Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity

Accepted Manuscript Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity

Lara A.B.C. Carneiro, Li Yu, Paul Dupree, Richard J. Ward PII: DOI: Reference:

S0141-8130(18)32960-X doi:10.1016/j.ijbiomac.2018.07.116 BIOMAC 10160

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

15 June 2018 14 July 2018 16 July 2018

Please cite this article as: Lara A.B.C. Carneiro, Li Yu, Paul Dupree, Richard J. Ward , Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity. Biomac (2018), doi:10.1016/j.ijbiomac.2018.07.116

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ACCEPTED MANUSCRIPT

Characterization of a β-galactosidase from Bacillus subtilis with Transgalactosylation Activity

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Lara A. B. C. Carneiroa, Li Yub, Paul Dupreeb and Richard J. Wardc†

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a. Departamento de Bioquímica e Imunologia, FMRP - Universidade de São PauloUSP, Ribeirão Preto, São Paulo, CEP 14049-900, Brazil.

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b. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.

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c. Departamento de Química, FFCLRP - Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, CEP 14040-140, Brazil

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Abstract

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† Corresponding author e-mail address: [email protected]

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Microbial β-galactosidases (EC 3.1.2.23) have applications in the production of galactooligosaccharides, which are established prebiotic food ingredients. The β-galactosidase from Bacillus subtilis (YesZ) was expressed as a heterologous protein in Escherichia coli, and presented an optimum activity at pH 6.5 and 40 °C. The catalytic constants Km and Vmax of the enzyme were 8.26 mM and 1.42 µmol.min-1.mg-1 against pNP-β-Dgalactopyranoside, respectively. Structural characterization revealed that YesZ is a homotrimer in solution, and homology modelling suggested that the YesZ conserves a Cys cluster zinc binding site. Flame photometry experiments confirmed the presence of bound zinc in the recombinant enzyme, and YesZ activity was inhibited by 1 mM zinc, copper and silver ions. Transgalactosylation activity of YesZ was observed with the 1

ACCEPTED MANUSCRIPT synthetic substrate p-NP-βGal in the presence of a D-xylose acceptor, producing a β-Dgalactopyranosyl-(1→4)-D-xylopyranose disaccharide. Analysis of this disaccharide by MALDI-ToF-MS/MS suggested a β-1,4 glycosidic linkage between a non-reducing galactose residue

and the xylose. The β-galactosidase YesZ from B. subtilis is a

candidate for enzymatic synthesis showing favorable thermostability (with residual activity of 50% after incubation at 30 °C for 25 h) and transgalactosylation activity.

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Key-words: β-galactosidase, Bacillus subtilis, GH42, transgalactosylation, β-D-

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galactopyranosyl-(1→4)-D-xylopyranose.

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ACCEPTED MANUSCRIPT Introduction The β-galactosidases (EC 3.2.1.23) are enzymes found in Carbohydrate Active Enzyme families (www.cazy.org [1]) GH1, 2, 35, 42, 50 and 59, and use a general double displacement mechanism to hydrolyze O-glycosidic bonds [2]. In the hydrolysis reaction, two acidic amino acid residues participate in the catalytic cycle, in which one residue functions as a general acid/base and the second as a nucleophile that polarizes the water molecule targeting the glycosyl-enzyme intermediate [3]. Transglycosylation

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reactions occur when either the same leaving group or another carbohydrate molecule acceptor attacks the covalent galactosyl-enzyme intermediate, resulting in the formation

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of an O-glycosidic bond between the glycosyl group and the acceptor molecule [4].

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Galacto-oligosaccharides (GOS) are well known prebiotics that enhance the growth of bifidobacteria and lactobacilli in the large intestine, and are included as food

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ingredients in order to reduce the growth of pathogenic microorganisms [5–8]. In addition, these non-digested carbohydrates GOS may also improve mineral absorption due to osmotic transfer of water into the large bowel [9]. Furthermore, human milk

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oligosaccharides are GOS components of human breast milk that have a prebiotic effect on the establishment of the intestinal microflora in human infants [10], and synthetic GOS have been included in infant milk formulas [11].

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The hydrolysis of the disaccharide β-D-galactopyranosyl-(1→4)-D-xylopyranose D-galactose

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by intestinal lactase to yield

and

D-xylose

is involved in adult-type

alactasia, and this reaction forms the basis of a clinical diagnostic test that is important both in pediatrics and gastroenterology [12]. The test is based on the fact that the

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disaccharide may act as a lactase substrate to generate the monosaccharides that are easily absorbed by the intestine, after which the D-xylose remains unmetabolized and is

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excreted in the urine. Oral administration of the disaccharide therefore results in urinary excretion of

D-xylose,

which can be quantified colorimetrically [13]. The chemical

synthesis of β-D-galactopyranosyl-(1→4)-D-xylopyranose involves a multistep pathway for the glycosylation of benzyl 2,3-O-isopropylidene-β-D-xylopyranoside with 2,3,4,6tetra-O-benzoyl-α-D-galactopyranosyl bromide [12]. In contrast, the synthesis of galactosyl compounds by enzymatic treatment is more straightforward, involving a single-step process using enzymes and reaction conditions to control the specificity of the glycosidic linkage in the final product [14]. Bacillus subtilis is widely considered to be a common soil organism, whose endospore formation ensures long-term survival in the environment. However, B. 3

ACCEPTED MANUSCRIPT subtilis can also adapt to the mammalian intestinal environment as part of its natural life cycle, where it may be recruited as an autochthonous member of the intestinal flora [15]. The β-galactosidase from B. subtilis (YesZ) is CAZy family GH42 enzyme [16], which catalyzes the hydrolysis of β-D-galactosides with retention of stereochemistry between products and reactants [17]. In this study, we have performed the biochemical characterization of the recombinant β-galactosidase YesZ from Bacillus subtilis and have demonstrated that the homotrimeric enzyme possesses a transgalactosylation activity

the

disaccharide

β-D-galactopyranosyl-(1→4)-D-xylopyranose.

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producing

The

disaccharide β-D-galactopyranosyl-(1→4)-D-xylopyranose transgalactosylation product

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was structurally characterized by MALDI-ToF-MS/MS analysis. In addition to the

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biochemical characterization of the β-galactosidase YesZ, a three dimensional structural model for the enzyme is proposed and analyzed in the light of its biochemical and

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biophysical properties. The YesZ presents high thermostability, and is a candidate for use in the enzymatic synthesis of galacto-oligosaccharides with applications in the

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food, healthcare, and pharmaceutical industries.

Material and Methods

The

synthetic

substrates

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Synthetic and natural substrates

pNP-β-D-galactopyranoside

(ONPβGal),

galactopyranoside

(pNPαGal),

pNP-β-D-glucopyranoside

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galactopyranoside

pNP-α-L-arabino

(pNP-βGal),

oNP-β-D-

(pNPβGlu),

pNP-α-D-

furanoside,

and

pNP-α-D-

xylopyranoside (pNPαXyl) and D-lactose were purchased from Sigma (Sigma Aldrich,

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St.Louis, MO, USA). Seed storage xyloglucan from Tamarindus indica was kindly provided by Dr. Marcos Buckeridge, University of São Paulo, Brazil. The β-Galactan

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from Lupinus angustifolius was kindly provided by Dr. Paul Dupree, University of Cambridge, United Kingdom. Cloning of B. subtilis β-galactosidase A fragment containing the DNA coding sequence of the β-galactosidase from B. subtilis (YesZ) was amplified by PCR from Bacillus subtilis subsp. subtilis 168 (ATCC® Number: 23857D-5) using genomic DNA extracted as previously described [18]. The PCR reaction used forward (TTA TAT CAT ATG AGA AAA CTG TAT CAT GGC GCT TGC) and reverse (TTA TAT CTC GAG GCT GTG ATT GTC AAA TTG AAT CAC ACG) primers containing NdeI and XhoI restriction sites (underlined), 4

ACCEPTED MANUSCRIPT respectively. The PCR conditions were: 94°C/2 min (1 cycle); 94°C/1 min, 55°C/30 s, 72°C/4 min (30 cycles); 72°C/10 min (1 cycle), and 1U of Pfu DNA polymerase (Promega Corp. Madison, WI, USA). The amplified fragment was cloned into pJET (Thermo Scientific®), according to the manufacturers instructions, and the DNA fragment liberated after digestion with the restriction enzymes NdeI and XhoI was subcloned into the expression vector pET22b. The resulting expression plasmid pET22b-Yes encodes the recombinant β-galactosidase fused to a C-terminal 6 × His-

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tag. The integrity of the planned construct was confirmed by automated DNA

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Overexpression of the β-galactosidase gene in E. coli

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sequencing.

E. coli Rosetta (DE3) pLysS transformed with the pET22b-Yes plasmid was grown for

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12 h in HDM medium (1.5 % tryptone, 2.5 % yeast extract, pH 7.5) containing 100 μg/mL ampicillin and 34 µg/mL chloramphenicol at 37°C. A 500 µL aliquot of this culture was used to inoculate 50 mL of HDM medium containing 100 μg/mL ampicillin

0.6,

whereupon

protein

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and 34 µg/mL chloramphenicol at 37°C. The cells were grown at 37 °C to an A600 nm of expression

was

induced

by

adding

isopropyl-D-

D

thiogalactopyranoside (IPTG) to a final concentration of 0.15 mM. Expression was performed at 18 °C and 150 rpm for 16 hrs. Cells were harvested by centrifugation at 4

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°C for 15 min at 6000 g, and the resulting cell pellet was subsequently resuspended in 10 mL of lysis buffer pH 8.0 (100 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, 2 mM PMSF, 1% Triton X-100). The cell suspension was sonicated for 6 min with a 10s

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on/10s off pulse cycle at 60 W (QSonica Q125, Newtown, CT, USA). Cell debris were

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removed by centrifugation (7000×g, 4°C, 20 min) to obtain the crude cell extract.

Protein purification The purification of the recombinant β-galactosidase (YesZ) was carried out by applying the crude cell extract onto a Ni-Sepharose column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with buffer (pH 8.0) containing 100 mM NaH2PO4, 500 mM NaCl and 20 mM imidazole. Protein elution was performed by washing the column with buffers containing imidazole concentrations of 40 mM and 250 mM. Fractions eluted with the higher imidazole concentration were dialysed against McIlvaine’s citrate-phosphate buffer buffer pH 6.5. Protein concentrations were measured by A280 nm using extinction coefficients obtained from ProtParam software [19]. 5

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Biochemical characterization The β-galactosidase activity was measured at 40 °C and pH 6.5 by spectrophotometric quantification of p-nitrophenol release at 410 nm using 4-nitrophenyl-β-Dgalactopyranoside (pNP-βGal) as substrate. Biochemical assays were performed using McIlvaine’s buffer and 4-nitrophenyl-β-D-galactopyranoside (pNP-βGal) as substrate. The effect of pH and temperature on YesZ activity was measured over the pH range 3.5

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- 8.0 and between temperatures of 10 – 55 °C. Kinetic parameters were determined over a pNP-βGal concentration range of 0.13 - 30 mM with McIlvaine’s buffer pH 6.5 at 40

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°C. The effect of ionic strength on YesZ activity was assessed with NaCl (0 – 400 mM)

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with McIlvaine’s buffer pH 6.5, using 8 mM pNP-βGal as substrate at 40 °C. The extinction coefficient for p-nitrophenol at 410 nm was taken as 18300 M-1.cm-1 [20],

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and one activity unity (U) was defined as the amount of enzyme that releases 1 µmol of

Natural substrate hydrolysis assays

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p-nitrophenolate per minute. All assays were performed in triplicate.

Lactose, tamarind xyloglucan and β-galactan of Lupinus angustifolius were tested as

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substrates for YesZ activity. In the lactose hydrolysis assay, YesZ was incubated with the disaccharide at 400 mg/mL in 50 mM ammonium acetate pH 6.5 at 40 °C for 16 h,

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and the product was analyzed by ESI-MS. Tamarind xyloglucan 5 mg/mL was initially hydrolyzed with the endo-β-(1,4) glucanase XegA from Aspergillus niveus as previously described [21], and the reaction subsequently heated (80 °C, 10 min) to

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inactivate the enzyme. The xyloglucan oligosaccharides were then incubated with YesZ in 50 mM ammonium acetate, pH 6.5 at 40 °C for 16 h, and the resulting

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oligosaccharides were analyzed by ESI-MS. The β-galactan of Lupinus angustifolius was hydrolyzed with 200 mM trifluoroacteic acid (TFA) for 2 h at 80 °C and lyophilized to remove TFA. The lyophilized oligosaccharides were diluted in 50 mM ammonium acetate pH 6.5 at 40 °C and incubated with YesZ for 16 h. The resulting oligosaccharides were analyzed by polysaccharide analysis carbohydrate gel electrophoresis (PACE) [22].

Thermal inactivation

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ACCEPTED MANUSCRIPT The rate constants, k, for the first-order enzyme inactivation reactions were determined from the slopes of the inactivation curves at the temperature (T) 30, 40, 50 and 60 °C using Equation 1, in which A0 is the initial enzyme activity and A is the activity after the time t. The values of k were used to generate a Arrhenius plots (Equation 2) from which activation energies (Ea) were estimated by linear regression [23]. The units for the equations below are: time in hours, temperature in K; Ea in J . mol-1; the gas constant R

(Equation 1) (Equation 2)

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was 8.314 J . K-1 . mol -1.

ESI-MS analysis

Electrospray ionization mass spectrometry (ESI-MS) experiments were performed using

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a Xevo TQ-S MS spectrometer (Waters Corporation, Milford, MA, USA) equipped with an electrospray ionization source operating in positive mode. The data were

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acquired in the full scan mode. Typical ESI conditions were; capillary voltage, 3.2 kV; cone voltage, 40 V; source temperature, 50 °C and desolvation temperature (N2), 250 °C. The software MassLynx 4.0 (Waters Corporation, Milford, MA, USA) was used for

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data acquisition, processing and analysis.

Sample preparation and MALDI-ToF-MS and ToF-MS/MS analysis Enzyme-treated samples were desalted using HyperSep Hypercarb cartridges (Thermo-

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Fisher), and the oligosaccharides were reductively aminated with 2-aminobenzoic acid (2-AA) as previously described [24]. The labeled carbohydrates were purified using

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GlycoClean S cartridges (Prozyme, San Leandro, CA, USA). For analysis using Matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToFMS), the labled oligosaccharides were mixed (1:1) with 2,5-dihydroxybenzoic acid (2,5DHB) matrix (20 mg/mL in 30:70 (v/v) acetonitrile : TFA 0,1 % in water) and spotted on ground steel plate for analysis by MALDI-ToF/ToF-MS/MS (4700 proteomics analyzer, Applied Biosystems, Foster City, CA, USA). Mildly-hydrolyzed dextran prepared by the same procedure was used for calibration. MALDI-CID spectra were acquired with an average 10000 laser shots/spectrum and using 1 kV collision energy.

Transgalactosylation reactions 7

ACCEPTED MANUSCRIPT The transgalactosylation reactions were performed at 40 °C in McIlvaine buffer pH 6.5. Reaction mixtures contained 5.3 mM of pNP-βGal, 8.7 x 10-4 U of enzyme and 50 mM of a saccharide acceptor (fructose, galactose, glucose, lactose, cellulose, xylose, methanol or glycerol). A control for each assay was prepared with heat-inactivated enzyme (100 °C/ 5 min). The transgalactosylation and hydrolysis products were analyzed by ESI-MS mass spectrometry or PACE.

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Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE) Analysis

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Oligosaccharide samples were dried and the derivatized with fluorophore 8aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and subsequently separated by

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acrylamide gel electrophoresis, as previously described [22]. PACE gel images were analyzed using the GelAnalyzer 2010 package (www.genanalyzer.com, Dr Istvan

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Lazar).

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Purification of YesZ using Size Exclusion Chromatography For the molecular weight determination of the recombinant YesZ, a protein sample obtained from nickel-affinity chromatography was concentrated and applied on a 10/30

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GL Superdex 200 column (GE Healthcare, São Paulo, Brazil) equilibrated with

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McIlvaine’s buffer pH 6.5. The elution volumes of the known protein standards βamilase, ovalbumin, conalbumin (Sigma Aldrich, St.Louis, MO, USA) were used to

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calibrate the column.

Purification of β-D-galactopyranosyl-(1→4)-D-xylopyranose using Biogel P-2

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The β-D-galactopyranosyl-(1→4)-D-xylopyranose disaccharide was purified by size exclusion chromatography using a Biogel P2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) column 96 cm in length and 1.2 cm in diameter, equilibrated with ammonium acetate 50 mM pH 6.5. The column was eluted at 40 µL/min and 1 mL fractions were collected for subsequent analysis by PACE.

Dynamic light scattering The molecular weight and the hydrodynamic radius of YesZ were estimated by dynamic light scattering (DLS) using a Zetasizer µV (Malvern Instruments Ltd, Malvern, UK). The sample was centrifuged at 10000 g for 5 min at 4 °C prior to the analysis, and 8

ACCEPTED MANUSCRIPT measurements were made using a 2 µL quartz cuvette. Hydrodynamic radius and molecular weights were estimated using the software supplied with the apparatus.

Circular dichroism spectroscopy Far-ultraviolet CD spectra were measured with a J-810 spectropolarimeter (JASCO Corporation, Tokyo, Japan) between 184 and 250 nm. Purified YesZ (0.1 mg/mL) in 10

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mM sodium phosphate buffer pH 7.4 at 25 °C was analyzed using a 1 mm path length cuvette. The final spectra are the averages of five individual accumulations, and the

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averaged spectrum was corrected for the buffer baseline by subtracting the respective blank spectra recorded without protein. Data were converted to mean residue molar

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ellipticity (θ; deg.cm2.dmol-1).

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Flame photometry

The zinc content of the heterologous YesZ was analyzed by flame photometry using a

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Spectrometer Analyst 800 (Perkin Elmer, Waltham, MA, USA) with an air-acetylene flame and a hollow-cathode lamp source emitting at 213.9 nm. A calibration curve was constructed using known concentrations of zinc ions. Prior to the analysis, the baseline

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signal was established using a 10 mM sodium phosphate buffer pH 7.4 without enzyme.

Molecular Modeling by Homology

A 3D-structural model of the YesZ from Bacillus subtilis was obtained by restraint-

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based modeling as implemented in the program MODELLER [25], using the atomic coordinates of the Bacillus circulans β-galactosidase (Bca-β-gal, PDB ID: 3TTS) [26]

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as the template.

Results and Discussion Cloning, expression and purification A DNA fragment encoding the YesZ open reading was successfully amplified by PCR using the Bacillus subtilis subsp. subtilis 168 genomic DNA as template and cloned into the pJET1.2/blunt cloning vector. The fragment was subcloned into the pET22b expression vector resulting in a ~2600 bp coding sequence including a (His)6 C-terminal extension to the YesZ. The recombinant enzyme was overexpressed as a soluble protein in E. coli Rosetta(DE3) pLysS, and purified by affinity chromatography on Ni-NTA 9

ACCEPTED MANUSCRIPT resin followed by size exclusion chromatography on a Superdex 200 column (Figure 1A). The final yield of purified protein was 95 mg per litre of culture. The enzyme used in our experiments was purified with a Superdex 200 column step. The SDS-PAGE of the

purified recombinant YesZ reveals a single protein band with an apparent molecular mass of ~75 kDa, in agreement with the theoretical molecular weight of 75164.0 Da calculated for the YesZ aminoacid sequene including the C-terminal extension. In constrast, size exclusion chromatography of the recombinant YesZ reveals a molecular

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mass of ~200 kDa (Suplementary Figure S2A), and this was corroborated by the dynamic light scattering (DLS) results, which showed a unimodal particle size

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distribution with a mean hydrodynamic radius of 5.9 nm that corresponds to a protein of

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215 kDa (Suplementary Figure S2B). Therefore, gel filtration and DLS experiments suggest that three 75 kDa YesZ monomers form a stable homotrimer in solution.

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Biochemical characterization

The influence of pH, temperature and ionic strength on pNP-galactosidase

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activity of the recombinant YesZ is presented in Figure 1. Maximum catalytic activity was observed at 40 °C, and at 35 and 45 °C the enzyme retained 84 % of the maximum activity (Figure 1B). The optimum pH was 6.5 and a mild decrease in the activity was

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observed up to pH 8.0, where 46 % of the maximum activity was observed (Figure 1C).

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The ionic strength was found to influence the enzymatic activity, with a maximum value at 250 mM NaCl (Figure 1D). The effect of different cations, SDS and EDTA was evaluated at pH 6.5 and 40

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°C (Figure 2). The pNP-galactosidase activity of YesZ decreased more than 75 % by the presence of 1 mM Zn2+, Cu2+ and Ag2+ ions. The presence of 10 mM Pb2+, Mn2+, Al+3,

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Ni2+, Co2+ or SDS resulted in a decrease of at least 40 % in the YesZ catalytic activity. In contrast, even at a concentration of 10 mM, Mg2+, Sr2+, Ca2+ or the chelating agent EDTA did not alter the YesZ activity. These results indicate that additional cations are not necessary for activating the enzyme. The effect of Zn2+ was similar to that on βgalactosidase from Lactobacillus delbrueckii, in which Zn2+ significantly inhibited the β-galactosidase activity at a concentration of both 1 and 10 mM [27]. Kinetic and thermodynamic characterization The activities of YesZ against several substrates were tested at 40 °C in 50 mM ammonium acetate buffer pH 6.5 (Table 1). The highest activity was towards pNP-β-D10

ACCEPTED MANUSCRIPT galactopyranoside (pNP-βGal), followed by oNP-β-D-galactopyranoside (oNPβGal). The other synthetic substrates tested (pNP-β-D-glucopyranoside (pNPβGlu), pNP-α-Dgalactopyranoside

(pNPαGal),

pNP-α-L-arabino

furanoside

and

pNP-α-D-

xylopyranoside (pNPαXyl) were not hydrolyzed. Natural substrates (xyloglucan oligosaccharides, β-galactan oligosaccharides and lactose) containing galactose residues with different glycoside bond linkages were also tested, however no galactose release was detected after incubation with YesZ (Table 1).

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The YesZ kinetic parameters determined for the hydrolysis of pNP-βGal are presented in Table 2. The Km, Vmax and kcat parameters were 8.26 ± 0.48 mM, 1.42 ±

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0.083 µmol.min-1.mg-1 and 1.58 s-1, respectively. The catalytic coefficient (kcat/Km) was

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0.19 mM-1. s-1, and the pNP-βGal hydrolysis followed negative cooperativity kinetics (Hill coefficient, nH = 0.88), indicating that binding of one ligand molecule to the

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catalytic site reduces the affinity for ligand binding to other protein sub-units in the trimer.

The effect of temperature on the enzyme activity is required for a full kinetic

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characterization [28] and hydrolysis of pNP-βGal was performed over a temperature range of 30 to 60°C. A YesZ residual activity of 50 % was observed after incubation for

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25 h, 12 h, 40 min and 3.5 min at 30, 40, 50 and 60 °C, respectively. A β-galactosidase from Kluyveromyces lactis maintained 50 % of its initial activity for 5 h at 30 °C [30].

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A 50% residual activity of a β-galactosidase from Bacillus megaterium was measured after 60 min at 50 °C [7]. A β-galactosidase from an Antarctic arthrobacter isolate retained 50% of its initial activity after 30 min at 30 °C [29].

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The inactivation curves were also analyzed to determine the pre-exponential factor (k0) and the activation energy (Ea), as detailed in “Materials and methods”

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section. With these two parameters, the inactivation rate constant (k) at any temperature (T) can be predicted using the Arrhenius equation [23]. The thermal inactivation of YesZ followed simple first-order kinetics, with a high rate of initial inactivation followed by a decline at moderate rates [31] (Suplementary Figure S1). The activation energy for the hydrolysis of pNP-βGal was 180.86 kJ.mol-1 and k0 was 2.02 x 1029 h-1. YesZ has higher Ea than the β-galactosidases from Rhizomucor sp. [32] and V. sinensis [33], which present Ea values of 114.3 and 43.12 kJ/mol, respectively, using pNP-βGal as the substrate. Both these enzymes have Km values in the range 0.5 – 0.7 mM, in contrast with YesZ, which presents a Km of 8.26 mM. The lower affinity of the YesZ for pNP-βGal may contribute to the high activation energy observed with this substrate. 11

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CD spectroscopy and hydrodynamic radius measurements The GH42 β-galactosidases all demonstrate (α/β)8 barrel architecture, with two glutamic acid residues in the active site acting as an acid⁄base catalyst and a nucleophile, respectively [26,34]. In the case of the YesZ, chemical modification of active-site residues has demonstrated that the Glu295 acts as a nucleophile and the Glu145 as the acid ⁄ base catalyst is [16] (Figure 3A). The far-UV CD spectrum of the purified YesZ

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presents both a positive band around 190 nm and a defined minimum at 208 nm, which are typical features of spectra from proteins containing α/β barrels (Figure 3B). This

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result is in agreement with the (β/α)8 barrel archetecture observed in the catalytic

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domain of the modeled three-dimensional structure of the YesZ (Figure 3C). The quality assessment of a homology model using graphical plots of Anolea

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(mean force potential) [35], GROMOS (empirical force field energy) [36] and QMEAN (Qualitative Model Energy Analysis) [37] indicated that the model derived from the Bacillus

circulans

(MAKSIMAINEN et al., 2012)

Bca-β-gal

structure

(PDB

code:

3TTS)

contains few regions with unfavorable energy,

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homotrimeric

therefore the model of the trimeric YesZ structure used the coordinates of Bca-β-gal structure as template (Figure 3C). The predicted homotrimeric structure of the YesZ

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(DLS) experiments.

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model is supported by the results from the gel filtration and dynamic light scattering The β-galactosidases from Thermus thermophilus A4 (A4-β-gal, PDB ID: 1KWG) [38] and Bca-β-gal present 24 and 31% amino acid identity with the YesZ,

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respectively. The 3D-structures of both the A4-β-gal and the Bca-β-gal contain a single occupied Zn2+ binding site. In the A4-β-gal, a metal-binding cluster is formed by

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Cys106, Cys150, Cys152, and Cys155, and the analogous residues are highly conserved in GH42 enzymes. Thus, zinc binding in the Bca-β-gal is coordinated by Cys115, Cys155, Cys157 and Cys160. An amino acid sequence alignment of both βgalactosidases with the YesZ sequence reveal that all the Cys residues involved in zinc coordination are conserved in the YesZ (Figure 4A). The capacity for zinc binding of purified YesZ was therefore evaluated by flame photometry, and the concentration of zinc measured in the YesZ sample revealed a 1.1:1 molar proportion with the polypeptide, demonstrating that each polypeptide chain binds a single zinc atom, and strongly suggests that the

conserved Cys cluster maintains its function for zinc

coordination in the YesZ structure (Figure 4B). 12

ACCEPTED MANUSCRIPT Three types of zinc binding sites are known in proteins: catalytic, co-catalytic, and structural [39]. Structural Zn2+ binding is coordinated by four amino acid side chains and contains no bound water molecules, and these sites typically include either four cysteines or two histidines in combination with two cysteines [39]. Previous studies with Bca-β-gal have demonstrated that zinc ions play no role in the catalytic cycle, however zinc binding stabilized the structure of the catalytic domain [26]. Here we have demonstrated that the YesZ retains activity even in the presence of 10mM of the

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chelating agent EDTA (Figure 2), suggesting that the enzyme does not require a metal ion cofactor for activity against pNP-βGal (see biochemical characterization). In

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contrast, a strong inhibitory effect of Zn2+ on YesZ catalytic activity on pNP-βGal was

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observed, and although this may be due to reduced flexibility of the catalytic domain on

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zinc binding, further studies are needed to establish the structural basis of this effect.

Transgalactosylation activity

The transgalactosylation activity of the YesZ was evaluated using the pNP-βGal

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substrate together with various monosaccharide acceptors. No transgalactosylation was observed in the presence of galactose, glucose, fructose, lactose, cellobiose, methanol or

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glycerol as acceptors (Figure S3). However, in the presence of xylose, ESI-MS analysis reveals the formation of a transgalactosylation product with a m/z of 335, which

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corresponds to the sodium adduct of the disaccharide formed between xylose (m/z = 150) and the galactose residue of pNP-βGal (m/z = 162) (Figure 5). The transgalactosylation product was not observed on incubation with an extract of E. coli

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cells transformed with the empty pET22b vector (Figure S4), nor in a reaction mixture that substituted active YesZ for the heat inactivated enzyme (Figure 5C).

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It is noteworthy that of all the saccharides tested, only xylose acts as an acceptor, demonstrating that the transgalactosylation reaction catalyzed by YesZ is highly specific. This is in contrast to the β-galactosidase from Bacillus megaterium (BgaBM) that displays a broad acceptor specificity for transgalactosylation with a variety of acceptors, and a maximum yield of 20 % for the transgalactosylation products using a 50 mM donor concentration [7]. Analysis of the gels from the PACE experiments in the YesZ transgalactosylation experiments (Figure S5), indicates that between 75-80% of the β-galactose available from pNP-βGal cleavage is incorporated into the disaccharide transgalactosylation product.

13

ACCEPTED MANUSCRIPT In order to increase the quantity of disaccharide product, the YesZ transgalactosylation reaction between Gal and Xyl was scaled-up using concentrations of the donor (pNP-βGal) and acceptor (xylose) of 50 mM and 500 mM, respectively. The product of the scaled-up reaction was fractionated by size exclusion chromatography using a Biogel P-2 column to obtain a sample enriched with the β-Dgalactopyranosyl-(1→4)-D-xylopyranose disaccharide (Figure S6A). The disaccharide was labeled with 2-aminobenzoic acid (2-AA) and analyzed by MALDI-ToF-MS. The

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ion corresponding to the 2-AA-labeled disaccharide (m/z 456.5) (Figure S6B) was selected for structural analysis using MALDI collision induced dissociation (MALDI3,5

A2 (m/z 229.2) and G1 (m/z 260.2) in the

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CID), which revealed the presence of ions

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fragmentation spectrum (Figure S6C) suggesting a (1,4) bond between galactose and xylose. In addition, the Y1 ion (m/z 294.2) identifies galactose as the moiety at the non-

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reducing end of the disaccharide. Since the YesZ is unable to cleave pNP-αGal (Table 1), and the enzyme belongs to the GH42 family that presents a retaining Koshland double-displacement catalytic mechanism [17], the bond between galactose and xylose

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residues was identified as a β-D-galactopyranosyl-(1→4)-D-xylopyranose linkage. The β-galactosidases are widespread in nature, and are present in plants, animals

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and microorganisms. The β-galactosidases from bacteria, yeasts and fungi have been extensively studied due to the ease of cell manipulation and high enzyme titres typically

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obtained from microorganism cultures [40]. The synthesis of GOS using lactose as substrate has been extensively studied, and different species produce β-galactosidases that demonstrate diverse catalytic specificities, resulting in variation in the GOS

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products of their transgalactosidase activities [40]. Examples include the GOS produced by the β-galactosidases from Lactobacillus reuteri [41] and Aspergillus oryzae [42] that

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primarily contain β-(1→3) and β-(1→6) glycosidic linkages. The β-galactosidase from Kluyveromyces lactis yields predominantly β-(1→4) and β-(1→6)-linked oligosaccharides [43]. Although the β-galactosidase from Bacillus circulans βgalactosidase can form β-(1→2), β-(1→3), β-(1→4) and β-(1→6) glycosidic bonds [44], a more recent study indicated that the β-(1→4)-linked GOS predominate [45]. Previous studies using O-nitrophenyl--D-galactopyranoside and acceptor

in

transgalactosylation

reactions

with

the

D-xylose

β-galactosidases

as

from

Saccharomyces fragilis and Escherichia coli produced selective β-(1→4)-linked GalXyl disaccharides in yields of up to 53% [46]. More recently, yields of up to 50% have been reported for the synthesis of β-D-galactopyranosyl-(1→4)-D-xylopyranose under 14

ACCEPTED MANUSCRIPT controlled reaction conditions using the β-galactosidases from Escherichia coli [47]. The high specificity for the D-xylose acceptor in the YesZ transgalactosylation reaction together with the high yield of the β-D-galactopyranosyl-(1→4)-D-xylopyranose is of biotechnological interest, since the disaccharide product is useful as a non-invasive clinical diagnostic tool for intestinal lactase activity in adult-type alactasia [12]. In conclusion, a -galactosidase from B. subtilis (YesZ) has been expressed,

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purified and characterized. The recombinant enzyme is expressed at high levels in E. coli, and is readily purified and presents good thermostability, with residual activity of 50% after incubation at 30 °C for 25 h. In addition, a specific transgalactosylation

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activity has been demonstrated, in which of all the common monosaccharides, only D-

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xylose could act as an acceptor, producing the disaccharide β-D-galactopyranosyl(1→4)-D-xylopyranose with high yield. These properties suggest that the β-

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galactosidase YesZ from B. subtilis is a good candidate for the enzymatic synthesis of substrates used for clinical diagnostics, and expands the toolbox of enzymes for GOS

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synthesis.

Acknowledgements

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This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo

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(FAPESP) grants 2012/24147-8 and 2010/18850-2, and CNPq (307652/2013-0). We are grateful to Profa. Dra. Marcia Veiga and Luís Eduardo Bernardes for their help with the flame photometry experiments.

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Author contributions

L.A.B.C.C. and R.J.W. designed the research; L.A.B.C.C. performed the experiments;

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L.Y. performed the disaccharide structural analysis; L.A.B.C.C., R.J.W., L.Y and P.D analyzed the data; R.J.W. and P.D contributed with reagents and analysis tools; L.A.B.C.C. and R.J.W. wrote the manuscript.

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ACCEPTED MANUSCRIPT Figure Captions

Graphical Abstract. Trimeric model structure of the YesZ and the simplified mechanism for the pNP-βGal hydrolysis and β-D-galactopyranosyl-(1→4)-D-xylopyranose synthesis.

Figure 1. Expression, purification and biochemical charcterization of B. subtilis YesZ. (A) SDS-PAGE (12.5 %) of YesZ purification. M) Protein Marker. 1) Flow through. 2) Wash with

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buffer A. 3) YesZ (75 kDa) eluted from Ni Sepharose column. 4) Purified YesZ eluted from Superdex 200 column. The arrow indicates the position of the purified recombinant protein. (B)

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Effect of temperature on the YesZ activity carried out at pH 6.5 and 8 mM of pNP-βGal, (C) Effect of pH on the YesZ activity using McIlvaine buffers (pH 5 – 8) at 40 °C and 8 mM of

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pNP-βGal; (D) effect of the ionic strength on the YesZ activity carried out at 40 °C, pH 6.5 and 8 mM pNP-βGal. Data points are the average of three independent sets of experiments and the

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error bars represent the mean ± SD. All assays were performed in triplicate.

Figure 2. Effect of different ions, EDTA and SDS on pNP-galactosidase activity of YesZ. The

activities were determined in triplicate.

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experiment was done in McIlvaine buffer pH 6.5 at 40 °C and 8 mM pNP-βGal. Enzymatic

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Figure 3. Tertiary and quaternary structure of the YesZ (A) 3D model of the monomer of β-

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galactosidase YesZ. The inset shows detail of the active site of the protein evidencing its catalytic residues. (B) Far ultraviolet circular dichroism spectrum of the YesZ. (C) YesZ

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trimeric structure. The images were prepared using the program Chimera 1.6.

Figure 4. (A) Segment of the amino acid sequence alignment between the amino acids sequences of Bacillus circulans β-galactosidase (PDB ID: 3TTS) and Thermus thermophiles β-

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galactosidase (PDB ID: 1KWG) with the YesZ sequence. Aligned Cys residues are coloured in yellow. (B) Ribbon representation of the YesZ, showing the putative metal-binding cluster of YesZ (panel (ii) in orange), Comparison of the occupied Zn2+ binding sites in the crystal structures of Thermus thermophiles β-galactosidase (panel (iii) magenta) and Bacillus circulans β-galactosidase (panel (iv) cyan). Sulfhydryl groups of the cysteine residues coordinating the metal ion are coloured in yellow.

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ACCEPTED MANUSCRIPT Figure 5. Mass spectra measured by using ESI/Na+ addition obtained for (A) 5.2 mM pNPβGal hydrolysis promoted by YesZ in the presence of 50 mM D-xylose; (B) in the absence of Dxylose; (C) control of reaction in the same conditions of (A) with heat-inactivated YesZ.

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Substrate* Relative Activity*4 (%) * pNPβGal 100,00 ± 2,75 * ONPβGal 66,35 ± 1,14 pNPβGlu* ND pNPαXyl* ND * pNPαGal ND pNPαAra* ND Xyloglucan oligosaccharides*2 ND *3 Lactose ND β-galactan oligosaccharides*2 ND

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Table 1. Relative activities of YesZ on different substrates.

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Initial concentrations were 4 mM*, 5 mg/mL*2 or 500 mg/mL*3. *4Values relative to pNP-βGal activity. ND: activity not detected after 16 h reaction at pH 6.5 in 50mM ammonium acetate and 40 °C.

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ACCEPTED MANUSCRIPT Table 2. Kinetic parameters of the B. subtilis β-galactosidase using a Michaelis-Menten model and pNP-βGal as substrate compared to other β-galactosidases.

Substrate pNP-βGal pNP-βGal oNP-βGal pNP-βGal pNP-βGal pNP-βGal

Km (mM) 8.26 2.74 13.7 3.28 0.041 0.66

kcat (s-1) 1.58 5.85 785 932 90 -

kcat/Km (mM-1.s-1) 0.19 2.13 57.3 284 2200 -

Organism (Ref.) Bacillus subtilis (this study) Thermotoga maritima [48] Bacillus licheniformis [49] Aspergillus aculeatus [50] Escherichia coli [51] Rhizomucor sp. [32]

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Vmax (µmol.mg-1.min-1) 1.42 351 299 22140

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Figure 7

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Figure 11