Improving the Activity of Immobilized Subtilisin bySite-Specific Attachment to Surfaces

Wei Huang, Jianquan Wang, Dibakar Bhattacharyya,† and Leonidas G. Bachas*

Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506-0055

Understanding the properties of immobilized proteins iscritical to the optimal design of biosensors, biosepara-tions, and bioreactors. The protease subtilisin BPN′ wasused as a model protein to study how the orientation ofimmobilized enzyme molecules on surfaces affects theircatalytic properties. To achieve this goal, a single cysteineresidue was introduced into the cysteine-free enzyme bysite-directed mutagenesis. This cysteine residue wasdesigned to be away from the active site of the enzyme.The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on severalsupports. This site-specific immobilization method leadsto ordered two-dimensional arrays of enzyme moleculeson the support surface with the active sites of the enzymeoriented toward the solution phase. Such oriented im-mobilized subtilisin demonstrated a higher catalytic ef-ficiency compared to subtilisin that was immobilized bya conventional method that leads to random immobiliza-tion.

Protein immobilization is of fundamental importance in manyapplications of bioanalytical chemistry. Among these are thedevelopment of biosensors, bioreactors, immunoassays, andbioseparations.1-4 Chemical immobilization methods involvecovalent bonding of protein molecules to insoluble supports and,thus, typically lead to a stronger binding and longer lifetime thanphysical methods of immobilization (e.g., adsorption). In chemicalimmobilizations, functional groups on the surface of a proteinmolecule, such as amino and carboxyl groups, are often used inthe attachment to the support. Since each protein moleculenormally has many such functional groups distributed on itssurface, the orientation of a protein molecule on the supportsurface is random with regard to the amino acid residue(s) servingas the site of attachment. If the site of attachment is close to theactive site (or the binding site) of the protein molecule, the activity(or binding capacity) of the protein molecule may be partially ortotally lost due to steric effects. This is one of the reasons that adecrease in activity (or binding capacity) is often observed inchemical immobilizations.5

Recently, efforts have been made to study the orientationeffects in protein immobilizations. The key to controlling theorientation of immobilized protein molecules on a support is toselectively attach the protein from a predetermined site on theprotein surface. Antibodies have been site-specifically immobilizedthrough SH groups on Fab′ fragments so that the binding site isexposed to the solution phase.6,7 Antibodies can also be site-specifically immobilized through the carbohydrate moiety on theFc region of the protein or through the selective binding of theprotein A or protein G to a specific site on the Fc region.8 Selectiveattachment to surfaces can also be achieved for a limited numberof proteins by controlling the chemical immobilization conditions.For example, horseradish peroxidase has been selectively bioti-nylated and immobilized by such an approach.9 However, for themajority of proteins that need to be immobilized, the functionalgroups used for the attachment can be found in more than onelocation on the protein surface. It is therefore difficult to achievesite-specific immobilization by simply changing the chemicalreaction conditions.

Genetic engineering provides a way to produce large amountsof desired proteins in simple organisms such as Escherichia colior yeast.10 It also makes it possible to modify the protein ofinterest on the DNA level in order to introduce desired propertiesto the protein.11 In general, two different approaches have beenused to modify proteins for controlled immobilization. An affinitytag can be attached to the protein of interest by a gene fusionapproach; the resultant fusion protein conjugate can then beimmobilized through the affinity tag on appropriately modifiedsurfaces.12,13 This method is limited because affinity tags can onlybe attached to the amino or the carboxy terminus of the proteinof interest. The other approach is to introduce a unique aminoacid residue with a specific side-chain functional group to theprotein molecule by site-directed mutagenesis. The proteinmolecule can then be attached through this functional group,leading to a controlled orientation on the surface. Cysteineresidues have been genetically introduced into glucose dehydro-genase and lactate dehydrogenase, and the modified enzymeshave been used in site-specific immobilization.14,15 However,

* Address correspondence to this author at (e-mail) bachas@pop.uky.edu,(phone) (606)257-6350, or (fax) (606) 323-1069.

† Department of Chemical and Materials Engineering, University of Kentucky,Lexington, KY 40506-0046.(1) Katchalski-Katzir, E. Trends Biotechnol. 1993, 11, 471-478.(2) Mazid, M. A. Bio/Technology 1993, 11, 690-695.(3) Scouten, W. H.; Luong, J. H. T.; Brown, R. S. Trends Biotechnol. 1995, 13,

178-185.(4) Taylor, R. F., Ed. Protein Immobilization: Fundamentals and Applications;

Marcel Dekker: New York, 1991.

(5) Srere, P. A.; Uyeda, K. Methods Enzymol. 1976, 44, 11-19.(6) Spitznagel, T. M.; Clark, D. S. Bio/Technology 1993, 11, 825-829.(7) Lu, B.; Smyth, R. M.; O’Kennedy, R. Analyst 1996, 121, 29R-32R.(8) Hoffman, W. L.; O’Shannessy, D. J. J. Immunol. Methods 1988, 112, 113-

120.(9) Rao, S. V. Ph.D. Dissertation, University of Kentucky, 1996.

(10) Glick, B. R.; Pasternak, J. J. Molecular Biotechnology: Principles & Applica-tions of Recombinant DNA; ASM Press: Washington, DC, 1994.

(11) Wiseman, A. J. Chem. Technol. Biotechnol. 1993, 56, 3-13.(12) Vishwanath, S. K.; Bhattacharyya, D.; Huang, W.; Bachas, L. G. J. Membr.

Sci. 1995, 108, 1-13.(13) Vishwanath, S. K.; Watson, R. C.; Huang, W.; Bachas, L. G.; Bhattacharyya,

D. J. Chem. Technol. Biotechnol. 1997, 68, 294-302.

Anal. Chem. 1997, 69, 4601-4607

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glucose dehydrogenase and lactate dehydrogenase are multisub-unit enzymes, and the supports used in these studies arecommercial porous agarose beads of different sizes. Under theseconditions, it is difficult to study the effect of orientation of theimmobilized protein molecules on their activity. A cysteineresidue has also been genetically introduced at the C-terminus ofdihydrofolate reductase, and the modified enzyme has beenimmobilized onto a polymer support and gold surfaces.16,17 Inanother study, Sligar and co-workers have genetically introducedsingle cysteine residues at different sites of the heme proteincytochrome b5 and studied the site-specific immobilization of theprotein.18 These mutant proteins were also used to investigatemacromolecular recognition events.

In the present study, subtilisin BPN′ is used as a model proteinto investigate site-specific immobilization of proteases. Proteasesare an important class of enzymes for biochemical analysis. Theyare used in the free or immobilized state to generate fragmentationprofiles of native and modified proteins, which aid the character-ization and identification of proteins.19,20 For example, Amankwaand Kuhr have immobilized the protease trypsin on the innersurface of a fused-silica capillary for on-line digestion of picomolequantities of proteins.21,22 This enzyme-modified capillary wasused in conjunction with capillary zone electrophoresis to yieldtryptic maps for the characterization and identification of proteins.Other applications of proteases include the peptide synthesis andthe enantioselective hydrolysis of chiral amino acid esters inorganic solvents.23,24 Subtilisin BPN′ is a single-chain, cysteine-free enzyme from Bacillus amyloliquefaciens. A single cysteineresidue was introduced by site-directed mutagenesis into subtilisinat a location away from the active site of the enzyme. Themodified enzyme was immobilized onto several supports, includingnonporous, uniformly sized silica particles through the introducedunique SH group on the enzyme molecule. This site-specificimmobilization yields two-dimensional arrays of enzyme moleculeson the support surface with the active sites uniformly orientedtoward the solution phase. The kinetic characteristics of thissystem were compared to that of a randomly immobilizedsubtilisin, where the enzyme molecules were attached throughthe NH2 groups of lysine residues and the N-terminus onto amino-functionalized particles.

EXPERIMENTAL SECTIONReagents. An expression vector (pSbt) containing the sub-

tilisin BPN′ gene from B. amyloliquefaciens and a protease-deficientstrain of Bacillus subtilis (trpC2, lys-3, metB10, npr, apr::cat) usedto express subtilisin were kindly provided by Dr. Philip N. Bryan

(University of Maryland).25,26 The oligonucleotides used formutagenesis were from the University of Kentucky Macromo-lecular Structure Analysis Facility (Lexington, KY). The cross-linker N-γ-maleimidobutyryloxysuccinimide ester (GMBS), Ell-man’s reagent [5,5′-dithiobis(2-nitrobenzoic acid)] and Reduce-Imm column were from Pierce (Rockford, IL). Affi-Gel 501organomercurial agarose was from Bio-Rad Laboratories (Her-cules, CA). Luria-Bertani (LB) medium was from BIO 101 (Vista,CA). The nonporous silica beads functionalized with aminogroups that were used to immobilize subtilisin were from BangsLaboratory (Carmel, IN) and had an average diameter of 0.8 µm(stock no. S008003AN); the amino-functionalized nonporous silicabeads used to test for nonspecific binding (also from BangsLaboratory) had an average diameter of 0.5 µm. Activated thiolSepharose 4B, thiopropyl Sepharose 6B, glutaraldehyde, ampicil-lin, kanamycin, dithiothreitol (DTT), Bis-Tris propane [1,3-bis[tris-(hydroxymethyl)methylamino]propane], N,N-dimethylformamide(DMF) (this was subsequently dried in our laboratory), thesubstrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPF-pNA),and all other reagents were from Sigma (St. Louis, MO). Thefollowing buffers were used: subtilisin buffer (0.0100 M phos-phate, pH 6.2), substrate buffer (0.100 M Tris-HCl, pH 8.6), andPBS buffer (0.0200 M phosphate, 0.150 M NaCl, pH 7.0).

Site-Directed Mutagenesis. All molecular biology experi-ments were performed according to standard protocols.27 Theserine 249 to cysteine (S249C) mutation was performed accordingto an oligonucleotide-directed mutagenesis protocol using theAltered Sites in vitro mutagenesis kit from Promega (Madison,WI). The EcoRI-SalI fragment of pSbt containing the subtilisingene was cloned into the pAlter-1 vector to produce a new vectordesignated as pLGB101. A primer with the nucleotide sequence5′-CACTCAAGTCCGCAGCTGTCTAGAAAACACCACTAC-3′ wasdesigned to introduce both a serine 249 to cysteine mutation anda silent mutation to incorporate a unique XbaI restriction site. Afterthe annealing reaction (performed according to the manufacturer’sinstructions), the DNA product was transformed into a mismatch-repair-minus strain of E. coli, BMH 71-18 mut S (thi, supE, ∆-(lac-proAB), [mutS::Tn10] [f ′, proA+B+, laqIqZ∆m15]), and thecolonies were selected on an LB plate containing 100 mg/Lampicillin. A single colony, which contains a plasmid that has aXbaI restriction site, was selected, and the plasmid is designatedas pLGB102. The desired mutation was confirmed by DNAsequencing. The EcoRI-SalI fragment of pLGB102 containingthe mutated subtilisin gene was cloned back into the expressionvector pSbt, and the new plasmid is designated as pLGB103.

Site-directed mutagenesis was also performed according to anoverlap extension protocol using the polymerase chain reaction(PCR).28 The primer 5′-GCAACGACTACGACGCCGCATGCAACG-GCTTTATC-3′ was designed to introduce a serine 145 to cysteine(S145C) mutation and simultaneously a silent mutation thatincorporates a unique SphI site, which can be used to verify themutation. A 1100-bp SplI-SalI fragment of pLGB101 was syn-thesized by overlap extension with PCR to introduce the desiredmutation and placed back into pLGB101 to form a new vector

(14) Persson, M.; Bulow, L.; Mosbach, K. FEBS Lett. 1990, 270, 41-44.(15) Kallwass, H. K. W.; Parris, W.; McFarlane, E. L. A.; Gold, M.; Jones, J. B.

Biotechnol. Lett. 1993, 15, 29-34.(16) Iwakura, M.; Kokabu, T. J. Biochem. 1993, 114, 339-343.(17) Vigmond, S. J.; Iwakura, M.; Mizutani, F.; Katsura, T. Langmuir 1994, 10,

2860-2862.(18) McLean, M. A.; Stayton, P. S.; Sligar, S. G. Anal. Chem. 1993, 65, 2676-

2678.(19) Cottrell, J. S. Pept. Res. 1994, 7, 115-124.(20) Gharahdaghi, F.; Kirchner, M.; Fernandez, J.; Mische, S. M. Anal Biochem.

1996, 233, 94-99.(21) Amankwa, L. N.; Kuhr, W. G. Anal. Chem. 1993, 65, 2693-2697.(22) Amankwa, L. N.; Kuhr, W. G. Anal. Chem. 1992, 64, 1610-1613(23) Reimann, A.; Robb, D. A.; Halling, P. J. Biotechnol. Bioeng. 1994, 43, 1081-

1086.(24) Ricks, E. E.; Estrada-Valdes, M. C.; McLean, T. L.; Iacobucci, G. A.

Biotechnol. Prog. 1992, 8, 197-203.

(25) Strausberg, S.; Alexander, P.; Wang, L.; Gallagher, T.; Gilliland, G.; Bryan,P. Biochemistry 1993, 32, 10371-10377.

(26) Fahnestock, S. R.; Fisher, K. E. Appl. Environ. Microbiol. 1987, 53, 379-384.

(27) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold SpringHarbor Laboratory Press: New York, 1989.

(28) Horton, R. M.; Pease, L. R. In Directed Mutagenesis: a Practical Approach;McPherson, M. J., Ed.; IRL Press: New York, 1991.

4602 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997

pLGB104. The EcoRI-SalI fragment of this vector was clonedinto the expression vector pSbt to form pLGB105. DNA sequenc-ing confirmed the S145C mutation in pLGB105. A nonspecificmutation, which corresponds to a glycine 98 to serine mutationwas also found.

The expression vectors pSbt, pLGB103, and pLGB105 wereused to express wild-type subtilisin, subt-1 (S249C), and subt-2(S145C/G98S), respectively.

Expression of Subtilisin. Competent B. subtilis was preparedaccording to a modified Groningen protocol.29 Specifically, theminimal growth medium (MGM) suggested by this protocol wassupplemented with 20 mg/L tryptophan, lysine, and methionineto maintain the growth of the protease-deficient strain of B. subtilis.The transformation of expression vectors into B. subtilis was doneaccording to the same protocol. Clones containing the plasmidwere selected on an LB plate containing 20 mg/L kanamycin.Expression of extracellular subtilisin was observed with theformation of a “halo” around each colony when grown on an agarplate containing 1% (w/v) of powdered milk. For large-scaleexpression of subtilisin, an overnight culture of the transformedB. subtilis was diluted (1:100) into LB media containing 20 mg/Lkanamycin and shaken in baffled flasks at 250 rpm and 37 °C for24 h.

Purification of Subtilisin. Extracellular subtilisin was pre-cipitated from the cell culture supernatant by adding ammoniumsulfate to 85% saturation. The precipitate was redissolved intosubtilisin buffer and dialyzed against this buffer overnight. ABioCAD Sprint perfusion chromatography system (PerSeptiveBiosystems, Framingham, MA) equipped with a POROS HS cationexchange column was used to further purify the subtilisin usingan elution buffer at pH 4.5. The elution step was done by runninga gradient of NaCl from 0 to 1.5 M. Subtilisin was eluted at0.25 M NaCl and was purified to over 95% homogeneity by thisprotocol as estimated from SDS-PAGE. The purified subtilisinwas dialyzed against subtilisin buffer and stored at 4 °C or afterlyophilization at -20 °C. The concentration of subtilisin wasdetermined by its absorbance at 280 nm.30,31 The average yieldof subtilisin using this protocol was 20 mg/L of culture.

Determination of the Number of Cysteines. Purifiedmutant subtilisin with one cysteine on each molecule tends toform an intermolecular disulfide linkage and does not react withEllman’s reagent. A Reduce-Imm column was used to reduce theintermolecular disulfide bond and regenerate the free sulfhydrylgroup. The prepacked column with 2 mL of gel was equilibratedwith 5 mL of subtilisin buffer, and activated with 10 mL of subtilisinbuffer containing 10 mM DTT. After the column was rinsed with20 mL of subtilisin buffer, 1 mL of subtilisin solution was loadedto the column, and the resultant mixture was incubated at roomtemperature for 1 h. Then, the reduced protein was eluted withsubtilisin buffer, and the 1-mL fractions collected were tested forprotein concentration. The fractions containing the highestconcentration of subtilisin were pooled. A volume of 500 µL ofthe freshly reduced subtilisin was mixed with 50 µL of 4 mg/mLEllman’s reagent and 950 µL of subtilisin buffer and was incubatedat room temperature for 15 min. The change in absorbance at

412 nm was measured, and the concentration of free sulfhydrylgroup was calculated. From this, the number of sulfhydryl groupsper molecule of protein was estimated.

Immobilization on Agarose Beads. The commerciallyavailable activated thiol Sepharose 4B, thiopropyl Sepharose 6B,and Affi-Gel 501 organomercurial beads were reswelled accordingto the manufacturers’ instructions and washed three times withthe subtilisin buffer. A volume of 1 mL of subtilisin buffercontaining 1 mg of freshly reduced mutant subtilisin was incubatedwith 500 µL of the gel. The reaction was continued at 4 °Covernight with gentle shaking. The suspension was then centri-fuged, and the beads were rinsed three times with the subtilisinbuffer. The coupling yield (percentage of the enzyme im-mobilized) was calculated from the residual activity in thesupernatant and the washing solutions. The beads were resus-pended in the subtilisin buffer and stored at 4 °C.

Random Immobilization on Silica Beads. The reaction wasperformed in a 1.5-mL microcentrifuge tube with the end flattenedon flame. A volume of 1 mL of a 10% (w/v) suspension ofnonporous silica beads with amino groups on the surface fromBangs Laboratory was washed three times with the PBS bufferand centrifuged. A fresh solution of 8% (v/v) glutaraldehyde inPBS was added to the beads and incubated at 4 °C on a rotaryshaker overnight. The beads were then centrifuged and rinsedthree times with PBS buffer. A volume of 1 mL of subtilisinsolution (0.50 mg/mL) in PBS was added to the beads in themicrocentrifuge tube, and the tube was vortexed. The reactionwas allowed to continue overnight at 4 °C on a rotary shaker.Then, the beads were centrifuged and rinsed three times withthe PBS buffer. The coupling yield was calculated from theenzymatic activity in the supernatant and washing solutions asmentioned above.

Site-Directed Immobilization on Silica Beads. A volumeof 1 mL of a 10% (w/v) suspension of silica beads functionalizedwith amino groups on the surface from Bangs Laboratory waswashed three times with dry DMF and centrifuged. To this tube,1 mL of a GMBS/DMF solution (containing 10 mg of GMBS)was added, and the tube was gently shaken on a rotary shaker at4 °C overnight. The beads were then centrifuged and washedthree times with the PBS buffer. A volume of 1 mL of PBSsolution containing 1 mg of freshly reduced mutant subtilisin wasadded to the tube, and the beads were resuspended by vortexing.The reaction continued overnight at 4 °C with gentle shaking.Finally, the beads were centrifuged and rinsed three times withPBS. The activity of subtilisin in the supernatant and the washingsolutions was measured and the coupling yield was calculated.

Activity Measurement and Kinetic Study. The substratesolution was added to a cuvette and mixed with the enzymesolution. The increase in absorbance at 412 nm was monitoredon a Perkin-Elmer (Norwalk, CT) Lambda 4 spectrophotometer.When the enzymatic activity of immobilized subtilisin was mea-sured, a CUV-O-Stir 333 magnetic cuvette stirrer from Hellma(Baden, Germany) was used to stir the mixture during thereaction. To determine the KM and kcat values of the homogeneousand immobilized enzyme, the initial rate of the hydrolysis of thesubstrate SAAPF-pNA for a range of substrate concentrations wasmeasured. The KM and kcat values were determined from Lin-eweaver-Burk plots.

pH Profile of the Homogeneous and Immobilized En-zyme. Aliquots of 100 mL of buffer AB (0.0500 M acetic acid,

(29) Harwood, C. R.; Cutting, S. M. Molecular Biology Methods for Bacillus;Wiley: Chichester, New York, 1990.

(30) Matsubara, H.; Kasper, C. B.; Brown, D. M.; Smith, E. L. J. Biol. Chem.1965, 240, 1125-1130.

(31) Estell, D. A.; Graycar, T. P., Wells, J. A. J. Biol. Chem. 1985, 260, 6518-6521.

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0.0500 M Bis-Tris propane) were adjusted to pH 5.0, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 with HCl or NaOH solutions. To15 mL of each of these aliquots, a volume of 37.5 µL of a substratestock solution (40 mM SAAPF-pNA in DMSO) was added to makethe substrate solution. At each pH value, a volume of 2.00 mL ofthe substrate solution of the corresponding pH was mixed with afixed amount of the homogeneous or immobilized enzyme in acuvette and the initial reaction rate was determined as describedabove.

RESULTS AND DISCUSSIONDespite the vast number of applications of immobilized proteins

and the rapid development of immobilization technology, muchhas yet to be understood about the effects of immobilization onthe properties of the proteins. The major factors that affectimmobilized proteins are as follows: (1) the interaction of theprotein molecules and the insoluble support surface; (2) theexternal and internal diffusion of the substrates and productsacross the interface and inside the pores of the support; (3) theorientation of the protein molecules on the surface. It has beenobserved that immobilization can significantly affect many proper-ties of proteins, including reactivity, kinetics, pH optimum, andstability.32,33 However, it is usually difficult to study these effectsbecause of the complexity of the conventional heterogeneousimmobilization systems that lead to multiple orientations ofproteins on the surface. Given the importance of immobilizedproteins in bioanalytical chemistry, it is desirable to investigateand control these factors in protein immobilizations.

In this study, we address the issue of the orientation ofimmobilized enzymes on a solid support by comparing undercontrolled conditions the site-specific immobilization and randomimmobilization of subtilisin BPN′. As shown in Figure 1A,conventional chemical immobilization that is based on the aminoand carboxyl groups of proteins can lead to randomly orientedprotein molecules on the support surface because of the multitudeof such groups on proteins. Thus, the orientation of each proteinmolecule on the surfaces depends on the particular amino acidresidue that serves as the site of attachment. Multiple attachmentthrough more than one site on the protein may also occur in aconventional immobilization, and in some cases, this may resultin denaturation of the protein. On the other hand, if the site ofattachment can be controlled and each enzyme molecule isattached from a location that is away from its active site and othereffector sites, the enzyme molecules can be oriented on thesupport surface in a way that their active sites uniformly face thesolution phase (Figure 1B). It is conceivable that enzymes

immobilized in this manner will have a higher specific activitythan randomly immobilized enzymes.

Subtilisin BPN′ is used as a model protein to demonstrate theadvantages of site-specific immobilization. For this purpose, aunique cysteine residue was introduced by site-directed mutagen-esis into subtilisin BPN′ at a location that is away from its activesite and that exerts a minimum effect to the other properties ofthe enzyme. Subtilisin enzymes are a group of extracellularalkaline proteases produced by Bacillus. Subtilisin BPN′ from B.amyloliquefaciens was first cloned in 1983 and has been the focusof extensive protein engineering studies.25,34-36 The enzymeconsists of a single polypeptide chain of 275 amino acids, with 11lysines and no cysteine residues. Since the three-dimensionalstructure of subtilisin BPN′ was first determined in 1969, muchdetailed information on its structural characteristics and enzymaticmechanism have been obtained.37-39 Wild-type subtilisin has atypical globulin structure, and the “catalytic triad” of serine 221,histidine 64, and aspartic acid 32 forms the active site. Further,subtilisin BPN′ has two calcium-binding sites, and the presenceof Ca2+ in these sites improves the stability of the enzyme. Forthe mutant enzymes of this study, a cysteine was introduced at alocation away from the active site and the calcium binding sites.Previous protein engineering work has shown that the loopregions (the connections between R-helices and â-sheets) of aprotein can tolerate more changes than the regions of distinctsecondary structures.40,41 Therefore, in order to minimize theeffect of the mutation on the properties of the enzyme, it isdesirable to introduce the cysteine residue in a loop region onthe surface of the protein. With the above considerations, wechose serine 145 as the mutation site to be changed to cysteine(Figure 2). A serine-to-cysteine mutation is considered a conser-vative mutation because chemically the only change is a substitu-tion of a side chain OH group with a SH group. Serine 249, whichis away from the active site but in an R-helix, was also selected asa mutation site (Figure 2). Our hypothesis was that site-specificimmobilization through different sites of the enzyme, even whenthey are away from the active site, can have different effects onits specific activity.

The site-directed mutagenesis was performed as explained inthe Experimental Section. The mutated subtilisin genes wereinserted into plasmid pSbt to form the expression vectorspLGB103 (expressing the S249C mutant) and pLGB105 (express-ing the S145C mutant) (Figure 3). Subtilisin BPN′ was secretedinto the culture medium when these vectors were used to expressthe protein in B. subtilis. This greatly facilitates the purificationof the enzyme. Both the newly purified mutant subtilisins (subt-1and subt-2) did not react with Ellman’s reagent. This is becausethe free sulfhydryl group on the surface of the enzyme tends to

(32) Goldstein, L. Methods Enzymol. 1976, 44, 397-443.(33) Clark, S. D. Trends Biotechnol. 1994, 12, 439-443.

(34) Wells, J. A.; Ferrari, E.; Henner, D. J.; Estell, D. A.; Chen, E. Y. NucleicAcids Res. 1983, 11, 7911-7925.

(35) Wells, J. A.; Estell, D. A. Trends Biol. Sci. 1988, 13, 291-297.(36) Takagi, H. Int. J. Biochem. 1993, 25, 307-312.(37) Wright, C. S.; Alden, R. A.; Kraut, J. Nature 1969, 221, 235-242.(38) Bott, R.; Ultsch, M.; Kossiakoff, A.; Graycar, T.; Katz, B.; Power, S. J. Biol.

Chem. 1988, 263, 7895-7906.(39) Branden, C.; Tooze, J. Introduction to Protein Structure; Garland Publish-

ing: New York, 1991; pp 231-246.(40) Freimuth, P. I.; Taylor, J. W.; Kaiser, E. T. J. Biol. Chem. 1990, 265, 896-

901.(41) Sowadski, J. M.; Foster, B. A.; Wyckoff, H. W. J. Mol. Biol. 1981, 150,

245-272.(42) Sayle, R. A.; Milner-White, E. J. Trends Biol. Sci. 1995, 20, 374-376.(43) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950.(44) Merritt, E. A.; Murphy, M. E. P. Acta Crystallogr. 1994, D50, 869-873.

Figure 1. (A) Random and (B) site-specific immobilization ofproteins. Protein molecules are shown as shaded shapes, with theindentations representing the active or binding sites.

4604 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997

form an intermolecular disulfide bond with a second subtilisinmolecule. A Reduce-Imm column was used to reduce theintermolecular disulfide bonds. The freshly reduced protein wasthen used in the determination of free sulfhydryl groups by usingthe Ellman’s reagent. From the change in the absorbance at 412nm, the average number of SH groups on each protein moleculewas calculated to be ∼1.0 (Table 1). These results proved that asingle cysteine residue was introduced into each of the mutantsubtilisins and that the SH group is on the surface of the enzymemolecule and accessible to chemical reactions.

The kinetic parameters of the mutant subtilisins were com-pared to that of the wild-type enzyme, which was expressed andpurified according to the same procedure (Table 1). The kineticparameters of the wild-type enzyme are consistent with thosereported in the literature.31 The mutant subtilisins demonstratedkcat values only slightly lower than that of the wild-type enzyme.Likewise, the KM values for the mutant subtilisins were onlyslightly higher than that of wild-type enzyme. These observations

suggest that the introduced serine-to-cysteine mutation hasminimal effect on the properties of subtilisin.

The mutant subtilisin subt-2 was immobilized first on differentcommercially available beads. These beads (activated thiolSepharose 4B, thiopropyl Sepharose 6B, Affi-Gel 501) are cross-linked agarose particles functionalized with sulfhydryl-reactinggroups and are commonly used in chromatography to purifypeptides and proteins that have accessible free sulfhydryl groups.These agarose particles are different in size, with diametersranging from 50 to 150 µm, and have extensive pore structures.For the Sepharose 4B and 6B beads, the functional groups onthe surface are SH protected with the 2-thiopyridyl group; the Affi-Gel 501 beads are functionalized with phenylmercuryl groups onthe surface. The mutant subtilisin can be directly immobilizedonto these supports after the intermolecular disulfide bond isreduced to generate a free sulfhydryl group on subtilisin.

It has been reported that porous supports have a significanteffect on the properties of the immobilized enzyme because ofthe internal diffusion of the substrates and products within thepores of the support.32 When the pH profiles of the immobilizedsubtilisin on activated thiol Sepharose 4B and thiopropyl Sepharose6B were compared to that of the homogeneous enzyme, broad-ened pH profile curves were observed (Figure 4). This result isconsistent with previous reports on immobilized enzymes.45,46 Theeffect has been attributed to substrate diffusion limitations insidethe pores of the support. If the intrinsic specific activity of theimmobilized enzyme is high, the substrate concentration de-creases inside the pores and the substrate may not reach enzymemolecules immobilized deep inside the pores. When the pH

(45) Itoyama, K.; Tanibe, H.; Hayashi, T.; Ikada, Y. Biomaterials 1994, 15, 107-112.

(46) Trevan, M. D. Immobilized Enzymes: An Introduction and Applications inBiotechnology; John Wiley & Sons: Chichester, U.K., 1980.

Figure 2. Structure of subtilisin BPN′.38 The active site and Ca2+

cations are shown. Serine 249 and serine 145 are highlighted withball and stick. Serines 249 and 145 were changed to cysteine residuesin the mutants of subtilisin, subt-1 and subt-2, respectively. Thecoordinate data for this structure were obtained from the Brookhavenprotein data bank. This graph was generated with the Rasmol 2.5,Molscript 1.4 and Raster3D 2.0 software.42-44

Figure 3. Map of the plasmid pLGB105.

Table 1. Kinetic Parameters of Subtilisin

KM (mM) kcat (s-1) no. of cysteinesa

wild-type 0.16 ( 0.01 51.3 ( 1.6 0subt-1 (S249C) 0.19 ( 0.01 45.7 ( 1.5 1.06subt-2 (S145C/G98S) 0.21 ( 0.02 48.5 ( 2.4 0.97

a Number of cysteine residues for each protein molecule determinedby using the Ellman’s reagent.

Figure 4. pH profiles of the homogeneous (free) subt-2 (2) andsubt-2 immobilized on activated thiol Sepharose 4B particles (b).

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conditions are changed and the intrinsic enzymatic activitybecomes lower, the substrate concentration gradient across thepores will be less steep. Consequently, more enzyme moleculesinside the pores could be reached by the substrate. Such anincrease in the amount of the enzyme molecules that participatein the reaction moderates the effect of the pH on the overallactivity of the immobilized enzyme. Further, it should be notedthat when subtilisin was immobilized on the nonporous silicaparticles, no broadening of the pH profile was observed comparedto the homogeneous enzyme (vide infra).

The broadened pH profile curve for the immobilized enzymealso indicates that the internal substrate diffusion has an effecton the apparent enzyme activity. The kcat/KM ratio is a measureof the catalytic efficiency of enzymes and was used to comparethe apparent kinetic parameters of the immobilized mutantsubtilisin (Table 2). Compared to the homogeneous enzyme(Table 1), lower kcat/KM values (as a result of higher KM and lowerkcat values) were observed for the mutant subtilisin that wasimmobilized on these porous agarose supports. The observationof lower kcat/KM values, compared to the corresponding homo-geneous enzyme, is quite common with immobilized enzymes.5,12,13

The results were also consistent with the porosity of the im-mobilization supports. For example, activated thiol Sepharose 4Bparticles are more porous than thiopropyl Sepharose 6B particles.Thus, the internal substrate diffusion effect is more pronouncedwith the former particles, which helps explain the higher kcat/KM

value obtained with the thiopropyl Sepharose 6B particles andimmobilized subt-2.

The porous agarose beads are not ideal for studying the effectof enzyme orientation on immobilization, and for comparing thesite-specific and random immobilization, because the apparentactivity of the immobilized enzyme does not necessarily reflectthe activity of each enzyme molecule due to the internal diffusionlimitation mentioned above. Therefore, uniformly sized, nonpo-rous silica particles were used as the support to study further theimmobilization of the mutant subtilisin. These particles arecommercially available and had NH2 groups on the surface. Forthe random immobilization, mutant subtilisin was attached to thesupport by cross-linking the amino groups on the protein and onthe support surface with glutaraldehyde in a two-step reaction.To accomplish this, the particles were first activated with glut-araldehyde, and after the excess glutaraldehyde was washed away,a solution of the mutant subtilisin in PBS was added to couplethe enzyme to the particles. Site-specific immobilization was

achieved by attaching the enzyme molecules to the supportthrough the genetically introduced single sulfhydryl group usingGMBS as the cross-linker. GMBS is a heterobifunctional linkerwith a N-hydroxysuccinimide (NHS) ester moiety at one end ofthe molecule and a maleimide moiety at the other end. Thisreaction was also conducted according to a two-step protocol. Inthe first step, the NHS ester part of the cross-linker was reactedwith the amino group on the support surface, leaving themaleimide functional group exposed to the solution phase. AtpH 7, the reaction between maleimides and sulfhydryl groups ishighly selective, forming a stable covalent bond.47 Therefore, inthe second step, the freshly reduced mutant subtilisin solutionwas mixed directly with the beads. The coupling yield for thisprotocol is typically 40-55%. It should be noted that the reactionof GMBS with the amine-functionalized particles was conductedin an anhydrous organic solvent because of the rapid hydrolysisof the NHS ester in water.

The pH profile of the site-specifically immobilized subtilisinon the nonporous beads was also studied. As shown in Figure 5,no broadening of the pH profile was observed in comparison withthe homogeneous enzyme. This indicates that the internaldiffusion effect, which caused the broadening in Figure 4, waseliminated in the case of the nonporous support. The kineticparameters of the site-specifically and randomly immobilizedenzyme were then determined and compared. The kcat/KM forthe randomly immobilized subt-2 was significantly lower than thatfor the site-specifically immobilized subt-2 (Table 2). The twoimmobilization systems used the same mutant enzyme andsupport. Moreover, the amount of immobilized subtilisin per unitweight of particles was equal to within experimental error. Themain difference in the two systems is in the orientations of theenzyme molecules. In the case of site-specific immobilization, themutant subtilisin is immobilized through the SH group on cysteine145, and the immobilized enzyme molecules have a uniformorientation on the surface with the active site facing the solutionphase (Figure 1B). In the random immobilization system, on theother hand, the enzyme molecules can be attached to the NH2

on the support through one or more of the 12 amino groups onthe enzyme. The orientation of the enzyme molecules on thesurface is therefore dependent on the point of attachment (Figure

(47) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,CA, 1996.

Table 2. Apparent Kinetic Parameters of ImmobilizedSubtilisin

supportkcat/KM

(10-3 M-1 s-1)immobilized

subtilisina (mg)

immobilized subt-2activated thiol Sepharose 4B 4.7 ( 0.6 0.46thiopropyl Sepharose 6B 16.1 ( 1.0 0.66Affi-Gel 501 4.8 ( 0.4 0.74derivatized silica (site-specific) 42.9 ( 2.8 0.45derivatized silica (random) 12.8 ( 1.1 0.46

immobilized subt-1derivatized silica (site-specific) 32.6 ( 3.8 0.54

a Amount of subtilisin immobilized on the support. For thiol-activatedSepharose beads and Affi-Gel 501, 500 µL of reswelled gel was used ineach immobilization. For the derivatized silica particles, 0.1 g ofparticles was used in each immobilization.

Figure 5. pH profiles of the homogeneous subt-1 (2) and subt-1immobilized site-specifically on nonporous silica particles (b).

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1A). If the amino group(s) used for the attachment is(are) closeto the active site of the enzyme, the active site may be stericallyblocked, and the activity may be totally or partially lost. It is alsopossible that more than one amino group on the protein may beattached at the same time, and this may distort the conformationof the enzyme and affect its activity. A similar improvement inthe catalytic properties of enzymes that have been oriented onsurfaces in a site-specific manner compared to random im-mobilization was observed in our laboratory using recombinantalkaline phosphatase fused to a peptidic affinity tag.13 It shouldbe noted that although the site-specific immobilization yieldshigher overall activity than the random immobilization, it accountsfor only 30% of the activity of the free enzyme. (The kcat of thesite-specifically immobilized subt-2 on derivatized silica was 14.5s-1, compared to 51.3 s-1 for the homogeneous subt-2.) This isbecause other factors, namely, the external diffusion and protein-surface interactions, may still play a role in the properties of theimmobilized enzyme.

It was also observed that site-specific immobilization throughdifferent sites of subtilisin has a different effect on its activity. Asshown in Table 2, site-specific immobilization of subt-1 throughthe genetically introduced cysteine 249 yielded a kcat/KM value of32.6 × 103 M-1 s-1, which is lower that the corresponding valuefor subt-2. This may be because cysteine 249 is in the middle ofan R-helical secondary structure, and attachment of subtilisin fromthis site affects the overall conformation of the enzyme. Therefore,although the introduction of a cysteine at position 249 did notaffect the kcat and KM values of the enzyme significantly (Table1), the interaction of the enzyme with the surface imposed byattachment through cysteine 249 reduces the catalytic efficiencyof immobilized subtilisin. Therefore, judicious selection of thesite of attachment is necessary in order to achieve maximum kcat/KM.

To properly evaluate the above data, it was necessary toestablish whether subtilisin demonstrates any nonspecific adsorp-tion on the amine-functionalized silica. For this, a volume of 500µL of a 10% (w/v) suspension of nonporous amine-functionalizedsilica beads was incubated with 1 mL (1.94 mg) of a subtilisinsolution in PBS buffer. The beads were centrifuged, the super-natant was collected, and the beads were further washed threetimes with PBS buffer. The absorbance of the supernatant andall wash solutions at 280 nm was used to determine the amountof subtilisin present. By comparing this amount of subtilisin tothe 1.94 mg used initially, it was found that nonspecific adsorptionwas <1%.

One concern when immobilized proteins are used in biosen-sors, bioreactors, and bioseparations is the long-term stability ofthe protein under normal operation conditions. Thus, the stabilityof the site-specifically immobilized subtilisin (subt-2) was evaluatedover a 25-day period both when stored at room temperature andat 4 °C (Figure 6). It was found that the stability of theimmobilized subtilisin was superior when it was stored at 4 °C,

with the enzyme maintaining ∼61% of its original activity after 25days. Subtilisin stored in solution at 4 °C showed a similarbehavior, with the enzyme retaining 62% of its activity after 25days. The difference in long-term stability was significant whenthe enzyme was stored at room temperature. There, the im-mobilized enzyme maintained 28% of its activity after 25 days,whereas the homogeneous enzyme was only 12% active.

In conclusion, the orientation effect in the immobilization ofenzymes was studied using genetically modified subtilisin. Site-specific immobilization led to a higher catalytic efficiency of theimmobilized enzyme than the conventional random immobiliza-tion. By genetically introducing specific amino acids at predefinedlocations of a protein structure, the protein molecules can be site-specifically attached, and thus, the orientation of the proteinmolecules on the surface can be controlled. It was also shownthat the selection of the site of attachment has a significant effecton the catalytic properties of the immobilized enzyme. Besidesthe applications of this approach in the improvement of thereactivity of the immobilized proteins, the ability to control theorientation of a protein molecule should find applications in manyother aspects of bioanalytical chemistry, including the develop-ment of optimized enzyme electrodes, biosensors, and self-assembled systems.48

ACKNOWLEDGMENTThis project was funded by grants from the National Science

Foundation (CTS 9307518) and NASA. We thank Y. Liu for theassistance in the preparation of the graph of the protein structure.We also thank Dr. P. N. Bryan for supplying the pSbt plasmidand the strain of B. subtilis used for expression of subtilisin.

Received for review April 11, 1997. Accepted September3, 1997.X

AC970390G(48) Frey, W.; Schief, W. R., Jr.; Pack, D. W.; Chen, C. T.; Chilkoti, A.; Stayton,P.; Vogel, V.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4937-4941. X Abstract published in Advance ACS Abstracts, October 1, 1997.

Figure 6. Stability, plotted as percentage of initial activity vs time,of site-specifically immobilized subtilisin (subt-2) on nonporous silicaparticles stored at 4 °C (0) or at room temperature (b). Thecorresponding stability data of homogeneous subt-2 stored at roomtemperature are also shown (∆).

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