Expression and Characterization of Windmill Palm Tree (Trachycarpus

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Expression and Characterization of Windmill Palm Tree (Trachycarpus fortunei) Peroxidase by Pichia pastoris Boting Wen, Margaret R. Baker, Hongwei Zhao, Zongjun Cui, and Qing Xiao Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017

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Journal of Agricultural and Food Chemistry

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Manuscript revised according to reviewers’ and editor’s comments for possible publication in

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Expression and Characterization of Windmill Palm Tree

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(Trachycarpus fortunei) Peroxidase by Pichia pastoris

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Boting Wen,1,2 Margaret R. Baker,1 Hongwei Zhao,1 Zongjun Cui,2 Qing X. Li1*

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1. Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,

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Honolulu, Hawaii 96822, USA

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2. College of Agronomy and Biotechnology, China Agricultural University, Beijing, China

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Corresponding author: Qing X. Li. Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Currently, commercial plant peroxidases are all native and are isolated from plants such as

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horseradish and soybean. No recombinant plant peroxidase products have been available on the

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commercial market. The gene encoding peroxidase was cloned from windmill palm tree leaves.

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The codon-optimized gene was transformed into Pichia pastoris for expression. The recombinant

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windmill palm tree peroxidase (rWPTP) expressed by P. pastoris showed high stability under pH

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2-10, temperature up to 70 °C, to many metallic salts and organic solvents. The substrate

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specificity of WPTP was determined, and among the substrates tested, 2, 2′-azino-bis (3-

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ethylbenzothiazoline-6-sulfonic acid) (ABTS) was most suitable for WPTP. The Michaelis

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constants with the substrates H2O2 and ABTS were 4.6 x 10-4 M and 1.6 x 10-4 M, respectively.

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The rWPTP expressed in P. pastoris may be a suitable enzyme for biosynthesis of polymers

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because of its high stability and activity under acidic conditions.

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Keywords: peroxidase, windmill palm tree, expression, substrate specificity, stability

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INTRODUCTION

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Peroxidases are enzymes that catalyze various redox reactions and are thus potentially

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useful for industrial and biomedical applications. Peroxidase can be used in wastewater and

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agricultural waste treatment, chemiluminescence assays, enzyme electrodes, organic and

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polymeric molecule synthesis and as a therapeutic agent.1-5 Palm tree peroxidases are

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constitutive, soluble enzymes that contain glycan groups covalently bound to several asparagine

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side-chains of a single polypeptide chain with about 300 amino acid residues. Palm peroxidases

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showed very high tolerance to elevated temperatures, extreme acidic and basic pH (a broad range 2


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of pH values) and high concentrations of hydrogen peroxide.6-9

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Native windmill palm tree peroxidase (WPTP) was isolated from leaves of the palm tree

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Trachycarpus fortunei. Native WPTP had a molecular mass of approximately 50 kDa and an

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isoelectric point of 3.5.10 The complete amino acid sequence and detailed glycosylation of native

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WPTP were recently determined by Baker et al.11, 12 Enzymes isolated directly from palm tree

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tissues are normally composed by a variety of isoenzymes, which is an impediment to fully study

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the structure and function.

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Peroxidase can be used in bioremediation,13, 14 and as destructors for lignin degradation,15 as

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reporter for food processing and diagnostic reagents,16, 17 and as catalysts for the synthesis of

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polymers, such as polyaniline.18-20 It is therefore important to obtain a large amount of single

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component enzyme by gene cloning and protein expression with microorganisms, which

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provides a new resource to study the stability and catalytic mechanisms as well as an economical

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production of unique peroxidase. Mature native WPTP is a highly glycosylated (glycan content

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21-29%)11 and heme-containing protein which multiplies the difficulty in expression. The native

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WPTP contains two Ca2+ centers per molecule as most plant peroxidases.21, 22 Ca2+ is required to

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maintain the structural integrity of the heme in peroxidase. Ca2+ cations are important for both

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the catalytic activity and thermal stability of the enzyme.23

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Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its sole

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carbon source. P. pastoris is as easy to manipulate as Escherichia coli or Saccharomyces

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cerevisiae, and is faster, easier, and less expensive to use than other eukaryotic expression

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systems and to produce foreign proteins at high levels, either intracellularly or extracellularly.24

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P. pastoris has several advantages over E. coli as an expression system which does not produce

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inclusion bodies and it promotes the correct folding of eukaryotic proteins. In addition,

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hyperglycosylation can interfere with protein folding. Glycoproteins expressed by P. pastoris

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have less mannose residues. The genetic traits of P. pastoris expression system are stable. The

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exogenous gene is integrated into the chromosome of P. pastoris. The exogenous gene is not

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easily lost because it is replicated with P. pastoris chromosome replication. Moreover, the

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genetic background of P. pastoris is clear, which is easy for genetic manipulations. Therefore, P.

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pastoris is suitable for expression of proteins25, including different peroxidases.26-29

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In the present study, the EasySelectTM Pichia expression kit with AOX1 promoter and α-

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factor secretion signal peptide were used for expression of WPTP. AOX1 is the strongest and

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strictest promotor in P. pastoris. AOX1 is an inducible promoter, which is strongly induced by

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methanol. The WPTP gene sequence (ca. 1 kb) was obtained with the rapid-amplification of

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cDNA ends (RACE) techniue.11 WPTP was suitable for expression in P. pastoris after codon

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optimization. The codons for the full length sequence of WPTP gene were optimized and were

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designed with a histidine tag followed by a flexible sequence at the C-terminal. The recombinant

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WPTP (rWPTP) was then purified to characterize its properties.

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MATERIALS AND METHODS Strains, plasmids and media. Gene sequence of WPTP was optimized by OptimumGeneTM

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(GenScript, the Biology CRO, Piscataway, NJ). The strain for expression was P. pastoris GS115

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(his4) with PICZαA as a vector (Invitrogen, Waltham, MA). Yeast extract-peptone dextrose

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medium (YPD), YPD (+Zeocin), buffered glycerol-complex medium (BMGY) and buffered

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methanol-complex medium (BMMY) were prepared according to Pichia media recipes

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(EasySelectTM Pichia expression kit). BMGY and BMMY per liter contained 1% yeast extract,

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2% peptone, 100 mM potassium phosphate (pH 6.0), 0.4 ppm biotin, 1.34% yeast nitrogen base,

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1% glycerol (BMGY) or 0.5% methanol (BMMY). DNA polymerase, T4 DNA ligase and

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restriction enzymes were purchased from New England BioLabs (Ipswich, MA).

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Cloning and sequence analysis of the WPTP gene. The WPTP gene was amplified with

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the primer pair (W-F-Xho I, W-R-Xba I-1/2) by PCR under the conditions: pre-denaturation at

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98 ℃ for 30 s; 5 cycles at 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s; 5 cycles at 98 °C for

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10 s, 50 °C for 15 s, and 72 °C for 30 s; 25 cycles at 98 °C for 10 s, 45 °C for 15 s, and 72 °C for

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30 s; followed by a final extension at 72 °C for 5 min. The PCR product was ligated with

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pPICZαA and then cloned into E. coli.

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The designed primers, W-F-XhoI was GTA TCT CTC GAG AAA AGA GAG GCT GAA

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GCT GAT TTG CAA ATC GGT TTC TAC AAC C; W-R-XbaI-1 was GTG ATG TGA ACC

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TCC TGA ACC TCC GGA AGC GGA GTT TAC TAC GGA GCA G, W-R-XbaI-2 was

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TCTAGA GGT ACC TCA ATG ATG GTG GTG GTG ATG TGA ACC TCC TGA ACC TCC.

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Transformation of Pichia pastoris. The plasmid was extracted from E. coli colonies with

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the antibiotic zeocin (25 µg/ml), linearized, and then electro-transformed into P. pastoris GS115

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with a gene pulser II (BioRad, Hercules, CA). According to the manufacturer’s protocol, the

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parameters were 1,500 V, 200 Ω, and 25 µF using a 0.2 cm cuvette. Transformants were selected

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on YPDS (supplemented with zeocin) plates after incubation for 3 d at 30 °C.

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Expression and purification. The transformants were selected for expression based on

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recombinant WPTP activity. The selected colony was inoculated into a 1-L baffled flask

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containing 200 mL of BMGY and cultured at 28 °C until the optical density of the culture

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reached 2–6 at 600 nm. The cells were harvested by centrifuging at 2000x g for 5 min at room

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temperature. After the supernatant was discarded, the cell pellet was resuspended in BMMY

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medium to an OD600 of 1.0, followed by addition of 0.5% (v/v) methanol every 24 h to induce 5



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rWPTP expression. Aliquots of the cultures were harvested every 24 h and peroxidase activity

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was determined. The expressed rWPTP was centrifuged at 2000x g for 15 min. The target protein

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was then purified by Ni2+ column chromatography (Ni-NTA agarose, Qiagen, Valencia, CA).

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After column purification, the eluent was successively applied into 15-mL and 0.5-mL

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centrifugal filter devices (30 K, Amicon® Ultra, EMD Millipore, Billerica, MA) for further

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concentration. The purified rWPTP was monitored by Reinheitszahl (RZ) values which were

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calculated as a ratio of absorbance at 403 nm and 275 nm (A403/A275).

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Enzyme assay. The activity of rWPTP was determined spectrophotometrically with ABTS

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as a substrate. An aliquot (0.5 µL) of enzyme solution, which the concentration was 3.6*10-6 M,

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was added into 200 µL of 10 mM citric acid-Na2HPO4 buffer (pH 3.0) containing 0.1 mM ABTS

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and 2 mM H2O2. The absorbance was measured at 405 nm at room temperature. One unit of

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peroxidase activity was defined as the amount of peroxidase oxidizing 1 µmole of the substrate

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in one minute. Specific activity was expressed as activity units per milligram of protein

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(unit/mg).

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified

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rWPTP was analyzed by SDS-PAGE, which was performed with a 3.75% stacking gel and 12%

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running gel on a mini protean apparatus (BioRad). The gel was run at 80 V for 15 min and then

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120 V until the loading dye near the bottom of the gel. The protein gel was stained with

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Coomassie Brilliant Blue R-250. The molecular weight of rWPTP was estimated with protein

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standard markers (Precision plus protein dual xltra standards, 250 KD, BioRad).

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Liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of tryptic and chymotryptic digests of rWPTP. In order to determine the validity of rWPTP amino acid sequence after codon optimization, 6


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rWPTP was digested for LC-MS/MS analysis. The SDS-PAGE band containing rWPTP was

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reduced with 10 mM of dithiothreitol for 30 min at 56 °C and then alkylated with 27.5 mM of

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iodoacetamide for 20 min at room temperature in the dark. The reduced and alkylated rWPTP

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was digested with 0.8 µg of trypsin or chymotrypsin in 25 mM of NH4HCO3 at 37 °C for 12 h.

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Peptides were dried via SpeedVac and resuspended in 5% acetonitrile with 0.1% trifluoroacetic

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acid (TFA). The tryptic digests were desalted by ZipTip pipette tips (C18, 10 µL, Millipore,

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Billerica, MA) prior to LC-MS/MS analysis. The ZipTip was first conditioned with 10 µL of

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100% acetonitrile and then equilibrated with 10 µL of 1% formic acid. The tryptic digests were

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loaded onto a ZipTip pipette tip, followed by washing with 10 µL of 1% formic acid. The

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column run-through was discarded. The tryptic peptides were eluted from the ZipTip with the

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elution solution (a mixture of 60% acetonitrile and 1% formic acid). The eluents were dried with

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a speed-vacuum concentrator (Vacufuge concentrator 5305, Eppendorf, USA) for 10 min. To the

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dried residues were added 50 µL of solution containing 0.1% formic acid and 5% acetonitrile.

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After sonication for 10 min, the solution was centrifuged at 4000x g for 1 min and then used for

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LC-MS/MS analysis.

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Tryptic and chymotryptic digests of rWPTP were separated on a Michrom Advance nanoLC

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system (Bruker Daltonics, Fremont, CA) equipped with a C18 nano-column (Michrom Magic

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C18AQ, 100 µm × 150 mm, 3 µm, 200 Å). Linear gradient elution was performed from 2% to

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95% acetonitrile in water with 0.1% formic acid, at a flow rate of 500 nL/min for 57 min.

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MS/MS analysis was performed on a maXis Impact QTOF mass spectrometer (Bruker). MS and

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MS/MS scans in the range of 50–3000 m/z were acquired in positive ion mode. The top 5

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precursor ions were selected for further fragmentation. The absolute and relative thresholds of

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precursor were set at 1000 and 5%. Exclusion was active after 3 spectra and the excluded spectra

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were released after 0.2 min. Line spectral data were then processed into peak lists by Bruker

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DataAnalysis 4.1, wherein spectra were deconvoluted and the peak lists were exported as Mascot

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Generic Files (MGF). A database search was conducted with Mascot 2.10 (Matrix Science,

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Boston, MA). The database was established in the laboratory.12 The database was searched at a

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peptide tolerance of 50.0 ppm and MS/MS tolerance of 0.1 Da. Oxidation of methionine was

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allowed as a variable modification and carbamidomethylation of cysteine was set as a global

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modification. We also used a peptide MOWSE score of 25 as a cut-off that was calculated by

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Mascot.

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Determination of catalytic constant values. The catalytic efficiency of rWPTP was

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determined via the Lineweaver-Burk plotting method of the “Bi-Bi ping-pong” mechanism. The

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Michaelis constants with the two substrates, ABTS and H2O2 were determined under varying

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concentrations of ABTS in the range of 0.02-10 mM in 10 mM citrate phosphate buffer and H2O2

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in the range of 0.05-10 mM. The required H2O2 solution used in this study was prepared daily.

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Determination of substrate specificity. The substrate specificity of rWPTP was

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determined under the conditions (optimum pH, concentrations of H2O2 and different substrates,

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and buffer concentration) optimized previously for palm peroxidases.4, 10

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Effect of pH on enzyme activity and stability. The optimum pH for rWPTP activity was

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determined within a pH range of 1.0-12.0 using 10 mM citrate phosphate buffer (pH 2.0-8.0),

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and 10 mM Na2CO3/NaHCO3 (pH 9.0-12.0). The pH 2 buffer was adjusted with HCl to obtain

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the pH 1 buffer for assay. The effect of pH on WPTP stability was determined by analyzing

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residual WPTP activity after incubation of the WPTP in the aforementioned buffers in the

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absence of the substrate ABTS at 4 °C for 24 h.

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Effect of temperature on enzyme stability. Thermal stability of rWPTP was evaluated via 8


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the assessment of residual activity after incubation of rWPTP in 10 mM citrate phosphate buffer

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(pH 6.0) at different temperatures (20-90 °C) for 1 h.

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Tolerance of rWPTP to metallic ions, denaturants and organic solvents. rWPTP activity

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was determined with ABTS reaction mixture (10 mM Tris-HCl) in the presence of various metal

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cations (Mg2+, Cu2+, Fe2+, Ca2+, Al3+, Mn2+, Zn2+, Fe3+, Na+, K+, and Co2+), denaturants [citric

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acid, SDS, EDTA, urea, and guanidine hydrochloride (Gu-HCl)], and organic solvents

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(methanol, ethanol, isopropanol, glycol, and DMSO) at a concentration of 5 mM, 20% and 20%,

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respectively. It is noteworthy that citric acid was grouped into denaturants because of the high

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concentrations tested. The remaining activity was measured and expressed as a percentage of the

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rWPTP activity in solutions without the added metal ions, denaturants or organic solvents.

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RESULTS AND DISCUSSION

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Expression and purification of rWPTP. After incubation of rWPTP transformants for 3 days,

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the white colonies were selected for mini-scale expression to determine the best strains for scale-

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up expression in a 1-L flask. After expression of rWPTP in P. pastoris for 3 days, the expressed

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rWPTP was centrifuged at 2000x g for 15 min. The target enzyme was purified with Ni-NTA

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resin column chromatography using the 15-mL and 1.5-mL centrifugal filter devices. The yield

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of rWPTP after each purification step was summarized in Table 1. After Ni-NTA resin column

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chromatographic purification, 58% of WPTP remained with a total amount of protein of 9.6 mg

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and specific activity of 0.96 U/mg. After concentration with the 15-mL and 1.5-mL centrifugal

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filter devices, WPTP, having a specific activity of 112 U/mg of protein, was obtained with an

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overall yield of 27%. The purified rWPTP (Fig. 1) had an RZ value of 0.5, which was similar to

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the RZ values (0.4-0.6) of HRP expressed in S. cerevisiae and P. pastoris27. The RZ value 9



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measures the heme content by using the aromatic amino acid content as a reference and is a

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measure of the purity of rWPTP.30 The final protein concentration of rWPTP was low, so larger

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scale fermentation would be required to obtain a greater amount of rWPTP.

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Molecular weight, glycosylation and amino acid sequences of rWPTP. The purified

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rWPTP showed only one band on SDS-PAGE (Fig. 1). The band was at around 43±2 kDa.

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Mature rWPTP has 306 amino acids in length (Fig. 2). The rWPTP expressed in P. pastoris in the

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present study had approximately 23.4% of glycan content, which is similar to the glycan content

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(21-29%) of mature native WPTP previously reported11.

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The single band observed by SDS-PAGE was isolated and digested by trypsin or

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chymotrypsin. The resulting peptides were analyzed by LC-MS/MS. The mass spectrometry

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analysis verified that the single band was rWPTP. A fragmentation spectrum from the LC-

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MS/MS analysis was shown in Fig. 2A. The b- and y-ions in the fragmentation spectrum verified

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the amino acid sequence of rWPTP residues 48 - 65. In total, 11 tryptic peptides and 4

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chymotryptic peptides provided 51.9% sequence coverage of rWPTP (Figure 2B). The average

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mass of native WPTP, which has 306 amino acid residues in length, is 32.2 kD (vs 43±2 kDa by

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SDS-PAGE).11 Fig. 2B shows 13 potential glycosylation sites that could be identified by the

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sequence Asn- Xxx- Ser/Thr (Xxx is any amino acid except Pro).

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Michaelis-Menten constant (Km) of rWPTP. The catalytic efficiency of rWPTP was

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determined in the “Bi-Bi ping-pong” mechanism.31, 32

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E + H2O2 → Compound I + H2O

(1)

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Compound I + AH2 → Compound II + AH˙

(2)

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Compound II + AH2 → E + AH˙ + H2O

(3)

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E is the enzyme in resting state. Compounds I and II are the oxidized intermediates of 10


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peroxidase. AH2 and AH˙ are the electron donor substrate and the radical product of its one

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electron oxidation, respectively.

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The Km constant of rWPTP with the substrates ABTS and H2O2 was measured (Fig. 3). A

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primary double-reciprocal plot of the initial rate of ABTS oxidation as a function of H2O2

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concentrations at fixed ABTS concentrations (0.02, 0.05, 0.1 and 0.2 mM) was presented in Fig.

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3A. The insert shows the secondary plot of 1/V vs. 1/[ABTS] which was used to calculate K ୅୆୘ୗ . ୫

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A double-reciprocal plot was obtained upon studying the effect of ABTS concentration on the

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initial rates of the oxidation reaction at different fixed H2O2 concentrations (0.1, 0.2, 0.3 and 0.6

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. The mM) (Fig. 3B). The insert shows the secondary plot of 1/V vs. 1/[ H2O2] to calculate K ୌଶ୓ଶ ୫

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Km is the substrate concentration at which the reaction rate is at half-maximum. A Km value is an

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inverse measure of the substrate’s affinity for the enzyme. A low Km value means high affinity.

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The Km value of H2O2 (K ୌଶ୓ଶ ) was 4.6*10-4 M, while the Km value of ABTS (K ୅୆୘ୗ ) was ୫ ୫

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1.6*10-4 M and the Vmax was 6.0*10-5 M/min. The lower the Km value is, the greater reactivity of

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the peroxidase is toward the substrate ABTS. The K ୌଶ୓ଶ and K ୅୆୘ୗ of Chamaerops excelsa ୫ ୫

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palm tree peroxidase (CEP) are 7.1*10-5 M and 6.2*10-4 M, respectively. The K ୌଶ୓ଶ and K ୅୆୘ୗ ୫ ୫

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of royal palm tree peroxidase (RPTP) are 1.8*10-3 M and 4.9*10-4 M, respectively.33 The K ୌଶ୓ଶ ୫

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value of rWPTP is 6.5- and 0.3-fold of that of CEP and RPTP, respectively. The K ୅୆୘ୗ of ୫

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rWPTP is one forth and one third of that of CEP and RPTP, respectively. The Km constants

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imply that WPTP has greater affinity than that of CEP and RPTP toward the substrate ABTS.

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Substrate specificity of rWPTP. As the “Bi-Bi ping-pong” mechanism is described in

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equations 1-3, the reaction with the lowest rate which limits the overall catalytic process is the

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one of Compound II with substrate (AН2). Similar approaches were used successfully.4, 34-35 The

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reduction of compound II is the rate-limiting step in catalysis.36 The values of the second-order

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rate constant (kapp) for the reaction between compound II and hydrogen donor substrates (AH2)

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were then used to evaluate WPTP catalytic efficiency. The constant was calculated by equation 4.

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Rate = kapp*[compound II]*[AH2]

(4)

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Because reaction 3 is rate-limiting, we used it to calculate the initial concentration of

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peroxidase instead of compound II concentration. rWPTP did not catalyze the reaction of the

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substrate ferulic acid (Table 2). The kapp value measured with ABTS was 16, 12, 8, 24 and 190

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times greater than that with ο-dianisidine, ο-phenylenediamine, guaiacol, pyrogallol, and

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catechol, respectively. The kapp value is related to the peroxidase catalytic capacity, which means

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that among the substrates tested, ABTS is the best substrate for rWPTP. Oxidation of pyrogallol

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and catechol catalyzed by rWPTP was approximately 10 times slower than that of aromatic

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amines and guaiacol; this characteristic was the same as native RPTP (Table 3). rWPTP can be

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used as an indicator to test the reaction with the concentration of H2O2 and ABTS as low as 1.5

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mM and 2.5 µM, respectively (data not shown).

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Comparison of the kapp values measured under the optimal conditions revealed that the

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efficiency of rWPTP catalysis depends strictly on the chemical nature of substrates (Table 3).

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Native WPTP is the peroxidase extracted from windmill palm tree leaves, while rWPTP is the

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peroxidase that was expressed in P. pastoris. The kapp value of rWPTP with ABTS was 52.8-,

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4.8-, 17.3-, 51.4- and 19.0- fold of that of soybean, horseradish, tobacco, peanut and alfalfa

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peroxidases4. The kapp value of rWPTP was 0.2-, 1.2-, 3.1-, 0.3-, 0.6-, 0.6- and 0.5-fold of that of

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native WPTP10, RPTP, soybean, horseradish, tobacco, peanut and alfalfa peroxidases4 toward ο-

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dianisidine, respectively, whereas it was, respectively, 29.1-, 0.6-, 40.0-, 3.3-, 50.0-, 7.3- and

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38.1-fold with ο-phenylenediamine as the substrate, and was 1.4-, 2.1-, 4.0-, 1.6-, 5.0-, 1.0- and

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0.2-fold with the substrate guaiacol. Only rWPTP and native RPTP can oxidize pyrogallol and

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catechol, phenolic compounds containing two and three hydroxy groups, respectively, which

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were oxidized by rWPTP more than 10 times slower than aromatic amines and guaiacol

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(monophenol). The data show a substrate specificity pattern of rWPTP that can oxidize aromatic

267

amines and phenols besides ferulic acid.

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Optimum pH with substrate ABTS. The purified rWPTP showed optimal catalytic

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activity to ABTS under extreme acidic conditions (pH 2.0–4.0) with 0.1 and 0.2 mM ABTS as

270

the substrate (Fig. 4). The maximal rWPTP activity measured with ABTS was observed at pH

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3.0, and the activities under pH 2 and 4 were about 93% of the maximum value. The results

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agreed with the fact that WPTP is an anionic peroxidase, with an isoelectric point of pI 3.5.10 The

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optimum pH of RPTP was 7. The maximum activity of AOPTP occurred at pH 3, however, the

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activities of AOPTP at pH 2.8 and 3.5 were 57% and 67% of the maximum value.4,34 The results

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indicate excellent suitability of rWPTP to catalyze the synthesis of polyaniline, which is most

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effective at a pH value below 4.1, 37

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pH and thermal stability of rWPTP. As shown in Fig. 5, the purified rWPTP retained

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90% of its activity at pH 10 and 100% of activity when it was stored in pH 2 buffer for 24 h. The

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purified rWPTP retained 75% of its activity even when it was stored at pH 12 for 24 h. Many

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plant peroxidases are labile at acidic pH conditions. HRP is unstable at pH below 4.5, and native

281

WPTP had a lower stability at acidic conditions.38-39 The purified rWPTP maintained greater than

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93% of its activity when it was stored at 60 °C and 87% activity at 70 °C. The activity of the

283

purified rWPTP declined dramatically to 40% when it was stored at 80 °C. Thermal inactivation

284

of native WPTP visibly occurred in the temperature range of 61-67 °C. The poor thermal

285

stability limits peroxidase’s large-scale catalysis applications, which is particularly true in

286

bioremediation and polyelectrolyte synthesis. Good thermal stability of rWPTP is therefore a

13



287

desirable property for polyelectrolyte synthesis.40 Comparison of substrate specificity and

288

stability profiles between rWPTP and other peroxidases indicates that catalytic features and

289

optimal performance conditions (acidic) of rWPTP offer good perspectives for its applications.

290

Tolerance of rWPTP to metallic cations, denaturants and organic solvents. Different

291

metallic ions (Table 4), denaturants and organic solvents (Table 5) have different effects on the

292

enzyme activity. The enzyme activity value measured without fortified metallic cations,

293

denaturants and organic solvents was taken as 100%. Mg2+, Cu2+, Fe3+, Ni2+, citric acid and urea

294

had no effect on the peroxidase activity. Fe2+ and Mn2+ highly inhibited the enzyme activity,

295

which might be due to their interactions with the heme prosthetic group. The metallic ions Ca2+,

296

Pb2+ and K+ had an activation effect. SDS, EDTA, 20% DMSO and 10% DMSO had inhibitory

297

effects on the activity, while 1% DMSO had negligible effects on the enzyme activity. DMSO at

298

1% or less can be therefore used as a carrier solvent in applications.

299

Page 14 of 32

rWPTP has good pH and thermal stability and shows optimum catalytic activity in the range

300

from pH 2.0 to pH 4.0. rWPTP can also maintain high activity under most metallic ions and

301

organic solvents. The results obtained in this study imply that the rWPTP expressed in P.

302

pastoris has a good application prospect. The future work would include optimization of

303

fermentation parameters at a large scale as well as investigation of potential applications of the

304

rWPTP.

305 306 307

Abbreviations Used rWPTP, recombinant windmill palm tree peroxidase expressed in P. pastoris; Native WPTP,

308

windmill palm tree peroxidase extracted from leaves of the palm tree Trachycarpus fortunei;

309

RPTP, royal palm tree peroxidase; CEP, Chamaerops excelsa palm tree peroxidase; AOPTP,

14


Page 15 of 32


310

African oil palm tree peroxidase; ABTS, 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid);

311

SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA,

312

Ethylenediaminetetraacetic acid; DMSO, Dimethyl sulfoxide; SDS, sodium dodecyl sulfate.

313 314

Acknowledgement

315

The work was supported in part by the National Institutes of Health Research Centers in

316

Minority Institutions Program (G12 MD007601) and China Postdoctoral Science Foundation

317

(2016M590155). BW was a China Scholarship Council scholarship recipient.

318 319

References

320

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323

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(2) Keum, Y. S.; Li, Q. X. Copper dissociation as a mechanism of fungal laccase denaturation by humic acid. Appl. Microbiol. Biotech. 2004a, 64, 588-592. (3) Keum, Y. S.; Li, Q. X. Fungal laccase-catalyzed degradation of hydroxy polychlorinated biphenyls. Chemosphere 2004b, 56, 23-30. (4) Sakharov, I. Y. Long-term chemiluminescent signal is produced in the course of luminol

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peroxidation catalyzed by peroxidase isolated from leaves of African oil palm tree. Biochem.

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(5) Wardman, P. Indole-3-acetic acids and horseradish peroxidase: a new prodrug/enzyme

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combination for targeted cancer therapy. Curr. Pharm. Des. 2002, 8, 1363-1374. (6) Alpeeva, I. S.; Niculescu-Nistor, M.; Castillo Leon, J.; Csöregi, E.; Sakharov, I. Y. Palm tree

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peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring.

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and characterization of polyelectrolyte complex of polyaniline and sulfonated polystyrene

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produced by palm tree peroxidase. Synth. Met. 2004, 142, 127-135.

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(8) Sakharov, I. Y.; Sakharova, I. V. Extremely high stability of African oil palm tree peroxidase. Biochim. Biophys. Acta. 2002, 1598, 108-114. (9) Zamorano, L. S.; Pina, D. G.; Arellano, J. B.; Bursakov, S. A.; Zhadan, A. P.; Calvete, J. J.;

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Sanz, L.; Nielsen, P. R.; Villar, E.; Gavel, O.; Roig, M. G.; Watanabe, L.; Polikarpov, I.;

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(10) Caramyshev, A. V.; Firsova, Y. N.; Slastya, E. A.; Tagaev, A. A.; Potapenko, N. V.;

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Lobakova, E. S.; Pletjushkina, O. Y.; Sakharov, I. Y. Purification and characterization of

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windmill palm tree (Trachycarpus fortunei) peroxidase. J. Agric. Food Chem. 2006, 54,

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(11) Baker, M. R.; Zhao, H. W; Sakharov, I. Y.; Li, Q. X. Amino acid sequence of anionic

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peroxidase from the windmill palm tree Trachycarpus fortunei. J. Agric. Food Chem. 2014,

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62, 11941–11948.

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(12) Baker, M. R.; Tabb, D. L.; Ching, T.; Zimmerman, L. J.; Sakharov, I. Y.; Li, Q. X. Site-

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specific N-glycosylation characterization of windmill palm tree peroxidase using novel tools

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for analysis of plant glycopeptide mass spectrometry data. J. Proteome Res. 2016, 15, 2026-

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2038. (13) Arseguel, D.; Baboulène, M. Removal of phenol from coupling of talc and peroxidase.

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produced by horseradish peroxidase in dioxane. Polym, J., Sci.: Part A: Polymer Chem.

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1991, 29, 1561-1574.

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(20) Caramyshev, A. V.; Lobachov, V. M.; Selivanov, D. V.; Sheval, E. V.; Vorobiev, A. K.;

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Katasova, O. N.; Polyakov, V. Y.; Makarov, A. A.; Sakharov, I. Y. Micellar peroxidase-

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catalyzed synthesis of chiral polyaniline. Biomacromolecules 2007, 8, 2549-2555.

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J.; Sanz, L.; Pérez, A.; Bursakov, S.; Roig, M. G. Crystal structure and statistical coupling

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analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia). J. Struct.

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Biol. 2010, 169, 226-242.

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(22) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Crystal structure of

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horseradish peroxidase C at 2.15 Å resolution. Nat. Struct. Biol. 1997, 4, 1032-1048.

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(23) Laberge, M.; Huang, Q.; Schweitzer-Stenner, R.; Fidy, J. The endogenous calcium ions of

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horseradish peroxidase C are required to maintain the functional nonplanarity of the heme.

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Biophys. J. 2003, 84: 2542-2552.

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(24) Cereghino, J. L.; Cregg, J. M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 2000, 24, 45-66. (25) Cummings, R. D.; Doering, T. L. Fungi. In: Essentials of Glycobiology. 2nd ed. Varki, A.;

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Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler,

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M. E., Eds. Cold Spring Harbor Laboratory Press: NY, 2009.

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(26) Huang, J. Z.; Lin, J. H.; Shi, Y. P.; Hu, M. R.; Tao, Y. Expression of soybean peroxidase from soybean in Pichia pastoris. Microbiol. China. 2014, 41, 1850-1855. (27) Morawski, B.; Lin, Z. L.; Cirino, P.; Joo, H.; Bandara, G.; Arnold, F. H. Functional

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expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris.

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Protein Eng. 2000, 13, 377-384.

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(28) Ciaccio, C.; Gambacurta, A.; Sanctis, G. D. E.; Spagnolo, D.; Sakarikou, C.; Petrella, G.;

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Coletta, M. rhEPO (recombinant human eosinophil peroxidase): expression in Pichia

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pastoris and biochemical characterization. Biochem. J. 2006, 395, 295-301.

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(29) Spadiut, O.; Rossetti, L.; Dietzsch, C.; Herwig. C. Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy. Protein Expr. Purif. 2012,

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86, 89-97. (30) Segura, M. de las M.; Levin, G.; Miranda, M. V.; Mendive, F. M.; Targovnik, H. M.;

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Cascone, O. High-level expression and purification of recombinant horseradish peroxidase

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isozyme C in SF-9 insect cell culture. Process Biochem. 2005, 40, 795–800.

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(31) Poulos, T. L.; Kraut, J. The stereochemistry of peroxidase catalysis. J. Biol. Chem. 1980, 255, 8199-8205. (32) Dunford, H. B. Horseradish peroxidase: structure and kinetic properties. In: Peroxidase in

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Chemistry and Biology. Everse, J.; Everse, K. E.; Grisham, M. B., Eds. Vol. II. CRC Press:

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Boca Raton, FL, 1991, 1-24.

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(33) Cuadrado, N. H.; Arellano, J. B.; Calvete, J. J.; Sanz. L.; Zhadan. G. G.; Textor. L. C.;

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Polikarpov. I. Palm peroxidases: The most robust enzymes. Curr. Top. Biochem. Res. 2011,

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13, 67-79.

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(34) Sakharov, I. Y.; Vesga B, M. K.; Galaev, I. Y.; Sakharova, I. V.; Pletjushkina, O. Y.

415

Peroxidase from leaves of royal palm tree Roystonea regia: purification and some properties.

416

Plant Sci. 2001, 161, 853-860.

417

(35) Ryabov, A. D.; Goral, V. N. Steady-state kinetics, micellar effects, and the mechanism of

418

peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide. J. Biol. Inorg.

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Chem. 1997, 2, 182-190.

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(36) Dunford, H. B. Ed. Heme Peroxidases. Wiley, New York, US. 1999, 962-965.

421

(37) Nagarajan, R.; Liu, W.; Kumar, J.; Tripathy, S. K.; Bruno, F. F.; Samuelson, L. A.

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Manipulating DNA conformation using intertwined conducting polymer chains.

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Macromolecules 2001, 34, 3921-3927.

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(38) Liu, W.; Cholli, A. L.; Nagarajan, R., Kumar, J., Tripathy, S., Bruno, F.F., Samuelson, L.

19



425 426

Enzymatically synthesized conducting polyaniline. J. Am. Chem. Soc. 1999, 121, 71– 78. (39) Berezin, I.V.; Ugarova, N.N.; Kerschengoltz, B.M.; Brovko, L. Yu. Effect of prosthetic

427

group of horseradish peroxidase on the enzyme stability, Biochem. (Moscow). 1975, 40: 297-

428

300.

429

(40) Zamorano, L.; Vilarmau, S. B.; Arellano, J. B.; Zhadan, G. G.; Cuadrado, N. H.; Bursakov,

430

S. A.; Roig, M. G.; Shnyrov, V. L. Thermal stability of peroxidase from Chamaerops excelsa

431

palm tree at pH 3. Int. J. Biol. Macromol. 2009, 44, 326-332.

432

20


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Page 21 of 32


433

Figures Captions

434

Fig. 1. SDS-PAGE of purified rWPTP.

435 436 437

Fig. 2. (A) LC tandem MS spectrum of a tryptic peptide of the parent ion at m/z 933.4. (B) Amino acid sequence coverage of rWPTP.

438 439

Fig. 3. Lineweaver–Burk plot of rWPTP kinetic data. The Michaelis constants with the two

440

substrates, ABTS and H2O2 were determined under varying concentrations of ABTS and

441

H2O2.

442 443

Fig. 4. Optimal pH for rWPTP catalytic activity with 0.1 and 0.2 mM ABTS as the substrate. The

444

optimum optimal pH was determined within a pH range of 1.0-12.0 using 10 mM citrate

445

phosphate buffer and 10 mM Na2CO3/NaHCO3.

446 447

Fig. 5. Stability of rWPTP activity under different pH and temperature conditions. The effect of

448

pH and temperature on WPTP stability was determined by analyzing residual WPTP activity

449

after incubation of the WPTP in different pH buffers at 4 °C for 24 h or at different

450

temperatures (20-90 °C) for 1 h.

21



Fig. 1.

Molecular Marker weight

rWPTP

250 150 100 75 50 37

22


Page 22 of 32

Page 23 of 32


Fig. 2A

Fig. 2B

23



Fig. 3

24


Page 24 of 32

Page 25 of 32


Fig. 4

25



Fig. 5

26


Page 26 of 32

Page 27 of 32


Table 1. Specific activity and yield of rWPTP expressed in P. pastoris after each purification step Procedure

Total

Specific

Total

Purification Yield

protein

activity

activity

(fold)

(%)

(mg)

(Units/mg)

(Units)

Crude enzyme

105.3

0.15

16.1

1.0

100

Ni-NTA resin

9.6

0.96

9.2

6.0

58

0.14

38.2

5.5

250

34

0.04

112

4.4

736

27

a

15-mL filter

0.5-mL filter a

a

15-mL and 0.5-mL filters stand for 15-mL and 0.5-mL centrifugal filter devices used, respectively.

27



Page 28 of 32

Table 2. Substrate specificity of rWPTP expressed in P. pastoris Substrate (AH2)

λ, nm

ε, M-1cm-1

[H2O2], mM

[AH2], mM

pH

[Buffer], M

kapp, M-1S-1

ABTS

414

31100

3

0.006

3

0.01

1.9*107

Ferulic acid ο-Dianisidine

318 420

6000 30000

1 9

0.1 0.17

5 5.2

0.04 0.09

— 1.2*106

ο-Phenylenediamine

445

11100

1

0.25

5

0.10

1.6*106

Guaiacol

470

5200

2.6

0.4

5.5

0.05

2.5*106

Pyrogallol

420

2640

4

5.5

6

0.025

8.0*105

Catechol

295

1700

5

25

5

0.04

1.1*105

28


Page 29 of 32


Table 3. Comparison of substrate specificity and the second-order rate constant (kapp) of native WPTP, rWPTP and the other peroxidases kapp (M-1S-1)

Substrate Native

rWPTP

RPTPb

Soybeanb

Horseradishb

Tobacoob

Peanutb

Alfalfab

WPTPa ABTS

6.0×107

1.9×107

5.2×107

3.6×105

4.0×106

1.1×106

3.7×105

1.0×106

Ferulic acid

3.0×106

－

6.3×107

－

－

－

－

－

6

1.2×10

6

9.7×10

5

3.9×10

5

4.3×10

6

2.0×10

6

2.0×10

6

2.4×106

ο-Dianisidine

5.0×10

ο-Phenylenediamine

5.5×104

1.6×106

2.9×106

4.0×104

4.9×105

3.2×104

2.2×105

4.2×104

Guaiacol

1.8×106

2.5×106

1.2×106

6.4×105

1.6×106

5.1×105

2.4×106

1.5×107

Pyrogallol

－

8.0×105

1.7×105

－

－

－

－

－

Catechol

－

1.1×105

2.3×105

－

－

－

－

－

a

The data for native WPTP which was extracted from leaves are cited from Caramyshev et al.10

b

The data for the six peroxidases are cited from Sakharov et al.4

29



Page 30 of 32

Table 4. rWPTP tolerance to various metallic cationsa Interference

Mg2+

Cu2+

Fe2+

Ca2+

Al3+

Ag+

Mn2+

Pb2+

Zn2+

Fe3+

Na+

K+

Co2+

Ni2+

WPTP activity

100%

100%

13%

101%

99%

98%

10%

103%

99%

100%

99%

102%

97%

100%

a,

The concentration of Mg2+, Cu2+, Fe2+, Ca2+, Al3+, Mn2+, Zn2+, Fe3+, Na+, K+, and Co2+ was 5 mM.

30


Page 31 of 32


Table 5. rWPTP tolerance to various denaturants and organic solventsa Interference

WPTP activity

Guanidine

Citric

-HCl

acid

99%

100%

SDS

51%

EDTA

86%

Urea

100%

Methanol

94%

Ethanol

92%

Isopropanol

96%

Glycol

93%

20%

10%

1%

DMSO

DMSO

DMSO

66%

81%

99%

The content of citric acid, SDS, EDTA, urea, guanidine HCl, methanol, ethanol, isopropanol, and glycol was 20%. The content of DMSO was 1%, 10% and 20%.

31



TOC graphic

Recombinant palm peroxidase expressed by P. pastoris

Catalytic activity

Thermal stability

32


Page 32 of 32

Expression and Characterization of Windmill Palm Tree (Trachycarpus

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