Efficient Expression of an Acidic Endo-polygalacturonase from

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Efficient expression of an acidic endo-polygalacturonase from Aspergillus niger and its application in juice production Jiaojiao Wang, Yuhong Zhang, Xing Qin, Lingyu Gao, Bin Han, Deqing Zhang, Jinyang Li, He Huang, and Wei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05109 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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Journal of Agricultural and Food Chemistry

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Efficient expression of an acidic endo-polygalacturonase from Aspergillus niger and its

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application in juice production

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Jiaojiao Wanga, b, #, Yuhong Zhanga #, Xing Qina, Lingyu Gaoc, Bin Hanc, Deqing Zhanga,

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Jinyang Lia, He Huangb *, Wei Zhanga *

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a

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100081, China

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b

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Technology, Nanjing 211816, China

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing

College of Biotechnology and Pharmaceutical Engineering, Nanjing University of

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c

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100093, China

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing

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#

These authors contributed equally to this work.

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*Corresponding author:

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* Wei Zhang

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Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12

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Zhongguancun South Street, Beijing 100081, China

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Fax: +86-10-82106141

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E-mail: [email protected]

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ACS Paragon Plus Environment


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*He Huang

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College of Biotechnology and Pharmaceutical Engineering, Nanjing University of

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Technology, No.30 South Puzhu Road, Nanjing 211816, China

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Fax: +86-25-58139942

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E-mail: [email protected]

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Running title: Expression of an acidic polygalacturonase from Aspergillus niger ZJ5

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ABSTRACT

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An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs

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synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was

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1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was

34

successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the

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pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris

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GS115 for heterologous expression. The polygalacturonase showed a maximum activity level

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of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic

38

properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were

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4.5 and 40°C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the

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volume of pear juice by 41.8% and improved its light transmittance three-fold.

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KEY WORDS: endo-polygalacturonase; Aspergillus niger; Pichia pastoris; juice production

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INTRODUCTION Pectin,

45

which

is

mainly

degraded

by

pectolytic

enzymes,

is

a

natural

46

high-molecular-weight complex heteropolysaccharide and a cell wall component. It is mainly

47

localized in plant cell walls and fruit lamella, consists largely of linear chains of α-(1,4)

48

glycosidic linked D-galacturonic acid residues and is partially esterified with methyl groups1.

49

Pectin is associated with many problems in the food industry, feed industry and textile

50

industry, such as the high viscosity of fruit juice. The addition of exogenous pectinases can

51

resolve these problems2.

52

Pectinases catalyze the hydrolysis of pectin substances and are generally divided into

53

several groups: pectate lyase (EC 4.2.2.2) and pectin lyase (EC 4.2.2.10), which work by the

54

mechanism of β-elimination; pectin methyl esterases (EC 3.1.1.11), which remove methoxyl

55

groups3; and exo-polygalacturonase (EC 3.2.1.67) and endo-polygalacturonase (EC 3.2.2.15),

56

which cleave α-(1–4)-linked glycosidic bonds between two non-esterified galacturonic acid

57

units

58

(exo-polygalacturonase)4. Among the pectic enzymes, polygalacturonases are the most

59

extensively studied and are commonly classified into family 28 of the CAZy

60

(Carbohydrate-Active enZYmes Database) glycosyl hydrolases based on sequence

61

similarity5-7.

in

either

a

random

(endo-polygalacturonase)

or

a

terminal

fashion

62

Pectinases have many applications in various industries, such as pectin treatment in the

63

food industry8, wastewater treatment in the paper and pulp industry, natural fiber treatment in

64

the textile industry2. In fact, microbial pectinases account for a considerable proportion of

65

global food enzyme sales. Almost all pectinases are produced from fungal sources, mainly

66

polygalacturonase from Aspergillus niger4. Biochemical and thermal characterizations of

67

polygalacturonases produced by fungi or bacteria have also been reported9, 10. However, most

68

scientific research has focused on polygalacturonases that have an optimal pH that is alkaline 4


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or neutral, but that lose stability under acidic conditions. For example, PGI from A. niger

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NRRL311, PelB from hyperthermophilic Thermotoga maritime12 and from a Bacillus isolate13

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had close to alkaline pH optima. Acidic pectinase has applications in the fruit juices industry,

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but there has been limit research on one or several polygalacturonase compounds with acidic

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pH optima, warranting efforts to discover new polygalacturonases with acidic properties.

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Against this background, an acidic endo-polygalacturonase gene, pga-zj5a, was cloned

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from the pectin-degrading strain A. niger ZJ5. We found it has a high expression level in

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Pichia pastoris GS115. Therefore the properties of its expression product, PGA-ZJ5A, were

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studied in the present study. Its high specific activity and stability over a wide pH range make

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PGA-ZJ5A potentially effective in juice clarification without pH adjustment. Its combination

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with other pectinases resulted in a higher juice clarification efficiency.

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MATERIALS AND METHODS

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Strains, plasmids and reagents. A. niger ZJ5 was isolated from a sample of forest soil

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from Yunnan, China. The ZJ5 strain has high ability to degrade pectin14. The plasmid pPIC9

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was used to construct the expression vector for heterologous expression in P. pastoris GS115

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cells (Invitrogen, Carlsbad, CA, USA). Fastpfu DNA polymerase, Escherichia coli Trans1-T1,

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and pEASY-Blunt simple vector (TransGen, China), T4-DNA ligase (New England Biolabs,

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MA, USA), endo-β-N-acetyl glucosaminidase H (Endo-H, New England Biolabs) and the

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restriction endonucleases (Fermentas, Vilnius, Lithuania) were obtained from commercial

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sources. Polygalacturonic acid from oranges (P3889), D-(+)-galacturonic acid, standard

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oligo-galacturonates, citrus pectin with 34% DE (degree of esterification), citrus pectin with

91

70% DE and citrus pectin with 85% DE were purchased from Sigma-Aldrich (San Diego, CA,

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USA). In accordance with the manual of the Pichia Expression Kit (Invitrogen, Carlsbad, CA,

93

USA), regeneration dextrose medium (RDB), minimal dextrose medium (MD), buffered 5



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94

glycerol complex medium (BMGY) and buffered methanol complex medium (BMMY) were

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prepared. All other chemicals used in this study were of analytical grade and commercially

96

available.

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Sequence analysis. Nucleotide sequence analysis, protein molecular mass and pI value

98

prediction were carried out by Vector NTI 10.0 software. BLAST search was performed at the

99

NCBI (National Center for Biotechnology Information) website. The signal peptide was

100

predicted using SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/)15. Potential N-

101

glycosylation sites were predicted based on the analysis of NetNGlyc 1.0 Server online

102

(http://www.cbs.dtu.dk/services/NetNGlyc/). Potential O-glycosylation sites were predicted

103

based

104

(http://www.cbs.dtu.dk/services/NetOGlyc/)16.

on

the

analysis

of

NetOGlyc

4.0

Server

online

105 106

Total RNA isolation, cDNA synthesis and PCR amplification of pga-zj5a. The A.

107

niger ZJ5 was grown in an pectinase-inducing medium containing 20 g/L pectin, 20 g/L

108

(NH4)2SO4, 1.0 g/L tryptone, 3.8 g/L KH2PO4, 3.3 g/L K2HPO4, 3.0 g/L NaNO3, 0.5 g/L KCl,

109

0.15 g/L CaCl2, 0.24 g/L MgSO4 and 0.01 g/L FeSO4. The medium was adjusted to pH 6.0,

110

then autoclaved at 121°C for 15 minutes. After incubation at 30°C for 48 hours, mycelia of A.

111

niger ZJ5 were frozen in liquid nitrogen and then powdered by grinding. Total RNA was

112

extracted using TRIzol (Invitrogen, Carlsbad, CA, USA), and cDNA was produced by reverse

113

transcription polymerase chain reaction. To amplify the pectinase gene, the primers were

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designed according to the genome sequence of Aspergillus kawachii IFO 430817, which was

115

very similar to the ZJ5 strain in microbial taxonomic status14. The pga-zj5a cDNA was cloned

116

using the specific primers A1 (5′-CGGAATTCGCTCCCGCTCCTTCTC-3′), containing an

117

EcoR

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(5′-ATAAGAATGCGGCCGCTTAGTGGTGGTGGTGGTGGTGGCAAGAAGCACTGG-3′

I

recognition

site

(underlined),

6


and

A2

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), containing a Not I recognition site (underlined) and a His-tag coding sequence. The PCR

120

parameters were as follows: denaturation at 95°C for 4 min; 30 cycles of 20 s at 94°C, 20 s at

121

55°C and 45 s at 72°C; followed by 10 min at 72°C. The resulting PCR product was purified

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and ligated into the pEASY-Blunt simple vector for sequencing. The obtained plasmid was

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named pET1S-PGA-ZJ5A.

124 125

Construction of the expression plasmid. The obtained plasmid, pET1S-PGA-ZJ5A,

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was digested with EcoR I and Not I and then ligated into the pPIC9 vector. The recombinant

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plasmid pPIC9-PGA-ZJ5A was transformed into Escherichia coli Trans1-T1, followed by

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DNA sequencing. A large amount of pPIC9-PGA-ZJ5A plasmid was obtained using the

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TIAN Prep Mini Plasmid Kit (Tiangen Biotech, Beijing, China).

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Expression of pga-zj5a in P. pastoris GS115. The pPIC9-PGA-ZJ5A plasmid was

132

linearized by Sal I and transformed into P. pastoris strain GS115 competent cells using an

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electroporator (Bio-Rad, Hercules, CA, USA), in accordance with the electroporation protocol.

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Transformants were cultured on RDB and grown for 48 h at 30°C. Positive transformants

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were screened based on polygalacturonase activity. These colonies were then transferred to 10

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mL BMGY medium and grown at 30°C for 48 h. The cells were collected by centrifugation

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and then cultured in 5 mL BMMY medium containing methanol. After 48 h induction, the

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culture supernatant was collected by centrifugation (12,000×g, 4°C, 10 min) for use in a

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polygalacturonase activity assay. The positive transformant exhibiting the highest

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polygalacturonase activity was selected for fed-batch fermentation in a 3 L fermenter. The

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entire procedure was carried out in accordance with the Invitrogen Pichia Expression Kit

142

manual.

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Aliquots of culture supernatant (10 µL) obtained at different fermentation times were 7



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subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The

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stacking and separating gels contained 5% and 12% polyacrylamide, respectively. Proteins

146

were visualized by Coomassie Brilliant Blue G-250 staining.

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Purification and analysis of recombinant PGA-ZJ5A. To purify the recombinant

149

PGA-ZJ5A, the induced culture supernatant was centrifuged at 8000×g for 20 min at 4°C to

150

remove cell debris and undissolved materials. The crude enzyme obtained after centrifugation

151

was applied to a Vivaflow 200 ultrafiltration membrane with a 10-kDa molecular weight

152

cut-off (Sartorius, Göttingen, Germany). The clear supernatant was purified on a His-Trap

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Sepharose XL 5 mL fast protein liquid chromatography column (GE Healthcare, CT, USA),

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pre-equilibrated with NTA0 buffer (20 mM Tris-HCl, pH 6.0, 0.5 M NaCl, 10% glycerol),

155

and eluted using a linear gradient of imidazole (0.0 – 0.5 M) in NTA0 buffer at a flow rate of

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4.0 mL/min. All purification steps were carried out at 4°C. Fractions showing

157

polygalacturonase activity were collected, and their purity was determined by SDS-PAGE. To

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determine the protein concentration, a Bradford assay kit (Bio-Rad) was used with bovine

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serine albumin as the standard.

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To remove N-glycosylation, purified recombinant PGA-ZJ5A was treated with 25 U/µL

161

Endo-H for 1 h at 37°C in accordance with the supplier’s instructions and then analyzed by

162

SDS-PAGE.

163

To identify the purified protein, the corresponding band was cut from the gel, digested

164

with trypsin and then analyzed by liquid chromatography/mass spectrometry. The conditions

165

are as follows. Instrument: HPLC-ESI-Q-Exactive (Thermo Fisher Scientific, Bremen,

166

Germany) and Easy-nLC 1000 (Thermo Fisher Scientific). Mobile phase: A. 0.1% Formic

167

acid in water; B: 0.1% Formic acid in Acetonitrile. Flow rate: 300 nL/min. Elution gradient:

168

from 3 to 8% B in 1 min, from 8 to 40% B in 5 min, from 40 to 85% B in 1 min and 85% B 8


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for 1 min. Mass spectrometry: Resolution, 70,000; Scan range, 350–1600.

170

Enzyme activity assay. PGA-ZJ5A activity was assayed by measuring the formation of

171

galacturonic acid using the 3,5-dinitrosalicylic acid (DNS) method18. The reaction system

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contained 450 µL 0.1% (w/v) polygalacturonic acid in 0.2 M Na2HPO4-NaH2PO4 buffer (pH

173

4.5) and 50 µL sample at 40°C for 10 min. The reaction was terminated by adding 750 µL

174

DNS, boiled for 5 min and cooled to room temperature. The absorption at 540 nm was

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measured. One unit (U) endo-polygalacturonase activity was defined as the amount of

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enzyme that released 1 µmol galacturonic acid from substrate per min under the above

177

conditions (pH 4.5, 40°C, 10 min). For every reaction, triplicate measurements were

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conducted and the mean value calculated.

179 180

Biochemical characterization of the purified recombinant PGA-ZJ5A enzyme.

181

Polygalacturonic acid was used as the substrate for the biochemical characterization of

182

purified recombinant PGA-ZJ5A. The optimal pH was determined at 40°C in 0.2 M

183

Na2HPO4-NaH2PO4 buffer within a pH range from 2.5 to 9.0. The enzyme stability at these

184

different pHs was determined by measuring the residual enzymatic activity under standard

185

conditions (pH 4.5, 40°C and 10 min) after pre-incubation at 37°C for 1 h.

186

The optimal temperature for PGA-ZJ5A activity was measured at temperatures from 15

187

to 60°C. Thermal stability was determined by assessing the residual activity under standard

188

conditions after incubation of the enzyme at 40, 45, 50, or 55°C for various durations.

189

To study the effects of chemical reagents and metal ions on the activity of purified

190

PGA-ZJ5A,

different

chemical

reagents

[sodium

dodecyl

191

trimethylammonium bromide (CTAB), or ethylenediaminetetraacetic acid disodium (EDTA)]

192

and metal ions [KCl, NaCl, CaCl2, CoCl2, NiSO4, MgCl2, MnCl2, ZnSO4, FeSO4,

193

Pb(CH3COO)2 and CdSO4,] were added to the reaction system to a final concentration of 1 or 9


sulfate

(SDS),

cetyl


194

10 mM. The residual enzyme activity was determined under the standard assay conditions.

195

The enzyme activity in the absence of reagent was set as 100%.

196 197

Substrate specificity and kinetic analysis. The substrate specificity of recombinant

198

PGA-ZJ5A was obtained at 40°C for 10 min in 0.2 M Na2HPO4-NaH2PO4 buffer (pH 4.5) by

199

measuring the enzyme activity towards polygalacturonic acid under standard conditions. The

200

Km and Vmax of PGA-ZJ5A were determined at different concentrations of substrate (0.02% to

201

2%). The enzyme assays were performed at various substrate concentrations for 10 min at

202

40°C in 0.2 M sodium phosphate buffer (pH 4.5). The kinetic parameters of PGA-ZJ5A were

203

determined using GraphPad Prism 5.01 Software.

204 205 206

Nucleotide sequence accession number. The nucleotide sequence for the pga-zj5a gene was deposited in the GenBank database under accession no. KU896780.

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Application of PGA-ZJ5A in the clarification of pear juice. Pear juice was extracted

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from Pyrus bretschneideri Rehder ‘Xuehua’ pears with 0.5% (w/v) ascorbic acid, followed by

210

filtration through eight layers of gauze (League, Beijing, China) to filter out the residue. The

211

pH and density of pear juice were 4.8 and 0.85 g/mL, respectively. In accordance with

212

previous experiments, PGA-ZJ5A was incubated in 50 mL pear juice at 40°C for 60 min.

213

Juice containing the same amount of inactive enzyme was used as a control.

214

To verify the application effect of PGA-ZJ5A, pear juice was treated with various

215

pectinases. PNL-ZJ5A, a pectin lyase from A. niger19, was used at 1 U/mL juice, and

216

PGA-ZJ5A was used at 5 U/mL juice. These pectinases in various combinations were added

217

to 50 mL pear juice and incubated at 40°C for 120 min. The pear juice was then filtered

218

through one layer of filter paper #4 (Whatman, Little Chalfont, UK), and the filtrate volume 10


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produced after 2 min was measured20. The light transmittance at 600 nm of the juice

220

supernatant was determined, and the viscosity was assessed using an SNB viscometer (NiRun,

221

Shanghai, China). All reactions were performed in triplicate.

222 223

RESULTS

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Cloning and sequence analysis of the pga-zj5a. The pga-zj5a gene was cloned from A.

225

niger ZJ5 by PCR using specific primers. The open reading frame (ORF) of pga-zj5a was

226

1089 bp long, encoding a polypeptide of 362 amino acids plus a stop codon. The deduced

227

PGA-ZJ5A protein contained a putative N-terminal signal peptide (residues 1–18) through

228

SignalP prediction. The molecular weight and isoelectric point of the mature protein were

229

predicted to be 37.04 kDa and pH 6.3, respectively. There were three potential

230

O-glycosylation sites (31Thr, 32Ser, 34Ser) and one potential N-glycosylation site

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(334Asn-Trp-Thr-Trp) in PGA-ZJ5A based on the analysis of NetOGlyc 4.0 Server and

232

NetNGlyc 1.0 Server online, respectively.

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The deduced PGA-ZJ5A amino acid sequence showed the highest identity (99%) with

234

the endo-polygalacturonase from Aspergillus kawachii IFO 4308 (GenBank Accession No.

235

GAA82222)17 and 83% identity with endo-polygalacturonase C from A. rambellii21 (GenBank

236

Accession No. KKK13564). However, both of these genes were identified by genome

237

sequencing, and their functions have not yet been determined yet.

238

Heterologous expression, purification and identification of PGA-ZJ5A. The pga-zj5a

239

gene was successfully expressed in P. pastoris GS115. The transformant with the highest

240

endo-polygalacturonase activity (1437 U/mL in shaker) was selected for fed-batch

241

fermentation in a 3 L fermenter. After growing in the fermenter for 132 h under optimal

242

conditions, the total yield of protein in the culture was 2.68 g/L, and showed a maximum

243

polygalacturonase activity of 10436 U/mL. SDS-PAGE analysis was performed on the 11



244

recombinant protein in the fermented supernatant with different incubation times (Fig. 1).

245

Recombinant PGA-ZJ5A protein was purified to electrophoretic homogeneity by His-tag

246

Ni2+ affinity chromatography (Fig. 2). The purified fractions that showed the highest protein

247

concentrations and polygalacturonase activity were collected for further analysis of their

248

activity. SDS-PAGE analysis showed that PGA-ZJ5A was represented by two bands around

249

41 kDa and higher (Fig. 2), which is higher than the calculated value (37.04 kDa). After

250

deglycosylation using Endo H, the purified PGA-ZJ5A showed a single band with a

251

molecular weight of approximately 40 kDa (Fig. 2). The observed variation in the apparent

252

molecular mass of PGA-ZJ5A revealed that N-glycosylation modifications occurred in

253

PGA-ZJ5A during its heterologous expression in P. pastoris. Other post-translational

254

modifications, such as O-glycosylation might also have occurred during heterologous

255

expression. This is consistent with glycosylation predictions of the PGA-ZJ5A.

256

To identify the purified protein, peptide sequences obtained by liquid chromatography/

257

mass spectrometry were compared to the deduced PGA-ZJ5A amino acid sequence

258

(Supplementary Fig. S1). The complete match of these sequences confirmed that the purified

259

protein was the target protein PGA-ZJ5A.

260

Characterization of purified recombinant PGA-ZJ5A. Recombinant PGA-ZJ5A

261

showed a preference for acidic conditions, with a pH optimum between 4.5 and 6.5 (Fig. 3A);

262

it also retained more than 50% of its maximum activity at pH 5.5 to 7.0. As shown in Fig. 3B,

263

it was also stable over a wide pH range, retaining over 50% of its initial activity after

264

pretreatment at pH 2.0 to 6.0, 37°C, for 1 h. The optimum temperature of PGA-ZJ5A was

265

40°C (pH 4.5), and more than 50% of the maximum activity was retained between 25 and

266

45°C (Fig. 3C). The enzyme was stable at 40°C but lost 50% of its initial activity after

267

incubation at 45°C for 1 h and 85% of its initial activity at 55°C for 5 min (Fig. 3D).

268

The effects of metal ions and chemical reagents on PGA-ZJ5A activity were evaluated at 12


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269

concentrations of 1 and 10 mM (Table 1). Of the ions and chemical reagents assessed, Pb2+

270

was the strongest inhibitor. Pb2+ at 1 and 10 mM caused a greater than 60% loss of activity. In

271

addition, 10 mM Mn2+, Ca2+ and Ni2+ inhibited activity by more than 50%. Other metal ions

272

and chemical reagents partially inhibited enzyme activity in a concentration-dependent

273

manner.

274

Substrate

specificity

and

kinetic

parameters.

The

enzyme

activity

with

275

polygalacturonic acid as the substrate was regarded as 100%. Purified recombinant

276

PGA-ZJ5A showed 67.3% relative activity towards pectins with a DE of 34%, 21.5% activity

277

towards pectins with a DE of 70% and 6.4% activity towards pectins with a DE of 85%.

278

When using polygalacturonic acid (P3889; Sigma-Aldrich) as the substrate, the apparent Km

279

and Vmax values of PGA-ZJ5A for polygalacturonic acid were 0.85 mg/mL and 1.871

280

µmol/min/mg, respectively. The purified recombinant PGA-ZJ5A showed a specific activity

281

of 6360.6 U/mg towards polygalacturonic acid.

282

Application of PGA-ZJ5A in pear juice production. PGA-ZJ5A showed considerable

283

potential for increasing the clarity of pear juice. To determine the optimal volume of enzyme

284

to be added to the juice, purified enzyme with 0, 1, 2, 5 and 10 U/mL juice was added to 50

285

mL pear juice. The results obtained after incubation at 40°C for 1 h are shown in Figure 4.

286

Simultaneous addition of PGA-ZJ5A increased the juice volume by 41.8%. The light

287

transmittance of pear juice was increased nearly three-fold. Considering the effect of the

288

enzyme on fruit juice, 5 U/mL juice was chosen as the optimal amount.

289

PGA-ZJ5A showed tremendous potential for the clarification of pear juice. The results of

290

clarification by purified PGA-ZJ5A and other pectinases are shown in Figure 5. Pectinase

291

treatments significantly increased the transmittance of pear juice. The use of PNL-ZJ5A

292

exhibited superior performance. The longer the incubation, the more effective the clarification,

293

while the best results being achieved at 120 min. The transmittance of juice treated with 13



294

PGA-ZJ5A alone changed from 57.4% at 15 min to 94.3% at 120 min, increasing by 64.3%.

295

The volume of pear juice treated with PGA-ZJ5A alone changed from 6.06 mL at 15 min to

296

10.37 mL at 120 min, increasing by 71.12%. When the fruit juice was treated with PGA-ZJ5A

297

and PNL-ZJ5A together, transmittance was enhanced by 18.1% compared to PGA-ZJ5A

298

alone, suggesting that PNL-ZJ5A has the ability to react with oligogalacturonic acids, the

299

enzymatic hydrolysis products of PNL-ZJ5A, which are also factors contributing to juice

300

transmittance15. The effect of the combination of PNL-ZJ5A and PGA-ZJ5A was maintained

301

over time. The same effect of the addition of PNL-ZJ5A was also reflected in the juice

302

volume. When the pear juice was treated with PGA-ZJ5A and PNL-ZJ5A, its volume

303

changed from 6.06 (PGA-ZJ5A alone) to 6.53 mL within 2 min, increasing by 7.7%,

304

suggesting that the combination of these two enzymes produced soluble substances in pear

305

juice decomposition and improved the filtering speed.

306 307

DISCUSSION

308

The remarkable properties of recombinant endo-polygalacturonase PGA-ZJ5A were high

309

specific activity, high expression level and broad acidic pH adaptability (pH 4.0–7.0). Several

310

polygalacturonases in commercial pectinase preparations have been purified and

311

characterized22. These polygalacturonases exhibit specific activities ranging from 25 to 4000

312

U/mg protein6. Endo-polygalacturonase I from Achaetomium sp. Xz8 showed extremely high

313

activity towards polygalacturonic acid (28,122 U/mg), with optimal activity at pH 6, which

314

makes it suitable for process pH neutral fruit23. In this study, an acidic PGA-ZJ5A with a

315

specific activity of 6360.6 U/mg was cloned from A. niger. Compared with PGase from

316

Mucor rouxii NRRL 1894 (specific activity, 1372.5 U/mg) 24, endo-polygalacturonase A from

317

A. niger JL-15 (specific activity, 2091.0 U/mg)25 and the exo-polygalacturonase from

318

Thermotoga maritima (specific activity, 1000 U/mg)12, PGA-ZJ5A showed higher specific 14


Page 14 of 35

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319


activity under acidic conditions.

320

In order to improve the yield of polygalacturonases, some acidic endo-polygalacturonases

321

have been expressed in Pichia pastoris, including PGA1 from Bispora sp. MEY-126 and

322

endo-PG I from Penicillium sp. CGMCC 166927. The optimal pH of PGA1 and endo-PG I

323

was observed in the pH 3.5. The acidophilic stability of these enzymes make them a potential

324

candidate for juice processing. But the expression level needs to be further increased. The

325

yield of PGA1 in P. pastoris was 50 U/mL26, that of endo-PG I was 6.2 U/mL27, whereas the

326

corresponding data of PGA-ZJ5A in this study was 10436 U/mL. The high expression level of

327

PGA-ZJ5A was helpful to reduce its production cost and promote its application in fruit juice

328

processing.

329

Compared with the endo-polygalacturonases from A. niger N40028, A. awamori29, A.

330

niger SC32330, and A. niger JL-1525, which show maximum activities at pH 5.0, PGA-ZJ5A

331

showed maximum activity at pH 4.5, close to the native pH of pear juice (pH 4.3–4.8), and

332

retained more than 80% activity at pH 6.5. More importantly, PGA-ZJ5A retained more than

333

70% activity at pH 2.0 to 4.5. This would enable use of PGA-ZJ5A in the production of

334

various fruit juices, such as litchi (pH 4.8), peach (pH 4.6) and watermelon (pH 6.5). In

335

addition, PGA-ZJ5A also showed potential in wolfberry extract. As the amount of enzyme

336

added increased, the viscosity of wolfberry juice decreased gradually (data not shown).

337

Different types of pectinase have different modes of action. To obtain better results

338

during their application, the mixing of different types of enzymes could be effective. Upon

339

use in combination with PNL-ZJ5A, more efficient juice clarification was achieved than with

340

PGA-ZJ5A alone. The differences in performance could be a result of a variety of factors,

341

such as the type of enzyme preparation, pear variety, treatment and pressing conditions. 15



342

Therefore, we predict that determining the optimal ratio of enzyme preparations would enable

343

better juice clarification to be achieved.

344

In summary, considering its high specific enzyme activity, high expression level, natural

345

adaptation to acidic conditions and specific potential for juice clarification, recombinant

346

PGA-ZJ5A is a candidate acidic enzyme with a wide pH range for use in fruit processing.

347 348

AUTHOR INFORMATION

349

Corresponding Authors

350

* (W. Z.) E-mail: [email protected]. Phone: +86-10-82106141. Fax: +86-10-82106141.

351

* (H. H.) E-mail: [email protected]. Phone: +86-25-58139942. Fax: +86-25-58139942.

352

AUTHOR CONTRIBUTIONS

353

#

354

version of the manuscript. J. W. and Y. Z. performed most of experiments and data analysis,

355

and drafted the manuscript. W. Z. and H. H. provided advice on experiments design, and

356

revised the manuscript. X. Q. constructed the cloning and expression plasmid. J. L. and D. Z.

357

carried out the experiments related to juice process. L. G. and B. H. preformed the

358

experiments of liquid chromatography/electrospray ionization tandem mass spectrometry.

359

FUNDING

360

This research was supported by the National High Technology Research and Development

361

Program of China (863 Program, Grants number 2012AA022207 and 2012AA022105).

362

NOTES

363

The authors declare no competing fancial interest.

J. W. and Y. Z. contributed equally to this paper. All authors have given approval to the final

16


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364

ABBREVIATIONS USED

365

DE, degree of esterification; RDB, regeneration dextrose medium; MD, minimal dextrose

366

medium; BMGY, buffered glycerol complex medium; BMMY, buffered methanol complex

367

medium; DNS, 3,5-dinitrosalicylic acid; SDS, sodium dodecyl sulfate; CTAB, cetyl

368

trimethylammonium bromide; EDTA, ethylenediaminetetraacetic acid disodium; SDS-PAGE,

369

sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

370

SUPPORTING INFORMATION

371

Supplementary table S1. Test data of endo-polygalacturonase activity in fermentor.

372

Supplementary figure S1. Identification of PGA-ZJ5A by liquid chromatography/mass

373

spectrometry.

374

Supplementary figure S2. Galacturonic acid standard curve.

375

17



376

377 378 379 380 381 382 383 384 385 386 387

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Kluskens L. D.; van Alebeek G. J.; Walther J.; Voragen A. G.; de Vos W. M.; van

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der Oost J. Characterization and mode of action of an exopolygalacturonase from the

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hyperthermophilic bacterium Thermotoga maritima. FEBS Journal 2005, 272, 5464-5473.

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Kobayashi, T.; Higaki, N.; Yajima, N.; Suzumatsu, A.; Hagihara, H.; Kawai, S.; Ito,

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S. Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a

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strain of Bacillus. Biosci. Biotech. Bioch. 2001, 65, 842-847.

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Gupta, R.; Paul Bennett, E.; Mandel, U.; Brunak, S.; Wandall, H. H.; Levery, S. B.; Clausen,

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H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell

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technology. The EMBO Journal 2013, 32, 1478-1488.

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Aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu.

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Eukaryot. Cell 2011, 10, 1586-1587.

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Rhizopus microsporus var. microsporus, and its potential for application in the brewing

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related aflatoxigenic Aspergillus species obtained from the Ivory Coast. Genome Biol. Evol.

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JL-15 endo-polygalacturonase A gene in Pichia pastoris and oligo-galacturonates production.

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Protein Expres. Purif. 2014, 94, 53-59.

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from Bispora sp. MEY-1 and its potential application in juice clarification. Process

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Biochemistry 2011, 46, 272-277.

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acidic and low-temperature-active endo-polygalacturonase from Penicillium sp. CGMCC

450

1669 with potential for application in apple juice clarification. Food Chemistry 2011, 129,

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constitutively expressed endopolygalacturonases of Aspergillus niger. Biochem. J. 2000, 345,

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637-644.

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characterization of an endo-polygalacturonase from Aspergillus awamori. Biosci. Biotech.

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Bioch. 2000, 64, 1729-1732.

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459

Secretory expression and characterization of an acidic endo-polygalacturonase from

460

Aspergillus niger SC323 in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 2015, 25,

461

999-1006.

Yang, J.; Luo, H. Y.; Li, J.; Wang, K.; Cheng, H. P.; Bai, Y. G.; Yuan, T. Z.; Fan, Y.

Yuan, P.; Meng, K.; Huang, H. Q.; Shi, P. J.; Luo, H. Y.; Yang, P. L.; Yao, B. A novel

Parenicova, L.; Benen, J.; Kester, H.; Visser, J. pgaA and pgaB encode two

Nagai, M.; Katsuragi, T.; Terashita, T.; Yoshikawa, K.; Sakai, T. Purification and

Zhou, H. X.; Li, X.; Guo, M. Y.; Xu, Q. R.; Cao, Y.; Qiao, D. R.; Cao, Y.; Xu, H.

21



462

FIGURE CAPTIONS

463

Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of PGA-ZJ5A

464

expressed in P. pastoris at different times. Lane M contained standard molecular weight

465

markers. Lane 1 contained the culture supernatant of pPIC9-PG/GS115 harboring pga-zj5a

466

before induction, while lanes 2–7 contained the culture supernatant of recombinant P. pastoris

467

harboring pga-zj5a after induction by methanol for 24, 36, 72, 84, 108 and 132 h, respectively.

468

The arrow indicates the recombinant protein.

469

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified

470

recombinant PGA-ZJ5A protein. Lanes: M, molecular mass markers; 1, culture supernatant of

471

recombinant P. pastoris harboring pga-zj5a; 2, purified recombinant PGA-ZJ5A protein; 3,

472

PGA-ZJ5A deglycosylated by treatment with Endo-H; 4, Endo-H enzyme from New England

473

Biolabs. The arrow indicates the recombinant protein.

474

Fig. 3. Characterization of purified recombinant PGA-ZJ5A. (A) Effect of pH on PGA-ZJ5A

475

activity. The recombinant PGA-ZJ5A activity was assayed at 40°C in buffers at pH 2.5–9.0.

476

(B) pH stability of PGA-ZJ5A activity. After incubating the enzyme at 37°C for 1 h in buffers

477

ranging from pH 2.0 to 9.0, the activity was determined in 0.2 M Na2HPO4-NaH2PO4 buffer

478

(pH 4.5) at 40°C. (C) Effect of temperature on PGA-ZJ5A activity measured in 0.2 M

479

Na2HPO4-NaH2PO4 buffer at pH 4.5. (D) Thermostability of recombinant PGA-ZJ5A.

480

Thermostability of PGA-ZJ5A was determined by measuring the residual activity after

481

pre-incubation at 40, 45, 50 and 55°C in 0.2 M Na2HPO4-NaH2PO4 buffer (pH 4.5) for

482

various periods. Each value in the panel represents the mean ± SD (n = 3).

22


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483

Fig. 4. Determination of the optimal amount of purified PGA-ZJ5A in juice. Different

484

dosages of purified enzyme (0, 1, 2, 5 and 10 U/mL juice) were added to 50 mL pear juice. (A)

485

Pear juice volume upon treatment with different dosages of purified PGA-ZJ5A. (B) The

486

transmittance of pear juice treated with different dosages of purified PGA-ZJ5A.

487

Fig. 5. Efficiencies of PGA-ZJ5A and other pectinases in the clarification of pear juice.

488

PNL-ZJ5A is a pectin lyase that was used at 1 U/mL juice. The purified PGA-ZJ5A was used

489

at 5 U/mL juice. Fresh pear juice without the addition of pectinase was used as a control.

23



Table 1 Effect of metal ions and chemical reagents on the activity of recombinant PGA-ZJ5A Relative activity (%)a

Metal ions and reagents

1 mM

10 mM

Control

100

100

SDS

87.43±1.7

44.19±1.3

CTAB

42.12±2.1

41.46±0.9

EDTA

82.82±0.7

43.86±1.2

ZnSO4

70.5±1.3

56.58±0.7

FeSO4

99.83±0.8

65.05±1.5

NiSO4

73.26±1.5

37.63±1.8

CdSO4

42.71±1.6

41.24±2.3

MgSO4

88.53±0.6

76.03±1.2

CaCl2

53.2±2.1

36.44±1.7

KCl

93.54±1.9

70.94±2.1

MnCl2

89.53±0.6

39.75±0.9

NaCl

97.64±2.1

85.85±2.4

MgCl2

85.86±1.5

68.28±0.7

CoCl2

91.63±2.1

50.89±1.6

Pb(CH3COO)2

35.19±2.9

22.77±1.3

a

Values represent the means of triplicate experiments relative to the untreated control samples.

SDS, sodium dodecyl sulfate; CTAB, cetyl trimethylammonium bromide; EDTA, ethylenediaminetetraacetic acid disodium. 24


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Figure 1

25



Figure 2

26


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Figure 3

27



Figure 4

28


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Figure 5

29



TOC Graphic

30


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89x52mm (200 x 200 DPI)




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61x47mm (600 x 600 DPI)



187x285mm (96 x 96 DPI)


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176x230mm (300 x 300 DPI)


Efficient Expression of an Acidic Endo-polygalacturonase from

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