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Biotechnology and Biological Transformations

Discovery and Characterization of a Novel Chitosanase from Paenibacillus
dendritiformis by Phylogeny-based Enzymatic Product Specificity Prediction
Huihui Sun, Xiangzhao Mao, Na Guo, Ling Zhao, Rong Cao, and Qi Liu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06067 • Publication Date (Web): 24 Apr 2018
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Page 1 of 24 Journal of Agricultural and Food Chemistry

1 Discovery and Characterization of a Novel Chitosanase from Paenibacillus dendritiformis by Phylogeny-based

2 Enzymatic Product Specificity Prediction

3 Huihui Sun1, Xiangzhao Mao2, Na Guo2, Ling Zhao1, Rong Cao*1, Qi Liu1

1
4 Department of Food Engineering and Nutrition, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery

5 Sciences, Qingdao 266071, China

2
6 College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

10

11 *Corresponding author: Professor Rong Cao

12 Address: Department of Food Engineering and Nutrition, Yellow Sea Fisheries Research Institute, Chinese Academy of

13 Fishery Sciences, Qingdao 266071, China

14 Tel.: +86-532-85830760

15 Email: caorong@ysfri.ac.cn

16

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17 ABSTRACT: In the process of genome mining for novel chitosanases by phylogeny-based enzymatic product specificity

18 prediction, a gene named Csn-PD from Paenibacillus dendritiformis was discovered. The enzyme was classified as a

19 member of the GH46 family of glycoside hydrolase based on sequence alignment, and it was functionally expressed in

20 Escherichia coli BL21 (DE3). The recombinant chitosanase was purified, and its molecular weight was estimated to be

21 31 kDa by SDS-PAGE. Csn-PD displayed maximal activity toward colloidal chitosan at pH 7.0 and 45 °C, respectively.

22 A combination of thin-layer chromatography and electrospray ionization-mass spectrometry results showed that Csn-PD

23 exhibited an endo-type cleavage pattern and hydrolyzed chitosan to yield (GlcN)2 as the major product. The unique

24 enzymatic properties of this chitosanase may make it a good candidate for (GlcN)2 production.

25 KEYWORDS: chitosanase, (GlcN)2, chitosan, Paenibacillus dendritiformis, product specificity prediction

26

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27 ■ INTRODUCTION

28 Chitosan, a linear polysaccharide composed of β-1,4 linked D-glucosamine (GlcN), is a totally or partially

29 deacetylated form of chitin, which is the second most abundant polysaccharide on earth1,2. Chitin can be commercially

30 obtained from the exoskeletons (shells) of crustacea, such as shrimp and crab, which are continuously generated in nature

31 and thus an almost unlimited renewable resource3. Chitosans have been reported to support a broad range of potential

32 applications in agriculture, biotechnology, medicine and environmental treatment4-8. However, the commercial use of

33 chitosan is restricted because of its poor solubility and high viscosity in neutral or basic aqueous media9-11. Therefore,

34 there is considerable interest in converting chitosan to chitooligosaccharides (COSs), which are water-soluble; these even

35 possess additional biological properties such as antimicrobial, antitumor, and immuno-enhancing activities12-15.

36 Chemical and physical methods can be used to produce COSs16,17. However, there are many problems with both

37 processes, such as requirements for high temperature and pressure, production of secondary compounds, high separation

38 cost, and low yields of COSs. In contrast, chitosan may be biologically converted to COSs under ambient and

39 environmentally friendly conditions. Chitosanases (EC 3.2.1.132) are enzymes that can hydrolyze chitosan into COSs

40 and glucosamine and are found in many kinds of organisms, including bacteria, fungi, and plants18.

41 Chitosanases can be grouped into six glycosyl hydrolase (GH) families, i.e. GH5, GH7, GH8, GH46, GH75 and GH80,

42 based on their amino acid sequences rather than their substrate specificities19. Families GH46, GH75, and GH80 are

43 believed to be more substrate specific (limited to chitosan), while chitosanases from families GH5 and GH8 also tend to

44 possess other glycoside hydrolase activities such as cellulose and licheninase20. Most chitosanases have been shown to be

45 the endo-type enzyme, which randomly cleave glycosidic bonds of chitosan to produce a mixture of oligosaccharides

46 with various degrees of polymerization (DP)18,21,22. There is to date no successful method to obtain COSs with a single

47 DP directly with one endo-chitosanase. Costly and time-consuming separation of COSs with different DPs, such as size

48 exclusion chromatography and ultrafiltration, is commonly employed, but is only practical on a small scale23,24. However,

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49 the biological functionalities of COSs have been shown to be strongly dependent on their DPs25-27. Thus, the production

50 of defined oligomers has generated significant interest both for fundamental structure-function relationship research and

51 for the development of chitosan and COS applications.

52 As far as we know, there have been several works that described the existence of exo-β-D-glucosaminidases

53 (belonging to GH2 and not classified into the chitosanase families listed above), that can hydrolyze chitosan to generate

54 glucosamine (GlcN)28-32. In another study, the endo-type chitosanase from Bacillus circulans MH-K1 was converted into

55 an exo-type chitosanase by inserting two surface loops; this produced (GlcN)2 as the dominant product, but, unfortunately,

56 the catalytic rate of the mutant was only 3% that of the wild-type chitosanase22.

57 In the present study, phylogeny-based enzymatic product specificity prediction (PEPSP) was introduced for the

58 efficient discovery of chitosanases to produce COSs with different DPs. A novel chitosanase, Csn-PD, belonging to

59 family GH46, was discovered from Paenibacillus dendritiformis. It was cloned and overexpressed in Escherichia coli

60 BL21 (DE3). The substrate specificity of Csn-PD and its stability as a function of temperature and pH were investigated.

61 ■ MATERIALS AND METHODS

62 Materials. All the three chitosanase genes extracted from the GenBank database, including Csn-PD from

63 Paenibacillus dendritiformis (WP_006679998.1), Csn-CAP from Staphylococcus capitis (OAN23142), and Csn-But from

64 Butyrivibrio sp. MC2013 (WP_026508362), were synthesized by Talen-bio Scientific (Shanghai) Co., Ltd. and inserted

65 into plasmid pET28a(+). The resulting plasmids were respectively transformed into E. coli BL21 (DE3) for expression of

66 the chitosanase. Chitosans with a degree of deacetylation (DDA) of 85% and 95% were purchased from Sigma Chemical

67 Co. (St. Louis, MO, USA). COSs with DP 2–6 were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao,

68 China). Other chemicals and reagents used were of analytical grade and purchased from local markets.

69 Database mining and sequence analysis. Searching for novel chitosanase gene sequences was performed using the

70 BLASTP program

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71 (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMSblastp&PAGE_TY

72 PE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome). The chitosanase from B. circulans MH-K1

73 (BAA01474) was chosen as the identifier to detect potential chitosanases to produce COSs with different DPs33. This

74 chitosanase has been studied extensively, and its product ((GlcN)1–(GlcN)5) was completely different from those of other

75 reported chitosanases22. Chitosanase sequences showing 20–80% amino acid identity were extracted from the GenBank

76 database. Chitosanases with experimentally defined product specificity belonging to family GH46 were chosen as a

77 priority to refine the phylogenetic analysis. Sequence alignments were conducted using Clustal W34. Phylogenetic

78 relationships among chitosanolytic enzymes were analyzed through a neighbor-joining method packaged in MEGA 6.035.

79 Expression of the three chitosanases genes in E. coli. Recombinant E. coli BL21 (DE3) cells for chitosanases

80 expression, including Csn-PD, Csn-CAP, and Csn-But, were cultivated in Luria-Bertani medium containing kanamycin

81 (50 µg/mL) at 37 °C in a shaker (200 rpm). When the OD600 of the culture reached 0.6, isopropyl-β-D-thio-

82 galactopyranoside was added to a final concentration of 1 mM. The induced cultures were further incubated for 16 h at

83 30 °C. Then, cells were harvested by centrifugation (10,000 × g, 10 min), washed twice with 0.85% NaCl solution, and

84 stored at −20°C.

85 Purification of Csn-PD. The obtained cell pellets were resuspended in sodium phosphate buffer (50 mM, pH 7.0).

86 Cell disruption was performed by sonication, and the debris was removed by centrifugation (10,000 × g, 30 min). The

87 resulting crude extract was loaded onto a Ni-NTA column (1 mL; Qiagen, Hilden, Germany) at a flow rate of 1 mL/min.

88 The column was equilibrated with buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Then the

89 column was washed with 20 mM imidazole in buffer A. Fractions containing the target protein were eluted with 200 mM

90 imidazole in buffer A. The eluted protein was desalted and concentrated by ultrafiltration. The crude extract and the

91 purified protein were analyzed by SDS-PAGE. Protein concentration was determined using the Bradford method. All

92 purification steps were carried out at 4 °C.

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93 Enzyme assay. Standard assays were performed in a reaction mixture (1 mL) containing sodium phosphate buffer (50

94 mM, pH 7.0), colloidal chitosan (1%, w/v, DDA of 85%), and an appropriate amount of chitosanase. The mixture was

95 incubated at 30 °C for 10 min and then the reaction was quenched in boiling water for 10 min. The reducing sugars

96 released were determined by the DNS method with minor modification36. All the experiments were performed in

97 triplicate. One unit (U) of the enzyme activity was defined as the amount of enzyme that produced 1 µmol of reducing

98 sugar per min under the above assay conditions using GlcN as the standard.

99 Characterization of purified Csn-PD. The optimum temperature for Csn-PD activity was determined by incubating

100 the enzyme with colloidal chitosan (1%, w/v, DDA of 85%) at 20–60 °C in 50 mM phosphate buffer (pH 7.0). The

101 thermostability of Csn-PD was determined by measuring the residual activity after the enzyme was incubated at different

102 temperatures (20–60 °C) for 30 min. The optimum pH for Csn-PD activity was determined at pH 4.0 to 8.0 (citrate buffer

103 pH 4.0 to 6.0, phosphate buffer pH 6.0 to 8.0). The residual chitosanase activity was also measured after the enzyme was

104 incubated in the above mentioned buffers at pH 4.0–8.0 at 30 °C for 30 min.

105 Substrate specificity of Csn-PD was determined at 30 °C for 30 min in 50 mM phosphate buffer (pH 7.0). The tested

106 polysaccharide substrates (1%, w/v) included colloidal chitosan, chitosan with DDAs of 85% and 95% respectively,

107 colloidal chitin, and carboxymethyl cellulose (CMC).

108 Hydrolytic properties of the purified Csn-PD. The hydrolytic properties of Csn-PD were investigated using

109 colloidal chitosan and COSs (DP 2–6) by analyzing the hydrolysis products by thin-layer chromatography (TLC) and

110 electrospray ionization-mass spectrometry (ESI-MS). The reaction mixture was centrifuged at 10,000 × g for 10 min,

111 then the supernatant was spotted onto a silica gel plate (Merck, Darmstadt, Germany), which was developed in a solvent

112 system containing propanol–25% ammonia–water (8:3:1, v/v/v). The oligosaccharide spots were visualized by spraying

113 0.1% ninhydrin reagent (dissolved in ethanol), followed by heating at 105 °C for 10 min. ESI-MS studies were performed

114 in positive-ion mode and conducted by Shanghai Micronmr Infor Technology Co., Ltd. Samples were analyzed by

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115 WATERS UPLC-MS SQD2 scanning with a ratio of mass to charge in the range of 50–2000 (m/z). In all ESI-MS

116 experiments, the scan mode was used under a capillary needle at 3 kV and the ion source temperature at 150 °C.

117 ■ RESULTS AND DISCUSSION

118 Discovery and identification of chitosanase Csn-PD by PEPSP. Based on the GenBank database mining and amino

119 acid sequence analysis, a total of 16 proteins, annotated as chitosanases, were chosen for analysis of their products

120 toward chitosan. Among them, 13 chitosanases, belonging to GH46, have been experimentally determined to produce

121 COSs with different DPs (Figure 1). Multiple sequence alignment (Figure 1A) showed that the catalytic domain of the

122 three novel chitosanases contained two key active site residues (Glu and Asp), which are conserved in all members of

123 GH46. These results indicated that chitosanases Csn-But, Csn-CAP, and Csn-PD were novel members of chitosanolytic

124 enzyme family GH46.

125 Based on the phylogenetic analysis (Figure 1B), the 13 chitosanases that have been studied previously clustered into

126 five groups. For example, the main products of the first six chitosanases are (GlcN)2 and (GlcN)337-42. The next four

127 chitosanases produce mixtures of (GlcN)2–643-46. The products of the chitosanases from Burkholderia gladioli, Bacillus

128 circulans MH-K1, and Bacillus coagulans include (GlcN)2–(GlcN)4, (GlcN)1–(GlcN)5, and (GlcN)3–(GlcN)5,

129 respectively22,47,48. The three novel chitosanases are distributed in different groups. Chitosanases Csn-But and Csn-CAP

130 clustered into the first group which can produce (GlcN)2 and (GlcN)3. However, chitosanase Csn-PD did not belong to

131 any of the groups.

132 To verify the accuracy of this prediction method and to confirm the product of the three novel chitosanases, the three

133 chitosanase genes Csn-PD, Csn-CAP, and Csn-But, were respectively expressed in E. coli. All three enzymes were active

134 toward colloidal chitosan. The results of TLC showed that the final products of chitosanases Csn-But and Csn-CAP were

135 (GlcN)2 and (GlcN)3 (Figure S1), which was in accordance with the prediction. Csn-PD produced only (GlcN)2. These

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136 results not only confirmed the accuracy of the PEPSP method, but also identified a novel chitosanase Csn-PD that could

137 produce COS with a single DP. Csn-PD was thus selected for further study.

138 Purification of chitosanase Csn-PD. The chitosanase Csn-PD from P. dendritiformis was successfully expressed in E.

139 coli as an active enzyme. Using the 6×His affinity tag, Csn-PD was purified to electrophoretic homogeneity by nickel

140 affinity chromatography. The purified Csn-PD was separated as a single protein band of approximately 31 kDa by SDS-

141 PAGE (Figure 2). The molecular mass is lower than those of the chitosanases from Janthinobacterium sp. 4239 (33

142 kDa)41 and Bacillus subtilis (36 kDa)46, but higher than those of most other reported bacterial chitosanases, e.g., from B.

143 circulans MH-K1 (29 kDa)33 and Amycolatopsis sp. CsO-2 (27 kDa)37. The specific activity of purified Csn-PD was 76.4

144 U/mg toward chitosan with DDA of 85%, which was much higher than the activity of CSN from Penicillium sp. D-118.

145 Characterization of the purified Csn-PD. The optimum reaction temperature of purified Csn-PD was at 45 °C, and

146 there was a decrease to 31.8% of the maximal activity at 60 °C (Figure 3A). Csn-PD could maintain >80% of the initial

147 activity after incubation at 20–50 °C. However, the residual activity of Csn-PD decreased sharply when it was incubated

148 at 60 °C for 30 min (relative activity 10%) (Figure 3B). The purified Csn-PD was very pH-sensitive, with the highest

149 activity at pH 7.0 in 50 mM phosphate buffer, some activity at acidic pH values, but inactivation at pH 8.0 (Figure 3C).

150 The enzyme was stable at pH 6.0–7.0 (retained >90% of the maximal activity after incubation) but showed a dramatic

151 decrease in activity after incubation at pH <6.0 and >7.0 (Figure 3D).

152 The specificity of the purified Csn-PD for chitin and chitosans with different DDAs is presented in Table 1. The

153 enzyme showed higher hydrolysis of chitosan and colloidal chitosan with DDA 95% than DDA 85%. However, it was

154 not active toward colloidal chitin or CMC. Csn-PD thus displayed strict substrate specificity, in accordance with most

155 reported chitosanases belonging to family GH4618,37,43,44,.

156 Hydrolytic properties of purified Csn-PD. The hydrolytic properties of Csn-PD toward colloidal chitosan were

157 investigated in detail. On incubation of colloidal chitosan with the purified recombinant enzyme at 30 °C for 1 h (Figure

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158 4), Csn-PD initially hydrolyzed the substrate to yield mainly (GlcN)2 and (GlcN)3 (0–0.5 h). (GlcN)3 was further

159 converted to (GlcN)2 with extension of the reaction time (0.5–1 h), but no GlcN was observed. ESI-MS analysis also

160 confirmed that (GlcN)2 was the dominant product. As shown in Figure 5A, apart from (GlcN)2, no other oligomers,

161 including GlcN, was detected, which indicated that (GlcN)2 was not hydrolyzed any more. Meanwhile, it was

162 demonstrated from Figure 5B that the products derived from hydrolysis of colloidal chitosan by Csn-PD were only

163 (GlcN)2. These results suggested that Csn-PD exhibited a random catalytic mode of action toward chitosan with a high

164 ability to form (GlcN)2. This property is similar to most chitosanases from microbes that belong to family GH46, which

165 yield COSs with various DPs by an endo-type catalytic action37,38,41.

166 COSs (DP 2–6) were also used as substrates to test the hydrolytic properties of Csn-PD. As shown in Figure 6, the

167 enzyme mainly hydrolyzed these COSs to (GlcN)2 and (GlcN)3 in the initial stage, but (GlcN)2 was not hydrolyzed at all.

168 (GlcN)3 was completely converted to (GlcN)2 in 4 h, with no GlcN production. This result implied that (GlcN)3

169 molecules must have combined then been hydrolyzed to produce (GlcN)2, rather than (GlcN)3 being cleaved to (GlcN)2

170 and GlcN, which was confirmed by the hydrolysis of (GlcN)5. (GlcN)5 was hydrolyzed to mainly yield equivalent

171 amounts of (GlcN)2 and (GlcN)3 in the initial stage (0–1 h), and the released (GlcN)3 was then further converted to

172 (GlcN)2 within 4 h. (GlcN)4 and (GlcN)6 were converted to (GlcN)2 in much less time than (GlcN)3 and (GlcN)5.

173 As far as we know, Csn-PD is the first reported chitosanase that exhibits an endo-type pattern and produces (GlcN)2 as

174 the dominant product. A mutant of B. circulans MH-K1 chitosanase functions as an exo-type enzyme with (GlcN)2 as the

175 dominant product22. Strangely, these two chitosanases share only 72% identity in amino acid sequence. Thus, the PEPSP

176 method we used. This method discovered a novel enzyme based on its product specificity toward chitosan, rather than the

177 sequence identity. Therefore, despite the low identity among these chitosanases, they were successfully clustered.

178 In this study, a novel chitosanase, named Csn-PD, was discovered from Paenibacillus dendritiformis by PEPSP, and

179 was successfully expressed in E. coli BL21 (DE3). The recombinant chitosanase was purified and biochemically

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180 characterized. It was most active at pH 7.0 and 45 °C. Csn-PD converted chitosan to, mainly, (GlcN)2, via an endo-type

181 mechanism. The excellent biochemical properties may make this enzyme a good candidate for (GlcN)2 production. More

182 broadly, this work shows that there is every possibility to screen diverse chitosanases for different product specificities.

183 Moreover, before the characterization of an enzyme, use of the PEPSP approach can save large amounts of experimental

184 labor, time, and cost. Better catalysts will be developed in this way for efficient production of pure COSs with specific

185 DP.

186 ■ ABBREVIATIONS

187 PEPSP: phylogeny-based enzymatic product specificity prediction; COSs: chitooligosaccharides; GH: glycosyl hydrolase;

188 DP: degree of polymerization; DDA: degree of deacetylation; CMC: carboxylmethyl cellulose; TLC: thin-layer

189 chromatography; ESI-MS: electrospray ionization-mass spectrometry.

190 ■ FUNDING

191 This work was supported by the China Postdoctoral Science Foundation (funded project No. 2017M612379), and the

192 Natural Science Foundation of Shandong Province (No. ZR2017BC095).

193 Notes

194 The authors declare that they have no competing interests.

195 Supporting Information

196 Figure S1. TLC analysis of hydrolysis products of Csn-CAP and Csn-But.

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257 glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases.

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300 characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101.

301 Appl. Microbiol. Biotechnol. 2000, 54, 354-360.

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305 Figure captions

306 Figure 1. Bioinformatic analysis of Csn-PD. (A) Multiple amino acid sequence alignment of Csn-PD and other

307 chitosanases belonging to family GH46. The typical catalytic sites (Glu and Asp) were emphasized with triangles. (B)

308 Neighbor-joining phylogenetic tree. Phylogenetic analysis was carried out using MEGA 6.0 software. The novel

309 chitosanases are in black frames. Groups with different product specificities are in red frames.

310 Figure 2. SDS-PAGE analysis of Csn-PD. Lane M, protein marker; Lane 1, whole cell lysate of recombinant Csn-PD;

311 lane 2, supernatant of Csn-PD cell lysate; lane 3, purified Csn-PD.

312 Figure 3. The Characterization of Csn-PD. (A) Effect of temperature on enzyme activity; (B) Effect of temperature on

313 enzyme stability. (C) Effect of pH on enzyme activity; (D) Effect of pH on enzyme stability.

314 Figure 4. TLC analysis of hydrolysis products of colloidal chitosan.

315 Figure 5. ESI-MS analyses of products derived from hydrolysis of (GlcN)2 and colloidal chitosan by Csn-PD. A: ESI-

316 MS analyses of products derived from hydrolysis of (GlcN)2 by Csn-PD. The (GlcN)2 was used as a control. (GlcN)2 (m/z

317 341 and +K+ m/z 379). B: ESI-MS analyses of products derived from hydrolysis of colloidal chitosan by Csn-PD.

318 (GlcN)2 (m/z 341 and +K+ m/z 379).

319 Figure 6. TLC analysis of hydrolysis products of chitooligosaccharides (DP 2–6).

320

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321 Tables

322 Table 1 Substrate specificity of the purified Csn-PD

Substrates DDA (%) Relative activity (%)

Chitosan 85 100

Chitosan 95 128.4

Colloidal chitosan 85 94.5

Colloidal chitosan 95 124.4

Colloidal chitin 0 0

CMC 0 0

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323 Figures

324 Figure 1.

325

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326 Figure 2.

327

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328 Figure 3.

329

330

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331 Figure 4.

332

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333 Figure 5.

334

335

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336 Figure 6.

337

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338

TOC Graphic

339

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