YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 Protein Expression and Purification xxx (2014) xxx–xxx 1

Contents lists available at ScienceDirect

Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 5 6

Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis

3 4 7

Q1

8 9 10 11 12 13 14 1 7 6 2 17 18 19 20 21 22 23 24 25 26

Luz Edith Casados-Vazquez a,b, Salvador Avila-Cabrera b, Dennis K. Bideshi c,d, J. Eleazar Barboza-Corona a,b,⇑ a

Universidad de Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, Irapuato, Guanajuato 36500, Mexico Department of Food, Irapuato, Guanajuato 36500, Mexico California Baptist University, Department of Natural and Mathematical Sciences, 8432 Magnolia Avenue, Riverside, CA 92504, United States d Department of Entomology, University of California, Riverside, Riverside, CA 92521, United States b

Q2

c

a r t i c l e

i n f o

Article history: Received 21 August 2014 and in revised form 12 November 2014 Available online xxxx Keywords: Endochitinase ChiA74 Bacillus thuringiensis Purification Characterization

a b s t r a c t ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 50 sequence coding for its secretion signal peptide (chiA74Dsp) into a cold shock expression vector (pCold I) for production of the enzyme in Escherichia coli BL21-Rosetta2. As a result, the N-terminal end of ChiA74Dsp ORF was fused to an artificial sequence of 28 amino acid, including a 6 histidine tag for purification of recombinant 6His tagged-ChiA74Dsp (rChiA74, 74 kDa). Along with a protein of 74 kDa, we co-purified its 55 kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40 °C. Most divalent cations (e.g. Ba+2 and Ca+2, Mn+2, Mg+2, Zn+2, Cu+2) at concentration of 10 mM reduced chitinase activity by 30%, and Hg+2 (10 mM) drastically inhibited ChiA74 activity by 75–100%. The Vmax, Km and kcat for rChiA74 were 0.11 ± 0.01 nmol/min, 2.15 lM ± 0.45 and 3.81 s1, respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected. Ó 2014 Published by Elsevier Inc.

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

45 46

Introduction

47

Chitin is a homopolymeric polysaccharide formed by b-(1 ? 4)linked units of N-acetylglucosamine (GlcNAc)1. It is ubiquitous in nature, occurring as structural components of insect exoskeleton, fungal cell wall, shells of terrestrial and marine crustaceans, and nanofibrils. It is also responsible for mimicking a variety of colors observed in bird plumage and butterfly wings [1,2]. Indeed, after cellulose, chitin is the most abundant polysaccharide in nature [3], and is a valuable natural resource for production of bioactive derivatives. In this regard, both chemical cleavage and enzymatic hydrolysis

48 49 50 51 52 53 54 55

⇑ Corresponding author at: Food Department, Life Science Division, Graduate Program in Biosciences, University of Guanajuato, Irapuato, Guanajuato 36500, Mexico. Tel./fax: +52 462 624 1889. E-mail address: [email protected] (J.E. Barboza-Corona). 1 Abbreviations used: GlcNAc, N-acetylglucosamine; ORF, open reading frame; Bt, Bacillus thuringiensis; FPLC, fast protein liquid chromatography; 4-MU-GlcNAc3, 4-methylumbelliferyl b-D-N,N0 ,N00 -triacetylchitotrioside; SDS–PAGE, SDS–polyacrylamide gel; OGS, oligosaccharides; TLC, thin layer chromatography; DP, degree of polymerization.

have been employed for generating these compounds that have potential medicinal, industrial and agronomical applications [4,5]. To this end, chitinolytic enzymes have received considerable attention. These enzymes, which play significant roles in the natural biology of prokaryotes and eukaryotes, are ubiquitous glycosyl hydrolases (enzyme family 18, 19, 24 and 49) classified as endochitinases that randomly hydrolyze chitin at internal sites, and exochitinases that remove GlcNAc units from the non-reducing end of the polymer [6,7–13]. In particular, chitinases of Bacillus thuringiensis (Bt) the most successful microbial biopesticide used worldwide, have received increasing attention over the last several years, primarily because they can (i) synergize insecticidal crystalline (Cry) proteins of Bt, (ii) be used to control phytopathogenic fungi, and (iii) be used to generate chitin-oligosaccharides with bioactive properties [10,14– 15]. Of 40 chitinase genes of B. thuringiensis reported (GenBank, www.ncbi.nlm.nih.gov/genbank/) [16], only 30% of the encoded chitinases have been partially characterized at the biochemical level [9,17–19]. To our knowledge the Vmax and Km have been determined only for an exochitinase from B. thuringiensis subsp. aizawai [10].

http://dx.doi.org/10.1016/j.pep.2014.11.015 1046-5928/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 2 76 77 78 79 80 81 82

L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx

Previously, we have partially characterized the physical and biochemical properties and potential applied versatility of ChiA74, a chitinase synthesized by Bt using crude preparations of the bacterial secretome [9]. Here, using purified recombinant rChiA74, we have further determined its Vmax, Km and kcat, using a fluorogenic chitin derivate. We also demonstrate that optimal activity of rChiA74 occurs at pH 7 and 40 °C.

83

Materials and methods

84

Cloning of ChiA74 and recombinant E. coli strains

85

110

The endochitinase chiA74Dsp gene lacking its secretion signal peptide sequence [15] was amplified using chiA74-34-Nter/HindIII (50 CCGCCGAAGCTTGATTCACCAAAGCAAAGTCAAAAA30 ) and chiA74-Cter/HindIII (50 GCCGCCAAGCTTCTAGTTTTCGCTAATGACGGCATT 30 ), forward and reverse primers, respectively. The HindIII restriction site is shown underlined whereas the stop codon is indicated in italics in the reverse primer. Gene amplification was performed using pEBchiA74Dsp as a template that harbors the chiA74Dsp under regulation of the cytA-p/STAB-SD expression system [15]. Conditions for amplification were as follow: an initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min, an extension at 72 °C for 2 min and final extension of 72 °C for 10 min. The amplicon (2 kbp) and cold shock expression vector (pCold I) (Takara Bio Inc, Otsu Shiga Japan) were digested with HindIII and then purified with the gel extraction kit (Qiagen, Valencia, CA, USA). The vector was dephosphorylated and the amplicon was ligated into pCold I overnight at 16 °C. Escherichia coli TOP10 was transformed with the recombinant plasmid (pCold I-chiA74Dsp) and selected with ampicillin (100 lg/ml). For endochitinase expression and purification of the recombinant 6-histidine tagged protein (hereafter rChiA74), calcium competent BL21-Rosetta2 E. coli was transformed with pCold I-chiA74Dsp. Briefly, rChiA74 contained the N-terminal end coded by ChiA74Dsp fused to a heterologous sequence of 28 amino acid that included a 6 histidine tag and Factor Xa cleavage sites coded by pCold I.

111

Protein structure homology modeling

112

117

Structural modeling and visualization of native ChiA74 and rChiA74 were performed using the I-TASSER server for protein 3D structure prediction [20,21]. The 3D structure of ChiA1 of Bacillus circulans was used for modeling [22,23]. Molecular graphics were performed with the UCSF Chimera package (www.cgl. ucsf.edu/chimera).

118

Expression and purification of rChiA74

119

Recombinant E. coli BL21 Rosetta2/pCold I-chiA74Dsp was grown overnight in 2.5 ml of Luria–Bertani broth supplemented with ampicillin (100 lg/ml) and chloramphenicol (34 lg/ml), and then transferred to 250 ml fresh medium supplemented with the same antibiotics. The culture was grown at 37 °C and 200 rpm to an OD600 of 04–0.6. Then the culture was incubated at 15 °C for 30 min, IPTG was added to a final concentration of 0.5 mM, and incubation was continued at 16 °C for 24 h at 200 rpm. The culture was centrifuged and the supernatant was discarded. The pellet was resuspended and incubated on ice for 30 min in 25 ml of buffer A (100 mM Tris–HCl pH 7, 500 mM NaCl, 10 mM imidazole) supplemented with 1 mg/ml lysozyme (final concentration). Then, the sample was sonicated ten times, 30 s each, at an amplitude of 30 Hz using a 20 kHz ultrasonic processor (Sonic and Materials, Inc., Newtown, CT 06470-1614 USA). The extract was centrifuged

86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

113 114 115 116

120 121 122 123 124 125 126 127 128 129 130 131 132 133

30 min at 13,000g and the supernatant was passed through a HiTrap Ni affinity column (GE Healthcare Bio-Sciences AB, Upsala Sweden) pre-equilibrated with buffer A. Unbound protein was removed with 40 ml of buffer A, 40 ml of buffer A-20 mM imidazole, and rChiA74 was finally eluted with 5 ml of buffer A-500 mM imidazole. Dialysis was carried out in buffer A without imidazole and protein concentration was determined using the Quick Start Bradford 1x Dye reagent (BioRad, Hercules CA, USA). Sample purified by Ni affinity was loaded onto a Superdex 200 10/300 GL (GE Healthcare life science) column previously equilibrated with buffer A (100 mM Tris–HCl pH 7.0, 150 mM NaCl) and rChiA74 was separated by fast protein liquid chromatography (FPLC) (Biologic Duo-Flow Pathfinder 20 System BioRad, Hercules CA, USA). Fractions of 1 ml were collected at a rate of 0.5 ml/min using buffer A and monitored at 280 nm. Fractions in the peak were collected and analyzed by 10% SDS–PAGE and zymograms. Endochitinase activity was determined in triplicate assays at 37 °C in 100 mM phosphate buffer, pH 7, using the substrate 4-methylumbelliferyl b-D-N,N0 ,N00 -triacetylchitotrioside (4-MUGlcNAc3) (Sigma) and purified rChiA74 at final concentrations of, respectively 2.5 lM and 0.7 nM, similar as previously described [9]. The amount of 4-MU released from the substrate was calculated fluorometrically (excitation at 360 nm and emission at 455 nm) with the Glomax Multi Jr. Detection System (Promega, Sunnyvale CA, USA) using a 4-MU standard curve. One unit (U) of chitinolytic activity was defined as the amount of enzyme required to releases 1 lmol of 4-methylumbelliferone in 1 h.

134

Western blot analysis using His-tag antibody

161

rChiA74 purified by Ni column was subjected to Western blot analysis using His-tag antibodies. rChiA74 was separated in 10% SDS–PAGE and then proteins were transferred from the gels onto PVDF membranes (Bio-Rad) using a semi-dry transfer system (Semiphor, Hoefer Instruments). Membranes were blocked for 2 h with 1% (w/v) casein in a blot buffer [100 mM NaCl, 10 mM Tris– HCl pH 7.6, 1 mM EDTA, 0.1% (v/v) Tween-20] and incubated for 3 h at room temperature with anti-His antibody. Membranes were washed with blot buffer and incubated for 1 h at room temperature with IgG coupled with anti-horseradish peroxidase in the blot buffer. Then five washes were carried out with blot buffer and two with blot buffer without Tween-20. Detection was performed with ECL Western blotting substrate (Thermo scientific).

162

SDS–PAGE and zymograms

175

Fractions containing rChiA74 were monitored by SDS–PAGE and zymogram to corroborate the presence and activity of the purified recombinant protein. Portions of each fraction were treated with Laemmli’s disruption buffer supplemented with b-mercaptoethanol. Identical samples were fractionated by electrophoresis in a 10% SDS–polyacrylamide gel (SDS–PAGE). One gel was stained with coomassie blue and the other was washed four times with casein-EDTA buffer [1% casein, 2 mM EDTA, 40 mM Tris–HCl, pH 9]. Finally the gel was equilibrated with 100 mM sodium acetate buffer, pH 5.0 for 20 min. The gel was covered with 1% low melting temperature agarose (Sigma) supplemented with the fluorogenic substrate (25 lM). After 5 min incubation at 37 °C, reactions were monitored with UV light and detection of chitinase activity was performed as previously described [9].

176

Effect of pH and temperature on chitinase activity

190

Optimal pH for endochitinase activity was determine by performing the standard activity assay at 37 °C with 1.25 lM of substrate and 0.8 nM of rChiA74. The pH of reaction buffers

191

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

163 164 165 166 167 168 169 170 171 172 173 174

177 178 179 180 181 182 183 184 185 186 187 188 189

192 193

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx

3

In addition, the heterologous 28 amino acids that included the 6 histidine-tag and Factor Xa cleavage sites in the N-terminal of rChiA74 are out of the catalytic center and were predicted to have little or no effect on the enzymatic activity of rChiA7 (Fig. 1).

250

Production, endochitinase ChiA74 purification and Western blot analysis

254

200

(100 mM) varied between 3 and 10 with increments of one pH unit, as follows: sodium citrate/citric acid (pH 3 and 4), sodium acetate/acetic acid (pH 5), phosphate buffer (pH 6 and 7), and Tris–HCl (pH 8 to 10). Optimal temperature was measured using the standard chitinase assay in phosphate buffer (pH 7.0) [9] at temperatures between 0 and 60 °C for 5 min; the increments were 5 °C for 0 to 20 °C and 10 °C for 20 to 60 °C.

201

Effects of divalent ions

256

202

208

After size-exclusion chromatography, purified rChiA74 (60 nM) was incubated with 1, 5 and 10 mM of salt solutions (BaCl2, SnCl2, HgCl2, CaCl2, MnCl2, MgCl2, CuCl2 and ZnCl2) for 10 min at room temperature in 100 mM acetate buffer pH 5. Seven microliters of the treated enzyme was used to perform the assays in triplicate at 37 °C using the substrate 4-MU-GlcNAc3 as explained above [9,24–25].

209

Determination of kinetic constants

210

Kinetics experiments were performed using purified rChiA74 and the 4-MU-GlcNAc3 substrate. Reaction mixtures were incubated at 37 °C in 100 mM phosphate buffer (pH 7.0), using 0.8 nM purified chitinase and 0.25, 0.5, 0.75, 1, 1.25, 2, 4, 8 lM of 4-MU-GlcNAc3. The amount of 4-MU released from the fluorogenic substrate was calculated spectrofluorometrically from a calibration curve using the same concentration of substrate with an excess of enzyme to convert all the substrate in product. The released fluorogenic product was measured fluorometrically, as described above. Assays were repeated in triplicate. Initial velocities were used to calculate kinetic constants (Vmax, Km, kcat) using GraphPad Prism Version 6.04 (www.graphpad.com).

As expected, a protein of 74 kDa (the deduced molecular mass rChiA74 that hydrolyzed the 4-MU-GlcNAc3 fluorogenic substrate) and also of a 55 kDa were observed by SDS–PAGE and zymogram analyses after Ni column affinity and size-exclusion chromatography (Fig. 2e). The 55 kDa protein is likely a processed derivative of rChiA74 (i.e. 74 kDa) because (i) E. coli BL21 does not synthesize a chitinase (see lane 2, Fig. 2a), and (ii) both proteins screened positive using the His-tag antibody in Western blot analysis (Fig. 2g and h). Similar functional processed species are known for other chitinases [9,17–18]. Samples from IPTG-induced cell culture had a 314-fold higher activity than samples from non-induced cells (data not shown). These activities were also in agreement with the signal intensities observed in zymograms, i.e. higher activity in samples under induction when compared with the activity in samples from non-induced cells (Fig. 2b). In addition, we could obtain a good quantity of soluble protein after cellular broken (Fig. 2a). When samples containing rChiA74 was loaded on HiTrap Ni affinity column, apparently there was a high affinity, and small amount of protein was lost in the washes (Fig. 2, lane 9). With the Ni affinity column we were able to obtain a yield and purification of 32% and 1.5-fold, respectively. In addition, when sample purified by Ni affinity was subjected to size-exclusion chromatography, we observed a yield of 11% without change in the purification (Table 1). Effect of pH and temperature on ChiA74 activity

280

Purified rChiA74 showed its maximum enzymatic activity at pH 7.0 (Fig. 3a). Additionally, activity was tested under a gradient of temperatures, where the highest activity was observed at 40 °C (Fig. 3b).

281

Effect of divalent cations on ChiA74 activity

285

We did not observe an increment in the chitinase activity using divalent cations compared with the control (Fig. 4). In fact, most of the divalent cations diminished the chitinase activity at 30% at concentration of 10 mM, and Hg+2 (1, 5,10 mM) reduced drastically the ChiA74 activity at values of 75–100%.

286

Kinetic constant values

291

The Vmax, Km and kcat for purified recombinant ChiA74, using the 4-MU-GlcNAc3 substrate, were 0.11 ± 0.01 nmol/min, 2.15 lM ± 0.45 and 3.81 s1, respectively (Fig. 5 and Table 2).

292

Generation of chitin-derived oligosaccharides

295

Using purified rChiA74 and colloidal chitin as a substrate, chitin-derived oligosaccharides with degree of polymerization of 2 (GlcNAc2) and 1 (GlcNAc) were detected, mainly the dimeric compound (i.e. N,N0 -diacetylchitobiose) (Fig. 6). Interesting, we repeated the assay three times on different days with distinct purified sample, and under these conditions of the assay, we were unable to detect the chitin-derived oligosaccharides with degrees of polymerization (DP) of 3, 5 and 6, as we reported previously using crude enzyme preparations [14].

296

194 195 196 197 198 199

203 204 205 206 207

211 212 213 214 215 216 217 218 219 220 221

222 223

Chitin hydrolysis with rChiA74 and thin layer chromatography analysis

239

Mixtures containing 2.0 U of rChiA74, 0.5% (w/v) colloidal chitin, and 100 mM phosphate buffer (pH 7.0) were incubated with continuous agitation (200 rpm) to avoid solid substrate precipitation at 37 °C for 2–12 h to generate chitin-derived oligosaccharides (OGS). Samples were centrifuged and non-dissolved chitin was discarded. Supernatants were concentrated in a DNA120 SpeedVac (ThermoSavant) and pellets were resuspended in 10 ll of double distilled water and 10 ll of methanol. OGS were analyzed by silica gel (Merk) thin layer chromatography (TLC) using 5:4:3 (v/v/v) n-butanol:methanol:16% aqueous ammonia as the mobile phase. Samples were visualized by staining with 20% (v/v) sulfuric acid in ethanol and incubated at 80 °C until chitin-derived oligosaccharides signals were developed. To determine the degree of polymerization (DP), molecular markers (Sigma) used were N-acetyl-D-glucosamine (GlcNAc); N,N0 -diacetylchitobiose (GlcNAc2) and N,N0 ,N00 -triacetylchitotriose (GlcNAc3) [14].

240

Results

241

ChiA74 and rChiA74 modeling

242

Models of the deduced 3-D structure of ChiA74Dsp was compared with rChiA74 based on sequence similarity with ChiA of B. circulans [22,23]. The data showed that both proteins did not have structural differences. Also, corresponding residues have the same coordinates in the untagged and 6 histidine-tagged structure, for example residues D-207, E-211, W-123 and W-137, which have been demonstrated to be important for the catalytic activity in ChiA1 of B. circulans [26], have essentially the same position.

224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

243 244 245 246 247 248 249

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

251 252 253

255

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279

282 283 284

287 288 289 290

293 294

297 298 299 300 301 302 303 304

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 4

L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx

(a) (I)$

cspAp) 6xHis

chiA74Δsp$ (II)$

(b)

(c)

Fig. 1. Schematic illustration and modeling of 6-histidine tagged-ChiA74Dsp (rChiA74). (a) The chiA74 orf was amplified without the sequence coding the signal peptide (chiA74Dsp) and inserted into the HindIII site of the pCold I vector in frame with heterologous sequences coding for 28 amino acids, including the 6 histidine tag, in the N-terminal end of rChiA74. The recombinant gene is under the control of the cspA promoter (cspAp). Lollipop indicates the putative transcriptional terminator site of the cspA gene. (I) and (II) indicate the position of chiA74-34-Nter/HindIII and chiA74-34-Cter/HindIII, forward and reverse primers used to amplify the chiA74Dsp orf (for sequences see materials and methods). (b) and (c) are the 3-D deduced structures of ChiA74Dsp and rChiA74, respectively. Aspartic acid in N-terminal is indicated in black. Sequence including 6His tagged and Factor Xa cleavage site are colored in green. Amino acids aspartic acid 207, glutamic acid 211, tryptophan 123 and 137 are shown as sticks, these residues have been reported to be important for catalysis in Bacillus circulans chitinase. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

a

b 1

kDa

2

3

4

5

6

7 8 9

1

2 3

4 5

6

7

8

9

180 115 82

49

c

mS/cm

e 1

g

f 2

1

h

2

AU

d 12 3 4

Min.Tenth

Fig. 2. Production in E. coli BL21 Rosetta2/pCold and purification of 6His tagged-ChiA74Dsp (rChiA74). (a) SDS–PAGE; protein standard (lane 1); non-induced cells (lane 2); IPTG induced cells (lane 3), cells pellet (lane 4) and supernatant (lane 5) following cell lysis. Preparations of sample (use in lane 5) was separated in a HiTrap Ni affinity column; flow through (lane 6); sample from first wash with buffer A (lane 7); sample from second wash with buffer A-20 mM imidazole (lane 8), and elution of the protein with buffer A-500 mM imidazole (lane 9). (b) Zymogram of samples fractionated in (a) using 4-MU-GlcNAc3 (Sigma) as substrate. (c) Chromatogram of purification by sizeexclusion chromatography using a Superdex 200 10/300 GL (GE Healthcare life science) column. (d) The four fractions of the peak obtained by size-exclusion chromatography were collected and analyzed by SDS–PAGE. (e) SDS–PAGE, (f) zymogram of sample purified by Ni affinity column (lane 1) and size-exclusion chromatography (lane 2). Western blot analysis (g) Sample purified by Ni affinity column, (h) detection using His-tag antibodies.

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 5

L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx Table 1 Purification of rChiA74 produced in Escherichia coli L21 Rosetta 2. Step b

Crude extract Niquel purificationc Size-exclusiond a b c d

Q5

Volume (ml)

Total activitya (U)

Total protein (mg)

Specific activity (Umg1)

Yield (%)

Purification (Fold)

25 5 1

3107 1002 341

65 13.5 4.53

47.80 74.20 75.23

100 32.24 10.97

1.0 1.55 1.57

One unit (U) was defined as the amount of enzyme required to release 1 lmol of 4-methylumbelliferone in 1 h [18]. Crude extract obtained after saline precipitation and dialysis. Purified enzyme following Hi Trap Ni affinity column chromatography. Fast protein liquid chromatography using a size-exclusion column (Superdex 200 10/300, GE Healthcare life science).

Fig. 3. Effect of pH (a) and temperature (b) on the enzymatic activity of rChiA74, as determined fluorometrically following hydrolysis of 4-MU-(GlcNAc)3.

0.10

0.08

0.06

0.04

0.02

0.00

0

Fig. 4. Effect of divalent cations on rChiA74 hydrolysis of 4-MU-(GlcNAc)3. Mix reaction not supplemented with cations was used as control.

305

Discussion

306

Previously, we partially characterized the physical properties that influence ChiA74 endochitinase activity in crude extracts prepared from recombinant E. coli DH5a producing the enzyme [9]. In addition, for applied purposes we have also expressed ChiA74 in a GRAS strain of E. coli and, importantly, showed that crude extract of the enzyme is useful for generating chitin-derived oligosaccharides with antibacterial activity [14]. We have also demonstrated that stable inclusion of ChiA74 could be produced in recombinant B. thuringiensis subsp. kurstaki HD1 harboring its native insecticidal proteinaceous crystalline (Cry) cassette, a promising result with commercial applications in insect biocontrol [15–16]. Altogether, these studies demonstrated the potential for applied use of ChiA74 in medical and food industries, in agronomy and insect pest

307 308 309 310 311 312 313 314 315 316 317 318

2

4

6

8

10

Fig. 5. Determination of Km of purified rChiA74 using the dependence of initial velocity on substrate concentration.

biocontrol. In particular, our primary purpose in the present study was to devise a strategy to overexpress N-terminal 6-histidine tagged recombinant rChiA74 to obtain a purified enzyme for basic and applied studies, specifically with the intent of using the enzyme to generate bioactive derivatives of chitin. The results obtained in this study demonstrate that purified rChiA74 produced in a heterologous expression system retains its native activity and does not require accessory proteins, metal cofactors or other host-derived factors for its function in vitro. The host bacterium produced an endochitinase of the expected size of 74 kDa [9], suggesting that the protein is structurally stable. We also observed a shorter functional chitinase of 55 kDa during the purification process using Ni-affinity column and size-exclusion chromatography (Fig. 2), a unique species also observed when ChiA74 was expressed in both B. thuringiensis and E. coli [9,15]. The 55 kDa is likely a C-terminal processed form of the 74 kDa rChi-

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 6

L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx

Table 2 Comparative analysis of the kinetic parameters of purified rChiA74 with other chitinases using 4-Mu-GlcNAc3. Chitinasea

Vmax (nmol/min)

Kmb (lM)

kcat (s1)

kcat/Km (s1/lM)

Refs.

ChiA74 ChiA1 Bc ChiA Ac BJL200-ChiA BJL200-ChiB BJL200-ChiC1 Chi Ba old EppoNPV Chic EppoNPV Chid

0.11 – –

2.15 3.6 129.3 4.2 6.8 79.8 4.07 20 14

3.81 1.5 0.667 67.4 56.8 2.0

1.77 4  101 5  103 16 8.4 0.025

This work [26] [32] [31] [31] [31] [33] [34] [34]

270

a Bc, Bacillus circulans WL-12; Ac, Aeromonas caviae; EppoNPV, Epiphyas postvittana nucleopolyhedrovirus; BJL200, Serratia marcescens BJL200; Ba old, suspension-cultured bamboo (Bambusa oldhamii). b For each enzyme, kinetic parameters were evaluated using optimal pH and temperature. ChiA74, pH 7, 37 °C; ChiA1 Bc, pH6, 37 °C; ChiA Ac, pH 6.5, 42.5 °C; BJL200-ChiA, BJL200-ChiB, pH 6.3; Chi Ba old, pH 3.0, 70 °C; EppoNPV Chi, pH 6. c Native. d Recombinant.

1

GlcNAc (GlcNAc)2

2

3

4

5

6 * *

(GlcNAc)3 Fig. 6. Thin layer chromatography (TLC) analysis of chitin-derived oligosaccharides produced by hydrolytic action of rChiA74 on colloidal chitin. Lane 1, molecular markers (Sigma): N-acetyl-D-glucosamine (GlcNAc), N,N-diacetylchitobioside (GlcNAc2), N,N,N00 -triacetylchitotriose (GlcNAc3); lanes 2–5, oligosaccharides (see *) produced from colloidal chitin treated with purified rChiA74; lane 6, mix reaction containing chitin but not rChiA74 treated as indicated in material and methods.

335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368

A74 based on Western blot analysis and because it is known that E. coli BL21 Rosetta2/pCold does not synthesize chitinase. Similar C-terminal processed ChiA71 and chitinase A1 have been reported from B. thuringiensis subsp. pakistani and B. circulans WL-12, respectively [9,17,26]. Previously, we reported that mature ChiA74 in crude extracts obtained from E. coli DH5a has an optimal activity at 55 °C [9], significantly different than that observed for rChiA74, i.e. 40 °C. The difference could be a result of factors present in crude extract secretome, including proteins that physically interact with ChiA74. No significant differences in the optimal pH range for crude extracts [9], and rChiA74 and dissolved inclusions produced in Bt [15], respectively, pH 6.5 and pH 7, were noted. It has been established that different divalent cations can enhance or reduce optimal activity of several microbial chitinases. Interestingly, we demonstrate that the endochitinase activity of rChiA74 is significanty reduced in the presence of metal such as Ba+2 and Ca+2, Mn+2, Mg+2, Zn+2, Cu+2, similar to chitinases from Pseudomonas sp, TKU015 and Streptomyces SP DA11 and Bacillus sp 33.26 [28]. Moreover, Hg+2 (1, 5, 10 mM) drastically reduced (75–100%) rChiA74 activity. Although we have not studied the mechanism of inhibition, it is probably that Hg+2 interact with S–S or –SH group of rChiA74, forming a chitinase-Hg complex, changing the conformation and reducing the activity of the enzyme, as has been observed with nitrogenase, b-N-acetylglucosaminidase of carp spermatozoa, and chitinases from Serratia sp. KCK, B. subtilis NPU 001 and Streptomyces aureofaciens CMUAc130 [29–30]. In addition, we determined the Vmax, Km, and kcat of rChiA74 using 4-MU-GlcNAc3 as the substrate. Previously, the Vmax and Km for an exochitinase of B. thuringiensis subsp. aizawai was determined using colloidal chitin as the substrate [10]. We were interested in determining the kinetic parameters of rChiA74 using the 4-MU-GlcNAc3 as substrate because we routinely use this

tetrameric chitin derivate in crude chitinase screening and assays. Comparison of the Km and the catalytic efficiency (kcat/Km) of rChiA74 with other chitinases whose constants were determined using the same substrate showed that rChiA74 has a higher affinity for the synthetic derivative than chitinases from B. circulans, Serratia marcescens BJL200, Epiphyas postvittana nucleopolyhedrovirus and Bambusa oldhamii, but the catalytic efficiency was lower than chitinases ChiA1 and ChiB from S. marcescens BJL200 [26,31–34]. When we assayed the ability of the rChiA74 to hydrolyze colloidal chitin, we detected mainly GlcNAc2 (N,N0 -diacetylchitobiose), and GlcNAc. This is very interesting, as we have previously showed that using crude extract of ChiA74, chitin-derived oligosaccharides with DP of 3, 5, and 6 are also produced. In different studies we have demonstrate the endochitinase activity of ChiA74 using fluorogenic substrates [9,14–15], but the result obtained here using different substrate, suggest that ChiA74 could have a dual activity (i.e. endochitinases/chitobiosidase). Similar endochitinases/chitobiosidase activities using chitin and fluorogenic substrates have been demonstrated with ChiA of S. marcescens BJL200 [27]. Regardless, these derivatives could have applied utility as GlcNAc2 has been used as an inhibitor of lysozyme and an immunopotentiator (http://www.chemicalbook.com/ChemicalProductProperty_EN_CB1689710.htm), whereas GlcNAc has been employed to treat the joint damage and the inflammatory bowel disease, among others [35]. In summary, apart from roles bacterial chitinase play in the natural biology of prokaryotes and eukaryotes, its derivatives have several potential applications in food biotechnology, agronomy and medicine. Based on previous studies, and the present work, it is clear that purified rChiA74 has a potential for use as an endochitinase for generating bioactive derivative of chitin, and we have elucidated both physical and biochemical parameters for the efficient use of this enzyme. In future studies, we plan to determine the three-dimensional structure of ChiA74 and to develop strategies to engineer the enzyme to optimize its applied value.

369

Acknowledgments

405

This research was supported by Grant SEP-CONACYT (156682) Q3 México to J.E. Barboza-Corona. S. Ávila-Cabrera and L.E. CasadosVázquez are undergraduate student and Postdoctoral researcher, Q4 respectively, and were supported by CONACYT, México.

406

References

410

[1] M.D. Lenardon, C.A. Munro, N.A. Gow, Chitin synthesis and fungal pathogenesis, Curr. Opin. Microbiol. 13 (2010) 416–423.

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404

407 408 409

411 412

YPREP 4611

No. of Pages 7, Model 5G

2 December 2014 L.E. Casados-Vazquez et al. / Protein Expression and Purification xxx (2014) xxx–xxx 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

[2] P. Morganti, Nanoparticles and nanostructures man-made or naturally recovered: the biomimetic activity of chitin nanofibrils, J. Nanomater. Mol. Nanotechnol. 1 (2012) 2. [3] S.A. Shaikh, M.W. Deshpande, Chitinolytic enzymes: their contribution to basic and applied research, World J. Microbiol. Biotechnol. 9 (1993) 468–475. [4] R. Tarsi, R.A.A. Muzzarelli, C.A. Guzman, C. Pruzzo, Inhibition of Streptococcus mutans adsorption to hydroxyapatite by low-molecular-weight chitosans, J. Dent. Res. 76 (1997) 665–672. [5] R.A. Muzzarelli, Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone, Carbohydr. Polym. 76 (2009) 167–182. [6] S. Adrangi, M.A. Faramarzi, From bacteria to human: a journey into the world of chitinases, Biotechnol. Adv. 31 (2013) 1786–1795. [7] R.G. Boot, G.H. Renkema, A. Strijland, A.J. van Zonneveld, J.M. Aerts, Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages, J. Biol. Chem. 270 (1995) 26252–26256. [8] R.G. Boot, E.F. Blommaart, E. Swart, K. Ghauharali-van der Vlugt, N. Bijl, C. Moe, A. Place, J.M. Aerts, Identification of a novel acidic mammalian chitinase distinct from chitotriosidase, J. Biol. Chem. 276 (2001) 6770–6778. [9] J.E. Barboza-Corona, E. Nieto-Mazzocco, R. Velázquez-Robledo, R. SalcedoHernández, M. Bautista, B. Jiménez, J.E. Ibarra, Cloning, sequencing and expression of the chitinase gene chiA74 from Bacillus thuringiensis, Appl. Environ. Microbiol. 69 (2003) 1023–1029. [10] L. Morales de la Vega, J.E. Barboza-Corona, M.G. Aguilar-Uscanga, M. RamírezLepe, Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi, Can. J. Microbiol. 52 (2006) 651–657. [11] P.H.B. Poulsen, J. Moller, J. Magid, Determination of a relationship between chitinase activity and microbial diversity in chitin amended compost, Bioresour. Technol. 99 (2008) 4355–4359. [12] M.W. Delpin, A.E. Goodman, Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster, ISME J. 3 (2009) 1053–1063. [13] M.S. Cretoiu, A.M. Kielak, W.A. Al-Soud, S.J. Sørensen, J.D. van Elsas, Mining of unexplored habitats for novel chitinases-chiA as a helper gene proxy in metagenomics, Appl. Microbiol. Biotechnol. 94 (2012) 1347–1358. [14] J.C. Castañeda-Ramírez, N.M. de la Fuente-Salcido, R. Salcedo-Hernández, F. León-Galván, D.K. Bideshi, J.E. Barboza-Corona, High-level synthesis of endochitinase ChiA74 in Escherichia coli K12 and its promising potential for use in biotechnology, Folia Microbiol. 58 (2013) 455–462. [15] J.E. Barboza-Corona, J.L. Delgadillo-Ángeles, J.C. Castañeda-Ramírez, U.E. Barboza-Pérez, L.E. Casados-Vázquez, D.K. Bideshi, M.C. del Rincón-Castro, Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74Dsp chitinase inclusions, Cry crystals and spores for applied use, Microb. Cell Fact. 13 (2014) 15. [16] E. Barboza-Corona, N.M. de la Fuente-Salcido, M.F. León-Galván, Future challenges and prospects of Bacillus thuringiensis, in: E. Sansinenea (Ed.), Bacillus thuringiensis Biotechnology, Ó Springer Science+Business Media B.V., 2012, pp. 367–384 (DOI 10.1007/978-94-007-3021-2_19). [17] S. Thamthiankul, S. Suan-Ngay, S. Tantimavanich, W. Panbangred, Chitinase from Bacillus thuringiensis subsp. pakistani, Appl. Microbiol. Biotechnol. 56 (2001) 395–401. [18] J.E. Barboza-Corona, D.M. Reyes-Rios, R. Salcedo-Hernández, D. Bideshi, Molecular and biochemical characterization of an endochitinase (ChiA-

[19]

[20] [21] [22]

[23] [24]

[25]

[26]

[27]

[28]

[29] [30]

[31]

[32]

[33]

[34]

[35]

7

HD73) from Bacillus thuringiensis subsp. kurstaki HD-73, Mol. Biotechnol. 39 (2008) 29–37. N.M. Rosas-García, J.M. Fortuna-González, J.E. Barboza-Corona, Characterization of the chitinase gene in Bacillus thuringiensis mexican isolates, Folia Microbiol. 58 (2013) 483–490. Y. Zhang, I-TASSER server for protein 3D structure prediction, BMC Bioinformatics 9 (2008) 40. A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unified platform for automated protein structure and function prediction, Nat. Protoc. 5 (2010) 725–738. T. Matsumoto, T. Nonaka, M. Hashimoto, T. Watanabe, Y. Mitsui, Threedimensional structure of the catalytic domain of chitinase a1 from Bacillus circulans WL-12 at a very high resolution, Proc. Jpn. Acad. Ser. B. 75 (1999) 269. M. Carson, D.H. Johnson, H. McDonald, C. Brouillette, L.J. DeLucas, His-tag impact on structure, Acta Crystallogr. D Biol. Crystallogr. 63 (2007) 295–301. C.F. Hobel, G.O. Hreggvidsson, V.T. Marteinsson, F. Bahrani-Mougeot, J.M. Einarsson, J.K. Kristjánsson, Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus, Extremophiles 1 (2005) 53–64. M. Zarei, S. Aminzadeh, H. Zolgharnein, A. Safahieh, M. Daliri, K.A. Noghabi, A. Motallebi, Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A, Braz. J. Microbiol. 42 (2011) 1017–1029. T. Watanabe, K. Kobori, K. Miyashita, T. Fujii, M. Sakai, M. Uchida, H. Tamaka, Identification of glutamic acid 201 and aspartic 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity, J. Biol. Chem. 268 (1993) 18567–18572. M.B. Brurberg, I.F. Nes, V.G.H. Eijsink, The chitinolytic system of Serratia marcescens, in: R.A.A. Muzzarelli (Ed.), Chitin Enzymology, Atec, Italy, 1996, pp. 171–180. M. Swiontek Brzezinska, U. Jankiewicz, A. Burkowska, M. Walczak, Chitonolytic organisms and their possible application in environmental protection, Curr. Microbiol. 68 (2014) 71–81. B.L. Vallee, D.D. Ulmer, Biochemical effects of mercury, cadmium, and lead, Annu. Rev. Biochem. 41 (1972) 91–128. B. Sarosiek, M. Pietrusewickz, J. Radziwoniuk, J. Glogowski, The effect of copper, zinc, mercury and cadmium o some sperm enzyme activities in the common carp/Cyprinums carpio L.), Reprod. Biol. 9 (2009) 295–301. B. Synstad, G. Vaaje-Kolstad, F.H. Cederkvist, S.F. Saua, S.J. Horn, V.G.H. Eijsink, M. Sorle, Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a One-step general chitinase purification method, Biosci. Biotechnol. Biochem. 72 (2008) 715–723. F.P. Lin, H.C. Chen, C.S. Lin, Site directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae, IUBMB Life 48 (1999) 199– 204. C.-J. Kuo, L.-C. Huang, Li-C. Liao, C.-T. Chang, H.-Yi. Sung, Biochemical characterization of a novel chitotriosidase from suspension-cultured bamboo (Bambusa oldhamii) cells, Bot. Stud. 50 (2009) 281–289. V.L. Young, R.M. Simpson, V.K. Ward, Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrosis (family Baculoviridae), J. Gen. Microbiol. 86 (2005) 3253–3261. J.K. Chen, C.R. Shen, C.L. Liu, N-acetylglucosamine: production and application, Mar. Drugs 8 (2010) 2493–2516.

Please cite this article in press as: L.E. Casados-Vazquez et al., Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.015

466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517

Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis.

ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect ...
1MB Sizes 3 Downloads 21 Views