Effect of N-Linked Glycosylation of Recombinant Windmill Palm Tree

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Effect of N-linked glycosylation of recombinant windmill palm tree peroxidase on its activity and stability Bo Fu, Margaret R. Baker, and Qing X. Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of Agricultural and Food Chemistry

Manuscript revised according to editor’s and reviewers’ comments for possible publication in Journal of Agricultural and Food Chemistry

Effect of N-linked glycosylation of recombinant windmill palm tree peroxidase on its activity and stability

Bo Fu, Margaret R. Baker, Qing X. Li*

Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States

Correspondence: *Qing X. Li Tel: (808) 956-2011 Fax: (808) 956-3542 Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Plant secretory peroxidases are valuable commercial enzymes. The windmill palm tree

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Trachycarpus fortunei produces one of the most stable and fastest peroxidases (WPTP)

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characterized to date, however, an economical source is needed. Pichia pastoris has been used as

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an expression system for WPTP and other peroxidases. However, yeast and plants synthesize

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different types of N-linked glycan structures and may differ the level of glycosylation at each site.

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Such non-native glycosylation can have unwanted consequences. Glycosylation site N256 was

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under-glycosylated in the wild-type (1.5%) compared to the native enzyme (55%); therefore, we

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mutated WPTP to promote glycosylation at this site (WPTP E254G). Glycosylation increased 4-

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fold, as measured by liquid chromatography-tandem mass spectrometry. The mutation did not

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change the substrate specificity and optimal pH- and thermo-stability ranges, but increased the

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catalytic activity 2-3 fold. In comparison with wild-type WPTP, WPTP E254G showed a shift of

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the most stable pH from 7 to 9, making it suitable for applications under alkaline conditions.

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KEYWORDS: Peroxidase; Windmill palm tree; Glycosylation; Glyco-engineering; Substrate

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specificity; Stability

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Introduction Plant secretory peroxidases (EC 1.11.1.7) are enzymes that catalyze the oxidation of a broad

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range of aromatic compounds by reducing hydrogen peroxide to water. The aromatic radical

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products of the reaction go on to polymerize, and result in a quantitative color change. Thus,

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peroxidases are frequently used in enzyme immunoassays, chemiluminescence assays,

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construction of biosensors, organic and polymer synthesis, and wastewater treatment in which

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polymerization of toxic phenolic compounds renders them insoluble and less toxic.1-6

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Palm is among the top 10 produce crops in the world. Palm plays important roles in daily

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life because it produces palm oil that has nutritional benefits and versatility.7 Palm oil is readily

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available and is the second largest vegetable oil in the world.8, 9 Nowadays, approximately 50%

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of traded oils is palm oil.8 Palm tree peroxidases, such as windmill palm tree peroxidase (WPTP),

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exhibit extremely high stability under a broader range of pH and at higher temperatures than the

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widely used horseradish peroxidase (HRP).10-12 Those characteristics make WPTP more robust

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and therefore more suited for applications requiring acidic or alkaline conditions and high

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temperature.

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Plant secretory peroxidases, including WPTP, are highly N-glycosylated. N-Linked glycans

34

play an important role on enzyme substrate affinity, catalytic turnover, and pH and temperature

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stability.13, 14 One potential mechanism for these phenomena is that N-glycosylation of a folded

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protein can reduce the backbone flexibility, and thus have a significant stabilizing effect on large

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regions of the backbone structure.15 Mature native peroxidase from the windmill palm tree

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Trachycarpus fortunei (WPTP) contains 306 amino acid residues. There are 13 N-linked

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glycosylation sites (N-X-S/T, X is not P) on WPTP and its carbohydrate content ranges from 21%

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to 29%.16 The function of these glycans has not been studied to date.

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Glyco-engineering to change properties of peroxidase and other enzymes has been

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performed in some studies. Glycosylation at individual sites showed different effects on the

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enzyme activity. In the majority of cases, elimination of glycosylation sites resulted in decrease

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in the enzyme activity and thermal stability.17-19 In the converse case, unglycosylated RNase A

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had greater than three times activity than its glycosylated variant RNase B.20 Less glycosylated

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variants of HRP from P. pastoris were engineered. Great differences were found in catalytic

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activity and stability among wild-type HRP and its variants.18 Four mutants carrying Asn to Ala

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substitutions in potential glycosylation sites of cellobiohydrolase I was produced by site-directed

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mutagenesis. Three of the mutants showed different activity from the wild type against natural

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and synthetic substrates.19 On the other hand, promoting glycosylation at naïve or

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underglycosylated sites often enhances the enzyme characteristics.21

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Pichia pastoris is an economical organism to produce industrial or commercial enzymes,

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however, differences from native glycosylation may have unintended consequences on the

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enzyme characteristics. Non-native glycosylation includes both differences in the glycan

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structure and the level of glycosylation at that site. We recently expressed WPTP in P. pastoris.22

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While exploring structural differences between native and recombinant WPTP, we noted that the

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degree of glycosylation at N256 was significantly lower in recombinant WPTP (1.5%) than

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native WPTP (55%).23 We hypothesized that changing the amino acid sequence to promote

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glycosylation of this site would restore some of the properties of the native enzyme. The amino

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acids flanking the glycosylation site in part determine whether or not that site will be

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glycosylated, perhaps by influencing the interactions between the nascent polypeptide and the

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oligosaccharyltransferase complex.24 It was observed that glycosylation sites with an acidic

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residue 1 or 2 amino acids upstream of the glycosylation site are unglycosylated more frequently

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than expected, whereas a non-polar amino acid upstream of the glycosylation site tends to

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promote glycosylation.24

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In the present study, a WPTP variant (WPTP E254G) was constructed via site-directed

67

mutagenesis. Recombinant wild-type WPTP and WPTP E254G were expressed in P. pastoris

68

and their glycosylation level was investigated. We determined and compared the catalytic

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activity, pH stability and thermal stability of the two enzymes. We found that WPTP E254G was

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more stable than the wild-type and had faster rates of catalysis. The results indicated that WPTP

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E254G has potential for applications requiring alkaline conditions.

72 73

Materials and Methods

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Strains, plasmids and media

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E. coli DH5α (Invitrogen, Waltham, MA) was used as the host strain for cloning vectors.

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pPICZαA vector (Invitrogen) was used as the vector for expression of WPTP in P. pastoris

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GS115 (his4) (Invitrogen). Yeast extract peptone dextrose medium containing sorbitol (YPDS),

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

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medium (BMMY) were prepared according to the recipes in EasySelectTM Pichia Expression Kit

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instruction (Invitrogen). Phusion High-Fidelity DNA polymerase, 5 × Phusion High-Fidelity

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buffer and Endoglycosidase H (Endo H) were purchased from New England Biolabs (Ipswich,

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MA, USA). Restriction enzymes SacI and DpnI were purchased from Fisher Bioreagents (Fair

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Lawn, NJ).

84 85 86

Construction of WPTP E254G In order to increase the glycosylation level at glycosylation site N256, Glu at site 254 was

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mutated to Gly to form WPTP E254G. The gene encoding WPTP E254G was produced by site-

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directed mutagenesis. Primers containing mutational bases were: 254-F 5ʹ-CT TTG GTT ACA

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GGA GCA AAC TTA TCA GCA GCC G-3ʹ, 254-R 5ʹ-C GGC TGC TGA TAA GTT TGC TCC

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TGT AAC CAA AG-3ʹ. Polymerase chain reaction (PCR) amplification was performed in a 50

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µL reaction volume with 5 × Phusion High-Fidelity buffer, 6% DMSO, 10 µM dNTPs mixture,

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10 µM of each primer, 1 U Phusion High-Fidelity DNA polymerase and 0.01 ng plasmid wptp-

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pPICZαA used as template. The amplification was performed at 98 °C for 2 min, then 30 cycles

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at 98 °C for 10 s, 55 °C for 30 s, 72 °C for 4 min, and an extension period of 10 min at 72 °C.

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DpnI was used for digestion of residual template. Ligation was performed with the CloneEZ

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PCR Cloning Kit (GenScript, Piscataway, NJ, USA) to produce plasmid wptpE254G-pPICZαA.

97 98 99

Transformation Plasmid wptpE254G-pPICZαA was transformed into E. coli DH5α. Plasmids were extracted

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from transformants using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and linearized

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using SacI. Linearized plasmid was electro-transformed into P. pastoris GS115.

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Electrotransformation was performed at 1500 V, 200 Ω, and 25 µF in a 0.2 cm cuvette. Gene

103

pulser II (BioRad, Hercules, CA) was used for electrotransformation. The transformants were

104

selected on an YPDS plate with 100 µg/mL zeocin.

105 106 107

Expression and purification The EasySelectTM Pichia expression kit was used for expression of wild-type WPTP and

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WPTP E254G according to the procedure previously described with some modifications.22 All

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transformants were expressed first in a small-scale system (10 mL). Each one of wild-type

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WPTP and WPTP E254G transformants were screened based on their highest activities toward 3,

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3ʹ, 5, 5ʹ-tetramethylbenzidine (TMB). The recombinant wild-type WPTP and WPTP E254G were

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individually inoculated in a 250 mL flask containing 100 mL BMGY culture medium and

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cultured at 28 °C until the optical density reached 4 at 600 nm. The cells were harvested by

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centrifuging at 6000g for 5 min, then transferred into 2-L flask containing 400 mL of BMMY

115

expression medium. Methanol was added into the medium at a final concentration of 0.5% every

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24 h to induce wild-type WPTP and WPTP E254G expression. The fermentation broths were

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collected by centrifuging at 6000g for 15 min, and purified with Ni-NTA agarose (Qiagen), then

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ultrafiltered with 15 mL (30 K) and 0.5 mL (3 K) centrifugal filter devices (Amicon Ultra, EMD

119

Millipore) for desalting and concentration.

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Purified wild-type WPTP and WPTP E254G were deglycosylated by Endo H at 37 °C for 16

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h. The deglycosylated products were analyzed by sodium dodecyl sulfate-polyacrylamide gel

122

electrophoresis (SDS-PAGE) which was performed on 12% polyacrylamide gel. Protein staining

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was performed with Coomassie brilliant blue R-250.

124 125

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of wild-type

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WPTP and WPTP E254G

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In order to validate amino acid sequences and determine glycosylation level at glycosylation

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site 256 of wild-type WPTP and WPTP E254G, they were in-gel digested and analyzed by LC-

129

MS/MS. The SDS-PAGE bands containing wild-type WPTP and WPTP E254G were cut for in-

130

gel digestion. The two proteins were reduced using 10 mM dithiothreitol at 37 °C for 40 min,

131

then alkylated using 55 mM iodoacetamide at room temperature for 45 min in dark. The reduced

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and alkylated proteins were digested with Asp-N (Promega, Madison, WI) at 37 °C for 16 h. The

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digests were extracted from the gel by adding ammonium bicarbonate, formic acid and

134

acetonitrile followed by bath sonication. The extracted peptides were concentrated with an

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Eppendorf Vacufuge plus (Eppendorf, Hauppauge, NY).

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Digests of wild-type WPTP and WPTP E254G were dissolved in 0.1% formic acid/5%

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acetonitrile/94.9% water. All chromatographic separations were performed on a nanoAdvanced

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LC (Bruker Daltonics, Billerica, MA). For each analysis, 5 µL of sample was injected and loaded

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onto a nano-trap column (3 µm 200 Å ProntoSIL C18AQ, NanoLCMS Solutions) at a flow rate

140

of 5 µL/min with 100% Solvent A (0.1% formic acid, 5% acetonitrile, 94.9% water (v/v/v)).

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After a washing period of a total volume of 20 µL, the trap column was placed in-line with an

142

analytical column (ProntoSIL C18AQ, 25 cm × 100 µm, 3 µm, 120 Å, NanoLCMS Solutions).

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The peptides were separated using a linear gradient of 5−45% Solvent B (0.1% formic acid, 5%

144

water, 94.9% acetonitrile (v/v/v)) at a flow rate of 500 nL/min over 120 min followed by an

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increase to 95% B and held at 95% B for 15 min before returning to initial conditions of 5% B

146

for 21 min.

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The samples were analyzed on an amaZon speed ETD ion trap mass spectrometer (Bruker

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Daltonics). Data were collected using a data-dependent method with a scanning window of

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400−1400 m/z. The averages and rolling averages were set as 5 and 2, respectively. Full-scan

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spectra were acquired in enhanced resolution mode (8,100 m/z s-1), and the top 10 most abundant

151

ions were selected for fragmentation in ultrascan mode (32,500 m/z s-1). For MS, maximum

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accumulation time was set to 50 ms and the accumulation target was 400,000, and for the

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MS/MS scans, maximum accumulation time was set to 100 ms and the accumulation target was

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500,000. The absolute and relative thresholds of precursor were set at 30,000 and 0.2%,

155

respectively, and an isolation width of 2.2 m/z was used. For tandem mass spectra the scan began

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at 100 m/z and ended at 2x the precursor mass. Exclusion was active after 2 spectra and the

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excluded spectra were released after 0.8 min. However, when the intensity of a current precursor

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was 5 times greater than that of the previous precursor, the current precursor would be

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fragmented. Each sample was injected and analyzed three times.

160 161 162

LC-MS/MS data analysis DataAnalysis 4.1 (Bruker) was used for peak list generation. The intensity threshold was

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100,000 au. The Apex algorithm (Bruker) was used to pick peaks with a peak width of 0.1 m/z

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for MS and MS/MS spectra, a signal to noise ratio of 0.1, relative intensity threshold of 0.1% of

165

the base peak and an absolute intensity threshold of 50.

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ProteinScape 3.1 (Bruker) was used for data search through Mascot 2.5.1 (Matrix Science,

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London, UK). Data were searched against a custom database containing WPTP wild-type and

168

mutant sequences as well as a list proteins including keratins, proteases, and other common

169

contaminants. Up to 2 missed cleavages were allowed for Asp-N peptides, global modification

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included carbamidomethyl Cys, and variable modifications included HexNAc on Asn and

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oxidation of Met. The precursor and fragmentation mass tolerances were both set to 0.6 Da for

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the monoisotopic mass.

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The EICs for detected and sequenced Asp-N peptides were obtained with an m/z tolerance

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of 0.5 Da. The mass spectrum was summed for major peaks in the EIC to identify the

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compound’s expected isotope pattern (i.e., correct monoisotopic m/z value and charge state). The

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peak area of the monoisotopic peak was used to calculate the relative abundance of glycosylation

177

in the glycosylated and unglycosylated moieties. The reported abundance is the average and

178

standard deviation of three replicates.

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179 180 181

WPTP structure modeling A three-dimensional (3D) structure of wild-type WPTP was constructed according to the

182

homology modeling method on SWISS-MODEL.25 The crystal structure of peroxidase from the

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palm tree Chamaerops excelsa (PDB accession: 4USC), which is 98% identical in amino acid

184

sequence to WPTP, was used as a template.26 Pymol (version 1.6 Schrödinger, LLC) was used

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for analysis of the WPTP structure and for graphical presentation. Secondary structure

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predictions of wild-type WPTP and WPTP E254G were conducted with PredictProtein, Jpred4

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and YASPIN.27-29

188 189 190

Determination of substrate specificity Substrate specificity of wild-type WPTP and WPTP E254G was studied with ABTS,

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guaiacol, o-dianisidine and o-phenylenediamine as substrates (Table 1). Optimal conditions for

192

catalysis of each substrate by wild-type WPTP and WPTP E254G were determined. The

193

optimum of pH was first resolved under pH values ranging from 2.2 - 8.0. Determination of

194

optimal concentration of buffer, substrate and H2O2 in the reaction medium was carried out

195

under a ranged from 0.01 - 0.1 mM, 0.01 - 7 mM, 0.1 - 20 mM, respectively. The changing rates

196

of absorbance at 414, 470, 420, 445 nm were measured for ABTS, guaiacol, o-dianisidine and o-

197

phenylenediamine, respectively. The extinction coefficient of each substrate was listed in Table 1.

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The substrate specificity of wild-type WPTP and WPTP E254G were determined under the

199

optimal condition of each substrate. The reactions were performed in 200 µL of citric acid-

200

Na2HPO4 buffer at 25 °C. The concentrations of wild-type WPTP and WPTP E254G in the

201

reaction buffer were 10-8 M.

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Effect of pH on enzyme activity and stability

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The pH stability of wild-type WPTP and WPTP E254G were measured. One microliter of

205

each enzyme (concentration of 10-4 M) was incubated with 99 µL of buffers with pHs ranging

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from 1 - 12 for 24 h at 4 °C. The buffers used were glycine-HCl (pH 1 - 3), citric acid-Na2HPO4

207

(pH 3 - 8), Tris-HCl (pH 8 - 9) and glycine-NaOH (pH 9 - 12). The assays were performed in

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200 µL 0.04 M citric acid-Na2HPO4 containing 0.04 mM ABTS and 1.4 mM H2O2 at 25 °C. The

209

final concentrations of wild-type WPTP and WPTP E254G in the reactions were 10-8 M. The

210

isoelectric point was predicted with ProtParam.30

211 212

Effect of temperature on enzyme activity and stability

213

The thermal stability of wild-type WPTP and WPTP E254G was studied. One microliter of

214

each enzyme (concentration of 10-4 M) was incubated with 99 µL of 10 mM Tris-HCl at a range

215

of temperatures (50 - 90 °C) for 1 h. The assays were performed as described above for the pH

216

stability study. The final concentrations of wild-type WPTP and WPTP E254G in the reactions

217

were 10-8 M. The data were analyzed according to the first-order reaction rate equation:31

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lnAt / A0=-kinact

219

where A0 and At are original enzyme activity and activity measured at moment t, respectively,

220

(1)

and kinac is inactivation constant.

221 222 223 224

Statistical analysis SPSS Statistics 23.0 (IBM, Armonk, NY) was used for the statistical analysis. Independentsample t-tests (two-tailed) were used at a significant level of 0.05 to compare differences

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between the values of residual activities of wile-type WPTP and WPTP E254G.

226 227

Results and Discussion

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Rationale for the choice of mutation and computational analyses to assess the impact of the

229

mutation

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Preliminary mass spectrometry analysis indicated that glycosylation site N256 in wild-type

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WPTP was underglycosylated (described below) compared to the native enzyme produced in

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plants.23 There is evidence, from HRP expressed in P. pastoris, that this site could be important

233

for thermostability and substrate binding; deletion of this site destabilized the enzyme and

234

increased the Km value for ABTS.18 We wanted to test the hypothesis that increasing the level of

235

glycosylation at this site would improve enzyme stability and modulate substrate binding. Two

236

residues upstream of N256 is an acidic Glu residue. Presence of an acidic residue (e.g., Asp or

237

Glu) in the -1 or -2 position of a glycosylation site is associated with underglycosylation,

238

whereas presence of a non-polar residue (e.g., Gly, Ala, Leu, Ile, Met, Phe, Tyr, or Tryp) is

239

associated with glycosylation at the glycosylation site.24 Therefore, mutation of E254 to a non-

240

polar amino acid should increase the glycosylation level of site N256. Gly was found in the -1

241

and -2 position of some glycosylated sites of native WPTP that have high glycosylation level.23

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Therefore, we mutated the Glu254 to Gly254 of WPTP to promote glycosylation.

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Computational homology modeling was used to assess potential unwanted consequences of

244

changing the amino acid sequence. The 3D structure of WPTP showed that E254 is located on

245

the loop between α-helix H and I (Fig. 3). Because E254 is not located in a restrictive secondary

246

structure, such as α-helix, it is presumed that mutation of this residue would not cause a change

247

in the secondary structure. This was confirmed with computational secondary structure

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prediction. Analysis with PredictProtein, Jpred4 and YASPIN, suggested that the E254G

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mutation itself would not change the secondary structure meaning it should not cause major

250

conformational changes to WPTP.

251 252 253

Expression and purification of wild-type WPTP and WPTP E254G Wild-type WPTP and WPTP E254G were successfully expressed in P. pastoris GS115. Ten

254

and 14 transformants of wild-type WPTP and WPTP E254G, respectively, were screened. The

255

two transformants (one of each wild-type and mutant) with the highest activity toward the

256

substrate TMB were selected for further study. Expression, driven by the AOX1 promoter, was

257

induced by methanol. After expression for 96 h, fermentation broth was collected and then

258

purified using Ni-NTA agarose followed by concentration and desalting using centrifugal filter

259

devices.

260

Following digestion with Endo H, both purified wild-type WPTP and WPTP E254G showed

261

two bands on an SDS-PAGE gel (Fig. 1). The lower band of each lane was Endo H whose

262

molecular weight is 29 kDa. The upper band in each lane was observed around 35 kDa, which is

263

slightly larger than the theoretical mass (32.2 kDa) of WPTP. The discrepancy in mass can be

264

attributed to the presence of N-acetylglucosamine (GlcNAc) which remains attached to

265

glycosylation sites after deglycosylation by Endo H.

266 267

LC-MS/MS analysis of wild-type WPTP and WPTP E254G

268

Extracted ion chromatography (EIC) was used to measure the relative abundance of

269

glycoforms in wild-type WPTP and WPTP E254G. Proteolytic digestion of Endo H digested

270

samples with AspN, which cleaves at the N-terminus of Asp, resulted in generation of a single

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271

peptide containing the glycosylation site N256 for both mutant and wild-type samples. In both

272

cases, the glycosylated version of the peptide eluted approximately 2 min before its

273

unglycosylated counterpart, which is expected for reverse phase chromatography of

274

glycopeptides. Comparison of the peak areas in the EIC of the glycosylated variant of wild-type

275

WPTP (EIC 1016.51, Fig. 2A) to the unglycosylated variant (EIC 914.97) revealed that wild-

276

type WPTP was underglycosylated at site N256 (1.5% ± 0.3%) compared to the native enzyme

277

(55% ± 10%).23

278

EIC revealed that the mutant had a 4.3-fold increase in glycosylation (6.4% ± 0.5%) relative

279

to the wild-type enzyme (1.5% ± 0.3%) (Fig. 2B). Each structure represented in the EICs was

280

verified by LC-MS/MS. Fig. 2C shows a representative fragmentation spectrum of a

281

glycopeptide containing the mutation (DAQALVTGAN*LSAAVKNNA, where * is a GlcNAc

282

residue remaining after Endo H digestion). The present work shows that mutation of acidic

283

amino acid residue at -1 or -2 position of glycosylation site could be a strategy to generate

284

proteins with a higher degree of glycosylation.

285 286 287

Substrate specificity of wild-type WPTP and WPTP E254G The substrate specificity of wild-type WPTP and WPTP E254G was analyzed with ABTS,

288

guaiacol, o-dianisidine and o-phenylenediamine as substrates under optimal conditions. The

289

conditions, including substrate concentration, H2O2 concentration, buffer concentration and

290

buffer pH, were optimized for each substrate (Table 1).

291 292 293

Peroxidases catalyze the oxidation of substrates by the “ping-pong” mechanism, in a threestep catalytic cycle:11, 32 E + H2O2 → compound I + H2O

(2)

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compound I + AH2 → compound II + AH

295

• compound II + AH2 → E + AH + H2O

•

(3) (4)

296

where E is the enzyme in resting form, and compounds I and II are the oxidized intermediates of

297

• peroxidase. AH2 and AH are the electron-donor substrate and free radical product, respectively.

298

The reduction of compound I (eq 2) is the rate-limiting step in WPTP catalysis.12 Therefore,

299

the values of the second-order rate constant (kapp) for the reaction between compound I and the

300

hydrogen donor substrate (AH2) were used to evaluate the catalytic efficiency of wild-type

301

WPTP and WPTP E254G using eq 5.12

302

rate = kapp[compound I][AH2]

303

Among the four substrates, the best substrate for both wild-type WPTP and WPTP E254G

(5)

304

was ABTS and the worst was guaiacol (Table 1). The catalytic efficiency of WPTP E254G

305

toward ABTS, guaiacol, o-dianisidine and o-phenylenediamine was 2.4, 3.0, 1.9 and 1.9-fold

306

higher than by wild-type WPTP (Table 1). The results demonstrated that the higher degree of

307

glycosylation at N256 of WPTP did not change the substrate specificity, but increased the

308

catalytic efficiency up to 3-fold toward those four substrates.

309

Glycosylation may have a direct or indirect effect on catalytic efficiency depending on the

310

distance from the active site. NMR studies of HRP with a bound substrate suggested that

311

residues within 12 Å of the heme iron could directly interact with the substrate.33 In order to

312

investigate the observed effects in the present study, we used the 3D homology model to measure

313

the distance from N256 to the active site. We found that N256 is 19.2 Å from the heme iron and

314

17.9 Å from the heme-binding site, H169. This suggests that site N256 is not likely to have a

315

direct effect on the catalytic efficiency.

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316

Glycans that are farther away may have an indirect effect by influencing the overall

317

conformation of the enzyme. For instance, cationic peanut peroxidase (cPrx) with site N60

318

glycosylated showed 1.4-fold higher activity than its non-glycosylated site-directed mutation

319

variant.17 This glycosylation site is located on the loop between α-helices B and C which is far

320

away from the active site. Glycosylation of this site was proposed to cause the flanking helices to

321

bend to a greater extent, leading to a more compactly folded active site.17, 34

322

In another example, enzymatic removal of all six N-linked glycans of Russell’s viper venom

323

factor X activator (RVV-X) (PDB accession: 2E3X), which are all distant from the active site,

324

resulted in almost total loss of the ability of RVV-X to activate factor X to factor Xa.35, 36

325

Deglycosylation resulted in changes to the secondary structure which significantly affected the

326

overall conformation of the enzyme.36 It is possible that the glycan attached to site N256 of

327

WPTP induces conformational changes that result in an enzyme with higher catalytic activity

328

than the unglycosylated variant.

329

Glycans that were beneficial for catalytic activity were also found in other enzymes.

330

Deleting the glycosylation site at N255 in HRP caused somewhat lower affinity to ABTS.18

331

N255 of HRP shares a similar position with N256 of WPTP in their 3D structures.37 Therefore,

332

the glycan attached to this specific site of WPTP may have an advantageous influence on

333

catalytic activity. The kcat and kcat/Km values of Rhizopus chinensis lipase (RCL) mutant

334

proRCLCN14Q that lost the N14 glycan were much lower than that of its wild-type,

335

proRCLCNQ, which suggests that the glycan attached to N14 of proRCLCNQ was beneficial for

336

its catalytic activity.38

337

Analysis of the homology model of WPTP as well as secondary structure prediction, as

338

previously mentioned suggest that the increased activity was due to the increased glycosylation

16


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339

level of site N256 of WPTP as opposed to the mutation itself. Resolving the crystal structure of

340

wild-type WPTP and WPTP E254G could help to better explain the observed effect of the

341

mutation and glycosylation on catalytic activity observed in this study.

342 343 344

Effect of pH on enzyme activity and stability Peroxidases are useful in various applications that demand acidic conditions, such as

345

synthesis of semiconductive polyaniline, and alkaline conditions such as some wastewater

346

treatment environments. To monitor irreversible inactivation due to buffer pH, the wild-type and

347

mutant enzymes were incubated in buffers with pH values varying from pH 1 to pH 12 for 24 h,

348

and then tested for residual activity.

349

The optimal pH range for both enzymes was the same; however, WPTP E254G had higher

350

activity in all buffers from pH 4 to pH 12 (Fig. 4). Wild-type WPTP was most stable at pH 7,

351

whereas WPTP E254G was most stable at pH 9. Both enzymes showed minimal activity from

352

pH 1 to pH 3. WPTP E254G maintained 66% of the maximal activity when it was stored at pH 5.

353

However, it showed higher stability under basic conditions, retaining as high as 80% of maximal

354

activity after it was stored at pH 12 for 24 h. The enhanced pH stability of WPTP E254G

355

compared to wild-type WPTP makes WPTP E254G more applicable in alkaline conditions, such

356

as alkali wastewater treatment.

357

Like WPTP, other palm tree peroxidases are maximally stable under neutral to slightly

358

alkaline conditions and tend to have a wide range of pH values in which they are stable. Royal

359

palm tree peroxidase was most stable at pH 8 and maintained above 75% of its maximal activity

360

from pH 4 – 10 under the experimental conditions in that study.10 African oil palm tree

361

peroxidase (AOPTP) has high stability from pH 2 to pH 12 at 25 °C, and maintained high

17



362

stability from pH 5 to pH 10 when the temperature increased up to 70 °C. The most stable pH

363

condition for AOPTP shifted from pH 7 to pH 8 when the temperature changed from 25 °C to

364

70 °C.31 In contrast, HRP is stable in a narrower range, from pH 4 - 8.39

365

In other cases, glycosylation was important for pH stability. When compared with wild-type

366

xylanase (Af-XYNA), its mutant N124T exhibited a narrower pH adaptation range and worse pH

367

stability.40 It is suggested that glycans attached to N124 play an important role on its pH

368

adaptation and pH stability although this site is far away from the active site. Glycans that

369

contributed to stability under alkaline conditions were found in human mast cell chymase. The

370

deglycosylated chymase was less stable than its wild-type counterpart from pH 9 to 10.41

371

The results indicated that the higher degree of glycosylation at site N256 may have

372

contributed to the observed stability under basic conditions compared with wild-type WPTP

373

which was most stable under neutral conditions. However, it is likely that the mutation itself of

374

Glu to Gly could have an impact on the observed results. The E254G mutation will cause a

375

change in the net charge relative to wild-type WPTP. The E254G mutation increases the

376

theoretical isoelectric point of wild-type WPTP from 5.54 to 5.79 of WPTP E254G. Therefore,

377

the observed shift of the most stable pH from 7 of wild-type WPTP to 9 of WPTP E254G might

378

be attributed to both increased glycosylation level and E254G mutation.

379 380 381

Effect of temperature on enzyme activity and stability The effects of temperature on wild-type WPTP and WPTP E254G were determined from 50

382

to 90 °C. The inactivation constants (kinac) of wild-type WPTP at 50, 60, 70, 80 and 90 °C were

383

0.052, 0.030, 0.014, 0.0006 and 0.0002, respectively, while the respective kinac of WPTP E254G

384

were 0.062, 0.034, 0.011, 0.0007 and 0.0003. These kinac values indicated no apparent difference

18


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Page 19 of 31


385

in thermal stability between wild-type WPTP and WPTP E254G. However, WPTP E254G had

386

1.2 - 2.5-fold higher catalytic activity than wild-type WPTP in the temperature range of 50-80 °C,

387

which the differences were statistically significant (p < 0.05) (Fig. 5). WPTP E254G retained

388

above 50% of its original activity under 50 °C - 70 °C, and declined to 13% after being stored at

389

80 °C for 1 h (Fig. 5).

390

Glyco-engineering has been useful in other studies to improve the thermostability of

391

industrial enzymes.41, 42 Addition of a glycosylation site in fungal cutinase heterologously

392

expressed in P. pastoris improved the thermal stability.42 It was thought that loss of enzyme

393

activity was due to aggregation upon unfolding at high temperature. One site on barley α-

394

glucosidase resulted in an increase in thermal stability; however, addition at another site did not

395

show any improvements.43 In both of those studies, the chosen site for addition of a

396

glycosylation site was based on homology to orthologous enzymes.

397

In some cases, glycosylation of a single site had effects on different properties.

398

Glycosylation at N255 of HRP was not only related to catalytic activity, but also had a notable

399

influence on enzyme thermal stability. Glyco-variant N255D of HRP produced in P. pastoris had

400

a significant decrease in thermal stability.18 Glycosylation of N124 of Af-XYNA played an

401

important role on both pH and thermal stability. When Af-XYNA and its counterpart mutant

402

N124T were incubated at 70 °C for 1 h, N124T had much lower activity than wild type. They

403

found that a higher degree of glycosylation resulted in higher thermal stability.40

404

The results obtained in this study indicated that the degree of glycosylation influenced

405

catalytic activity, as well as activity under stringent pH and temperature conditions. The study

406

implies a strategy to generate desirable proteins with a higher degree of glycosylation by glyco-

407

engineering. The most stable pH condition of WPTP E254G differing from wild-type WPTP

19



408

provides potential broader application of WPTP in alkaline wastewater treatment.

409 410

ABBREVIATIONS

411

WPTP, windmill palm tree peroxidase; ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate

412

acid); HRP, horseradish peroxidase; YPDS, yeast extract peptone dextrose medium; BMMY,

413

buffered methanol-complex medium; BMGY, buffered glycerol-complex medium; TMB, 3, 3ʹ, 5,

414

5ʹ-tetramethylbenzidine; Endo H, Endoglycosidase H; LC-MS, liquid chromatography-mass

415

spectrometry; GlcNAc, N-acetylglucosamine; EIC, extracted ion chromatography; SDS-PAGE,

416

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

417 418

ACKNOWLEDGMENT

419

We are grateful to Dr. Ivan Yu. Sakharov of Lomonosov Moscow State University for helpful

420

suggestion and discussion.

421

FUNDING

422

This work was supported by the National Institutes of Health Research Centers in Minority

423

Institutions Program grant no. G12 MD007601 and by the USDA National Institute of Food and

424

Agriculture Hatch project HAW5032-R,managed by the College of Tropical Agriculture and

425

Human Resources, University of Hawaii at Manoa and grant no. 2018-67012-28082.

426

NOTES

427

The authors declare no conflict of interest.

20


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428

References

429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470

1. Cooper, V. A.; Nicell, J. A. Removal of phenols from a foundry wastewater using horseradish peroxidase. Water Res. 1996, 30, 954-964. 2. Gorton, L.; Lindgren, A.; Larsson, T.; Munteanu, F. D.; Ruzgas, T.; Gazaryan, I. Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors. Anal. Chim. Acta 1999, 400, 91-108. 3. Sakharov, I. Y. Long-term chemiluminescent signal is produced in the course of luminol peroxidation catalyzed by peroxidase isolated from leaves of african oil palm tree. Biochemistry (Moscow) 2001, 66, 515-519. 4. Caramyshev, A. V.; Evtushenko, E. G.; Ivanov, V. F.; Barcelo, A. R.; Roig, M. G.; Shnyrov, V. L.; van Huystee, R. B.; Kurochkin, I. N.; Vorobiev, A.; Sakharov, I. Y. Synthesis of conducting polyelectrolyte complexes of polyaniline and poly(2-acrylamido-3-methyl-1propanesulfonic acid) catalyzed by pH-stable palm tree peroxidase. Biomacromolecules 2005, 6, 1360-1366. 5. Kawakita, H.; Hamamoto, K.; Ohto, K.; Inoue, K. Polyphenol polymerization by horseradish peroxidase for metal adsorption studies. Ind. Eng. Chem. Res. 2009, 48, 4440-4444. 6. Zhang, H.; Lu, Y.; Ushio, H.; Shiomi, K. Development of sandwich ELISA for detection and quantification of invertebrate major allergen tropomyosin by a monoclonal antibody. Food Chem. 2014, 150, 151-157. 7. Ong, A. S.; Goh, S. H. Palm oil: a healthful and cost-effective dietary component. Food Nutr. Bull 2002, 23, 11-22. 8. Schroeder, M. T.; Becker, E. M.; Skibsted, L. H. Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil. J. Agric. Food Chem. 2006, 54, 34453453. 9. Zhang, L.; Wu, G.; Wu, Y.; Cao, Y.; Xiao, L.; Lu, C. The gene MT3-B can differentiate palm oil from other oil samples. J. Agric. Food Chem. 2009, 57, 7227-7232. 10. Sakharov, I. Y.; Vesgac B, M. K.; Galaev, I. Y.; Sakharova, I. V.; Pletjushkina, O. Y. Peroxidase from leaves of royal palm tree Roystonea regia: purification and some properties. Plant Sci. 2001, 161, 853-860. 11. Sakharov, I. Y.; Vesga Blanco, M. K.; Sakharova, I. V. Substrate specificity of african oil palm tree peroxidase. Biochemistry (Mosc) 2002, 67, 1043-1047. 12. Caramyshev, A. V.; Firsova, Y. N.; Slastya, E. A.; Tagaev, A. A.; Potapenko, N. V.; Lobakova, E. S.; Pletjushkina, O. Y.; Sakharov, I. Y. Purification and characterization of windmill palm tree (Trachycarpus fortunei) peroxidase. J. Agric. Food Chem. 2006, 54, 9888-9894. 13. Nagai, K.; Ihara, Y.; Wada, Y.; Taniguchi, N. N-Glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology 1997, 7, 769-776. 14. Guo, S.; Skala, W.; Magdolen, V.; Briza, P.; Biniossek, M. L.; Schilling, O.; Kellermann, J.; Brandstetter, H.; Goettig, P. A Single glycan at the 99-loop of human kallikrein-related peptidase 2 regulates activation and enzymatic activity. J. Biol. Chem. 2016, 291, 593-604. 15. Wormald, M. R.; Dwek, R. A. Glycoproteins: glycan presentation and protein-fold stability. Structure 1999, 7, R155-R160.

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16. Baker, M. R.; Zhao, H.; Sakharov, I. Y.; Li, Q. X. Amino acid sequence of anionic peroxidase from the windmill palm tree Trachycarpus fortunei. J. Agric. Food Chem. 2014, 62, 11941-11948. 17. Lige, B.; Ma, S.; van Huystee, R. B. The effects of the site-directed removal of Nglycosylation from cationic peanut peroxidase on its function. Arch. Biochem. Biophys. 2001, 386, 17-24. 18. Capone, S.; Pletzenauer, R.; Maresch, D.; Metzger, K.; Altmann, F.; Herwig, C.; Spadiut, O. Glyco-variant library of the versatile enzyme horseradish peroxidase. Glycobiology 2014, 24, 852-863. 19. Dotsenko, A. S.; Gusakov, A. V.; Volkov, P. V.; Rozhkova, A. M.; Sinitsyn, A. P. N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity. Biotechnol. Bioeng. 2016, 113, 283-291. 20. Rudd, P. M.; Joao, H. C.; Coghill, E.; Fiten, P.; Saunders, M. R.; Opdenakker, G.; Dwek, R. A. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 1994, 33, 17-22. 21. Culyba, E. K.; Price, J. L.; Hanson, S. R.; Dhar, A.; Wong, C. H.; Gruebele, M.; Powers, E. T.; Kelly, J. W. Protein native-state stabilization by placing aromatic side chains in Nglycosylated reverse turns. Science 2011, 331, 571-575. 22. Wen, B.; Baker, M. R.; Zhao, H.; Cui, Z.; Li, Q. X. Expression and characterization of windmill palm tree (Trachycarpus fortunei) peroxidase by Pichia pastoris. J. Agric. Food Chem. 2017, 65, 4676-4682. 23. Baker, M. R.; Tabb, D. L.; Ching, T.; Zimmerman, L. J.; Sakharov, I. Y.; Li, Q. X. Sitespecific N-glycosylation characterization of windmill palm tree peroxidase using novel tools for analysis of plant glycopeptide mass spectrometry data. J. Proteome Res. 2016, 15, 20262038. 24. Petrescu, A. J.; Milac, A. L.; Petrescu, S. M.; Dwek, R. A.; Wormald, M. R. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology 2004, 14, 103-114. 25. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: a webbased environment for protein structure homology modelling. Bioinformatics 2006, 22, 195201. 26. Bernardes, A.; Textor, L. C.; Santos, J. C.; Cuadrado, N. H.; Kostetsky, E. Y.; Roig, M. G.; Bavro, V. N.; Muniz, J. R.; Shnyrov, V. L.; Polikarpov, I. Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa. Biochimie 2015, 111, 58-69. 27. Yachdav, G.; Kloppmann, E.; Kajan, L.; Hecht, M.; Goldberg, T.; Hamp, T.; Honigschmid, P.; Schafferhans, A.; Roos, M.; Bernhofer, M.; Richter, L.; Ashkenazy, H.; Punta, M.; Schlessinger, A.; Bromberg, Y.; Schneider, R.; Vriend, G.; Sander, C.; Ben-Tal, N.; Rost, B. PredictProtein--an open resource for online prediction of protein structural and functional features. Nucleic Acids Res. 2014, 42, W337-W343. 28. Drozdetskiy, A.; Cole, C.; Procter, J.; Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 2015, 43, W389-W394. 29. Lin, K.; Simossis, V. A.; Taylor, W. R.; Heringa, J. A simple and fast secondary structure prediction method using hidden neural networks. Bioinformatics 2005, 21, 152-159. 30. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein identification and analysis tools on the ExPASy server. John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press 2005, 571-607.

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31.Sakharov, I. Y.; Sakharova, I. V. Extremely high stability of African oil palm tree peroxidase. Biochim. Biophys. Acta. 2002, 1598, 108-114. 32. Cuadrado, N. H.; Arellano, J. B.; Calvete, J. J.; Sanz, L.; Zhadan, G. G.; Polikarpov, I.; Bursakov, S.; Roig, M. G.; Shnyrov, V. L. Substrate specificity of the Chamaerops excelsa palm tree peroxidase. A steady-state kinetic study. J. Mol. Catal. B: Enzym. 2012, 74, 103108. 33. Veitch, N. C. Aromatic donor molecule binding sites of haem peroxidases. Biochem. Soc. Trans. 1995, 23, 232-240. 34.Imperiali, B.; Rickert, K. W. Conformational implications of asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA 1995, 92, 97-101. 35. Takeda, S.; Igarashi, T.; Mori, H. Crystal structure of RVV-X: an example of evolutionary gain of specificity by ADAM proteinases. FEBS Lett. 2007, 581, 5859-5864. 36. Gowda, D. C.; Jackson, C. M.; Kurzban, G. P.; McPhie, P.; Davidson, E. A. Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure. Biochemistry 1996, 35, 5833-5837. 37. Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szoke, H.; Henriksen, A.; Hajdu, J. The catalytic pathway of horseradish peroxidase at high resolution. Nature 2002, 417, 463-468. 38. Yang, M.; Yu, X. W.; Zheng, H.; Sha, C.; Zhao, C.; Qian, M.; Xu, Y. Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris. Microb. Cell Fact. 2015, 14, 40. 39. Ryan, B. J.; Carolan, N.; O'Fagain, C. Horseradish and soybean peroxidases: comparable tools for alternative niches? Trends Biotechnol. 2006, 24, 355-363. 40. Chang, X.; Xu, B.; Bai, Y.; Luo, H.; Ma, R.; Shi, P.; Yao, B. Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris. PLoS One 2017, 12, e0171111. 41. Takao, K.; Takai, S.; Shiota, N.; Song, K.; Nishimura, K.; Ishihara, T.; Miyazaki, M. Lack of effect of carbohydrate depletion on some properties of human mast cell chymase. BBA- Gen. Subjects 1999, 1427, 74-81. 42. Shirke, A. N.; Su, A.; Jones, J. A.; Butterfoss, G. L.; Koffas, M. A.; Kim, J. R.; Gross, R. A. Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation. Biotechnol. Bioeng. 2017, 114, 63-73. 43. Clark, S. E.; Muslin, E. H.; Henson, C. A. Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley alpha-glucosidase. Protein Eng. Des. Sel. 2004, 17, 245-249.

23



FIGURE CAPTIONS Figure 1. SDS-PAGE analysis of purified deglycosylated wild-type WPTP and WPTP E254G. Molecular weight protein markers from top to bottom are 250, 150, 100, 75, 50, 37, 25 and 20 kDa. A shows deglycosylated wild-type WPTP; B shows deglycosylated WPTP E254G; C shows glycosidase endoglycosidase H (Endo H). Figure 2. Extracted ion chromatograms of (A) m/z 914.97 and m/z 1016.51 of the unglycosylated and glycosylated peptide, respectively, containing glycosylation site N256 on wild-type WPTP and (B) m/z 878.96 and m/z 980.50 of the unglycosylated and glycosylated peptide, respectively, containing glycosylation site N256 on WPTP E254G. (C) Fragmentation spectrum of parent ion at m/z 980.94 (+2) was interpreted to be the peptide (DAQALVTGAN*LSAAVKNNA, where * is a GlcNAc residue remaining after EndoH digestion) from an AspN digest of WPTP E254G. The glycopeptide fragmented into b- (red), y- (blue), and b-17- (pink) ions. The parent ion, after neutral loss of water (-18 Da), was also observed (light blue). Figure 3. Position of amino acid residue Glu254 of wild-type WPTP (A) and Gly254 of WPTP E254G (B) on the protein structure. The α-helices are in cyan. Side chains of Glu254 and Asn256 are in sticks, where atom C, O and N are in cyan, red and blue, respectively. The main chain of Gly254 is in magenta. Figure 4. Catalytic efficiency of wild-type WPTP and WPTP E254G after storage in buffers with pHs ranging from 1 - 12 for 24 h. Figure 5. Catalytic efficiency of wild-type WPTP and WPTP E254G after incubation at different temperatures for 1 h. Values with different lower case letters were significantly different (p < 0.05).

24


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Figure 1. SDS-PAGE analysis of purified deglycosylated wild-type WPTP and WPTP E254G. Molecular weight protein markers from top to bottom are 250, 150, 100, 75, 50, 37, 25 and 20 kDa. A shows deglycosylated wild-type WPTP; B shows deglycosylated WPTP E254G; C shows glycosidase endoglycosidase H (Endo H).

25



Figure 2. Extracted ion chromatograms (EIC) of (A) m/z 914.97 and m/z 1016.51 of the unglycosylated and glycosylated peptide, respectively, containing glycosylation site N256 on wild-type WPTP and (B) m/z 878.96 and m/z 980.50 of the unglycosylated and glycosylated peptide, respectively, containing glycosylation site N256 on WPTP E254G. (C) Fragmentation spectrum of parent ion at m/z 980.94 (+2) was interpreted to be the peptide (DAQALVTGAN*LSAAVKNNA, where * is a GlcNAc residue remaining after EndoH digestion) from an AspN digest of WPTP E254G. The glycopeptide fragmented into b- (red), y(blue), and b-17- (pink) ions. The parent ion, after neutral loss of water (-18 Da), was also observed (light blue).

26


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Page 27 of 31


Figure 3. Position of amino acid residue Glu254 of wild-type WPTP (A) and Gly254 of WPTP E254G (B) on the protein structure. The α-helices are in cyan. Side chains of Glu254 and Asn256 are in sticks, where atom C, O and N are in cyan, red and blue, respectively. The main chain of Gly254 is in magenta.

27



Figure 4. Catalytic efficiency of wild-type WPTP and WPTP E254G after storage in buffers with pHs ranging from 1 - 12 for 24 h.

28


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Page 29 of 31


Figure 5. Catalytic efficiency of wild-type WPTP and WPTP E254G after incubation at different temperatures for 1 h. Values with different lower case letters were significantly different (p < 0.05).

29



Page 30 of 31

Table 1. Optimal conditions for substrate specificity determinations and comparison of substrate specificity (kapp) between wild-type WPTP and WPTP E254G substrate (AH2)

λ, nm

߳, M-1 cm-1

[AH2], mM

[H2O2], mM

pH

kapp (M-1 s-1) [buffer]2, M wild-type WPTP WPTP-E254G

ABTS1

414

31100

0.04

1.4

2.4

0.04

1.3×104

3.1×104

guaiacol

470

5200

5

2

5.2

0.1

2.7×102

8.0×102

o-dianisidine

420

30000

0.1

4.5

5

0.09

3.3×103

6.2×103

o-phenylenediamine 445

11100

0.7

0.8

5.4

0.09

5.1×102

9.8×102

1 2

ABTS: 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) Citrate-Na2HPO4 buffer was used.

30


Page 31 of 31


TABLE OF CONTENT GRAPHIC

31


Effect of N-Linked Glycosylation of Recombinant Windmill Palm Tree

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