Transgalactosylation in a Water-Solvent Biphasic Reaction System with {beta}-Galactosidase Displayed on the Surfaces of Bacillus subtilis Spores

The ever-increasing industrial demand for biocatalysis necessitatesinnovations in the preparation and stabilization of biocatalysts.In this study, we demonstrated that ß-galactosidase(ß-Gal) displayed on Bacillus spores by fusion tothe spore coat proteins (CotG) may be used as a whole-cell immobilizedbiocatalyst for transgalactosylation in water-solvent biphasicreaction systems. The resulting spores had a specific hydrolyticactivity of 5 x 10³ U/g (dry weight) of spores. The ß-Galwas tightly attached to the spore surface and was more stablein the presence of various organic solvents than its nativeform was. The thermostability of the spore-displayed enzymewas also increased, and the enzyme was further stabilized bychemically cross-linking it with glutaraldehyde. With spore-displayedß-Gal, octyl-ß-D-galactopyranoside was synthesizedat concentrations up to 27.7 mM (8.1 g/liter) with a conversionyield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100mM octanol dissolved in phosphate buffer and ethyl ether, respectively.Interestingly, the spores were found to partition mainly atthe interface between the water and solvent phases, and theywere more available to catalysis between the two phases, asdetermined by light microscopy and confocal fluorescence microscopy.We propose that spore display not only offers a new and facileway to construct robust biocatalysts but also provides a novelbasis for phase transfer biocatalytic processes.

INTRODUCTION

Top

Introduction

Biocatalysis carried out in organic solvents, as well as inaqueous environments, is being used increasingly in biochemicalindustries (22). Using organic solvents as the reaction mediaincreases the solubility of hydrophobic substrates and/or changesthe kinetic equilibrium, enhancing the productivity of the process,but it requires stabilization of the enzymes, such as immobilization(12, 15). Enzyme immobilization can frequently be used to improveproduct separation, operational stability, and reusability (1).However, during the immobilization process, the enzyme activityand natural properties are lost, and the reusability of theimmobilized enzyme is also limited. As these issues can be overcomeby using whole cells, whole-cell biocatalysis has been widelyadopted for the commercial synthesis of a variety of compoundsranging from bulk chemicals to valuable pharmaceuticals (7,22) and for bioremediation (23). Compared with the isolatedenzymes, whole-cell biocatalysts can be much more readily andinexpensively prepared on an industrial scale. Owing to theirdiversity and ease of handling, microbial cells have been mostcommonly utilized for whole-cell biocatalysis. One of the technicalproblems in whole-cell biocatalysis is mass transfer limitation,since the cell membrane and wall act as a permeability barrier,which means that a permeabilization step is required. Chemicalpermeabilization processes may also result in other problems,such as enzyme inactivation and the release of cellular components.A more innovative solution to this problem would be to immobilizethe target enzyme on the surface of bacteria. Microbial surfacedisplay of enzymes has been suggested as a cost-efficient alternativeto whole-cell biocatalysts, which obviates the need for enzymeproduction and immobilization (3, 10, 26).

During our efforts to develop solvent-stable whole-cell biocatalysts(9), we became aware of the natural robustness of bacterialspores in the presence of heat, solvent, pH, oxidizing agents,salts, etc. (19), and we developed a spore surface display system(13, 14). Here, we demonstrate that ß-galactosidase(ß-Gal) displayed on spores may be used as a whole-cellimmobilized biocatalyst for transgalactosylation in water-solventbiphasic reaction systems. ß-Gal is an enzyme thathydrolyzes lactose to glucose and galactose, and it also catalyzesa transglycosylation reaction in water-organic solvent biphasicreaction systems (18). However, because of the low stabilityof the enzyme in solvents and a strong limitation in mass transferbetween water and solvent phases separating enzymes and substrates,it was difficult to obtain a significant yield of glycosylationproducts (18). We addressed these problems with spore-displayedß-Gal and more efficiently produced alkyl-ß-galactosides,a group of nonionic surfactants which exhibit antimicrobialactivity and are used as drug intermediates (16). Interestingly,the spores were partitioned mainly at the interface betweenthe water and solvent phases, and they were more available forcatalysis between the two phases.

MATERIALS AND METHODS

Top

Materials and Methods

Construction of plasmids and strains.
In order to display ß-Gal on the spore surface, agenetic fusion of cotG from Bacillus subtilis and lacZ fromEscherichia coli was constructed. By inserting BamHI and SalIsites and removing the stop codon, the cotG DNA was amplifiedfrom B. subtilis using two PCR primers (5'-GCCTTTGGATCCAGTGTCCCTAGCTCCGAG-3'and 5'-AAAAGACGTCGACTTTGTATTTCTTTTTGACTA-3'). The amplifiedfragments were cleaved with restriction enzymes and insertedinto pDG1728 (6). The lacZ gene was also PCR amplified and insertedin frame at the end of cotG to generate pCotG-LacZ, which couldbe integrated into the amyE sites by double-crossover homologousrecombination events. The plasmids were transformed into themultiple-protease-deficient B. subtilis strains DB104 (nprR2nprE18 ${Delta}$ aprA3) (11) and WB700 (nprE nprB aprE mpr bpr vpr) (R.Ye, L. P. Yang, and S. L. Wong, presented at the InternationalSymposium on Recent Advances in Bioindustry, 1996).

Sporulation and spore purification.
The recombinant B. subtilis cells were cultivated in GYS medium[2 g/liter (NH₄)₂SO₄, 2 g/liter yeast extract, 0.5 g/liter K₂HPO₄,1 g/liter glucose, 0.41 g/liter MgSO₄·H₂O, 0.08 g/literCaCl₂·2H₂O, 0.07 g/liter MnSO₄·5H₂O] at 37°Cand 250 rpm for 24 h. Spores were prepared as described previously(20). Spores were collected from 100 ml of a culture in thestationary phase by centrifugation (5,000 rpm) and were resuspendedin 1 ml of 20% Renografin (sodium diatrizoate; Sigma-Aldrich).The suspension was gently layered on 10 ml of 50% Renografinin a 15-ml centrifuge tube and was then centrifuged for 30 minat 12,000 rpm. The resulting pellets contained very pure spores.

Determination of ß-Gal activity.
ß-Gal activity was determined spectrophotometricallyat 405 nm, as previously described (20). In brief, the reactionmixture contained 3 µl of MgCl₂ (1 mM) and ß-mercaptoethanol(45 mM) and 267 µl of O-nitrophenyl-ß-D-galactopyranoside(ONPG) (3 mM) dissolved in 0.1 M sodium phosphate buffer (pH7.5). The purified spore-displayed or free form of ß-Gal(30 µl) was then added to the reaction mixture. Afterincubation at 37°C for 30 min, the reaction was stoppedby addition of 500 µl of 1 M Na₂CO₃, and the absorptionat 405 nm of the liberated O-nitrophenol was determined. Oneunit of ß-Gal activity was defined as the amount ofenzyme that hydrolyzed 1 µmol of ONPG in 1 min at 37°C.

Confocal fluorescence microscopy of microemulsions.
The interfacial localization of ß-Gal-displaying sporesin two-phase reaction systems was analyzed by confocal fluorescencemicroscopy. For immunofluorescence staining of spores, the sporeswere probed first with anti-ß-Gal antibody and thenwith fluorescein isothiocyanate-labeled secondary antibodies.Fluorescence-labeled spores were dispersed in a two-phase reactionsystem, which was followed by a short ultrasonic treatment toobtain microemulsions. Microemulsions were observed with a confocalfluorescence microscope (LSM510 META; Zeiss).

Biphasic transgalactosylation reaction system.
For the two-phase reaction system, lactose (100 mM) was dissolvedin an aqueous buffer solution (200 µl; 20 mM phosphate,pH 7.0), and octanol (100 mM) was dissolved in organic solvents(500 µl). The reactions were carried out in capped vialsor microtubes to avoid evaporation of volatile organic solventsand were initiated by addition of 1 mg (5 U) of spore-displayedenzyme or 5 U of free-form enzyme; this was followed by vigorousmixing in order to create a microemulsion at room temperature(25°C). Samples were removed and analyzed by reverse-phasehigh-performance liquid chromatography with isocratic elutionwith acetonitrile-water (1/1, vol/vol). An octadecyl silicacolumn (25 cm by 4.6 mm [inside diameter]) was used with a flowrate of 0.8 ml/min, and the samples were detected with a refractive,index detector. The transgalactosylation product, octyl-ß-D-galactopyranoside,was prepared from the reaction mixture by evaporation of theorganic solvents and was purified by silica gel thin-layer chromatography(ethyl acetate-methanol, 20:1 [vol/vol]). The chemical structuresof the synthesized products were confirmed by ¹H and ¹³C nuclearmagnetic resonance (NMR) spectroscopy. The octyl-ß-D-galactopyranoside¹H NMR (CD₃OD, 300 MHz) and ¹³C NMR (CD₃OD, 75.4 MHz) spectrawere as follows: for ¹H NMR, ${delta}$ 0.9 (3H, t), 1.3 (10H, m), 1.6(2H, m), 3.5 (1H, dd), 3.6-3.8 (5H, m), 3.9 (2H, m), 4.4 (1H,d, J 7.2 Hz); and for ¹³C NMR, ${delta}$ 14.4 (C-8'), 23.7 (C-7'), 27.1(C-6'), 30.4 (C-5'), 30.6 (C-4'), 30.8 (C-3'), 33.0 (C-2'),70.8 (C-1'), 62.5 (C-6), 76.6 (C-5), 70.3 (C-4), 75.1 (C-3),72.6 (C-2), 105.0 (C-1, ß-bond). The molecular weightwas determined by electrospray ionization mass spectrometry[m/z 315.3 (M+Na)⁺, 607.5 (2 M+Na)⁺].

RESULTS AND DISCUSSION

Top

Results and Discussion

Spore display and stability of ß-Gal in solvents.
For display of ß-Gal on the surfaces of B. subtilisspores, we used the previously selected CotG protein (14), a23.9-kDa outer coat protein, as an anchoring motif. The expressionvector containing the cotG-specific promoter was used to expressß-Gal as a CotG-ß-Gal fusion protein. Becauseproteases may destabilize target enzymes, we used the multiple-protease-deficientstrain B. subtilis DB104 (11) or WB700 (Ye et al., presentedat the International Symposium on Recent Advances in Bioindustry,1996). The recombinant B. subtilis cells were cultivated forefficient sporulation in GYS medium at 37°C and 250 rpmfor 24 h. Sporulation was initiated about 9 h after inoculation,and a sporulation efficiency of >95% was typically obtainedfor both recombinant B. subtilis strains. The concentrationsof spores in the cultures were in the range from 5 x 10⁸ to7 x 10⁸ spores/ml. The spores were collected from the culturesin the stationary phase by centrifugation, and levels of purityup to 99.4% were obtained by centrifugation using Renografin(see Materials and Methods). The enzyme activity of ß-Galdisplayed on the spores was 5 x 10³ U/g (dry weight) of sporesbased on the hydrolysis of ONPG (20). Notably, once the CotG-ß-Galfusion proteins were expressed and assembled on the spore surface,after incubation for several days they were not detached orreleased into the buffer solution. Several physicochemical treatments,such as vigorous mixing, ultrasonic vibration, and detergenttreatment, did not detach the displayed enzymes. The enzymeswere extracted only by treatment with a hot alkaline sodiumdodecyl sulfate solution (data not shown).

Because the stability of spore-displayed ß-Gal enzymesin the presence of organic solvents is critical, we first evaluatedthe stability of enzymes with the commonly used organic solvents(Table 1). Equal volumes of various organic solvents, such asn-hexane, ethyl ether, toluene, ethyl acetate, acetonitrile,and ethanol, were mixed with phosphate-buffered saline (pH 7.5)solutions containing purified spores displaying ß-Gal at a level of 20 U/ml. After 1 h of incubation at 37°C,the remaining ß-Gal activities were determined. Theresults showed that the spore-displayed ß-Gal wasstable in the presence of nonpolar hydrophobic organic solvents.ß-Gal displayed on spores of multiple-protease-deficientstrain B. subtilis WB700 was more stable than ß-Galdisplayed on DB104 spores. Even though ß-Gal was displayed,it was unstable in the presence of water-miscible polar solvents,such as acetonitrile and ethanol. Throughout this work, ethylether was used.

View this table:
[in this window]
[in a new window]

TABLE 1. Solvent stabilities of spore-displayed ß-Gal on different B. subtilis spores and free-form enzymes in various organic solvents after 1 h of incubation at 37°C

Not only was the stability in the presence of organic solventsimproved by displaying enzymes on spores (Table 1), but thethermal stability was also improved. Incubation for 2 h at 40°Ccompletely inactivated free-form ß-Gal, whereas morethan 45% of the initial activity of the spore-displayed enzymeremained (Fig. 1) when it was expressed in B. subtilis WB700strain. The thermostability was also affected by the host proteasebackground, as shown in Fig. 1. We tried to further increasethe stability of displayed enzymes by chemical cross-linkingwith glutaraldehyde. After spores of B. subtilis WB700 werecross-linked with 2% (wt/vol) glutaraldehyde at room temperaturefor 24 h, an activity of 3,050 U/g of spores was obtained fora preparation that initially had an activity of 5,000 U/g ofspores (enzyme activity yield, 61%). Then the residual enzymeactivities of spore-displayed enzymes were determined duringincubation at 37°C in a phosphate-buffered saline solution,as shown in Fig. 2. The residual enzyme activity of cross-linkedspore-displayed ß-Gal on spores was stably maintainedfor the first 2 h and slowly decreased to 1,754 U/g (57.5% ofthe initial activity) over 18 h, while the activity of spore-displayedß-Gal decreased rapidly to 916 U/g (18.3% of the initialactivity). These analyses revealed that chemical modificationcombined with genetic immobilization on spores could be an excellentmethod for enzyme stabilization.

View larger version (11K):
[in this window]
[in a new window]

FIG. 1. Thermostability of free-form ß-Gal ( ${blacksquare}$ ) and spore-displayed ß-Gal on spores of B. subtilis DB104 ( ${blacktriangledown}$ ) and WB700 (•). The residual hydrolytic activities of the enzymes were determined after incubation for 1 h at 42°C.

View larger version (10K):
[in this window]
[in a new window]

FIG. 2. Stability of displayed enzymes on spores of B. subtilis WB700 due to chemical cross-linking with glutaraldehyde (0.5%). ${blacktriangleup}$ , spore-displayed ß-Gal; ${blacksquare}$ , cross-linked spore-displayed ß-Gal. The residual hydrolytic activities of the enzymes were determined.

Transgalactosylation with spore-displayed ß-Gal.
Figure 3 shows a schematic diagram of the biphasic transgalactosylationsystem, in which lactose and spore-displayed enzymes are dissolvedin the phosphate-saline buffer phase (pH 7.0), while octanolis dissolved in the ethyl ether solvent phase. Vigorous mixingor a short ultrasonication resulted in a microemulsion and completelydispersed the spores, probably because of the spores' hydrophobicity.The microemulsion, which was stably maintained for an overnightreaction, should have increased the interfacial area, thus enhancingthe transgalactosylation reaction at the interface. For theinitial trial, the water content (water activity) was not optimizedfor transgalactosylation. The general water content (10 to 30%,vol/vol) for transgalactosylation with ß-Gal in thewater-solvent biphasic reaction system was used in our experiments(24). It is obvious that this water content is desirable toincrease the equilibrium of the hydrolase-based transglycosylationsynthetic reaction. However, the minimum amount of water isusually added since the hydrolase enzymes are not stable atlow water contents and the sugar substrate should be dissolved.Figure 4 shows a typical conversion profile obtained with 5U/100 µl of each of the enzymes. With spore-displayedß-Gal, up to 27.7 mM (8.1 g/liter) octyl-ß-D-galactopyranosidewas synthesized with a 27.7% (wt/wt) conversion yield from 100mM lactose and 100 mM octanol in 24 h, but with the free ß-Gallactose neither was hydrolyzed nor produced octyl-galactopyranoside.We analyzed the reaction mixtures by a thin-layer chromatography,as shown in Fig. 5. In the aqueous phase of the spore-displayedß-Gal reaction, glucose, galactose, and lactose wereobserved, while only lactose was present in the aqueous phaseof the free enzyme reaction. The end product was also confirmedby ¹H and ¹³C NMR spectroscopy, which revealed the ß-configurationanomeric structure of octyl-galactopyranoside (data not shown).

View larger version (20K):
[in this window]
[in a new window]

FIG. 3. Schematic diagram of the biphasic transgalactosylation reaction system. The water-to-solvent phase volume ratio was 2:5, and the reaction was carried out in capped vials at room temperature (25°C).

View larger version (9K):
[in this window]
[in a new window]

FIG. 4. Time course of transgalactosylation with free-form ß-Gal ( ${blacktriangleup}$ ) and spore-displayed ß-Gal ( ${blacksquare}$ ).

View larger version (9K):
[in this window]
[in a new window]

FIG. 5. Thin-layer chromatography analysis of reaction mixtures after 24 h of incubation. (a) Lanes 1 to 3, standard sugars, including lactose (lane 1), glucose (lane 2), and galactose (lane 3); lanes 4 and 5, reaction mixtures from aqueous phases with spore-displayed ß-Gal (lane 4) and free ß-Gal (lane 5). (b) Lane 1, reaction mixture from organic phase with spore-displayed ß-Gal; lane 2, reaction mixture from organic phase with free ß-Gal.

Localization of spores at water-solvent interfaces.
Enhanced reactions were expected not only due to the enzymestabilization with spore display but also due to the localizationof spore enzymes at the water-solvent interface. Interface-bindingenzymes are highly desirable for two-phase reactions becausethey can provide maximum accessibility to substrates dissolvedin both phases across the interface (25, 28). We observed sporesof the B. subtilis WB700 control strain without expression ofß-Gal and spores of the strain displaying ß-Galin microemulsions with a light microscope to determine how theywere dispersed in the reaction mixture. Quite surprisingly,spores were found to almost adhere on the surfaces of solventdroplets (Fig. 6A) and were found to be localized at the interfacesin water-solvent systems (Fig. 6B and D). To observe the interfacepartitioning more closely with a confocal fluorescence microscope,the spores were fluorescently stained by using primary anti-ß-Galantibodies and secondary fluorescein isothiocyanate-labeledanti-mouse immunoglobulin G antibodies. As shown in Fig. 6C,the spores were clearly localized at the interface. However,no fluorescence signals of control spores were observed withthe fluorescence microscope after they were stained with primaryand secondary antibodies as described above (data not shown).The hydrophobicity of the spore surface should have facilitatedlocalization at the interface between the two phases or adherenceto the surfaces of solvent droplets. Once present at the interphaseeither through localization or through surface adherence, spore-displayedenzymes should be more available for catalysis than the biocatalystsdissolved in either the water or solvent phase. The findingsare remarkable because the transgalactosylation by ß-Galis essentially an interphase catalytic reaction which transfersthe galactosyl moiety from lactose present in the water phaseto the octanol acceptor dissolved in the solvent phase. Thus,the biocatalysis reaction enhanced by spore display resultednot only from the stabilization of ß-Gal by sporedisplay but also from the increased availability of biocatalystslocalized at the interface. Despite the fact that the orientationof spore-displayed ß-Gal at the interface of the emulsioncould not be determined precisely, spores might be freely mobileat the interface of the emulsion, which might enhance theirchance to face both phases. In this connection, it is notablethat in vitro interfacial self-assembly of ß-Gal,when ß-Gal was conjugated with polystyrene polymer,improved the catalytic efficiency more than 145-fold for a transgalactosylationbetween water and toluene phases (28). Such polymer-conjugatedß-Gal can efficiently self-localize at the interfacesmainly due to the hydrophobicity of the polymer, resulting avery high rate of production of hexyl galatoside from hexanoland lactose. Thus, spore-displayed ß-Gal and polymer-conjugatedß-Gal have technological advantages in common, includingself-binding to the interface in a solvent-water reaction system,high specific activity, high transgalactosylation activity,increased stability of the enzymes, etc. Spore-displayed ß-Galis comparable to polymer-conjugated ß-Gal in termsof industrial-scale applications of enzymes, as described below.Chemical processes under harsh conditions are inevitable forimmobilization of ß-Gal on synthetic resins or bypolymer conjugation. Spore-displayed ß-Gal, however,can be made available by simple fermentation at a mild temperatureand atmospheric pressure, resulting in a naturally folded enzyme.One possible drawback is that display of ß-Gal onspores requires a specific protein expression system and a low-proteasebackground for the Bacillus host cells in order to avoid enzymedegradation during sporulation of the culture, which could besolved by using protease-negative strains.

View larger version (74K):
[in this window]
[in a new window]

FIG. 6. Determining the localization of spores at the interfacial surfaces of microemulsions by observing them with various microscopes. Scale bars = 10 µm. (A) Light microscopy of spore of B. subtilis WB700 displaying ß-Gal adhering on the surface of microemulsions. (B) Light microscopy of spores of B. subtilis WB700 displaying ß-Gal localized at the interface of the water and solvent phases. (C) Confocal fluorescence microscopy of microemulsion of fluorescently labeled spore of B. subtilis WB700 displaying ß-Gal using primary rabbit anti-ß-Gal antibodies and secondary fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin G antibodies. (D) Light microscopy of control spores of B. subtilis WB700 localized at the interface of the water and solvent phases.

Factors affecting process development.
The economic feasibility of biocatalytic processes depends onbiocatalyst availability, volumetric productivity, and stability(22). Spore-displayed biocatalysts can be prepared much morereadily and inexpensively, like a whole-cell biocatalyst, becausemethods for industrial-scale production of bacterial sporesby conventional high-cell-density fermentation have alreadybeen established. High volumetric productivity (space-time yield),which influences the capital costs of the process, requiresa high specific activity of the biocatalyst (U/g biocatalyst)(24). Quite encouragingly, we obtained a specific hydrolyticactivity of 5 x 10³ U/g (dry weight) of spores, which is higherthan the activity of ß-Gal immobilized on phenol-formaldehyderesins, on which the specific hydrolytic activities are 6 to $~$ 160 U/g (27). The high specific activities of spore-displayedenzymes are best explained by the high ratio of surface areato volume of spores, which reflects their extremely small size(diameter, ca. 1 µm). It was observed that the smallerthe particle size of the support, the greater the activity ofthe immobilized enzyme, which is typical of heterogeneous catalystsdue to a decrease in diffusion resistance and an increase inspecific volume as the particle size decreases (1, 2). The enzymeloading capacity for spore display may be further improved byconstructing fusions to multiple coat proteins or by modification,overcoming the limited productivity due to the low enzyme loadingin a conventional immobilization technique (1, 5).

Another notable aspect of the spore display immobilization systemis its reusability. After the reaction, spores can be separatedusing standard microfiltration processes. At each cycle of thetransgalactosylation reaction, more than 87% of the initialactivity was recovered. Furthermore, in the presence of nutrients,including the so-called germinants such as sugars and aminoacids, spores can germinate into vegetative cells and sporulateagain upon nutrient deprivation. Thus, when denatured or deactivated,spores displaying enzymes can be simply regenerated by regrowthof the culture, followed by sporulation, as shown in our previouswork (21).

In this study, we carried out the experiments on a small scale(reaction volume, 1 ml). However, with the high specific activityof spore-displayed enzymes, as described above, we expect highproductivity for production of alkyl galactoside with spore-displayedß-Gal. We are currently trying to optimize the productionat the kilogram scale.

Conclusions.
Based on the results obtained here, we propose that spore displaynot only offers a new and facile way for constructing robustbiocatalysts but also provides a basis for the novel phase transferbiocatalytic processes. Since our previous results showed thatdiverse enzymes, such as cellulase, subtilisin, lipase, etc.,can be displayed (14), the use of spore-displayed enzymes isnot specific for the ß-Gal reaction in phase transfercatalysis but is more generally applicable to other biocatalysisreactions. Furthermore, by expanding spore display motifs, suchas CotB (8), CotC (17), and CotG (13) of B. subtilis and InhA(21) and Cry1A (4) of Bacillus thuringiensis, and broadeningdisplayable target proteins for antigen (17), streptavidin (13),green fluorescent protein (4), antibody (4), and a fragmentof tetanus toxin (8), spore display technology can be used ina wide array of biotechnological fields in addition to biocatalysis,including biosensors, alternative vaccines, nanobiomaterials,and high-throughput library screening.

ACKNOWLEDGMENTS

This research was financially supported by the National ResearchLaboratory program of the Ministry of Commerce, Industry, andEnergy and the Ministry of Science and Technology, Republicof Korea, and by the Cooperative Research Program of the KoreaResearch Council of Fundamental Science and Technology.

We thank S. H. Park and S. K. Choi for providing Bacillus strainsand plasmids and for discussions. We also thank B. K. Kim andJ. H. Kim for their collaboration in the early phase of developmentof spore display technology.

FOOTNOTES

* Corresponding author. Mailing address: Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oundong, Yusong, Daejeon 305-333, Korea. Phone: 82-42-860-4483. Fax: 82-42-860-4488. E-mail: jgpan{at}kribb.re.kr.

Published ahead of print on 22 December 2006.

S.J.K. and H.-C.J. contributed equally to this work.

REFERENCES

Top