Construction of Xylose Dehydrogenase Displayed on the Surface of

Dec 13, 2011 - Construction of Xylose Dehydrogenase Displayed on the Surface of Bacteria Using Ice Nucleation Protein for Sensitive d-Xylose Detection...
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Construction of Xylose Dehydrogenase Displayed on the Surface of Bacteria Using Ice Nucleation Protein for Sensitive D-Xylose Detection Bo Liang,† Liang Li,† Marco Mascin,*,‡ and Aihua Liu*,† †

Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road Qingdao, 266101, People’s Republic of China ‡ Dipartimento di Chimica, Universita degli Studi di Firenze, 50019 Sesto Fiorentino, Italy S Supporting Information *

ABSTRACT: A novel method was developed to detect D-xylose (INS 967) sensitively and selectively, which is based on a xylose dehydrogenase (XDH) cell-surface displaying system using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. With coenzyme NAD+, the XDH-displayed bacteria facilitates the catalysis of the oxidization of xylose and the resultant NADH can be detected spectrometrically at 340 nm. The fusion protein was characterized by proteinase accessibility, Western blot, and enzyme activity assays. The established XDH surface displaying system did not inhibit the growth of the recombinant Escherichia coli strain. The XDH was mainly displayed on the surface of host cells, which is of high XDH activity and high D-xylose specificity. The optimal temperature and pH of cell displayed XDH were found at 30 °C and pH 8.0, respectively. The XDH-displayed bacteria can be used directly without further enzyme extraction and purification, and it improved the stability of the enzyme. Moreover, the cell-surface-displayed-protein-based approach showed a wide linear range (5−900 μM) and a low detection limit of 2 μM of D-xylose. More importantly, the recombinant cells could be used for precise detection of D-xylose from the real samples such as foods and degradation products of lignocellulose. The method shown here provides a simple, rapid, and low-cost strategy for the sensitive and selective measurement of D-xylose. In addition, the XDHdisplayed bacteria showed an interesting response in developing electrochemical biosensors. Thus, the genetically engineered cells may find broad application in such biosensors and biocatalysts. Similarly, this type of genetic approach may be used for the expression of other intracellular enzymes of interest for certain purposes.

C

rapid, accurate analysis of xylose would be of significant interest in genetics, human nutrition, food technology, and pharmacology, as well as many bioprocesses including biofuel fields.1,7,8 To date, many analytical methods including high-performance liquid chromatography (HPLC),9,10 capillary electrophoresis,11 HPLC/mass spectrometry,7,12 Raman spectroscopy,13 and near-infrared spectroscopy14 have been developed for the determination of xylose. However, these techniques have experienced practical challenges, including complex processes of sample handling, time consumption, lower selectivity, insensitivity, and expensive instrumentation. Recently, an indirect competitive immunoassay of xylose was reported with improved sensitivity;8 nevertheless, the procedure is

arbohydrates widely exist in living organisms, which occur in plant storage organs, plant cell wall, glycoproteins, various glycosides, and starch.1 They play different roles based on their structure with function as carbon and energy sources, as well as the different physiological and/or therapeutic effects.1,2 Xylose (INS 967) is an important carbohydrate in our daily products and lives, which could be used as a perfect sweetener, nourishment, and therapeutical agent for diabetics,3 and can be made as a softener, surfactant, and plasticizer in various industries such as paint, toothpaste, tobacco, foundry, and gunpowder.4 On the other hand, it is vital to establish an efficient bioconversion of cellulosic biomass and its hydrolysis to ethanol in a bioenergy industry.1,5,6 Thus, both fermentability and bioconversion efficiency of xylose are important factors in identifying superior engineering strains and scaling-up production. Xylose must be monitored for metabolic engineering in the microbial production of bioethanol.5 Therefore, a © 2011 American Chemical Society

Received: September 22, 2011 Accepted: November 28, 2011 Published: December 13, 2011 275

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detect D-xylose. To the authors’ best knowledge, this is the first report on the functional surface display of XDH using P. borealis INP for sensitive and selective D-xylose detection. Finally, the use of this newly prepared cell-displayed XDH for possible biosensing application is discussed.

complicated. Electrochemical detection of xylose was also developed: for example, a pulsed amperometric detection on an electrophoretic microchip with a lower detection limit of 20 μM xylose15 and a potentiometric biosensor based on a fieldeffect transistor utilizing entire Gluconobacter oxidans cells, which exhibit a lower limit of 0.5 mM xylose.16 Obviously, currently, detection sensitivity using the electrochemical method is also not satisfactory. Therefore, it is important to establish a monitoring method to detect xylose rapidly, sensitively, and selectively. The development of an enzyme-based detection system can play a crucial role in this area. However, the practical applications of xylose detection by intracellular protein could be limited by the cost of purification and the stability of the enzyme. The strategy of bacteria surface expression of XDH is an ideal method to handle this issue. Among several routes for D-xylose degradation in microorganisms, D-xylose metabolism catalyzed by xylose dehydrogenase (XDH) (EC 1.1.1.175) occurs through a distinct pathway that differs from the classical 17−19 D-xylose pathway in most bacteria. An archeal XDH for Dxylose degradation has been identified in the halophilic archaeon Haloferax volcanii and Haloarcula marismortui, respectively.20,21 XDH could convert D-xylose to D-xylonolactone, with NAD+ as its cofactor, which is shown in eq 1. The Dxylose metabolic pathway in Caulobacter crescentus involves several enzymes, of which XDH expressed in the cytoplasm of E. coli strain BL21(DE3) pLysS exhibited high selectivity and enzyme activity.22

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EXPERIMENTAL SECTION Bacterial Strains, Plasmids, and Culture Conditions. P. borealis DL7 and C. crescentus NA1000 strains were kindly gifted by Drs. Virginia K. Walker (Department of Microbiology and Immunology, Queen’s University, Canada) and Joseph C. Chen (Department of Biology, San Francisco State University, USA), respectively. The N-terminal domain of the inaPb gene (INP) and the xylB gene (XDH) were amplified from the genomic DNA of P. borealis DL7 and C. crescentus NA1000, respectively. E. coli DH5α (F−φ80 lacZΔM15Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk−,mk+)supE44λ-thi-1 gyrA96 relA1 phoA) was used for constructing recombinant plasmids. E. coli BL21 (DE3) (F− ompT hsdSB(rB−mB−) gal dcm(DE3)) was used as the host for recombinant protein expression. All primers used in this study for the construction of recombinant plasmids are listed in Table S1 in the Supporting Information. Plasmid pET28a (+) was used as the parent vector for the construction and expression of fusion protein. The details for the construction of plasmid pTInaPbN-Xdh are schematically shown in Figure S1 in the Supporting Information. Strain C. crescentus NA1000 was grown under aerobic conditions with shaking (200 rpm) at 30 °C in a PYE medium. P. borealis DL7 was inoculated in 10% tryptic soy medium at 22 °C. E. coli was grown in Luria−Bertani (LB) medium composed of 5 g/L yeast extract, 10 g/L tryptone, and 10 g/ L NaCl supplemented with ampicillin (100 μg/mL) or kanamycin (30 μg/mL) at 200 rpm and 37 °C. The strains harboring expression vectors were grown to an optical density at 600 nm (OD600) value of 0.5, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM) at 25 °C for 24 h. The procedure for the cell fractionation was summarized in the Supporting Information. SDS-PAGE and Western Blot Analysis. An equal volume of each fraction (cytoplasm, outer membrane, and inner membrane) from the cells harboring the expressing vectors pTInaPb-N/Xdh and pET28a (+) (as control sample) were mixed with loading buffer, boiled for 5 min, and resolved by 12% (wt/vol) SDS−polyacrylamide gel electrophoresis (SDSPAGE). Protein samples were electroblotted onto PVDF membrane and allowed to incubate with mouse Anti-His monoclonal antibody (Tiangen, PRC). Subsequently, samples were probed with Antimouse IgG secondary antibody conjugated with alkaline phosphatase. After washed with TBST, BCIP/NBT (Tiangen, PRC) was added to detect antigen−antibody conjugates. The reaction was quenched with distilled water. XDH Activity Assay. After 24 h of IPTG induction, cells were harvested and diluted to unit cell density (OD600 = 1.0) with 50 mM PBS buffer (pH 8.0). XDH activity was measured by an increase in absorption at 340 nm, suggesting the generation of NADH.22 For each assay, 100 μL of cells (OD600 = 1.0) were added to 50 mM PBS buffer containing 5 mM Dxylose and 4 mM NAD+. Activity was expressed in units (1 μmol NADH generated per min) per entire OD600 cells at 30 °C. Proteinase Accessibility Assay. E. coli harboring pTInaPb-N/Xdh and pET28a (+) (as control sample) were

XDH

xylose + NAD+ ⎯⎯⎯⎯⎯→ xylonolactone + NADH

(1)

Ice nucleation protein (INP) is an outer membrane protein of several plant pathogenic bacteria, which could accelerate the formation of ice crystal in supercooled water. It is composed of three distinct domains, namely, N-terminal, C-terminal, and central repeating domains. The N-terminal domain is relatively hydrophobic and links the protein to the out membrane via a glycosylphosphatidylinositol anchor.23 Ice nucleation bacteria are related to the genus Pseudomonas,24 Erwinia,25,26 and Xanthomonas.27 INP was widely used for the surface display of heterologous proteins. For example, green fluorescent protein (GFP) was displayed on the surface of E. coli and P. putida by fusing the truncated InaK and InaQ proteins.28,29 On the other hand, the N-terminal region of INPs can be used as an anchoring motif to display the Chitinase 9230 and heterologous phosphate-binding protein (PBP).29 Recently, INP from Pseudomonas borealis had been reported, which offered new avenues for ina genes.31 This gene, designated as inaPb, was divergent from the well-known INP genes in the first 500 bp, which was of ∼66% identity with InaV and InaK of P. syringae.32,33 Although four INP genesinaV, inaK, inaQ, and inaX originating from P. syringae and X. campestris were used for bacteria surface display,34−37 there is no report on the surface display with inaPb from P. borealis. Previous studies showed that INP was suitable for use in a bacterial surface display system and the N-terminal domain appeared to be the only domain for successful surface anchoring.37 Therefore, it is prerequisite to investigate whether an N-terminal domain of INP from P. borealis could serve as a potential anchoring motif. In the present work, a novel entire-cell biocatalyst was developed by the fusion of INP to XDH. Because of its high XDH activity and D-xylose specificity, the recombination strain has been proven to be useful to develop a facile method to 276

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Figure 1. (A) SDS-PAGE and (B) Western blotting analysis of E. coli cells harboring the expressing vectors pTInaPb-N/Xdh and pET28a (+). (Lane M, protein standard markers; lane 1, outer membrane fraction of surface displayed strain; lane 2, inner membrane fraction of surface displayed strain; lane 3, cytoplasmic fraction of surface displayed strain; lane 4, outer membrane fraction of control strain; lane 5, inner membrane fraction of control strain; and lane 3, cytoplasmic fraction of control strain.)

Preparation of Modified Electrode. The bare glassy carbon electrode (GCE, 3 mm in diameter) was polished carefully with 1.0-, 0.3-, and 0.05-μm alumina slurry, and was sonicated in anhydrous ethanol and water, respectively. Finally, the GCE was thoroughly rinsed with distilled deionized water. A quantity of 7.5 μL of XDH-displaying bacteria aqueous dispersion was added on the inverted GCE, followed by syringing 5 μL of Nafion solution (0.05 wt %). Before use, the modified electrodes were washed repeatedly with Milli-Q water (Millipore, Bedford, MA, USA) to remove the loosely combined modifiers. A Nafion/GCE was prepared for comparison. Apparatus and Measurements. SDS-PAGE and electroblotting were performed using Biorad Mini-PROTEAN Tetra Electrophoresis System and Biorad Mini Trans-Blot Cell (BioRad Laboratories, Inc., USA), respectively. The UV−Vis spectra were performed using a Beckman Coulter Model DU 800 UV/ vis spectrophotometer (Beckman Coulter, Inc., USA) at room temperature. The Agilent 1200 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Inc., USA) was used to separate and analyze D-xylose in all samples. Electrochemical measurements were performed using a Model CHI660D Potentiostat (CH Instruments, Chenhua, Shanghai, PRC). The electrochemical response was measured in a conventional three-electrode system using a Nafion/GCE or a chemically modified GCE as the working electrode, a Pt wire auxiliary electrode, and an Ag/AgCl (3.3 M KCl) electrode as the reference electrode. All potentials were reported in this context with respect to this reference. All measurements were performed at room temperature (∼23 °C).

centrifuged and washed three times with PBS buffer, then adjusted to an OD600 value of 1.0. Samples were treated with Proteinase K (0.1 mg/mL) at 37 °C. After 1 h of incubation, Proteinase K treated and untreated cells were assayed for XDH activity, as described above. Optimization of Conditions Affecting Enzymatic Activity. The enzymatic activity was measured to establish the optimal temperature and pH for the XDH-displaying E. coli. To determine the optimum temperature, the enzyme activity was measured at temperatures of 20−50 °C with a constant pH of 8.0. To determine the optimum pH, all activities were measured at 30 °C by varying the pH values of the buffer solution. Stability Study of Resting Culture. Cells containing plasmid pTInaPb-N/Xdh were grown in a LB medium for 24 h, harvested, washed with 50 mM PBS buffer (pH 8.0), resuspended in the same buffer, and incubated at 4 °C. At the same time, the crude enzyme solution of C. crescentus NA1000 (the origin strain) was extracted and incubated under the same conditions. Over the duration of one month, 0.5 mL of each sample solution was taken to check the enzymatic activity daily. Samples were centrifuged and assayed for XDH activity as described above. Substrate Specificity of Cell-Surface-Displayed XDH. E. coli cells harboring the expressed vectors pTInaPb-N/Xdh and pET28a (+) (as control sample) were harvested after 24 h of IPTG induction and then the optical density at 600 nm (OD600) was adjusted to 1.0 with PBS buffer (pH 8.0). D-xylose (0.1 mM) and cellobiose, D-fructose, D-sucrose, D-maltose, Dglucose, D-mannose, D-galactose, D-xylitol, D-ribose (each 10 mM), or L-arabinose (1 mM) was added to the cell reaction buffer, separately, and the absorption spectra in the wavelength range of 310−410 nm were measured. In another experiment, the absorption spectrum of 0.1 mM D-xylose mixed with the above nine saccharides (each 10 mM) and L-arabinose (1 mM) in the same buffer was also checked to investigate the interference of other saccharides on D-xylose detection. Detection of D-Xylose Using Cell-Surface-Displayed XDH. Detection of D-xylose was carried out at 30 ◦C in PBS buffer (50 mM, pH 8.0) containing NAD+ (1 mM), recombination strain (100 μL cell, OD600 = 1.0) and varying D-xylose concentration (0−1000 μM). UV−visible absorption spectra were measured. All the experiments were repeated at least three times.

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RESULTS AND DISCUSSION Construction of XDH Surface Display System. Both InaK from P. syringae KCTC1832 and InaV from P. syringae, INA5 had been used for cell surface display of heterologous proteins.38,39 However, there was no report about practical applications using an InaPb-N anchoring motif. Although both P. borealis INP and P. syringae INP are functionally similar, there is only ∼66% amino acid sequence homology. This difference is mainly from the critical N-terminal domain, which interacts with the phospholipids moiety of the outer membrane.40,41 In order to investigate the feasibility of targeting XDH fusion onto the cell surface of E. coli, the truncated InaPb protein from 277

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Table 1. XDH Activity of Different Subcellular Fraction of E. coli Harboring Expression Vectors XDH Activitya (U/OD600) plasmid

entire cells

cell lysate

cytoplasm

outer membrane

inner membrane

pTInaPbN-Xdh pET28a (+)

1.89 ± 0.03 0.11 ± 0.02

2.07 ± 0.14 0.14 ± 0.03

0.17 ± 0.02 0.084 ± 0.005

1.61 ± 0. 11 0.021 ± 0.003

0.25 ± 0.03 0.019 ± 0.002

The values shown represent the average of three repetitive measurements plus−minus standard deviation. One unit of enzyme activity is 1 μmol NADH generated per minute per entire OD600 cell. a

P. borealis DL7 was used as a surface-anchoring motif. A recombinant plasmid pTInaPbN-Xdh was constructed in the way that the xylB gene was fused into the N-terminus gene of an INP and cloned to pET28a (+). The recombinant plasmid pTInaPbN-Xdh was then transformed into E. coli BL21 (DE3) (see Figure S1 in the Supporting Information). The plasmid could be digested into a ∼1300 bp strip by a Nco I/Hind III double enzyme. E. coli harboring expression vectors were grown to an OD600 value of 0.5 before induction with 0.5 mM IPTG. After incubation at 25 °C for 24 h, cells were harvested and fractionated. Surface Localization of InaPb-N/XDH Fusion in Cell. Expression of InaPb-N/XDH in E. coli was verified by SDSPAGE and Western blot analysis (Figure 1). Subcellular fractionated samples were probed with Anti-His antibody. In contrast to the control sample, most InaPb-N/XDH proteins were found in the outer membrane fraction due to the fusion construct. A specific band corresponding to InaPb-N/XDH at ∼50 kDa was detected in the outer membrane fraction, and the molecular weight was almost identical to the predicted size. Proteinases cannot penetrate the outer membrane; therefore, only surface-exposed proteins could be degraded by proteinases.28 The proteinases K accessibility assay can be used to provide evidence for the surface localization of InaPb-N/XDH protein. After treatment with proteinases K, the enzymatic activity and Western Blot analysis experiments were carried out separately. The XDH activity for pTInaPbN-Xdh-carrying cells was lost by 91%. Using mouse Anti-His monoclonal antibody, no target protein was detected in the outer membrane fraction. However, the enzyme activity for control sample just decreased slightly. These results suggested the proper construction of InaPb-N/XDH fusion. Taken together, the InaPb-N/XDH fusion was displayed on the cell surface successfully. To the best of the authors’ knowledge, this is the first report on the functional display of XDH on bacterial cell surface. XDH Activity of Recombination Strain. The XDH activity can be detected on the basis of eq 1. The coenzyme of NAD+ was reduced to NADH, which shows a typical absorption peak at 340 nm. The XDH activity of entire cells and subcellular fraction samples is summarized in Table 1. The entire-cell activity of E. coli harboring plasmid pTInaPbN-Xdh was 17-fold higher than that of the control. Approximately 90% of total XDH activity was found in the total membrane fraction of InaPb-N/XDH fusion-expressing cells. Furthermore, the XDH activity was mainly contributed from the outer membrane fraction (77% of the cell lysate activity) (Table 1). These results suggested the correct translocation of fusion protein on the outer membrane of host cells and efficient surface display of XDH on E. coli. In the context, INP has been widely used to display heterogeneous enzymes and develop entire-cell biocatalysts with high efficiency. For instance, GFP was displayed on the surface of E. coli and P. putida with InaQ− N as the anchoring motif, and more than 40% of the total GFP intensity was detected in the total membrane fractions.29 For

organophosphorus hydrolase (OPH) surface display system, over 80% OPH activity was obtained in the membrane fractions of E. coli.34 In above two cases, both GFP and OPH were displayed on the surface of bacteria using P. syringae INPs (InaQ, InaV, and InaK), which have low homology with InaPb−N. Therefore, it is obvious that InaPb−N was more effective as an anchoring motif than P. syringae INP. The growth profile of strain E. coli cells harboring the expressing vectors pTInaPb-N/Xdh and pET28a (+) (as control sample) is shown in Figure 2. The OD measurements

Figure 2. Time course for the growth E. coli cells harboring the expressing vectors pTInaPb-N/Xdh and pET28a (+). Error bars represent the standard error of three replicates.

at 600 nm showed a steady increase in bacterial mass. After incubation for 32 h, both cultures reached the same final cell density, which indicates that no growth inhibition was observed for the recombinant E. coli strain. The inhibition of cell growth often occurs when the foreign protein is displayed on the surface of the host cells.42 Similar results were obtained by other groups,28,30,34,36 which demonstrate that the surface display system with the N-terminal domain of INP did not affect cell growth. Thus, this approach is proven to be efficient and stable for foreign protein expression. Effects of Conditions on the Surface Displaying XDH. Temperature and solution pH affect enzymatic activity to a great extent. Therefore, it is important to examine the effect of temperature and pH on the enzymatic characteristics of surfacedisplayed XDH. The related results are shown in Figure 3. The E. coli-displaying XDH was optimally active at ∼30 °C and was fairly stable at 40 °C (Figure 3A). The entire cell can retain over 90% of its relative activity within pH 6.0−8.0 (Figure 3B). The optimum enzyme activity was obtained within pH 6.0−8.0 at 22−33 °C, indicating that the displayed XDH retained relatively higher activity at a wide range of temperature and pH value, which makes the recombination E. coli strain an attractive candidate for further real applications. 278

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Table 2. Substrate Specificity of Cell Surface Displayed XDH substrate

concentration (mM)

absorbance at 340 nm

D-xylose

0.1 1 10 10 10 10 10 10 10 10 10

0.374 ± 0.028a 0.031 ± 0.007a 0 0 0 0 0 0 0 0 0

L-arabinose D-glucose D-ribose D-galactose D-mannose D-sucrose cellobiose D-xylitol D-fructose D-maltose

a

All values were obtained as the average of three repetitive measurements plus−minus standard deviation.

concentration of L-arabinose (1 mM) is 10-fold higher than that of D-xylose (Table 2). Usually, the concentration of Dxylose is much higher than that of L-arabinose in the actual degradation products of lignocelluloses and food samples,43which means no interference from L-arabinose with regard to the detection of D-xylose in real samples. In another test, a solution containing D-fructose, D-sucrose, D-maltose, Dglucose, D-mannose, D-galactose, D-xylitol, D-ribose (each 10 mM), and L-arabinose (1 mM), as well as D-xylose (0.1 mM), exhibited almost the same absorbance as the case of D-xylose (0.1 mM) only. In other words, higher concentrations of other tested saccharides did not affect the detection of D-xylose. Taken together, the cell-surface-displayed XDH showed high specificity for D-xylose, which can be developed for the accurate detection of D-xylose in real samples. In addition, it should be mentioned here that the N-terminal region of InaPb, which is an anchoring motif of XDH displayed on the surface of E. coli, could not affect the catalytic domain of XDH itself, which is similar to the case for substrate specificity with cytoplasmicexpressed XDH in the previous study.22 Detection of D-Xylose Using Cell-Surface-Displaying XDH. The UV−visible absorption spectra of PBS buffer (50 mM, pH 8.0) solution containing NAD+ (1 mM) and recombination strain (100 μL cell, OD600 of 1.0), as well as different concentrations of D-xylose are shown in Figure 4A. The calibration curve for D-xylose was obtained to plot the absorbance at 340 nm with D-xylose concentration (Figure 4B), from which the absorbance value is linear with a D-xylose concentration in the range of 5−900 μM with a correlation coefficient of 0.999. The low detection limit was 2 μM (S/N = 3). The recombinant strain was utilized to determine D-xylose in several products of lignocellulose degradation samples and food samples. To evaluate the performance of the proposed method, four samples were collected from actual lignocellulose degradation process and foods (beer and peaches). The peach was initially treated with a tissue tearor homogenizer. For the proposed method, the samples were filtered through a 0.22-μm membrane, and the filtrate was collected and diluted with 50 mM PBS buffer (pH 8.0) solution before measurement. For HPLC separation and detection, the sample was further derivatized using 1-phenyl-3-methyl-5-pyrazolone, and then filtered through a 0.22-μm membrane. The results were shown in Table 3. Apparently, the results are in good agreement with those achieved using HPLC method, which demonstrated that the recombinant cells were capable of precise detection of D-

Figure 3. Effects of (A) temperature and (B) pH on surface-displayed XDH.

Stability of Cultures Expressing INP-XDH Fusion Protein. To monitor the stability of INP-XDH fusion protein, the XDH activity of the entire cell from E. coli harboring pTInaPb-N/Xdh and crude enzyme solution from C. crescentus NA1000 were measured for one month. The entire-cell activity of E. coli declined by ∼24% during the one-month period. In contrast, ∼80% XDH activity was decreased for the crude enzyme solution from C. crescentus NA1000 (see Figure S2 in the Supporting Information). These results demonstrated that the recombinant strains were more stable than the cytoplasmicfree XDH from the original strain, considering the long-term stability and simplification of the protein purification step. Substrate Specificity of Cell-Surface-Displaying XDH. As shown previously, the cell-harboring pTInaPb-N/Xdh strain exhibited good activity and stability to D-xylose. Here we further investigated the substrate specificity of the E. coli harboring pET28a (+) and cell harboring pTInaPb-N/Xdh. The activity of XDH to saccharides including cellobiose, Dfructose, D-sucrose, D-maltose, D-glucose, D-mannose, Dgalactose, D-xylitol, D-ribose (each 10 mM) or L-arabinose (1 mM), as well as D-xylose (0.1 mM), was measured. Obviously, the E. coli harboring pET28a (+) exhibited no catalytic effect on the above-mentioned saccarides (data not shown); that is, the E. coli has no specificity to the tested saccarides. No absorbance peak at 340 nm was detected when cell harboring pTInaPb-N/ Xdh was mixed well with cellobiose, D-fructose, D-sucrose, Dmaltose, D-glucose, D-mannose, D-galactose, D-xylitol, and Dribose (each 10 mM), separately. However, a strong absorbance was detectible when D-xylose (0.1 mM) was present (see Table 2). In the case of L-arabinose (1 mM) as a substrate, obvious absorbance at 340 nm due to the catalysis of the cell displayed XDH was observed. The absorbance value for L-arabinose was just one-twelfth of that value for D-xylose, although the 279

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of-concept of the XDH-displayed bacteria (XDH-bacteria) for electrochemical biosensing applications, the XDH-bacteria was immobilized on the GCE surface. The voltammetric behavior of the Nafion/XDH-bacteria/GCE in D-xylose solution was examined by differential pulse voltammetry. The differential pulse voltammograms of the Nafion/XDH-bacteria/GCE in a NAD+-containing buffer solution in the absence or presence of 1 mM D-xylose are shown in Figure 5A. An oxidization peak at

Figure 4. (A) The UV−visible absorption spectra of solution with different concentration of D-xylose, the XDH surface-displayed cells (100 μL cell, OD600 of 1.0) and 1 mM NAD+. For the clarity of spectra for the lower concentrations, the spectra for high concentration (800, 900, 950, and 1000 μM xylose) were not shown. (B) Calibration curve for D-xylose. Error bars represent the standard error of three replicates.

Table 3. D-Xylose Concentrations of Four Samples Determined Using the Described Method and HPLC Method D-Xylose

Concentrationa

a

Figure 5. (A) Differential pulse voltammograms of Nafion/GCE (curve a) and Nafion/XDH-bacteria/GCE (curve b) in the presence of 1 mM D-xylose and 1 mM NAD+ in 0.1 M PBS (pH 7.4). The differential pulse voltammetry conditions: amplitude, 0.05 V; pulse width, 0.2 s; sampling width, 0.0167; and pulse period, 0.5 s. (B) Current−time curve for Nafion/bacteria-XHD/GCE at +0.7 V with increasing concentration of D-xylose in 10 μM steps.

xylose in a complex compounds. Compared with HPLC and other traditional methods, our strategy is characteristic of simple sample treatments, simple instrumentation, and high sensitivity and selectivity. Potential Biosensing of D-Xylose Using E. coliDisplayed XDH. The possible applications of this E. colidisplaying XDH may be interesting. In order to show the proof-

+0.7 V appeared in the presence of 1 mM D-xylose (line b in Figure 5A), which is from the direct oxidization of NADH, the product of the coenzyme of NAD+ during the XDH-displaying bacteria catalyzing the oxidization of D-xylose, as shown in eq 1. The current−time curve was obtained with the Nafion/XDHbacteria/GCE, via the successive addition of D-xylose, using amperometry at an applied potential of ∼0.7 V (Figure 5B). The oxidation current increased after the addition of D-xylose and reached a steady-state value of 95% within 5 s (Figure 5B). Thus, the XDH-bacteria-modified electrode exhibited good response to D-xylose, which indicates that the bacteriadisplaying XDH may find potential application in exploring amperometric enzymatic biosensor. Similarly, other types of

sample

this method

HPLC method

relative error (%)

#1b #2c #3d #4e

84.87 ± 1.45 mM 3.18 ± 0.27 mM 1.81 ± 0.13 mM 0.019% ± 0.002%

83.30 ± 2.13 mM 2.91 ± 0.22 mM 1.67 ± 0.16 mM 0.018% ± 0.002%

+1.9 +9.2 +8.4 +6.5

All values were obtained as the average of three repetitive measurements plus−minus standard deviation. b#1 represents the degradation product of straw after physicochemical processes. c#2 represents the fermentation liquor of degradation bacteria, which could utilize microcrystalline cellulose and xylan as the carbon sources for growth. d#3 represents the local Tsingtao beer. e#4 represents the local peach; here, the content of D-xylose is shown in terms of mass concentration.

280

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biosensors such as fiber-optic fluorescence-based biosensors could be developed using the XDH-displayed bacteria.

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CONCLUSIONS Using InaPb-N as its anchoring motif from P. borealis DL7, for the first time, we successfully displayed XDH on the cell surface of E. coli BL21 (DE3) without any inhibition on the host growth. The newly prepared XDH-displayed bacteria were found to have interesting applications in establishing a novel sensitive method for the detection of D-xylose. This approach exploited the catalysis of the oxidization of xylose to xylonolactone by the bacteria-displayed-XDH with coenzyme NAD+ and the resultant NADH being detected spectrometrically at 340 nm, which showed a wide linear range (5−900 μM) and a low detection limit of 2 μM of D-xylose. The prepared bacteria-displaying XDH is of high XDH activity and stability, compared with the free enzyme. In addition, the bacteriadisplayed XDH can be used directly without enzyme-extracting and purifying. Furthermore, good results were obtained using this approach to measure D-xylose in real samples such as degradation products of lignocelluloses, food, and fruits. In view of the proposed strategy is specific, sensitive, simple, rapid, and cost-effective, which holds great potential in on-site detection of xylose in many fields. Considering all of the abovementioned excellent properties, the genetically engineered cells may have a broad application in biosensors and biocatalysts, which occurs in this laboratory. Similarly, this type of genetic approach may be used for the expression of other intracellular enzymes for certain purposes, which could be used for other enzyme/analyte of interest (i.e., glucose oxidase/ glucose).

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ASSOCIATED CONTENT * Supporting Information All primers used for the construction of recombinant plasmids, the details for the construction of plasmid pTInaPbN-Xdh, cell fractionation and time course analysis of the XDH activity of the entire cell. This material is available free of charge via the Internet at http://pubs.acs.org. S

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AUTHOR INFORMATION Corresponding Author *E-mails: [email protected] (A.L.), [email protected] (M.M.).

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ACKNOWLEDGMENTS This research was supported in part by the Hundred-TalentProject (No. KSCX2-YW-BR-7) and the Knowledge Innovation Project in Biotechnology (No. Y11211110S), Chinese Academy of Sciences.

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dx.doi.org/10.1021/ac202513u | Anal. Chem. 2012, 84, 275−282

Analytical Chemistry

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