectively. The values for each experiment are put into the following equation: 1þcos h
ðÞc l¼2c LW sÂc LW l
ÀÁ1=2
þcþsÂcÀl
ÀÁ1=2
þcÀsÂcþl
ÀÁ1=2
whistle flo ridah i
cheap的反义词
:
ð1ÞThe total surface energy c TOT is regarded as the sum of Lifschitz–van der Waals and the Lewis acid and ba components:
c TOT¼c LWþc AB;ð2Þwhere c LW designate
d Lifschitz–van der Waals interaction,reflecting th
e long-range interactions(including the dispersive interaction,the dipole–dipole interaction,and dipole-induced dipole interaction, which is dominated by the dispersion),was calculated from the measured diiodomethane contact angles,and c AB designated such acid–ba interactions as hydrogen bonding,and c+and cÀrefer to proton and electron donating character,respectively.The method equations were solved using Phoneix150software package oper-ated under Windows XP(Surface Electro Optics,Korea).
2.4.Activation of p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes
Activation of p(HPMA)and p(HPMA-co-PEG-MA)-1-3mem-branes were described previously(Yavuz et al.,2009).p(HPMA)
476G.Bayramoglu et al./Bioresource Technology102(2011)475–482
and p(HPMA/PEG-MA)-1-3membranes disks(about4.0mL)were soaked in0.1M phosphate buffer pH7.5for2h,and then im-merd in50ml epichlorohydrin at25°C for24h.The membranes were then washed with acetone,acetic acids(0.1M)and phos-phate buffer(0.1M,pH7.0),dried under reduced pressure at 25°C,and stored in dry form at4°C until u.
2.5.Immobilization of CPO onto activated p(HPMA)and p(HPMA-co-PEG-MA)-1-3
Activated p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes disks(4.0mL)were swollen in phosphate buffer(pH7.5,50mM, 100mL)for18h and placed in a reactor(length6cm;diameter 1.2cm).The enzyme solution(2mg/mL)was prepared in same phosphate buffer(15ml).It was introduced to the reactor with a peristaltic pump(ISMATEC,IPC Model)through the lower inlet. The immobilization reaction was performed at15°C with a 20ml/hflow rate.The effect of enzyme coupling time on the immobilization capacity and on the enzyme activity were studied with activated membranes at15°C and at different reaction cou-pling time between4and24h.Non-covalently bound enzyme was removed by washing the membranesfirst with1.0M saline solution(20ml)and then with citrate buffer(50mM pH3.0).
2.6.Determination of immobilization efficiency
cafe是什么意思
The amount of immobilized CPO on the membranes was deter-mined by measuring the initial andfinal concentrations of protein within the immobilization medium using Coomassie Brilliant Blue as the method described by Bradford,1976.A calibration curve constructed with BSA solution of known concentration(0.05–0.50mg/ml)was ud in the calculation of protein in the enzyme and wash solutions.
2.7.Activity assay of free and immobilized CPO
The activity of chloroperoxida was determined as described previously by Thomas et al.(1970).The method is bad on the de-crea in absorbance at278nm accompanying the conversion of monochlorodimedone(MCD)to dichlorodimedon(DCD).The activity assay was carried out in citrate buffer solution(50mM, pH 3.0)containing MCD(0.1mM),KCl(20mM)and H2O2 (0.25mM)at25°C.The reaction was initiated with addition of 20l l CPO solution.After predetermined time,the reaction was stopped by the addition of0.1ml of1.0M NaOH.The CPO activity was calculated by the initial reaction rate m(mol MCD consumed per unit of time),which is calculated from the slope of changes in absorbance versus time.The activities of the free and immobi-lized enzymes were monitored with MCD assay at pH3.0and were t to100%at t=0.One unit of chloroperoxida catalyzed the conversion of1.0l mol of monochlorodimedon to dichlorodime-don per min at pH3.0and25°C in the pr
ence of KCl and H2O2.
The same assay medium was ud for the determination of the activity of the bio-conjugated-CPO on the membranes.The enzy-matic reaction was started by the introduction of CPO bio-conju-gated-CPO(5membrane disks)into the assay medium and was carried out at25°C in a shaking water bath for5.0min.After re-moval of disks,the remaining absorbance of monochlorodimedone was measured at278nm using a UV/Vis spectrophotometer(PG Instrument Ltd.,Model T80+;PRC).Effects of pH and temperature on CPO activity were studied over the pH range2.0–7.0and the temperatures range20–60°C,respectively.The results of pH and temperature are prented in a normalized form with the highest value of each t being assigned the value of100%activity.Each t of experiments was carried out in triplicate,the arithmetic mean values and standard deviations were calculated and the mar-gin of error for each data t was determined according to a confi-dence interval of95%using the Excel for Windows.
2.8.Determination of the kinetic parameters of the free and
bio-conjugated-CPO
The kinetic parameters,K m and V max values of the free and immobilized CPO(bio-conjugated-CP
O)were determined by mea-suring initial rates of the reaction with MCDM(0.02–0.5mM)as substrate in citrate buffer(50mM,pH3.0.)at25°C.The K m and V max values for the free and immobilized CPO were calculated from Lineweaver–Burk plots by using the initial rate of the enzymatic reaction data:
I=V¼K m=V maxÁ1=½S þ1=V max;ð3Þwhere[S]was the concentration of substrate,V and V max repre-nted the initial and maximum rate of reaction,respectively.K m was the Michaels constant.
2.9.Operational stability of bio-conjugated-CPO
The operational stability of the bio-conjugated-CPO was exam-ined under batch operation mode at25°C at intervals of30min. After each activity measurement,the bio-conjugated-CPO was p-arated from medium and washed three times with acetate buffer (3.0ml,50mM,pH3.0)and then fresh reaction medium was intro-duced onto the bio-conjugated-CPO.By this way,the next activity measurement was carried out.
2.10.Storage and thermal stability
Storage stability of the free and bio-conjugated-CPO was inves-tigated by measuring their activities after being stored at4°C for a two month period and the remaining activity measurement was performed once a week.
Thermal stability of the free and bio-conjugated-CPO was car-ried out by measuring the residual activity of the enzyme expod to two different temperatures(45and55°C)in citrate buffer (50mM,pH3.0)for120min.A sample was removed at15min time interval and assayed for enzymatic activity.
3.Results and discussion
3.1.Properties of p(HPMA)and p(HPMA-co-PEG-MA)
Poly(hydroxypropyl methacrylate)belongs to a class of poly-mers known as hydrogels,which swell in contact with water. The copolymer of hydrophilic p(HPMA-co-PEG-MA)-1-3mem-branes prepared in this study swell in water,but do not dissolve. Compared with p(HPMA)(49%),the water swelling ratio of the p(HPMA-co-PEG-MA)-1-3membranes significantly increas up to96%.The following possible factors may contribute to this result: (i)incorporating PEG actually introduces more hydrophilic poly-mer chains into the polymer backbone,which can attract more water molecules into polymer structure;(ii)the prence of PEG in the hydrogel structure could reduce the crystalline region of polymer.Thus,the water molecules penetrate into the polymer chains more easily,resulting in an improvement of polymer water swelling in aqueous solutions.
The scanning electron microscope(SEM)micrographs given in Supplementary Figs.S2(A)and S2(B)show the surface structure of the p(HPMA)and p(HPMA-co-PEG-MA)-3membranes,respec-tively.As en from the surface photograph,the homo-polymer p(HPMA)membrane has a porous surface structure,and the mi-cro-pore dimensions are around in the range of1–5l m.On the
G.Bayramoglu et al./Bioresource Technology102(2011)475–482477
other hand,the microstructures of the copolymer p(HPMA-co-PEG-MA)-3membranes have a smooth and den surface,and porous microstructure is disappeared.
The p(HPMA-co-PEG-MA)membrane had the characteristic stretching vibration band of hydrogen-bonded alcohol(O A H) around3445cmÀ1(Supplementary Fig.S3(A)).Among the charac-teristic vibration band of both HPMA and PEG-MA is the methylene vibration at2935cmÀ1.The vibration at1741cmÀ1reprents the ester configuration of both HPMA and PEG-MA in the copolymer structure.In addition,veral bands appeared in thefingerprint re-gion for alkylene glycol units between1611and1170cmÀ1on the p(HPMA-co-PEG-MA)copolymer structure.The peaks were as-signed to the A CH2scissoring band of alkylene glycol units at 1426cmÀ1and the anti-symmetric and symmetric stretching bands(A C A O A C A)of alkylene glycol units at1170and 1110cmÀ1,respectively.
header什么意思Other characteristic band at1278cmÀ1 originates in the C A O A C bond of PEG.The characteristic bands of protein(amide II bound)occur at1635and1538cmÀ1were ob-rved in p(HPMA-co-PEG-MA)-3-CPO copolymer surface which confirmed the conjugation of CPO on the membrane.In addition, following conjugation of CPO on the membrane(Supplementary Fig.S3(B)),there appears a broadened A NH2stretching vibration band at around3400cmÀ1overlapped the A OH band of the copolymer.
3.2.Evaluation of the contact angles studies
Polymeric membrane surfaces were characterized by contact angle procedures to determine the effect of functional groups to the protein adsorption performance of the different type ligands carrying affinity membranes(Arica et al.,2004).It was expected changes in surface properties of the p(HPMA)membrane after copolymerization with PEG carrying ,PEG-MA)and chloroperoxida bio-conjugation.The p(HPMA) p(HPMA-co-PEG-MA)-1-3and p(HPMA-co-PEG-MA)-3-chloroper-oxida bio-conjugated membrane gave quite different surface characteristics.The highest contact angles were obtained with water whereas diiodomethane gave the lowest contact angles for all the studied membrane.p(HPMA)membrane can be defined as relatively hydrophobic compared to PEG carrying copolymer mem-branes(Table1).After copolymerization of PEG carrying
macro-monomer with HPMA,the surface became less hydrophobic due to the incorporation of more hydrophilic alkylene glycol unit,as verified by the lower water contact angle.Upon increa the PEG ratio in the copolymer structures,the copolymer membrane sur-face became relatively hydrophilic(from p(HPMA-co-PEG-MA)-1 to p(HPMA-co-PEG-MA)-3formulation)due to the increa of the density of a pre-wetting water layer on the copolymer surface caud by hydrogen bonding.Thus,the copolymer surface with higher PEG density gave lower water contact angle value presum-ably due to the better surface coverage.An opposite trend was ob-rved for diiodomethane contact angle values for the tested membrane samples compared to water contact angles trends.A de-crea in the water contact angle value for p(HPMA-co-PEG-MA)-3-enzyme conjugated surface compared to enzyme-free counter-part may due to the prence of the less hydrophilic amino acid residues of the enzyme connected to PEG chains via epichlorohy-drin coupling(Table1).
wowod
Information about surface properties of a material can be ob-tained after the calculation of surface free energy using contact an-gle values of different test liquids(van Oss et al.,1986).It should be noted that the surface property of a material is the most important factor when enzyme immobilization is contemplated.In this study, surface free energies of the membrane samples were determined using the acid–ba method of van Oss’et al.,consisting of the sum of the Lifschitz–van
der Waals(c LW)and the acid–ba com-ponents(c AB)applies for all investigated membranes at different values(Table2).As can be en in the table,p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes emed to exhibit mainly Lewis basic character(cÀ).The basic properties of the membranes surface were incread as the ratio of PEG increas in the copoly-mer structure(from5.90to26.61mN/m).This could be an impor-tant obrvation becau the stability of an enzyme could be enhanced through reducing interacting force around of the enzyme molecule.Thus,the native conformation of enzyme could be maintained by creation of an aqueous microenvironment around the enzyme with Lewis basic character.This comfortable microen-vironment around the enzyme could also reduce the interactions between host enzyme and products in the reaction medium.The results indicated that the dispersive and polar , hydrophobic and Lewis acid/ba groups)of the membrane surface can be strongly controlled by changing the surface PEG density of the membranes.It was obrved that the Lifschitz–van der Waals, Lewis acid-basic ,c LW,c AB and c)of the CPO-conju-gated membrane was also significantly different compared to its enzyme-free counterpart.The incorporation of PEG in the copoly-mer structure and conjugation of PEG with CPO followed all the variations of the dispersive(c LW)and polar components(c AB)of the materials.Thus,all the parameters should be effective in determining the activity of the bio-conjugated-CPO on the mem-brane surface.
会计年终总结
3.3.Immobilization of CPO onto p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes
The covalent immobilization of CPO on activated p(HPMA)and p(HPMA-co-PEG-MA)-1-3membranes was carried out by incubat-ing membranes with CPO in phosphate buffer(pH7.5,50mM)and the results are prented in Table3.For this purpo,the hydroxyl groups of the supports,were modified with epichlorohydrin bear-ing free oxirane groups.The CPO solution is brought in contact with the membrane quiescently in a reactor,and the reactor was operated dead end mode.The reactions of oxirane groups of the carriers with different nucleophilic groups on the protein surface (e.g.,amino,hydroxy or thiol moieties)can be suitable to immobi-lize enzymes by forming extremely strong linkages(condary amino bonds,ether bonds and thioether bonds).Under neutral and alkaline conditions,the surface amino groups of an enzyme are principally responsible for binding to the oxirane groups.CPO has three lysine residues on the surface(Lys:112,145and211) (Aburto et al.,2005),which will readily conjugate with oxirane groups of support with minimal chemical modification of the en-zyme.As reported previously,the lysine residues are expod on the surface of enzyme and located on the opposite side of the substrate access to the heme group(Sundaramoorthy et al., 1998).This phenomenon may also provide additional advantage for the side directed conjugation of CPO on the oxirane groups con-taining membranes.Concerning enzymatic activity,all CPO-conju-
Table1
Contact angle values of water,glycerol and diiodomethane on the p(HPMA)and p(HPMA-co-PEG-MA)-1-3membrane.
Membrane compositions Test liquids and their surface tensions c(l)
Water
(c l=71.3)
(h°)
Glycerol
(c l=64.0)
(h°)
Diiodomethane
(c l=50.8)
new money(h°)
p(HPMA)80.3±1.871.1±0.713.4±0.2
p(HPMA-co-PEG-MA)-164.3±1.263.8±1.618.0±0.3
p(HPMA-co-PEG-MA)-261.9±1.163.2±1.523.4±0.6
p(HPMA-co-PEG-MA)-358.3±0.962.1±1.427.1±0.5
p(HPMA-co-PEG-MA)-3-enzyme67.6±0.456.4±1.328.7±0.4
478G.Bayramoglu et al./Bioresource Technology102(2011)475–482
内部控制gated membranes were able to halogenate monochlorodimedone. An other obrvation was that the bio-conjugated-CPO on the PEG carrying copolymer ,p(HPMA-co-PEG-MA)-1-3)has shown higher activity than PEG free membrane formula-tion(Table3).It should be also noted that as the ratio of co-mono-mer(PEG-MA)incread in the membrane structure,the specific activity of the CPO was enhanced.The high enzyme activity can be resulted from the large surface area of thefibrous PEG chains, which have highly swellable oxygen rich groups.Since,the ex-pected electros
tatic interaction between PEG and bio-conjugated enzyme is minimum on the PEG containing support for deforma-tion of native configuration of the enzyme.Thus,a diminutive elec-trostatic interaction was expected between PEG chains with Lewis ba character and surface carboxyl groups of the acidic CPO. Hence,the activity of CPO on the polymer p(HPMA)was lower than PEG-conjugated ones,and PEG free p(HPMA)surface should be provided less comfortable environment and/or caud some con-formational alterations on the enzyme structure.
The effect of coupling time on the immobilization efficiency and retained activity of the CPO was studied and exemplified with p(HPMA-co-PEG-MA)-3-CPO conjugate in Fig.1.The amount of the bio-conjugated-CPO on the p(HPMA-co-PEG-MA)-3incread with increasing coupling time up to16h whereas a decrea in activity yield was obrved at a prolonged incubation time up to 24h.As time prolonged,a large amount of CPO was conjugated
onto the membranes,on the basis of protein determinations,but the specific activity of the preparations was low(Fig.1).When the coupling time incread from4to24h,the amount of bio-con-jugated-CPO incread from0.85to3.11mg/mL on the p(HPMA-co-PEG-MA)-3membrane and reached a plateau value after16h coupling reaction time.In view of the obrvations,p(HPMA-co-PEG-MA)-3membranes were ud for CPO-conjugation in the rest of the experiment with a16h coup
agonling reaction time.
3.4.Effect of pH and temperature on the catalytic activity
The effect of pH on the activity of the free and(HPMA-co-PEG-MA)-3-conjugated preparation for chlorination of monochloro-dimedone to dichlorodimedone in the prence of H2O2was exam-ined in the pH range2.0–7.0at25°C and the results are prented in Fig.2.The optimum pH for the chlorination reaction of conju-gated-CPO is around pH3.0as shown in Fig.2,which is comparable to that of the free CPO.Upon immobilization,the pH profiles of the CPO display slightly improved stability on both sides of the opti-mum pH value,which means that the immobilization method pre-rved the enzyme activity.As an advantage,the conjugated-CPO on membrane reveals enzymatic activity in a wider pH range com-pared to the free CPO.This makes the conjugated-enzyme more suitable for u.Thus,the catalytic performance of the conjugated CPO was not significantly affected too much by the applied immo-bilization protocols(Arica et al.,2004;Cadena et al.,2010).
The effect of temperature on the free and conjugated-CPO activ-ities was investigated by using monochlorodimedone and H2O2as substrate as shown in Fig.3.Relative enzyme activity as calculated by assigning the maximum value(100%)of the each preparation.
At low temperatures,the catalytic activity of both free and con-jugated-CPO incread with the ri of the temperature atfirst and after a maximum(at around35°C for both preparations),de-cread at a higher temperature.On the other hand,the tempera-ture profile of the conjugated-CPO was broader at higher temperature compared to the free counterpart.As it was evident from thefigure,the conjugated-CPO reveals a better heat-resis-tance than that of the free enzyme.The immobilization procedure
Table2
Surface free energy parameters(mN/m)of p(HPMA)and p(HPMA-co-PEG-MA)-1-3membrane according to the van Oss hod.
Membrane compositions c Total(mN/m)c LW(mN/m)c AB(mN/m)c+(mN/m)cÀ(mN/m)Polarity(%)
p(HPMA)51.8949.43 2.460.26 5.90 4.74 p(HPMA-co-PEG-MA)-152.3748.34 4.030.2119.067.69 p(HPMA-co-PEG-MA)-250.6446.71 3.930.1822.117.76 p(HPMA-co-PEG-MA)-349.4045.38 4.020.1526.618.13 p(HPMA-co-PEG-MA)-3-enzyme47.9944.75 3.240.2510.72 6.75
Table3
Effect of membrane composition on enzyme loading and recovered activity.
Membrane composition Monomer ratio(v/v)HPMA:
PEG-MA Enzyme loading
(mg protein/mL membrane)
Enzyme activity*
(units/mL membrane)
Recovered activity(%)
p(HPMA)0.0:1.0 3.74388.037
p(HPMA-co-PEG-MA)-1 2.5:0.5 3.52473.848
p(HPMA-co-PEG-MA)-2 2.0:1.0 3.27696.976
p(HPMA-co-PEG-MA)-3 1.5:1.5 3.05709.883
*The activity of the conjugated enzyme preparations were determined by monitoring continuously the initial reaction rate m(moles of MCD consumed per unit of time), which is calculated from the slope of changes in absorbance at278nm at25°C.
G.Bayramoglu et al./Bioresource Technology102(2011)475–482479
could protect the enzyme active conformation from distortion or damage by heat exchange.One of the main reasons for enzyme immobilization is the anticipated increa in its stability to various deactivating factors due to restricted conformational mobility of the molecules after immobilization(Bayramoglu et al.,2008;Ma-teo et al.,2007).Therefore,the immobilized enzyme could work in harsh environmental conditions with lower activity loss com-pared to the free one.
3.5.Kinetic parameters of free and bio-conjugated-CPO
Kinetic constants were evaluated from the plot of the reaction rate versus monochlorodimedon concentration in Lineweaver–Burk coordinates.The K m values of the free and conjugated-CPO were determined to be34.6and47.2l M,respectively,and the K m values of enzyme preparations were in the same order of mag-nitude.The apparent K m for the conjugated enzyme was incread by about0.8-fold compared to the free enzyme.This indicated that there was no significant conformational change of the enzyme ac-tive site after immobilization.The V max values of the free and con-jugated-enzyme were found to be287.5and245.2U/mg protein. The V max value of the immobilized enzyme decread about1.17-fold compared to the free enzyme.However,K m and V max values of the free and immobilized.
CPO for MCD is in the same order of magnitude.This indicates that the catalytic function of CPO was not very much impaired by this immobilization method.A comparison with the results ob-tained by Wang et al.(2009)for CPO immobilized covalently on the polymer coated magnetic nano-particles,the K m values of the free and immobilized-CPO for monochlorodimedon were determined as26.1and of27.7l M,respectively.The K m value of the enzyme was not significantly changed upon immobilization.This indicated that there was no significant conformational change of the enzyme active site after immobilization(Wang et al.,2009).In other study, Caldariomyces fumago CPO was immobilized in the mesoporous sil-icate material,the K m and V max values of the free and immobilized CPO were calculated as148and108l M and693.8and361.8U/mg protein,respectively(Han et al.,2002).The reported K m and V max values are the same order of magnitude as reported in the pre-nted work.Some deviations of the kinetic constants may result from a certain environmental difference between the free and con-jugated-enzymes,due to the impod conformational and the dif-fusion effect.In addition,the immobilization process does not also control the proper orientation of the immobilized enzyme on the support.This improperfixation and/or the change in the property of the active sites might hinder the active site for binding of ,H2O2and MCD)to the conjugated-CPO molecules.
The efficiency factor g can be calculated from the maximum reaction rates of the immobilized enzyme over that of the free counterpart:
g¼m immobilized=m free;ð4Þwhere m immobilized was the reaction rate of the immobilized enzyme and m free that of the free enzyme.From this calculation,conjugated enzyme system provided an efficiency factor of0.853for the conju-gated CPO.The ratio V max/K m defines a measure of the catalytic effi-ciency of an enzyme–substrate pair.In this study,the catalytic efficiencies(V max/K m)of the free and conjugated-CPO were found to be83.1and51.9,respectively.The catalytic efficiency of free CPO was decread about1.6-fold upon immobilization.
3.6.Thermal stability of the free and conjugated-enzymes
Thermal stability experiments were carried out with free and conjugated-enzyme,which were incubated in the abnce of sub-strate at two different temperatures.Fig.4shows the heat inactiva-tion curves between45and55°C for the free and conjugated-enzyme.At45°C,the conjugated-CPO prerved lost about7%of its initial activity whereas the free enzyme lost about34%of its ini-tial activity during a120min incubation period.At55°C,the con-jugated-CPO and free CPO retained their activity about to a level of 53%and11%,respectively.The conjugated-CPO was inactivated at a much
slower rate than that of the free form.An increa in the rigidity of the conjugated-enzyme conformation resulting from the formation of additional covalent bonds increas the thermal stability of the conjugated-enzyme.As was evident from thefigure, the conjugated enzyme possd a better heat-resistance than that of the free enzyme.This may be explained by that the immo-bilization procedure could protect the enzyme active conformation from distortion or damage by heat exchange.One of the main rea-sons for enzyme immobilization is the anticipated increa in its stability to different deactivating force due to restricted conforma-
480G.Bayramoglu et al./Bioresource Technology102(2011)475–482
