biochemical analysis and kinetic modeling of the thermal inactivation of mbp-fused heparinase i:...

ARTICLE

Biochemical Analysis and Kinetic Modeling of theThermal Inactivation of MBP-Fused HeparinaseI: Implications for a ComprehensiveThermostabilization Strategy

Shuo Chen,1,2 Fengchun Ye,1 Yang Chen,1 Yu Chen,1 Hongxin Zhao,1 Rie Yatsunami,2

Satoshi Nakamura,2 Fumio Arisaka,2 Xin-Hui Xing1

1Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;

telephone: þ86-10-62794771; fax: þ86-10-62794771; e-mail: [email protected] School of Bioscience and Biotechnology, Tokyo Institute of Technology,

Nagatsuta, Midori-ku, Yokohama, Japan

Received 4 January 2011; revision received 7 March 2011; accepted 14 March 2011

Published online 28 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23144

ABSTRACT: Enzymatic degradation of heparin by heparinlyases has not only largely facilitated heparin structuralanalysis and contamination detection, but also showed greatpotential to be a green and cost-effective way to produce lowmolecular weight heparin (LMWH). However, the commer-cial use of heparinase I (HepI), one of the most studiedheparin lyases, has been largely hampered by its low pro-ductivity and extremely poor thermostability. Here wereport the thermal inactivation mechanism and strategicthermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield,solubility and activity. Biochemical studies demonstratedthat the thermal inactivation of MBP-HepI involves anunfolding step that is temperature-dependently reversible,followed by an irreversible dimerization step induced byintermolecular disulfide bonds. A good consistency betweenthe kinetic modeling and experimental data of the inactiva-tion was obtained within a wide range of temperature andenzyme concentration, confirming the adequacy of theproposed inactivation model. Based on the inactivationmechanism, a comprehensive strategy was proposed forthe thermal stabilization of MBP-HepI, in which Ca2þ

and Tween 80 were used to inhibit unfolding while sitemutation at Cys297 and DTT were employed to suppressdimerization. The engineered enzyme exhibits remarkablyimproved storage and operational thermostability, forexample, 16-fold increase in half-life at its optimum tem-perature of 308C and 8-fold increase in remaining activity of95% after 1-week storage at 48C, and therefore shows greatpotential as a commercial biocatalyst for heparin degrada-tion in the pharmaceutical industry.

Biotechnol. Bioeng. 2011;108: 1841–1851.

� 2011 Wiley Periodicals, Inc.

KEYWORDS: heparinase; inactivation; stabilization; unfold-ing; dimerization; kinetic modeling

Introduction

Heparin, a highly sulfated glycosaminoglycan (GAG)polysaccharide, has been in clinical use as a criticalanticoagulant for over 70 years (Casu and Lindahl, 2001;Lever and Page, 2002). Compared to heparin, low molecularweight heparin (LMWH), a heparin derivative obtainedfrom chemical or enzymatic degradation of heparin(Linhardt and Gunay, 1999), shows improved pharmaco-kinetics, bioavailability, and safety, allowing subcutaneousadministration in the out-of-hospital setting and thus beingsuggested to replace unfractionated heparin in the majorityof thromboembolic therapies (Hirsh et al., 2001; Sundaramet al., 2003). Recently, enormous attention has been paid to arapid onset side effect associated with heparin therapy thatled to angioedema, hypotension, and multiple deaths. Theadverse clinical event, which was believed to be caused bycontaminated heparin or LMWH, has raised an urgentdemand for precise heparin assays (Guerrini et al., 2008,2009; Liu et al., 2009). In the analysis of heparin structureand detection of heparin contaminants, heparin lyaseenzymes play an important role due to their uniquespecificity and high efficiency to digest unfractionatedheparins into small heparin components, as only smalloligosaccharides are amenable to current separationand structure elucidation methods, such as capillaryelectrophoresis, LC–MS, and NMR spectroscopy

Correspondence to: X.-H. Xing

Shuo Chen’s present address is Department of Chemistry and Biotechnology, School

of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656,

Japan.

Contract grant sponsor: National Natural Science Foundation of China

Contract grant number: 20836004; 20676071

Contract grant sponsor: Tokyo Tech–Tsinghua University Joint Graduate Program

� 2011 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 108, No. 8, August, 2011 1841

(Guerrini et al., 2008; Korir and Larive, 2009; Xiao et al.,2010).

Heparin lyases, or heparinases, first isolated fromPedobacter heparinus (formerly Flavobacterium heparinum)(Yang et al., 1985), are enzymes that can cleave heparin andheparan sulfate at the glycosidic linkages between hexosa-mines and uronic acids via a b-elimination mechanism(Ernst et al., 1995). In all three types of heparinases,heparinase I (HepI) is the most well-studied and widely usedone. Besides analyzing heparin and heparin-like GAGs asmentioned above, HepI is also an important tool inneutralizing the heparin concentration in human blood(Ameer et al., 1999; Linhardt et al., 1984) and producingLMWH as an improved anticoagulant (Pervin et al., 1995;Ye et al., 2009). An advantage of enzymatic degradation ofheparin is that the reaction conditions are milder and thereaction can be conducted without the complication ofcompeting side-reactions possibly associated with conven-tional chemical degradation (Korir and Larive, 2009).However, the commercial use of HepI, no matter in the labanalysis of heparin or in the industrial production ofLMWH, has so far been largely hampered by its lowproductivity and poor stability (Korir and Larive, 2009;Sasisekharan et al., 1993; Shpigel et al., 1999).

Our laboratory is involved in a continuing effort towardthe cost-effective and industry-applicable catalysis withheparinase I through improving its productivity andstability. To realize the soluble expression of HepI inE. coli, we fused HepI to maltose-binding protein (MBP)and succeeded in its high-yield production and easypurification (Chen et al., 2005, 2007). We further provedthat MBP-HepI is able to effectively degrade heparin intoLMWH with a narrow polydispersity and anti-factor Xaand IIa activities that meet the standard of EuropeanPharmacopoeia 5.0 (Ye et al., 2009).

However, fusion with MBP failed to overcome the poorstability of HepI. The crude enzyme of MBP-HepIunfavorably exhibited very poor thermostability at itsoptimum temperature of 308C with a half-life of only10min (Kuang et al., 2006). Lohse and Linhardt (1992)reported that, HepI when once being stored for a short periodat 48C or frozen once could retain only 50% and 45% of itsinitial activity, respectively. They also found that bovineserum albumin (BSA) addition could improve the enzymethermostability (Lohse and Linhardt, 1992). It is not hard tounderstand the lack of studies on the stability of HepIregarding the fact that the crystal structure of heparinse I fromBacteroides thetaiotaomicron (PDB code: 3IKW) was justreported very recently (Han et al., 2009) and all of theprevious research on HepI from P. heparinus was based on itspredicted secondary structure. Lacking mechanistic founda-tion, strategic stabilization of HepI remains a problem.

As is known, many genetic and chemical approacheshave been used for enzyme stabilization. For selection ofthe proper stabilization approach, it is always necessary toidentify the inactivation mechanism and accordingly studypossible enzyme–stabilizer interactions to enhance the

rigidity and inhibit the inactivation of the enzyme. In thisstudy, we first gained insight into the thermal inactivation ofMBP-HepI by a combination of biochemical investigationsand kinetic modeling. Then a mechanism-based compre-hensive stabilization strategy was proposed and proved tobe successful. This work takes an important step towardthe commercial use of HepI. To the best of our knowledge,this is also the first stability study on a glycosaminoglycanlyase, whose medical applications are well known butstability properties are rarely reported.

Materials and Methods

Materials

Heparin sodium salt (140 IU/mg) was purchased fromNacalai Tesque (Kyoto, Japan). 8-Anilino-1-naphthalene-sulphonate (ANS) was from Sigma-Aldrich (St Louis, MO).Amylose Resin High Flow was from New England Biolabs(Ipswich, MA). DEAE-Toyopearl 650M was from Tosoh(Tokyo, Japan). Sephacryl S-300 was from GE Healthcare(Piscataway, NJ). Crude MBP-HepI was obtained from E.coli TB1 [pMHS] by the procedure we reported (Chen et al.,2007).

Purification of MBP-HepI

Three steps of chromatography were performed at 48C forthe enzyme purification. Crude enzyme from the disruptionof cultivated cells was first purified by amylase affinitychromatography as we previously reported (Chen et al.,2005). MBP-HepI purified without concentration wassubjected to a DEAE-Toyopearl 650M column (0.7 cm�15 cm) pre-equilibrated with 20mM Tris–HCl buffer(pH 7.4). The column was eluted by a NaCl linear gradientof 0–500mM in the same buffer at 0.5mL/min flow rate.MBP-HepI without concentration was then applied to aSephacryl S-300 column (1.5 cm� 75 cm) equilibrated withthe same buffer as above. Fractions were collected at a flowrate of 6mL/h. Fractions with heparinase activity werepooled and dialysed against 20mM Tris–HCl bufferovernight.

Enzymatic Activity Assay

Heparinase activity was measured according to the UV232 nm method (Bernstein et al., 1988). The enzymaticreaction was carried out at 308C using heparin as substratein Tris–HCl buffer (pH 7.4) (containing 25 g/L heparin,40mM NaCl, 3.5mM CaCl2, and 17mM Tris–HCl).Heparin degradation was monitored by UV absorbance at232 nm on a UV-1206 Spectrophotometer (Shimadzu,Japan) and the activity was calculated using a molarextinction coefficient of 3,800M�1 cm�1. One internationalunit was defined as the amount of protein that can form1mmol unsaturated uronic acid per minute at 308C.

1842 Biotechnology and Bioengineering, Vol. 108, No. 8, August, 2011

Native SDS–PAGE

Enzyme samples were processed without adding 2-mercap-toethanol and boiling. All other steps followed the generalprocedure of Laemmli method (Laemmli, 1970). Thestacking gel of 5% acrylamide was used. The electrophoresiswas carried out at 48C.

Homology Modeling of P. heparinus heparinase I

A three-dimensional model structure of P. heparinus HepIwas built using Modeller (Eswar et al., 2006; Fiser et al.,2000; Marti-Renom et al., 2000; Sali and Blundell, 1993)based on the known crystal structure of B. thetaiotaomicronHepI (BtHepI, PDB code: 3IKW). Structural representationswere generated using PyMOL. An initial sequence alignmentwith the template was performed by ClustalW (Chennaet al., 2003), and the figure was prepared with ESPript(Gouet et al., 1999).

Chemical Modification and Spectroscopic Analysis

The UV spectra of MBP-HepI after 358C incubation for 0, 1,3, and 5min and following 48C incubation for 1 h were takenon the UV-1206 Spectrophotometer to show the proteinstructure change during the ongoing inactivation process.

ANS binding study was performed on an F-2500Fluorescence Spectrophotometer (Hitachi, Japan). Themolar ratio of protein to ANS was 1:50. Enzyme–ANSmixtures were incubated at 48C for 5min, or 358C for 5min,or 358C for 5min followed by 48C for 1 h for comparison.After incubation, the ANS emission of the mixtures wasscanned between 400 and 600 nm with an excitationwavelength of 380 nm.

Far-UV circular dichroism (CD) measurements wereperformed on a Jasco 500C Spectropolarimeter (Jasco,Japan) equipped with a thermostatically controlled holder.

Kinetic Modeling

The mathematical model of the inactivation scheme wasformed by a set of equations as follows.

Reaction rate equations:

dN

dt¼ k�1U�k1N (1)

dU

dt¼ k1N�k�1U�2k2U2 (2)

dD

dt¼ k2U

2 (3)

Ra ¼ N

N0(4)

Mass conservation:

N þ U þ 2D ¼ N0 (5)

Temperature dependence (Arrhenius equation):

lnki ¼ � EiR

1

Tþ lnAi i ¼ 1;�1; 2ð Þ (6)

Initial conditions:

t ¼ 0;N ¼ N0;U ¼ 0;D ¼ 0 (7)

N, U and D stand for the concentration of the protein in thenative, unfolded and dimer form, respectively. ki stands forthe rate constant, Ei for the activation energy and Ai for thepre-exponential factor of the ith reaction in the inactivationmodel. R stands for the standard gas constant. N0 stands forthe initial concentration of the active enzyme. Ra stands forthe residual activity of the enzyme. The fitting with theexperimental data and simulation were conducted with thesoftware MATLAB.

Site-Directed Mutagenesis

The C297S mutation was introduced by the QuikChangeTM

Site-DirectedMutagenesis Kit Stratagene (La Jolla, CA). Theprimers used are shown as follows.

50-primer: 50-CCCTAAAGATTCCTGGATTACTTTT-GACGTCGCCATAG-30

30-primer: 50-CTATGGCGACGTCAAAAGTAATCC-AGGAATCTTTAGGG-30

Effect of Additives on Enzyme Stability

Solutions of 0.6mg/mL native MBP-HepI with variousconcentrations of Tween 80 (0.001% and 0.01%w/v), TritonX-100 (0.001% and 0.01% w/v), trehalose (10%, 20%, and30% w/v), a-lactose (10%, 20%, and 30% w/v), sucrose(10%, 20%, and 30% w/v), Ca2þ (0.1, 0.5, 1, and 2mM),dextran (0.001%, 0.01%, 0.1%, and 1% w/v), glycerol (20%,30%, and 40% w/v), PEG 20000 (5%, 10%, and 20% w/v),PEG 8000 (5%, 10%, and 20% w/v) and DTT (0.5mM) wereincubated at 308C, and the enzymatic activity wasperiodically assayed.

Results and Discussion

Purification and Thermostability of MBP-HepI

MBP-HepI was purified to apparent homogeneity forinactivation studies (Fig. 1). All purification steps were

Chen et al.: Inactivation and Stabilization of MBP-HepI 1843

Biotechnology and Bioengineering

conducted at 48C. The specific activity of the purifiedenzyme was 149 IU/mg, and the yield was 81.3%, both to bethe highest level so far (130 IU/mg, Lohse and Linhardt,1992; 43%, Ernst et al., 1996). Furthermore, a single bandfor monomers in the native SDS–PAGE after purification(lane 1, Fig. 3) confirmed the complete separation of proteinaggregates possibly forming in the expression and purifica-tion processes.

Thermal stability of the purified MBP-HepI wascomprehensively examined. As shown in Figure 2, theenzyme exhibited poor thermostability with a non-first-order inactivation kinetics in a wide temperature range.The inactivation showed strong temperature dependence(Fig. 2a, Table I). It took less than 1min for MBP-HepI tolose 97% of its activity at 708C; while at 308C, the optimumtemperature of the enzyme, the half-life of MBP-HepIwas about 10min. In addition, the inactivation showedstrong concentration dependence. Acceleration of theinactivation with the increase of enzyme concentrationimplies multi-molecular interactions in the thermalinactivation (Fig. 2b).

Intermolecular Disulfide Bond-Induced Dimerization ofMBP-HepI

The change in molecular size is always one of the mostimportant clues to explore the protein inactivationmechanism (Iyer and Ananthanarayan, 2008; Lenckiet al., 1992). Various methods to detect protein size changeshave been employed in protein inactivation studies,including dynamic light scattering (Baptista et al., 2003;Liu et al., 2003), analytical ultracentrifugation (Kurganovet al., 2000; Urano et al., 2006), native SDS–PAGE (Kembleand Sun, 2009), etc. In our study, native SDS–PAGE of theinactivated MBP-HepI gave a dimer band and the bandturned darker with the progress of the inactivation (lanes 1–7, Fig. 3), clearly revealing the formation of dimers whenMBP-HepI became inactivated. The dimer band disap-peared by adding 2-mercaptoethanol into the inactivatedenzyme sample (lane 8, Fig. 3), suggesting that the dimers

Figure 2. Experimental and simulated time courses of the thermal inactivation of

MBP-HepI. Experimental data are shown in points, and simulated data are shown in

solid lines. a: Time courses of the residual activity of MBP-HepI (0.6 mg/mL) incubated

at different temperatures: 308C (*), 358C (&), 388C (^), 408C (~), 458C ( ). b: Time

courses of the residual activity of MBP-HepI at different concentrations incubated at

358C: 0.15 mg/mL (~), 0.3 mg/mL (&), 0.6 mg/mL (*). [Color figure can be seen in the

online version of this article, available at http://wileyonlinelibrary.com/bit]

Figure 1. SDS–PAGE after different steps in the purification of MBP-HepI.

Std, protein standards; Lane 1, total E. coli protein containing MBP-HepI; 2, after

purification by affinity chromatography; 3, after purification by anion exchange

chromatography; 4, after purification by gel filtration chromatography.


came from the formation of intermolecular disulfide bonds.In order to examine the reversibility of the heat-induceddimerization, cooling treatment at 48C was applied to thepartially inactivated MBP-HepI. No obvious change in thedimer band was observed after cooling treatment (lane 5,Fig. 3), proving the irreversibility of the dimerization or theformation of disulfide bonds. But we surprisingly observed arecovery of the enzymatic activity during the coolingtreatment, which will be discussed later.

The above results indicated that there was an irreversibledimerization step induced by disulfide bonds in theinactivation of MBP-HepI. Although in most cases proteinaggregation is due to intermolecular interactions between itshydrophobic regions, the formation of intermoleculardisulfide bonds, especially if one of the thiol groups islocated at or near the active site, can be another vital factorto facilitate aggregation and destroy the enzymatic activity(Lencki et al., 1992). There are two cysteine residues inMBP-HepI, both located in the heparinase region that

possibly contribute to the formation of disulfide bonds. Toreveal the positions of the two cysteine residues inP. heparinus HepI (PhHepI), a homology model of theenzyme was built using the known crystal structure ofB. thetaiotaomicron HepI (BtHepI, PDB code: 3IKW) as thetemplate. Sequence alignment showed that PhHepI shares68% identity with the template, and the only two cysteines inPhHepI are both conserved in BtHepI which has as many assix cysteines (Fig. 4a). As expected, PhHepI displayed asimilar structure to that of BtHepI, consisting of a b-jellyrolldomain harboring a long and deep substrate binding grooveand a thumb-resembling extension (Fig. 4b). Although thetwo cysteine residues Cys135 and Cys297 are too far apartfor intramolecular disulfide bond formation, they are bothexposed at the surface of the protein, revealing strongtendency to form intermolecular disulfide bonds (Fig. 4band c). It has been reported that one of the cysteines, Cys135,plays a critical role in the endolytic cleavage of heparin byPhHepI, while the other Cys297 does not show any essentialfunction (Sasisekharan et al., 1995). The nucleophilic aminoacid Cys135, surrounded by a positively charged environ-ment, is believed to be involved in the abstraction of the C5proton on the uronate of the disaccharide repeat unit ofheparin polysaccharides, and initiate the elimination-baseddepolymerization of heparin (Sasisekharan et al., 1995).Thus the formation of disulphide bonds at Cys135 willprobably force it to lose the right functioning as anucleophile, leading to the quick irreversible loss of theenzymatic activity.

The heat-induced dimerization of HepI provides a goodexplanation to Lohse and Linhardt (1992)’s finding that thethermostability of HepI can be improved by adding BSA.The addition of an inert protein probably helped reduce themolecular collision and thereby inhibited the dimerizationand inactivation of HepI.

However, dimerization is not the only or even the mainprocess in the inactivation of MBP-HepI, as for all theinactivated enzyme samples in Figure 3, even for that withthe residue activity as low as 2.6%, dimer only counted for asmall portion of the total enzyme content, revealing thatmost of the enzyme was inactivated in its monomeric statethrough unfolding, a detailed study of which is presented inthe next part.

Figure 3. Native SDS–PAGE of MBP-HepI (0.6 mg/mL) at different inactivation

degrees after 358C incubation. Std, protein standards; Lane 1, Ra (Residual activity)

100%; 2, Ra 62.8%; 3, Ra 40.2%; 4, Ra 36.0%; 5, the protein in 4 after cooling treatment

for 1 h, Ra 53.9%; 6, Ra 19.2%; 7, Ra 2.6%; 8, reducing SDS–PAGE of the protein in 7 (add

2-mercaptoethanol and boil during sample processing).

Table I. Reactivation of MBP-HepI (0.6mg/mL) inactivated under different conditions by subsequent

incubation at 48C for 1 h.

Inactivation

temperature (8C)Inactivation time

(min)

Residual activity after

inactivation (%)

Residual activity after

cooling treatment (%)

30 5 62.2� 3.2 87.3� 4.1

35 4 49.2� 2.5 70.6� 3.6

40 3 44.0� 2.8 61.9� 4.0

45 2 28.1� 1.8 43.5� 1.6

60 1 9.2� 0.7 20.9� 2.0

70 1 2.5� 0.3 2.1� 0.4

75 1 1.4� 0.2 1.1� 0.1

90 1 0.5� 0.1 0.5� 0.1



Temperature-Dependent Reversible Unfoldingof MBP-HepI

As mentioned above, the activity of inactivated MBP-HepIcould partially recover at 48C. Further investigation showedthat the reactivation tended to complete within 1 h. In a

detailed study (Table I), we found that the activity of MBP-HepI at different inactivation degrees after heat treatmentbelow 708C could all be partially recovered at 48C. But whenthe temperature for incubation was above 708C, the enzymedid not show any reactivation no matter how long itwas placed at 48C. The strong reactivation behavior of

Figure 4. Homology model of PhHepI. a: Alignment of the amino acid sequences of PhHepI and BtHepI. Identical and similar amino acid residues are shown by white letters

on a red background and red letters on a white background, respectively. Two cysteines conserved in PhHepI and BtHepI are indicated by gray stars. b: Superposition of the

structure model of PhHepI (cyan) and the crystal structure of BtHepI (gray, PDB code: 3IKW). Conserved cysteines in the two proteins are indicated by markers in red and blue,

respectively. c: Surface representation of the structure model of PhHepI. Cys135 and Cys297 on the surface are shown in red.


inactivated MBP-HepI implied a highly reversible unfoldingprocess in its inactivation. A simple UV spectroscopicanalysis was carried out to confirm this.

The UV spectrum of inactivated MBP-HepI (0.03mg/mL) at a low inactivation degree showed obvious blue shiftand intensity increase of the absorbance around 280 nmcompared with the native enzyme (Fig. 5a), indicating theformation of an unfolded protein intermediate with moretryptophans exposed. And after subsequent cooling treat-ment at 48C, the UV spectrum exhibited remarkablerecovery toward that of the native state (Fig. 5a), clearlyindicating the reversibility of the unfolding.

The temperature-induced reversible unfolding of MBP-HepI was further confirmed by ANS modification analysis(Fig. 5b). After 358C incubation, the exposure of hydro-phobic regions in the protein, happening along with theunfolding, resulted in more ANS binding. And a following48C treatment led to decreased ANS fluorescence, indicatingthe decrease of exposed hydrophobic regions and therefolding of the protein.

A remaining question is the temperature dependence ofthe reversibility of the unfolding. As mentioned above, the

unfolding of MBP-HepI was partially reversible below 708Cbut not above 708C. So what makes the difference betweenthe unfolding below and above 708C? To answer thisquestion, we looked into the secondary structure change ofMBP-HepI in its inactivation by far-UV CD spectrum.

Surprisingly no secondary structure change was observedin the inactivation ofMBP-HepI at 358C (Fig. 5c), indicatingthat the inactivation of the enzyme active site preceded itsextensive structural change (Tsou, 1995). Furthermore, inthe process of raising the temperature, no obvious secondarystructure change was observed until 708C, and the enzymeinactivated above 708C did not show any recovery in its CDspectrum during cooling treatment at 48C (Fig. 5d). Thusthe secondary structure of MBP-HepI changed irreversiblyabove 708C, which happened to be the same temperaturefor the enzyme to undergo irreversible inactivation. Thisparticular coincidence implied that the irreversible lossof the enzyme activity above 708C was due to extensiveunfolding of its secondary structure which ruled outthe possibility of tertiary refolding. Whereas below 708C,there was no change in the secondary structure of MBP-HepI, and the inactivation of MBP-HepI only resulted from

Figure 5. Changes in the structural properties of MBP-HepA during thermal inactivation. a: UV profiles of MBP-HepI (0.03 mg/mL) after 358C incubation for 0, 1, 3, 5 min and

following 48C incubation for 1 h. b: Fluorescence spectra of ANS in the presence of 0.03 mg/mL MBP-HepI [ANS:MBP-HepI¼ 50:1 (mol/mol)] after 48C incubation for 5min, 358Cincubation for 5 min and following 48C incubation for 1 h. c: Far-UV CD spectra of MBP-HepI (0.6 mg/mL) during the thermal inactivation at 358C. d: Far-UV CD spectra of MBP-HepI

(0.6 mg/mL) during the thermal inactivation in a temperature rising process.



unfolding of its tertiary structure, especially its active sitegeometry, while the reactivation accordingly resulted fromthe refolding of its tertiary structure. To our knowledge,a similar pattern of the reversible thermal inactivation thatshows structural changes only on the protein tertiary but notsecondary structure is rarely reported. A known case is latexproxidase from Euphorbia characias reported by Mura et al.(2005).

Kinetic Modeling Based on the Experimentally DerivedInactivation Pathway

To investigate the mechanism of enzyme inactivation,kinetics-based simulation of the effect of environmentalfactors on the enzymatic activity provides an alternativeapproach (Ladero et al., 2006; Sadana, 1988). From theabove experimental studies, we have derived a seriespathway for the inactivation of MBP-HepI below 708C,

N ��! ��k1

k�1U �!k2 D Dimerð Þ

in which N stands for native, U for unfolded and D for dimerof MBP-HepI. Only the native enzyme (N) shows enzymaticactivity. In order to make a multi-temperature evaluation ofthe above model, Arrhenius equation (Eq. 6) was employedto constrain the temperature dependence of the inactivationrate constant ki.

A comparison between the inactivation curve derivedfrom kinetic modeling and the experimental data is shownin Figure 2. The proposed series inactivation model was ableto fit the experimental data adequately within a widetemperature range, from the enzyme optimum temperatureof 308C at which the inactivation was relatively slow to 458Cat which the enzyme lost its activity very fast (Fig. 2a).The inactivation rate constants at different temperaturesobtained from the modeling are presented in Table IIa.Moreover, at different enzyme concentrations, the simula-tion also showed good consistency with the experimental

data (Fig. 2b), further proving the adequacy of the proposedinactivation model. The estimated activation energies in theinactivation model are presented in Table IIb.

The modeling has provided a kinetic view for thetemperature-induced reactivation of MBP-HepI. Accordingto our calculation, the unfolding of MBP-HepI hasan obvious higher activation energy (200.1 kJ/mol) thanrefolding (149.2 kJ/mol), which makes unfolding moresensitive to the temperature change. So when thetemperature was lowered from 35 to 48C, the rate constantof unfolding decreased by 6000 times while that of refoldingdecreased by only 600 times (Table IIa). This explains whyMBP-HepI underwent reactivation during the coolingtreatment at 48C after incubation at 358C.

Mechanism-Based Strategy for the ThermalStabilization of MBP-HepI

The above-presented inactivation biochemistry provides amechanistic basis for improving the thermostability ofMBP-HepI. Approaches for inhibiting the two inactivationsteps, naming the unfolding and dimerization, were studied,respectively and then combined for a comprehensivestabilization strategy.

Considering the strong reversibility of the unfolding ofMBP-HepI, we tried to shift the unfolding equilibrium tofavor the native structure of the enzyme. We noted that,in some cases, enzymes showing reversibility in thermalinactivation exhibit a requirement of metal ions for therecovery of enzymatic activity, such as glucose dehydro-genase (Geiger and Gorisch, 1989), lignin peroxidase (Nieand Aust, 1997), and latex peroxidase (Mura et al., 2005).Recently it has become clear that, there is a Ca2þ ion boundin the hinge region between the b-jellyroll domain and thethumb domain of HepI that aids the structural integrity ofthe enzyme (Han et al., 2009), implying that the loss of Ca2þ

will probably accompany the unfolding of HepI. Thus weintentionally added Ca2þ intoMBP-HepI and found that theresidual activity of MBP-HepI after 30min incubation at308C was successfully increased to 1.5 times upon theaddition of 1mM Ca2þ, the concentration of which wasoptimized (Fig. 6a).

We also tried various other stabilizing additives to inhibitthe unfolding of MBP-HepI, including surfactants (Tween80 and Triton X-100), sugars (trehalose, a-lactose, sucrose,and dextran), and polyols (glycerol, PEG 20000, and PEG8000). It has been reported that these additives exertstabilizing effects in a way that the intrinsic conformationalstability of the protein molecule itself is not increased butits unfolding is greatly disfavored due to the inductionof the preferential hydration of the protein (Iyer andAnanthanarayan, 2008; O’Fagain, 2003). As shown inFigure 6a, all additives except PEG improved the thermo-stability of MBP-HepI, in which surfactants induced betterstabilization effects than sugars and polyols. Notably,0.001% Tween 80 brought a dramatic threefold increase

Table II. Estimated parameters in the thermal inactivation model of

MBP-HepI.

Inactivation

temperature (8C) k1 (min�1) k�1 (min�1) k2 (M�1min�1)

(a) Rate constants

4 2.95� 10�2 6.50� 100 1.49� 101

30 4.87� 101 1.63� 103 9.76� 101

35 1.77� 102 4.26� 103 1.35� 102

38 3.76� 102 7.48� 103 1.64� 102

40 6.16� 102 1.08� 104 1.86� 102

45 2.06� 103 2.66� 104 2.52� 102

Process Parameter Value (kJ/mol)

(b) Activation energies

Unfolding E1 200.1

Refolding E�1 149.2

Dimerization E2 50.7

Values derived from the best fit are listed in the table.


in the residual activity of MBP-HepI after 308C incubationfor 30min (Fig. 6a).

Approaches to inhibit the disulfide bond-induceddimerization of MBP-HepI were also studied. In a previousstudy, researchers successfully improved the monodispersityand activity of MBP-HPV E6 fusion protein by substitutingall the cysteines in HPV E6 to serines to avoid the formationof intermolecular disulfide bonds (Nomine et al., 2001). Inour case, regarding that the mutation at Cys135 would fullyabolish the enzymatic activity (Sasisekharan et al., 1995), weintroduced cysteine-to-serine substitution at Cys297. As

anticipated, the mutant showed improved thermostabilityalthough not remarkable (Fig. 6b). Further investigationshowed that, the single mutation of C297S reduced but didnot completely prohibit dimerization, as there was still someformation of intermolecular disulfide bonds betweenCys135 itself, indicated by native SDS–PAGE (data notshown). As an alternative way, DTT was added into MBP-HepI and the residual heparinase activity after 308Cincubation was also improved (Fig. 6b).

Taking into account the above results, a comprehensivethermostabilization strategy was proposed, in which Ca2þ

Table III. Mechanism-based comprehensive strategy for the thermal stabilization of MBP-HepI (0.6mg/mL).

Modification

RA after 30min, 308Cincubation (%)

Half-life at

308C (min)

RA after 1 week,

48C storage (%)

RA after a single

freeze-thawing (%)

Control 23.1� 1.2 10 11.3� 2.4 48.1� 1.2

C297S 30.2� 1.1 13.5 21.2� 1.2 53.2� 2.2

0.5mM DTT 40.1� 2.1 19 30.9� 2.3 55.1� 1.9

1mM Ca2þ 33.6� 1.5 15 34.9� 1.8 56.7� 2.8

0.001% Tween 80 71.3� 2.8 �90 76.2� 2.6 84.7� 3.1

C297Sþ 0.5mM DTT 43.1� 1.3 22 35.1� 1.9 60.5� 2.2

C297Sþ 0.5mM DTTþ 1mM Ca2þ 50.7� 2.9 29.5 45.6� 2.9 67.8� 3.0

C297Sþ 0.5mM DTTþ 1mM Ca2þþ 0.001% Tween 80 87.9� 2.7 �160 95.1� 2.1 97.3� 1.5

RA, residual activity.

Figure 6. Strategies for the thermal stabilization of MBP-HepI (0.6 mg/mL): residual activity of MBP-HepI after incubation at 308C for 30min with different modifications.

a: Attempts to inhibit protein unfolding by additives. b: Attempts to inhibit disulfide bond-induced dimerization by site-directed mutagenesis and DTT addition. The concentration

shown for each additive has been optimized for maximum residual activity.



and Tween 80 were used to inhibit unfolding while sitemutation at C297 and DTT were employed to suppress thedimerization. As shown in Table III, the thermostability ofMBP-HepI was improved upon the addition of each newmodification into the stabilization strategy, and finallyreached the highest when all four modifications wereincluded. The addition of Tween 80 makes the largestcontribution to the stabilization, in agreement with ouranalysis that unfolding makes up the larger portion in theinactivation rather than dimerization. The thermostabilizedenzyme preparation showed a half-life of 160min at itsoptimum temperature of 308C (Table III), 16 times of thatof the original enzyme preparation. And when stored at 48Cfor 1 week or frozen once, the engineered enzymepreparation maintained more than 95% of its activity(Table III), 2–8 times of that before modification, muchmore stable than the original HepI as well (Lohse andLinhardt, 1992).

In summary, we proposed and proved a comprehensivestrategy for the thermal stabilization of MBP-fused HepIbased on its inactivation mechanism derived frombiochemical analysis and kinetic modeling. The engineeredenzyme of MBP-HepI has advantages over HepI producedby other systems for its high storage and operationalstabilities, as well as high-level functional expression, high-yield purification, and high specific activity (Table IV).Therefore, it shows great potential as a commercialbiocatalyst for heparin degradation in the pharmaceuticalindustry.

The authors would like to thank Dr. Minze Jia from the Institute of

Biophysics, Chinese Academy of Sciences for assistance in protein

structure analysis and helpful discussion. This work was supported by

the National Natural Science Foundation of China (Nos. 20836004

and 20676071). Shuo Chen was supported by the Tokyo Tech–

Tsinghua University Joint Graduate Program.

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