Chinese Journal of Chemistry, 2009, 27, 1291—1294 Full Paper

* E-mail: jyc@snnu.edu.cn Received November 8, 2008; revised February 12, 2009; accepted March 10, 2009. Project supported by the National Natural Science Foundation of China (No. 20876094).

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Activation Function of Chloroperoxidase in the Presence of Metal Ions at Elevated Temperature from 25 to 55 ℃

GAO, Qianga(高强) JIANG, Yucheng*,a,b(蒋育澄) GAO, Xiaoqina(高晓勤) HU, Manchenga,b(胡满成) LI, Shunia,b(李淑妮) ZHAI, Quanguoa,b(翟全国)

a School of Chemistry and Materials Science, Shaanxi Normal University, Xi'an, Shaanxi 710062, China

b Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi'an, Shaanxi 710062, China

The investigation and comparison of chlorination activity of chloroperoxidase (CPO) from Caldariomyces fumago in metal ion solutions to those in pure buffer indicated that CPO could be effectively activated by some al-kaline-earth metals and transition metals. The obtained maximum relative activity of CPO was 1.33 time at 75 μmol•L－1 Ca2＋, 1.37 time at 90 μmol•L－1 Mg2＋, 1.34 time at 90 μmol•L－1 Ni2＋, and 1.27 time at 105 μmol•L－1 Co2＋ at 25 ℃. Moreover, the CPO stability against temperature was improved in the presence of the above metal ions. At 55 ℃, CPO could retain only about 40% of activity whereas 75% and 81% of activity were maintained in Mg2＋ and Ca2＋ media, respectively. It was suggested that the metal ions bind to the acid-base catalytic groups Glu183, His105 and Asp106 around the active site of CPO, and activate CPO by both an enrichment of substrate concentration and the conformational change of CPO, which are favorable to the substrate access. The analysis of kinetic parameters indicated that the activation was mainly due to an increase in kcat values. The affinity and speci-ficity of CPO to substrates were also improved in these metal ion media. The results in this work are promising in view of industrial applications of this versatile biological catalyst.

Keywords chloroperoxidase, metal ion, catalytic activity, thermal stability, activation mechanism

Introduction

Chloroperoxidase (CPO, EC 1.11.1.10), a hame- containing monomeric glycoprotein isolated from the marine fungus Caldariomyces fumago, is one of the most versatile enzymes in the peroxidase super family. It catalyzes chlorination of activated C—H bond, as well as peroxidase, catalase and cytochrome P450 reac-tions. Most importantly, CPO can catalyze a variety of synthetically useful reactions with high regio- and enan-tioselectivity at the expense of hydrogen peroxide or other organic hydroperoxide without the need of cofactors.1 The broad activities of CPO were attributed to its unique structural feature with a proximal thiolate ligand to the hame iron and a distal glutamic acid resi-due as acid-base catalyst rather than a histidine com-monly found in most peroxidases.2-4

Although CPO has been studied for a wide range of applications from synthesis of fine chemicals to detoxi-cation of environmental pollutants,5-7 the industrial ap-plication of this enzyme has been hampered by instabil-ity at high temperatures, low water solubility of many organic substrates of synthetic interest, and deactivation at high concentrations of hydrogen peroxide.8 So, a lot of methods have been developed to enhance the CPO activity and its thermal stability, such as increasing the

initial enzymatic activity in reverse micellar systems,9 microencapsulating CPO as a recyclable catalyst,10 and immobilizing CPO on some mesoporous or macromo-lecular materials.11-14

In this work, the effects of metal ions on CPO chlorination activity at 25 ℃ and at elevated tempera-tures were investigated. The result indicated that Ca2＋, Mg2＋, Co2＋, and Ni2＋ could enhance chlorination ac-tivity of CPO at 25 ℃, and meanwhile, they could also improve the CPO thermal stability in a temperature range from 35 to 55 ℃.

Experimental

Materials

Chloroperoxidase was isolated from the growth me-dium of C. fumago according to the method established by Morris and Hager with minor modifications, using acetone in the solvent fractionation step.2 The enzyme had a specific activity of 4980 U/mL based on the stan-dard MCD assay (Rz＝1.06).

Calcium chloride, magnesium chloride, manganese chloride, cobalt chloride, nickel chloride, strontium chloride, barium chloride and hydrogen peroxide (30% in aqueous solution) were purchased from Xi'an

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© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Chemical and Medicine Co. Ltd. Monochlorodimedon (MCD) was obtained from Fluka. All chemicals were of analytical grade unless otherwise indicated.

Enzyme assay and other optical measurements were performed on a UV-1700 UV-Vis spectrophotometer (Shimadzu). Temperature was controlled using a CS501 super constant temperature controller.

CPO chlorination activity assay

CPO chlorination activity was determined by MCD assay according to the method reported previously (Scheme 1).15 Typically, the chlorination activity is tested in 0.1 mol•L－1 potassium phosphate buffer (pH＝

2.75) containing 0.1 mmol•L－1 MCD, 20 mmol•L－1 KCl and 2 mmol•L－1 H2O2. CPO activity was evaluated by the specific initial reaction rate ν (moles of MCD consumed per unit of time), which was calculated from the plot slope of absorbance versus time. All sets of ex-periments were reproduced several times under the identical operating conditions in order to increase the accuracy of the findings, each data point of a set of re-sults was obtained at least three times with the discrep-ancy being below 5%.

Scheme 1 Chlorination of MCD to DCD catalyzed by CPO

Investigation of effect of metal ions

CPO was incubated with metal ions (Ca2＋, Mg2＋, Sr2＋, Ba2＋, Co2＋, Zn2＋, Mn2＋ and Ni2＋) at 25 ℃ for the chlorination activity assay. The final volume was always 3 mL and the concentration of metal ions varied from 1 to 300 μmol•L－1.

The thermal stability of CPO in the presence of the above metal ions was investigated in phosphate buffer (pH＝2.75) from 35, 40, 45, 50 to 55 ℃. Prior to heat treatment, CPO was incubated with the relevant metal ions for 30 min at 25 ℃. The thermal stability was ex-pressed as residual activity of CPO at an elevated tem-perature.

Kinetic parameter determination of MCD chlorina-tion by CPO

The kinetic parameters, Km and Vmax values of the CPO with and without Ca2＋, Mg2＋, Co2＋and Ni2＋were determined by measuring the initial rates of the MCD chlorination in 0.1 mol•L－1 potassium phosphate buffer (pH＝2.75) at 25 ℃. The Km and Vmax values in pure buffer and in metal ion media were obtained by linear regression analysis of the double-reciprocal Lineweaver-Burk plots.

[ ] 11 1mmax

max

S

Kν V

V

⎧ ⎫⎪⎨ ⎬⎪ ⎭⎩

－

－ －

＝ ＋

where [S] is the concentration of substrate, ν and Vmax represent the initial and maximum rates of reaction, re-spectively.

Results and discussion

The effect of metal ion concentration on activity of CPO at 25 ℃ was shown in Figure 1. Ca2＋, Mg2＋, Co2＋ and Ni2＋ were found to have a positive effect on CPO activity. The results are expressed as relative ac-tivity (the ratio of chlorination rate in the presence of metal ion to that in pure buffer). Initially, the relative activity increased rapidly with the increase of metal ion concentration. With the increased concentration of metal ion, the relative activity began dropping after it reached the maximum. The obtained maximum relative activity of CPO was 1.33 time at 75 μmol•L－1 Ca2＋, 1.37 time at 90 μmol•L－1 Mg2＋, 1.34 time at 90 μmol• L－1 Ni2＋, 1.27 time at 105 μmol•L－1 Co2＋. Sr2＋ and Ba2＋ could also only improve the activity of CPO slightly at 25 ℃ (data not shown).

Figure 1 Effect of the concentration of metal ions on the activ-ity of CPO. CPO was incubated with Ca2＋(●), Mg2＋(■), Co2＋(▼) and Ni2＋(▲) for 60 min at 25 ℃. The relative activity means the ratio of activity of CPO in media containing metal ions to that in pure buffer.

The effect of incubation time on the activity of CPO was carried out in the presence of Ca2＋ (Figure 2). It was found that at a lower Ca2＋ concentration (15 or 30 μmol•L－1), the relative activity of CPO increased lin-eally with incubation time (0—2 h). However, at a higher Ca2＋ concentration, extended exposure of CPO to metal ion would result in a slower increase and even dropping of activity of CPO.

Metal ions (Ca2＋, Mg2＋, Sr2＋, Ba2＋, Co2＋, Zn2＋, Mn2＋ and Ni2＋) were tested to examine their effect on the thermal stability of the CPO from 35, 40, 45, 50 to 55 ℃. Incubation time was 30 min. Results were ex-pressed as residual activity (ratio of activity at an ele-vated temperature to that at 25 ℃ in pure buffer). However, as shown in Figure 3, only some of the above metal ions have positive effect. For example, CPO could

Chloroperoxidase Chin. J. Chem., 2009 Vol. 27 No. 7 1293

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 2 Effect of incubation time on the activity of CPO in Ca2＋ media. The concentration of Ca2＋ was 15 (■), 30 (●), 45 (▲), 60 (▼) and 75 (◆) μmol•L－1, respectively.

Figure 3 Effect of metal ions on the thermal stability of CPO at elevated temperatures. CPO was incubated with Mg2＋(●), Ca2＋

(■), Co2＋(▼), Ni2＋(▲) and blank ( ) for 30 min.◆

retain only about 40% of activity at 55 ℃ whereas 57%, 59%, 75% and 81% of CPO activity were main-tained in Ca2＋, Mg2＋, Co2＋and Ni2＋ media, respec-tively. At a lower temperature, the improvement of thermal stability of CPO is more obvious, especially in the presence of Ca2＋, Mg2＋. CPO could retain nearly 98% activity in Ca2＋ media while there was only 67% residual activity in pure buffer at 40 ℃, and even 103% residual activity could be obtained at 35 ℃ in Ca2＋ media. These results indicate that some metal ions really have activation effect on CPO at room temperature, and even at an elevated temperature. However, we found that Mn2＋ had negative effect on the thermal stability of CPO, which lost its activity totally at 55 ℃ in Mn2＋ media.

Why can these metal ions activate CPO? In the CPO catalytic cycle, hydrogen peroxide binds to the pentaco-ordinate ferric resting state FeIII to generate an interme-diate (compound I), a porphyrin π cation radical con-taining FeIV. Then, compound I reacts with Cl－ as an electron donor (Scheme 2). The crystal structure of CPO revealed a small channel above the heme, which allows direct substrate access to ferriporphyrin. CPO can

modulate the position of the substrates (relative to the heme) by adjusting the size/shape of this channel. In this work, Ca2＋, Mg2＋, Ni2＋ and Co2＋ could drill through the channel and bound to amino acid residues Glu183, His105 and Asp106, which acted as acid-base catalytic groups and positioned directly adjacent to the sub-strate-binding site.15 These metal ions with positive charge would attract Cl－ to result in an enrichment of Cl－ around the active site so as to enhance the reaction rate. On the other hand, the binding of metal ions to the oxygen atom in the porphyrin loop would cause conformational change of CPO and make the heme active center be more flexible, which are favorable to the substrate access. In addition, the analysis and comparison of UV-Vis spectroscopy of CPO in the presence of metal ions and in pure buffer indicated that the Soret band of native CPO at 398 nm had no obvious change, which demonstrated that these metal ions did not disturb the active-site of CPO. They improve the enzyme catalytic performance simply by changing the microenvironment around the active-site. This observation also supported the above mechanism about the activation of CPO by metal ions.

Scheme 2 Chlorination catalytic cycle diagram of CPO. It pro-ceeds via an oxyferryl porphyrin π-radical cation intermediate, CPO-compound I.

Moreover, the effect of the metal ions on kinetic pa-rameters of MCD chlorination catalyzed by CPO was investigated. All data points obey to Michaelis-Menten kinetics and can be correlated in the Lineweaver-Burk plot for an estimation of the kinetic parameters reported in Table 1. The Km values determined in metal ion solu-tions are all much lower than that in pure buffer. Meanwhile, the kcat values and the enzyme specificity, kcat/Km are obviously higher in the presence of the metal ions than those in pure buffer. These results indicated that both the affinity and specificity of CPO to sub-strates were also improved in these metal ion solutions.

Conclusion

The catalytic activity and thermal stability of CPO at an elevated temperature were improved in the presence

1294 Chin. J. Chem., 2009, Vol. 27, No. 7 GAO et al.

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 1 Kinetic parameters of the MCD chlorination catalyzed by CPO in pure buffer and in Ca2＋, Mg2＋, Co2＋, Ni2＋solutions

Medium Km /(10－3 mol•L－1) kcat/s－1

(kcat/Km)/

(mol－1•s－1•L)

Buffer 0.244±0.03 1970.1±18.9 8074.2±13.9

Buffer＋Ca2＋

Buffer＋Mg2＋

Buffer＋Co2＋

Buffer＋Ni2＋

0.218±0.04

0.226±0.02

0.231±0.01

0.204±0.04

2574.3±11.2

2403.5±15.4

2251.1±17.8

2754.8±28.2

11808.7±21.8

10635.2±34.2

9745.1±15.6

13503.9±37.1

of the metal ions: Ca2＋, Mg2＋, Co2＋and Ni2＋. Metal ions bind to the acid-base catalytic groups Glu183, His105 and Asp106 around the active site of CPO, and activate CPO through both an enrichment of substrate concentration and the conformational change of CPO, which are favorable to the substrate access. The analysis of kinetic parameters indicates that the activation of CPO is mainly due to an increase in kcat values, and both the affinity and specificity of CPO to substrates are also improved in these metal ion solutions.

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