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PEDS Advance Access originally published online on February 1, 2006
Protein Engineering Design and Selection 2006 19(4):169-173; doi:10.1093/protein/gzj016
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Increased thermal and organic solvent tolerance of modified horseradish peroxidase

Jian-Zhong Liu1, Teng-Li Wang, Ming-Tao Huang, Hai-Yan Song, Li-Ping Weng and Liang-Nian Ji

Biotechnology Research Center and Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, Zhongshan University, Guangzhou 510275, P.R.China

1 To whom correspondence should be addressed. Biotechnology Research Center, Zhongshan University, Guangzhou 510275, P.R.China. Email: lssljz{at}zsu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Acknowledgements
 References
 
Horseradish peroxidase (HRP) was modified by maleic anhydride and citraconic anhydride. The thermal and organic solvent tolerances of native and modified enzyme were compared. These chemical modifications of HRP increased their thermostability both in aqueous buffer and some organic solvents, and also enhanced their tolerances of some organic solvents. We have studied the unfolding of native and modified HRP by heat to determine the conformational stability. The temperature at the midpoint of thermal denaturation (Tm) was increased upon modification. Both enthalpy change ({Delta}Hm) and entropy change ({Delta}Sm) for unfolding of modified enzyme at Tm were decreased compared with native enzyme. Circular dichroism studies proved that these modifications changed the conformation of HRP. The improvements of stability are related to side chain reorientations of aromatics upon both modifications.

Keywords: chemical modification/horseradish peroxidase/organic solvent/thermostability


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Conclusions
 Acknowledgements
 References
 
Horseradish peroxidase (HRP, EC 1.11.1.7 [EC] ) catalyzes the oxidation of aromatic compounds by hydrogen peroxide or alkyl hydroperoxide. It was widely applied in the synthesis of fine chemicals and polymer, and the removal of toxic phenolics from wastewater (Klibanov et al., 1983Go; Colonna et al., 1999Go).

A stabilized HRP is required in its industrial applications. The inherent liability of many enzymes in organic solvents or at elevated temperatures limits their roles as potential reaction catalyst even though certain enzymes exhibit marked storage stability in organic solvents. Enhancement of functional stability will clearly broaden the range of enzyme applications. Over the years, several techniques have been developed to ameliorate this loss of catalytic function, including lyophilization in the presence of lyoprotectants and excipients such as KCl, crown ethers, cyclodextrins and molecular imprinters (Lee and Dordick, 2002Go), the use of site-directed mutagenesis and directed evolution (DeSantis and Jones, 1999Go; Hult and Berglund, 2003Go) or chemical modification (DeSantis and Jones, 1999Go; Davis, 2003Go). Chemical modification has now reemerged as a powerful complementary approach to site-directed mutagenesis and directed evolution (DeSantis and Jones, 1999Go). Chemical modification of HRP surface has been performed to improve its stability. Acetic acid N-hydroxysuccinimide ester (AA-NHS) (Miland et al., 1996aGo) and bifunctional N-hydroxysuccinimide ester (EG-NHS) (Ryan et al., 1994Go; Miland et al., 1996bGo) were successfully employed to modify HRP to increase HRP's stability in organic solvents. Our previous papers reported that modification of HRP by phthalic anhydride improved HRP's stability and catalytic activity in aqueous buffer (Liu et al., 2002Go; Song et al., 2003Go) and in solvents (Song et al., 2005Go). In this paper, other modification agents of {epsilon}-amino group of lysine residues, such as citraconic anhydride (CA) and maleic anhydride (MA), were used to modify HRP. The changes of stability and activity after modification were investigated. The aim was to understand how these modifications affect enzyme activity, stability and structure.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Conclusions
 Acknowledgements
 References
 
Reagent

HRP was purchased from Shanghai Lizhu Dong Feng Biotechnology Co. Ltd and had a specific activity of 250 purpurogallin units/mg and RZ = 3.0. CA was purchased from Alfa Aesar. MA (analytical grade) was obtained from Guangzhou Chemical Reagent Factory. All other reagents were of analytic grade.

Chemical modification

Chemical modification was based on our previous method (Liu et al., 2002Go; Song et al., 2003Go, 2005Go). An aliquot of 25 µl of 50% (v/v) CA in 0.1 M phosphate buffer (pH 7.4) or 50 µl of 2 mM MA in 0.1 M phosphate buffer (pH 7.4), and 2 ml of 1 mg/ml HRP in 0.1 M phosphate buffer (pH 7.4) were mixed. The reaction proceeded at 4°C for 1 h and was then dialyzed against 0.1 M phosphate buffer (pH 7.4) at 4°C to removal excess reagent.

The degree of modification was estimated by the method of Snyder (Snyder and Sobocinski, 1975Go).

Peroxidase activity assay

The enzyme activity was assayed by colorimetric method (Song et al., 2005Go). Reaction mixture containing 10 mM phenol, 0.2 mM hydrogen peroxide and 2.4 mM 4-aminoantipyrin (4-AAP) in a total volume of 3.0 ml was incubated at 30°C. All reagents were dissolved in 0.01 M phosphate buffer (pH 7.0). The reaction was then started by adding 0.1 ml of diluted enzyme solution, and the initial increase in absorbance was monitored at 510 nm during 1 min. Under such conditions, the rate of formation of colored product which absorbs light at a peak wavelength of 510 nm was calculated using a molar extinction coefficient of 7100/M/cm. One unit of peroxidase activity was defined as the amount of the enzyme consuming 1 µmol of hydrogen peroxide per minute under the assay conditions.

HRP concentration was estimated from its Soret absorbance (molar extinction coefficient at 402 nm = 102/M/cm) (Song et al., 2005Go).

Thermostability assay

Native and modified HRP preparations were incubated in 0.1 M phosphate buffer (pH 7.0) at 50°C. Aliquots of each sample were withdrawn at different times and assayed for enzymatic activity under the standard conditions as stated above.

Catalytic stability in organic solvents

Organic solvent profiles of HRP samples were carried out at room temperature with exposure times of 1 h. The solvents used were dimethylformamide (DMF), tetrahydofuran (THF) and dimethylsulfoxide (DMSO). Reaction mixtures were set up with increasing percent volumes of organic solvent in 0.1 M phosphate buffer (pH 7.4) in 10% (v/v) increments. Hundred microliters were withdrawn from each reaction mixture and assayed under the standard conditions as stated above.

Thermal unfolding

Thermal denaturations of native and modified HRP were monitored by heme absorption using Shimadzu UV2450 at pH 7.4 (0.1 M phosphate buffer). The temperature was raised from 25 to 95°C in steps of 2°C with an equilibration time of 2 min at each temperature. The reversibility of thermal denaturation was checked by cooling the denaturated sample and subsequent reheating. The reversibility usually exceeded 95%. The enzyme concentration was ~5 µM in 0.1 M phosphate buffer (pH 7.4).

The denaturation curves were plotted with the absorbance of the enzyme at 402 nm against temperature, and further analyses of the data were performed as described by Pace et al. (1990)Go. From the denaturation curves, a two-state N{leftrightarrows}U unfolding mechanism was assumed, and consequently, for any of the points, only the folded and unfolded conformations were present at the temperature. Thus, if fN and fU represent the fraction of protein present in the folded and unfolded conformations, respectively, fN + fU = 1.

The equilibrium constant between the unfolded and native states is given by

Formula 1(1)
The standard Gibbs free energy of denaturation ({Delta}Gd) was calculated using Equation (2).

Formula 2(2)
where R is the universal gas constant (8.31 J/mol/K) and T is the absolute temperature. fU and, hence, K may be calculated by recording the change in absorbance at 402 nm as follows:

Formula 3(3a)

Formula 4(3b)
where A is the observed absorbance at 402 nm, and AN and AU are the values of absorbance of the native and unfolded conformation, respectively. The values of AN and AU in transition region were obtained by extrapolating from the pre- and post-transition regions.

The temperature at the midpoint of thermal denaturation, Tm, was obtained as the temperature at which {Delta}Gd = 0 from the plot of {Delta}Gd versus T. The slope of such a plot at Tm yielded {Delta}Sm, the change in entropy. The enthalpy change for unfolding at Tm, {Delta}Hm, was calculated using the equation

Formula 5(4)

CD spectra

CD experiments were carried out using a Jasco J810 spectropolarimeter. CD in the UV region (200–250 nm) was monitored with a cell of 2 mm path length with enzyme concentration of ~1.25 µM in 50 mM phosphate buffer (pH 7.4). CD from near-UV region to visible region (250–700 nm) was monitored using a cell of 2 mm path length with enzyme concentration of ~1.25 µM in 50 mM phosphate buffer (pH 7.4). The CD data were expressed in terms of mean residue ellipticity, [{theta}.], in deg cm2/dmol. CD spectra reported in this paper were an average of three scans recorded at a speed of 100 nm/min and a resolution of 1 nm, corrected by subtracting the appropriate blank runs on HRP-free solutions. The secondary structure percentage predictions were made using the K2D software (http://www2.umdnj.edu/cdrw.jweb).


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Conclusions
 Acknowledgements
 References
 
As indicated in Figure 1, treatments of HRP with both modification agents resulted in a dramatic enhancement of its thermostability in aqueous buffer. After the exposure for 3 h at 50°C, native HRP retained only ~30% activity in aqueous buffer. HRPs modified by MA and CA, however, retained ~74 and 82% activity, respectively. Our previous paper reported that phthalic anhydride modification enhanced the thermostability of HRP ~10-fold (Liu et al., 2002Go). A few papers have also reported various chemical modifications to enhance thermostability of HRP. Treatment of HRP using bis-succinimides (EG-NHS) and AA-NHS0 also enhanced thermostability of HRP (Ryan et al., 1994Go; Miland et al., 1996aGo). Chemical modification of {alpha}-amylase using CA brought about a dramatic enhancement of thermal stability (Khajch et al., 2001Go).


Figure 1
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  		Fig. 1.. Thermostability of native and modified HRP at 50°C in aqueous buffer. Native HRP (closed square); HRP modified by maleic anhydride (open square); HRP modified by citriconic anhydride (open circle). The values represent the mean of three independent sets of experiments with SD of <5%.

 
HRP contains six lysine residues: Lys65, Lys84, Lys149, Lys174, Lys232 and Lys241 (Welinder, 1979Go). The modification degrees of amino groups from HRP by CA and MA were determined as ~56.1 and 46.7% of the native enzyme, respectively. This result indicated that three of the six lysine {epsilon}-amino groups from native enzyme were modified. Thus, the greater stability of MA-HRP and CA-HRP likely arises from these modifications neutralizing positive lysine charges and then forming tighter binding of a structural calcium ions as in the case of phthalic anhydride (Liu et al., 2002Go; Song et al., 2005Go), AA-NHS and EG-NHS modifications (Ryan et al., 1994Go; Miland et al., 1996aGo,bGo).

The temperature dependence of the absorbance at 402 nm is shown in Figure 2. For native HRP and modified HRP, a typical two-state transition was observed. Pina et al. (2001)Go applied detailed differential scanning calorimetry, steady-state tryptophan fluorescence, far-UV CD and absorbance in the soret band to monitor the thermal denaturation of HRP, and all these independent experimental data supported the conclusion that the thermal denaturation of HRP at pH 3.0 can be described by a two-state model. However, the thermal unfolding of the secondary structure of HRPc monitored by UV–CD appeared to be a two-state process, and the thermal unfolding of the tertiary structure of HRPc monitored by soret-CD and steady-state tryptophan fluorescence showed the existence of one intermediate (Chattopadhyay and Mazumdar, 2000Go).


Figure 2
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  		Fig. 2.. Change in absorbance of native HRP (closed square), HRP modified by maleic anhydride (open square) and HRP modified by citriconic anhydride (open circle). The continuous lines are fits to a simple two-state model.

 
Based on two-state model, the denaturation curves were analyzed using Equations (1)Go–(3) and {Delta}Gd was plotted against T to obtain Tm and {Delta}Sm (Table I). Thus, {Delta}Hm was calculated from Equation (4). From Table I, it is clear that Tm at pH 7.4 was increased from 62.2 to 69.3°C (for MA) and 70.2°C (for CA) after modification. These results also indicate that the thermal stability of HRP was enhanced after modification. From Table I, it is clear that the thermostabilization of HRP after modification is mostly accompanied by a decrease in {Delta}Sm and {Delta}Hm as in the case of charge neutralization by acetylation of the amino groups of {alpha}-amylase (Urabe et al., 1973Go), charge reversal of carboxyl groups of glucoamylase (Munch and Tritsch, 1990Go) and ß-glucosidase (Rashid and Siddiqui, 1998Go), charge neutralization of carboxyl groups of ß-glucosidase (Rashid and Siddiqui, 1998Go), and protein engineered T4 lysozyme (Matthews et al., 1987Go). The reason for further decreases in {Delta}Sm brought about by these modifications could be due to the elimination of repulsion between positively charged lysine residues thus decreasing the flexibility of an external loop (Matthews, 1993Go; Vieille and Zeikus, 1996Go), and thereby stabilizing both modified HRPs.


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  		Table I.. Thermodynamic parameters for thermal denaturation of native and modified HRP in 0.1 M phosphate buffer (pH 7.4)

 
As shown in Figure 3, MA-HRP and CA-HRP showed a greater tolerance of DMF, DMSO and THF at room temperature. In our previous paper (Song et al., 2005Go), HRP modified by phthalic anhydride also had a greater tolerance of DMF at 30°C. Some papers also reported that chemical modification improved the stability of HRP in some organic solvents. Acetylated HRP by acetic acid N-hydroxysuccinimide had a greater tolerance of THF at 25°C (Miland et al., 1996aGo). The cross-linked EG-HRP showed a greater tolerance of DMF and THF at 25 and 60°C (Miland et al., 1996bGo). Results that we are interested in are the activations of 10% THF on MA-HRP and CA-HRP, and 10% DMSO on CA-HRP. The activation of methanol was also found in acetylated HRP (Miland et al., 1996aGo) and cross-linked EG-HRP (Miland et al., 1996bGo). In our previous paper, we also reported the activation of methanol, acetonitrile on phthalic anhydride modified-HRP (Song et al., 2005Go). Khmelnitsky et al. (1988)Go have reported numerous examples of enzyme activation by moderate concentrations (10–30%) of solvents. The stability of native and modified HRP in some organic solvents was compared at 30°C (Figure 4). It is clear that MA-HRP and CA-HRP showed a greater stability in 50% DMSO and THF at 30°C. From Figures 1, 3 and 4 we can also find that CA-HRP had a greater stability than MA-HRP both in aqueous buffer and some organic solvents.


Figure 3
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  		Fig. 3.. Effects of solvents on native HRP (closed square), HRP modified by maleic anhydride (open square) and HRP modified by citriconic anhydride (open square) at room temperature for 1 h. The values represent the mean of three independent sets of experiments with SD of <5%.

 

Figure 4
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  		Fig. 4.. Thermostability of native and modified HRP at 30°C in 50% of organic solvents. Native HRP (closed square); HRP modified by maleic anhydride (open square); HRP modified by citriconic anhydride (open circle). The values represent the mean of three independent sets of experiments with SD of <5%.

 
CD spectra in UV and UV-Vis regions provide information on the structure of protein and prosthetic heme of HRP (Strickland, 1968Go). Spectroscopic data used for detection of changes in HRP structure are as follows: (i) Far-UV (190–260 nm) CD: changes in secondary structure of the protein. (ii) Near-UV (250–300 nm) CD: changes in tertiary structure of the protein. (iii) CD at 350–450 nm: dissociation of the prosthetic heme from the protein and the structural changes of the protein surrounding the heme. (iv) Absorption at 350–700 nm: changes of the structure and environment of the heme. Figure 5 shows CD of native and modified HRP in water. Native and modified HRP have identical CD spectra with negative bands at 208 and 220 nm, which agreed with the previous results (Strickland et al., 1968Go). The spectral shapes of both modified HRPs are almost identical to those of native HRP. The percentages of secondary structure elements calculated using K2D software are summarized in Table II. No significant changes in the {alpha}-helix content were observed by both modifications. In near-UV region (250–350 nm), native and modified HRP have identical CD spectra with a positive band at ~275 nm. The intensity of the CD band at ~275 nm increased after modification (Figure 5B). The ~2-fold increase at the 275 nm peak upon CA treatment was observed, suggesting very small side chain reorientations of aromatics. In visible region, no change of CD spectra at 403 nm and in 500–700 nm was detected after modification (data no shown). It indicates that there is no change of the structure and environment of the heme after modification. In our previous paper (Song et al., 2005Go), HRP modified by phthalic anhydride showed a higher {alpha}-helix content. Thus, the enhancements of stability in aqueous buffer and some organic solvents are related to side chain reorientations of aromatics upon both modifications.


Figure 5
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  		Fig. 5.. (A and B) CD spectra of native and modified HRP in 50 mM phosphate buffer (pH 7.4).

 

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  		Table II.. The percentage of secondary structure elements of native and modified HRP, estimated from CD spectra using K2D software (http://www2.umdnj.edu/cdrw.jweb)

 

   Conclusions
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
Acknowledgements
 References
 
Chemical modification of HRP by MA and citriconic anhydride increased their thermostability both in aqueous buffer and some organic solvents, and also enhanced their tolerances of some organic solvents. CD spectra proved that these modifications changed the conformation of HRP. The improvements of stability are related to side chain reorientations of aromatics upon both modifications.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Acknowledgements
References
 
We are grateful to National Natural Science Foundation of China and the Natural Science Foundation of Guangdong Province for their financial support.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Acknowledgements
 References
 
Chattopadhyay,K. and Mazumdar,S. (2000) Biochemistry, 39, 263–270.[CrossRef][Medline]

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Davis,B.G. (2003) Curr. Opin. Biotechnol., 14, 379–386.[CrossRef][Web of Science][Medline]

DeSantis,G. and Jones,J.B. (1999) Curr. Opin. Biotechnol., 10, 324–330.[CrossRef][Web of Science][Medline]

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Khajeh,K., Naderi-Manesh,H., Ranjbar,B., Moosavi-Movahedi,A. and Nemat-Gorgani,M. (2001) Enzyme Microb. Technol., 28, 543–549.[CrossRef][Web of Science][Medline]

Khmelnitsky,Y.L., Levashov,L.V., Klyachko,N.L. and Martinek,K. (1988) Enzyme Microb. Technol., 10, 710–724.[CrossRef]

Klibanov,A.M., Tu,T.M. and Scoot,K.P. (1983) Science, 221, 259–261.[Abstract/Free Full Text]

Lee,M.-Y.J. and Dordick,S. (2002) Curr. Opin. Biotechnol., 13, 376–384.[CrossRef][Web of Science][Medline]

Liu,J.-Z., Song,H.-Y., Weng,L.-P. and Ji,L.-N. (2002) J. Mol. Catal. B Enzym., 18, 225–232.[CrossRef]

Matthews,B.W. (1993) Annu. Rev. Biochem., 62, 139–160.[CrossRef][Web of Science][Medline]

Matthews,B.W., Nicholson,H. and Becktel,W.J. (1987) Proc. Natl Acad. Sci. USA, 84, 6663–6667.[Abstract/Free Full Text]

Miland,E., Smyth,M.R. and Ó'Fágáin,C. (1996a) Enzyme Microb. Technol.,19, 63–67.

Miland,E., Smyth,M.R. and Ó'Fágáin,C. (1996b) Enzyme Microb. Technol.,19, 242–249.[CrossRef]

Munch,O. and Tritsch,D. (1990) Biochim. Biophys. Acta, 1041, 111–116.[CrossRef][Medline]

Pace,C.N., Shirley,B.R. and Thomson,J.A. (1990) In Creighton,T.E. (ed.), Protein Structure and Function: A Practical Approach. IRL Press, Oxford, pp. 311–330.

Pina,D.G., Shnyrova,A.V., Gavilanes,F., Rodriguez,A., Leal,F., Roig,M.G., Sakharov,I.Y., Zhadan,G.G., Villar,E. and Shnyrov,V. (2001) Biochemistry, 268, 120–126.

Rashid,M.H. and Siddiqui,K.S. (1998) Process Biochem., 33, 109–115.[CrossRef]

Ryan,O., Smyth,M.R. and Ó'Fágáin,C. (1994) Enzyme Microb. Technol., 16, 501–505.[CrossRef][Web of Science][Medline]

Snyder,S.L. and Sobocinski,P.Z. (1975) Anal. Biochem., 64, 284–288.[CrossRef][Web of Science][Medline]

Song,H.-Y., Liu,J.-Z., Xiong,Y.-H., Weng,L.-P. and Ji,L.-N. (2003) J. Mol. Catal. B Enzym., 22, 37–44.[CrossRef]

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Received July 23, 2005; revised September 17, 2005; accepted January 3, 2006.

Edited by Adrian Goldman


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