Peroxidases have been shown to be used widely in clinical diagnosis enzyme immunoassays, histological chemistry, cancer therapy, and in the development of biosensors. Peroxidase can trigger the conversion of nontoxic indole-3-acetic acid (Auxin) to a toxic product and induce the apoptosis of tumor cells (36, 37). Peroxidase can be coupled with oxidase in a bi-enzyme assay system for the determination of various analytes, including biogenic amines, glucose, cholesterol, uric acid and many more metabolites.
Peroxidase have been proven to be superior in the chromogen-based chemiluminescence analysis. Luminescent analysis is 102-103 times more sensitive than that of the equivalent spectrophotometric method. However, chemiluminescence enhancer needs to be added to the HRP-based chemiluminescent system to increase the efficiency of oxidative degradation of chromogen. Interestingly, CIP or ARP can be used to trigger chemiluminescence reactions in the absence of chemiluminescence enhancer and shown to have higher catalytic efficiency than that of HRP (29).
Peroxidases were also shown in the removal of the phenolic wastes in the sewage waste water. The peroxidases from horseradish (HRP), Arthromyces ramosus (ARP) and soybean (SBP) effectively were used frequently to remove the phenolic compounds from solutions (38-41).
Although CIP shows higher catalytic activity than HRP, the enzyme is not very stable under thermal conditions. The oxidative stability of Coprinus cinereus peroxidase could be
enhanced by substituting single and multiple amino acids near the substrate channel or active centre of the enzyme (42). In addition, the stability of CIP was shown to be significantly improved by combining with random mutagenesis and in vivo DAN shuffling (43).
I-6 Aims of this study
The recombinant CIP is intended to be expressed in E. coli strain BL21(DE3). The expressed recombinant CIP will be characterized by its activity, the kinetics of CIP to various substrates, and its thermal stability.
II. Materials and methods II-1 Materials
2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 4-aminoantipyrine (4-AAP), DMSO, MBTH, DMAB, DTT, and hemin were purchased from Sigma. Glycerol, Na2HPO4, NaH2PO4, NaOH, KH2PO4, and sodium acetate were purchased from SHOWA.
Trptone, and yeast extract powder were purchased from USB. Acetone was purchased from C-ECHO. Ammonium sulfate and K2HPO4 were purchased from Riedel-de Haen. Urea was purchased from J.T. Baker. Agarose was purchased from gene bank. NaCl, Tris, and glycine were purchased from AMRESCO. EDTA was purchased from Mallinckrodt.
SDS-b, acrylamide, and oxidized glutathione were purchased from MD Bio. Bis-tris was purchased from MD biomedical. Acetone was purchased from PANREAC. Ammonium persulfate, and CaCl2 were purchased from YAKURI. Imidazole was purchased from Fluka.
Phenol was purchased from biobasic Inc. Bradford reagent was obtained from Bio-Rad.
Protein MW marker were purchased from MBI. All restriction enzyme were purchased from the NEB. Taq Dna polymerase and pGEM-T easy vector were purchased from invitrogen.
II-2 Instruments
Instrument Company
Spectrophotometer HITACHI U-1100 and
HITACHI U-3010, America
Orbital shaking incubator YIH DER/CM570R
PCR machine Applied Biosystem/GeneAmp 9700
FPLC system Amersham Bioscience ÄKTA prime
plus
Centrifuge Beckman, Avanti J-E centrifuge
CD spectropolarizer
II-3 Construction of bacterial expression plasmid
To obtain a construct expressing CIP in E. coli BL21(DE3) cDNA of the CIP gene (1035 bp) from pET30b(+) (44-46) was amplified by polymerase chain reaction (PCR) with the following primer set:
L: 5’-AAGCTTCAGGGTCCTGGAGGAGGCG - 3’ (sense)
R:5’-AAGCTTAGGAGCAGGAGCGAGGGAGG -3’ (antisense)
The primer pair contains a Hind III restriction site at 5’ end. The PCR product was inserted into an pGEM-T easy vector via “cohesive” ends and transformed into E. coli strain DH5α.
The transformed DH5α clones were plated on a LB/ampicillin/X-gal agar plated and incubated at 37 ℃ for over-night. Selected single colonies were amplified by incubating in 1 mL of LB/ampicillin medium and shaking with 150 rpm at 37 for 12 h.℃ Plasmid was isolated from 1 mL of E. coli cultures by a microprep protocol (GENEAID) as suggested by manufacture. The purified plasmid was checked with its size by digestion with
restriction enzyme Hind III (1035 bp) to check the size and sequence by DNA sequencing to confirm the absence of any mutation. The cleavage of Hind III yields a fragment of 1035 bp containing the cDNA of CIP. This fragment was then inserted into the same sites of the expression vector pET30b(-S), a S-tag removal variant, to give a final plasmid pET30b(-S)-CIP with a length of 6309 bp. This constructed plasmid was checked with restriction enzymes cleavage at Hind III (or EcoRI-XhoI) shown in Fig. 1A. The plasmids were used for the expression of recombinant CIP.
II-4 Expression of Coprinus cinereus peroxidase
The expression vector pET30b(-S)-CIP transformed E.coli strain BL21(DE3) was first amplified in a 5 mL culturing tube, then transferred to a 500 ml flask containing 250 ml LB medium containing 25 µg/µL kanamycin. The bacterial culture was then shake at 37 ℃at a speed of 200 rpm. Expression of recombinant CIP was initiated by adding 50 µM IPTG (isopropyl-β-pthiogalactopyranoside) in the mid-log phase of cell growth (0.5-0.7 absorbance unit at 600 nm), followed by cultivation at 37 and 200 rpm ℃ for 10-20 hours.
The biomass was collected by centrifugation at 6000 rpm for 20 min at 4 .℃
II-5 Isolation of inclusion bodies
The bacterial pellet was resuspend in 5 mL of 50 mM Tris-HCl buffer, pH 8.5 containing 10 mM DTT and 2 M NaCl, followed by sonication (on 2 s, off 1 s, 30 %) for 9 min, followed by incubation at 25 for ℃ 1.5 h. Repeat the above process once. The inclusion body were collected by centrifugation at 6000 rpm for 15 min. The inclusion bodies was resuspension in 50 mM Tris-HCl buffer (pH 8.5), and centrifugation at 6000 rpm for 15 min. Repeat the washing two more times. Finally, the inclusion body was resuspended in 50 mM Tris-HCl buffer (pH 8.5) and stored at 4 .℃
II-6 Renaturation of recombinant CIP
The inclusion bodies of recombinant CIP were completely dissolved in 6 M urea with gentle stirring for 1 hour at 4℃. The final concentration of recombinant CIP was adjusted to 0.5 mg/mL by 6 M urea.
The dissolved CIP apoprotein (0.5 mg/mL) was then added drop-by-drop to the refolding buffer (1.8 M urea, 5 mM calcium chloride, 5 µM hemin and 5 % glycerol in 50 mM Tris-HCl buffer, pH 9.5) in a ratio of 1:10 with gentle stirring. The CIP solution was incubated at 4 for 8℃ -16 h with a gentle stirring. The refolding medium was optimized by testing with various concentrations of urea (1.5-2.4 M), DTT (0-1 mM), GSSG (0-1 mM), calcium chloride (0-100 mM), hemin (0-20 µM) or KCl (0-100 mM) in fixed 5 % glycerol in 50 mM Tris-HCl buffer at pH 9.5. The enzyme activity of CIP at different stages was measured (as described in section II. 8) to determine the efficiency of renaturation.
II-7 Purification of recombinant CIP
The purification of renatured CIP was performed first by precipitation with 60%
ammonium sulfate. The precipitated CIP was collected by centrifugation at 15,000 rpm for 30 min. The pellet was then dissolved in 50 mM Tris-HCl buffer (pH 8.5) and filtered through a 0.45 µm filter to undissolved particulates. The filtrate (< 5 mL) was applied to a Hiprep 16/60 sephacryl S-200 column equilibrated with 50 mM Tris-HCl buffer, pH 8.5 containing 150 mM NaCl with a flow rate of 0.5 ml/min. Eluent was collected on a fraction collector with 4 mL per fraction. Fractions containing CIP were determined by activity assay. Fractions with peroxidase activity were then pooled and concentrated on a Amicon spin column (15 mL) to a volume of 1 mL. To further purify the enzyme, CIP can be applied onto the same column. Active fractions were finally pooled and stored at 4 or ℃
-20 ℃ in 30% glycerol.
II-8 Peroxidase activity assay
The enzyme activity of recombinant CIP was assayed on a U3010 spectrophotometer.
The activity assay was performed by adding 1 µL of 2.86 ng/µL recombinant CIP in 399 µL reaction mixture (100 mM sodium acetate buffer, pH 5.2 with 0.5 mM ABTS (ammonium 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonate)) and 5 mM hydrogen peroxide) and incubating at 25 for 1℃ min. The extinction coefficient (εABTS) of oxidized ABTS at O.D.
405 nm is 36.8 mM-1cm-1 (47). The enzyme activity of recombinant CIP can also be determined by a colorimetric assay using phenol and 4-AAP as substrates. The activity assay was performed by mixing 1µL of 71.4 ng/µL CIP with 399µL reaction mixture (phenol, 2.4 mM 4-AAP and 0.2 mM H2O2 inphosphate buffer pH 7.4) at 25 ℃ for 1 min.
The formation of 4-AAP-phenol complex after reaction can be determined at O.D. 510 nm (48). The enzyme can also be measured by a colorimetric assay using MBTH and DMAB (MBTH-DMAB, ε = 18.7 mM-1cm-1) at 590 nm at 25 ℃ (49). The activity assay was performed by mixing 1µL of 221 ng/µL CIP with 399µL reaction mixture (MBTH, 2 mM DMAB, 0.2 mM H2O2 in 0.5 M phosphate-citrate buffer, pH 4.0) at 25 for 1 min.℃
II-9 Protein concentration determination
Protein concentration was determined by Bradford protein assay (Bio-rad) using bovine serum albumin as a standard. To a 1-mL assay mixture, 1µL protein solution was mixed with 200 µL Bradford reagent and 799 µL d.d. H2O, at room temperature. The resulting brown-blue mixture was determined at O.D. 595 nm.
II-10 Gel electrophoresis of protein
SDS-PAGE was performed on a Bio-rad Mini protein II apparatus. Proteins (10 µg) was mixed with 5X sample buffer and heated at 95 for 5 min. The ℃ protein sample (10 µg) was loaded onto 10 % SDS polyacryamide gel and subjected to electrophoresis at 70 V for 30 min. The electrophoresis was further performed at 100 volts for another 140 min. After electrophoresis, the gel was stained with stained solution (0.1 % comassie brilliant blue R250, 10 % acetic acid and 50 % methanol) for 1 h, followed by incubating in destaining solution I (10 % acetic acid and 50 % methanol) for 30-60 min. Finally, the gel was incubated in destaining solution II (7 % acetic acid and 5 % methanol) for 2 hours or until background of gel was clear.
II-11 Catalytic properties of recombinant CIP
II. 11.1 Determination of optimal pH and temperature
The enzyme activity was measured at different pH values, such as 2.2, 3, 4, 5, 6, 7, 8, 9 and 10, for enzymes stock (286 ng/µL) in buffer at pH 8. The enzyme was added to an assay buffer in cuvette which was thermostated by means of a circulating bath. The enzyme was treated by varying the temperature from 16 to 40 (16, 25, 35, and 40 ).℃ ℃ ℃
The enzyme was incubated at eleven different pH as 2.2, 3, 4, 5, 6, 8, 9 , 10 , 11 and 12 at 25 for a 7℃ -day period, followed by enzyme activity assay toward ABTS. The buffer were: 0.2 M Gly-HCl at pH 2.2 - 3; 0.2 M sodium acetate at pH 4 - 5; 0.2 M K-phosphate at pH 6 - 8; 0.2 M Tris-HCl at pH 9-10.
II. 11.2 Kinetic parameters of recombinant CIP
The kinetics of recombinant CIP (in the reaction buffer (10 mM phenol, 2.4 mM 4-AAP, 0.1 M phosphate buffer at pH 7.4)) was studied in the presence of various concentration of hydrogen peroxide (0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM,
0.3 mM, 0.4 mM, 0.5 mM, 0.8 mM, 1.2 mM, 1.6 mM, 2.0 mM). The rate of product generation was monitored at O.D. 510 nm and calculated using linear regression on the linear portion of the plot of absorbance vs. reaction time. The rate of hydrogen peroxide consumption in the activity assay is calculated according to single line (d[H2O2]/dt = dA510ε-1L-1/dt).
A plot of [H2O2] versus activity gives a non-linear curve and fitted on software SIGMA plot based on following equation (Eq. 6) (50)
R = (α+β/ [H2O2]+γ[H2O2])-1 (Eq. 6)
These parameters (α, β, γ) can be substituted into the following equation:
a/α = b/β = c/γ (Eq. 7)
where a (model parameter, s) and α (model parameter, dimensionless) control the rates of aromatic substrate utilization, b (model parameter, molL-1s) and β (model parameter, molL-1) control the rates of hydrogen peroxide utilization, and c (model parameter, mol-1L s) and γ (model parameter, mol-1L) control the rates of inhibition through compound III formation.
a = KCa/(1+β/ α / [H2O2]s+γ[H2O2]s/α )( Eq. 8)
where K is a proportional constant (As = -K×d [H2O2]/dt; As, Activity measured at the standard hydrogen peroxide concentration (UL-1)), Ca is the proportionality constant relating the molar concentration of peroxidase to the corresponding activity (i.e., Ca = Eo/As), [H2O2]s is the standard concentration of H2O2 (2 × 10-4M). The values of a, b, and c
the one-electron oxidation of Compound I and Compound II, respectively (Fig. II-1).
The hydrogen peroxide concentration that results in maximum peroxidase activity [H2O2]maxR can be calculated as:
[H2O2]maxR = (β/γ)1/2 = (b/c)1/2 (Eq. 11)
In addition, the parameters were also determined by Lineweaver-Burk plot, as shown below:
1/V = Km/Vmax × 1/[S] + 1/Vmax (Eq. 12) The curve fitting was performed using SIGMA plot (50).
II-12 Spectrophotometric studies of the stability of recombinant CIP
The recombinant CIP (63.2 U in 10 µL) was incubated first in 50 mM Tris-HCl buffer, pH 8.0 at 25 , 35 , 55 and 80 for 5 ℃ ℃ ℃ ℃ - 20 min. After incubation,
recombinant CIP was then assay for the remainant enzyme activity. The effect of Ca2+ ion on the heat-denaturation/reactivation of CIP was also studied. CIP (63.2 U) was treated at 55 for 5 min in the presence of mock, 1 mM EDTA, or 1 mM EDTA and then add 1 mM ℃ CaCl2 at 4 for 20 min. The enzyme activity of treated CIP was then measured for its ℃ remainant enzyme activity. The time-dependent absorption of recombinant CIP at O.D. 404 nm upon thermal treatment/reactivation was also determined on U3010 spectrophotometer (Hitachi).
The 1 mL protein solution (63.2 U CIP) was placed in a cuvette that was thermostated in the chamber of Hitachi U3010 spectrophotometer with a circulating water bath. The heat-denaturation of CIP was directly monitored at 404. The stability of recombinant CIP
was analyzed based on the method proposed by Tams and Welinder (51). Accordingly, a plot of ∆A versus time (s) was generated.
∆A = (At-A∞)/(A0- A∞) (Eq. 13)
Where, At is the absorbance at time t during heat denaturation; A0 is the absorbance at the beginning of the experiment; A∞ is the absorbance of protein solution at the end of the heat denaturation. The experimental curve was fitted using SIGMA plot based on first-order expression as presented in Eq.14 (monophasic reaction) and Eq. 15 (for biphasic reaction):
∆A = exp(-t/τ) (monophasic reaction) (Eq. 14)
∆A = P1exp(-t/τ1) + P2exp(-t/τ2) (biphasic reaction) (Eq. 15)
where τ 1 and τ 2 are time constants for unfolding. The time constant exhibits an inverse
relationship with the rate constants of unfolding ku and folding kf . τ = 1/(ku+kf) (Eq. 16)
When folding of protein is arrested (EDTA) , kf = 0, then τ = 1/(ku) (Eq. 17)
The activation free energy (∆G≠u) can be calculated according the Eyring assumption:
∆G≠u = -RTln(kuh)/(kbT) (Eq. 18)
Where, h is a plank constant, 6.626 ×10-34 J s; kb is a Boltzmann constant, 1.3806505 × 10−23 J/K, R is universal constant, 8.3145 J/mol, T is absolute temperature (K) (51).
II-13 Thermodynamic properties of CIP
The effect of 4 mM EDTA on the heat denaturation of CIP could be monitored at 404 nm on U3010 spectrophotometer. The rate constants were derived from the major phase of the biphasic reactions or monophasic reactions (Eq. 14 and Eq. 15). A plot of lnku versus 1/T (in Kelvins) would yield a slope of –Ea/R. Where, Ea is the activation energy for denaturation; R is the molar gas constant.
lnku = lnA – Ea/RT (Eq. 19) where A is the frequency factor.
As the activation enthalpy ∆H≠u≒ Ea, the contribution from the activation entropy could be calculated from the Eq.10:
T∆S≠u =∆H≠u -∆G≠u (Eq. 20)
II-14 Scanning calorimetry
Heat capacity measurements was carried out on a CSC Nano II Differential Scanning Calorimeter (N-DSC II, Model 6100). Exhaustive cleaning of cells was performed before each experiment. The enzyme solution was dialyzed against 50 mM Tris-HCl, pH 8.5, and
the dialyzate was used as reference. The scan rate 1 K/min was employed. An over-pressure of 3 atm was kept over the liquid in the cells throughout the scans. The heat capacity Cp was measured as a function of temperature between 25 and 80 . The area ℃ ℃ under the DSC curve corresponds to the molar enthalpy change ∆Hcal for the phase transition. The phase transition temperature (Tm), or called melting temperature, for recombinant CIP was obtained from DSC curves.
II-15 Circular dichroism (CD)
CD experiments were carried out using a Jasco J715 spectropolarimeter. CD in the UV region (190-260 nm) was monitored with a cell of 1 mm path length. CD spectra reported in this study were an average of sixteen scans recorded at a speed of 200 nm/min and a resolution of 1 nm, corrected by subtracting the appropriated blank runs on recombinant CIP free solutions. The percentages of secondary structure elements was calculated using SELCON3 software.
III. Results and discussion
III-1 Expression of recombinat Coprinus cinereus peroxidase
The recombinant CIP was expressed in E. coli BL21(DE3) by inducing bacterial culture (O.D.600 nm = 0.8) with 0 or 50 µM IPTG at 37 for 10 or 20 h.℃ After induction, bacteria were collected, lysed with sonication and assayed for the presence of recombinant and the peroxidase activity. The electrophoresis of E. coli inclusion body after 50 µM IPTG induction is shown in Fig. 2A. Inclusion body formation in recombinant E. coli cells overexpressing CIP. The peroxidase activity from refolding of inclusion body was also tested. However, the bacterial extracts after 10 or 20 h IPTG induction were not much different between each other. The activity of recombinant CIP in E. coli appeared without the induction of IPTG and cannot be enhanced dramatically by increasing concentration of IPTG. It could be due to the constitutive expression of exogenous in E. coli BL21 (DE3).
This result suggests a deficiency in the suppression of recombinant protein expression in their system.
III-2 Refolding of inclusion bodies
The inclusion bodies was first solubilizd in 6 M urea to make a concentration of 0.5 mg/mL. The solubilized CIP was then subjected to renaturation by diluting in a refolding buffer (1.8 M urea in 50 mM Tris-HCl buffer, pH 9.5 containing 5 mM CaCl2, 5 µM hemin, 0.1 mM DTT, 0.5 mM oxidized glutathione and 5 % glycerol) in a ratio of 1:9 at 4 for a ℃ period of time (0-8 h). The peroxidase activity of the renatured CIP was monitored by transferring 1 µL aliquote of folding mixture to a reaction mixture (399 µL) for enzyme activity assay. As shown in Fig. 3, the recombinant CIP renatured in a time-dependent manner with a maximum reactivation occurred at about 7 h.
The effect of DTT, GSSG, urea, KCl, calcium ion and hemin in the refolding of CIP
was investigated. CIP contains four intramolecular disulfide bonds, which may be perturbed by DTT. DTT also prevents the proteins from unwanted oxidation. However, the renaturation of CIP was significantly perturbed by DTT (Fig. 4A). The refolding of CIP was rapidly suppressed by DTT. GSSG is required for the renaturation of manganese peroxidase (MnP) and tobacco anionic peroxidase (TOP) (24, 52). However, in HRP, the increase concentrations of DTT and GSSG results in decrease of its enzymic activity.
Interestingly, GSSG was also found to suppress the renaturation of recombinant CIP (Fig.
4B). The activity of CIP in refolding mixture decreases with increasing concentrations of GSSG from 0.1 to 1.0 mM. The effect of urea concentration in the renaturation of recombinant CIP was also tested. The highest enzyme activity could be observed in the refolding buffer containing 1.8 M urea (Fig. 4C). The urea concentration in renaturation buffer below or above 1.8 M may affect the refolding process, resulting in low enzyme reactivation. Ionic strength, as indicated by KCl concentration (0-100 mM), had no or little effect on the reanturation of CIP (Fig. 4D). The renaturation of the recombinant CIP requires hemin and Ca2+ ion. Maximal activity after renaturation occurred when renaturation buffer containing 5 µM hemin (Fig. 4F) or 10 mM CaCl2 (Fig. 4E). The concentration of Ca2+ ion higher than 10 mM exhibited less effect in the renaturation of recombinant CIP. The yield of active CIP during renaturation was more sensitive to the hemin concentration. The activity of renatured CIP quickly decreased with hemin higher than 5 µM (Fig. 4F). It is possible that the hydrophobic property of hemin molecule may interfere with the proper folding of CIP. Temperature also affect the renaturation of CIP.
The renaturation of CIP at 25 ℃ or 35 exhibited a much lower renaturation ℃ (53). Hemin is added after refolding to the apoenzyme restores the enzyme activity of recombinant CIP (Fig. 6). A similar result was also observed in HRP (54). However, in the case of tobacco anionic peroxidase, hemin should be added during the refolding (34).
III-3 Purification of recombinant CIP
The renatured recombinant CIP was purified from impurities come with inclusion body by first precipitates with 60 % ammonium sulfate followed by sizing exclusion column. The precipitation of recombinant CIP with ammonium sulfate helps to concentrate the enzyme and partly remove impurities. The 60 % ammonium sulfate precipitate may help refolding of recombinant CIP. A marked increase in the specific activity of CIP could be observed (Table 2). In the case of the MnP, the removal of 2 M urea after renaturation by dialysis also increases the specific activity of renatured MnP (55). In contrast, a significant activity lost was observed in recombinant tobacco anionic peroxidase precipitating with ammonium sulfate (52). The precipitated protein was then further purified on sizing exclusion column. The elution profile of recombinant CIP showed that most protein was eluted in fraction 9 (60-64 ml), where the maximal peroxidase activity appeared (Fig. 7-1).
The absorption spectra of several fractions including f1, f3, f5, f7, f9 and f11, between 700 nm and 350 nm. The fractions with higher specific activity exhibited a Soret band at 404 nm. While the recombinant CIP in fractions 3, 5 and 7, presumably have higher molecular weight than that in fraction 9, had a Soret band in a range of 410 ～ 408 nm. This result is consistent with observation that the aggregated TOP exhibits a Soret band at 409 nm. The aggregated CIP in fractions 3-7 also exhibited a low specific activity. The fractions 8-10 were collected and concentrated on Amicon Ultra-4 (centrifugal filter devices). The concentrated CIP was then reloaded on second sizing exclusion column S-200 (Amersham Biosciences, HiPrep 16/60) for further purification. The eluant was collected with 2 or 4 mL per fraction (Fig. 8-1). The high CIP activity appeared in fractions 9 and 10 (Fig. 8-1).
The absorption spectra of fractions 2-11 from S-200 column was also scanned between 700 nm and 300 nm (Fig. 8-2). The fractions (9 and 10) with higher activity had Soret band at 404 nm. The RZ (Reinheitszahl) values, as determined by the ratio of A404 nm (λMax of
Soret band) /A280 nm of isolated CIP in ammonium sulfate precipitation, first gel filtration column and second gel filtration column was 1.7, 2.9 and 3.3, respectively (Table 2). The increasing RZ values along with the purification process suggests the increasing purity of the purified CIP. SDS-PAGE analysis was also performed to evaluate the purity of the purified CIP (Fig 10). Table 2 summarizes the parameters regarding to recombinant CIP during purification. After ammonium sulfate precipitation, the specific activity of the recombinant CIP increased about 5-fold. The removal of urea after precipitation may be the
Soret band) /A280 nm of isolated CIP in ammonium sulfate precipitation, first gel filtration column and second gel filtration column was 1.7, 2.9 and 3.3, respectively (Table 2). The increasing RZ values along with the purification process suggests the increasing purity of the purified CIP. SDS-PAGE analysis was also performed to evaluate the purity of the purified CIP (Fig 10). Table 2 summarizes the parameters regarding to recombinant CIP during purification. After ammonium sulfate precipitation, the specific activity of the recombinant CIP increased about 5-fold. The removal of urea after precipitation may be the