Biochemical Characterization and Substrate Degradation Mode of a

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Biochemical Characterization and Substrate Degradation Mode of a Novel #-Agarase from Catenovulum agarivorans Jie Liu, Zhen Liu, Chengcheng Jiang, and Xiangzhao Mao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03073 • Publication Date (Web): 27 Aug 2019 Downloaded from pubs.acs.org on August 28, 2019

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Journal of Agricultural and Food Chemistry

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Biochemical Characterization and Substrate Degradation Mode of a Novel

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α-Agarase from Catenovulum agarivorans

3

Jie Liu1, Zhen Liu1, *, Chengcheng Jiang1, Xiangzhao Mao1, 2, *

4

1

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

5 6

2

Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China

7 8 9 10

* Corresponding author: Prof. Xiangzhao Mao, Dr. Zhen Liu

11

Prof. Xiangzhao Mao

12

Address: College of Food Science and Engineering, Ocean University of China,

13

Qingdao 266003, China

14

Tel.: +86-532-82032660

15

Fax: +86-532-82032272

16

E-mail: [email protected]

17

Dr. Zhen Liu

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Address: College of Food Science and Engineering, Ocean University of China,

19

Qingdao 266003, China

20

Tel.: +86-532-82031360

21

E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract

23

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase,

24

which is an important enzyme for efficient saccharification of agarose or preparation

25

of bioactive oligosaccharides from agarose. Although many β-agarases have been

26

reported and characterized, there are only a few studies on α-agarases. Here, we

27

cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately

28

200 kDa belonging to glycoside hydrolase (GH) family 96 from Catenovulum

29

agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37°C

30

and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as

31

agarobiose (A2), agarotetraose (A4) and agarohexaose (A6), in which A4 is the

32

dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose

33

(A3), and A6 into A2 and A4, but cannot act on A2, A3 or A4. This is the first report

34

to characterize the α-agarase action on agarooligosaccharides in detail. Therefore,

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CaLJ96 has potential for the manufacture of bioactive AOSs.

36

Keywords：α-Agarase; Agarose; Agarooligosaccharides; GH96; Degradation mode


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Introduction

38

Agar is a polysaccharide separated from the cell wall of red algae such as

39

Gelidium and Gracilaria1. As one of the three major polysaccharides in seaweed

40

industry, agar is widely used in food, medicine, biotechnology and many other fields2.

41

As a main component of agar, agarose is a repeating disaccharide linear chain

42

composed

43

3,6-anhydro-α-L-galactose (L-AHG)3. Agarase is a glycoside hydrolase which

44

hydrolyzes agar or agarose to produce agar oligosaccharides4. Compared with other

45

polysaccharides, the agar oligosaccharides have higher solubility and lower viscosity,

46

which could extend its industrial application. Furthermore, agar oligosaccharides have

47

been found to have many physiological functions, such as anti-oxidation, anti-viral

48

effect, anti-tumor, immune enhancement and moisturizing whitening5-8.

of

alternating

monosaccharide

residues

of

β-D-galactose

and

49

According to the hydrolyzing site, agarase was defined into two types: α-agarase

50

and β-agarase. The former cleaves the α-1,3-glycosidic bond and produces

51

agarooligosaccharides (AOSs) with β-D-galactose as non-reducing end; the latter acts

52

on the β-1,4-glycosidic bond and produces neoagarooligosaccharides (NAOSs) with

53

L-AHG as non-reducing end9-10. Compared with α-agarase, there are already many

54

studies focusing on β-agarase. Until now, three α-agarases have been reported, which

55

are AgaA from marine bacterium Alteromonas agarlyticus GJ1B11, AgaA33 of

56

Thalassomonas sp. JAMB-A33 from marine sediments12 and AgaD of Thalassomonas

57

sp. LD5 from coastal aquifer sediments13. With agarose as substrate, the main

58

hydrolysates of AgaA, AgaD and AgaA33 are agarotetraose (A4), with a small



59

amount of agarobiose (A2) and agarohexaose (A6). However, the substrate

60

degradation mode of α-agarase is still unclear.

61

In this study, a novel α-agarase named CaLJ96 from Catenovulum agarivorans,

62

which is a gram-negative, strictly aerobic and chemo-organotrophic bacterium14, was

63

cloned and expressed, the biochemical properties and hydrolysis pattern toward

64

different substrates by CaLJ96 were studied. As far as we know, this is the first report

65

of extensive study on the hydrolysis mode of α-agarase.

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Materials and Methods

67

Materials and Culture Conditions

68

The whole gene sequence of calj96 was analyzed (Tsingke, Qingdao, China).

69

Escherichia coli DH5α for gene cloning and E. coli BL21(DE3) for gene expression

70

were purchased from Tiangen (Tiangen Biotech, Beijing, China). Agarose for

71

degradation was purchased from Sigma (VetecTM Reagent Grade, St. Louis, Missouri,

72

USA). Tryptone and yeast extracts for fermentation media were purchased from

73

Oxoid (Basingstoke, England). The E. coli strains were cultivated at 37°C in

74

Luria-Bertani (LB) medium (0.5% yeast extract, 1% tryptone, and 1% NaCl)

75

containing 50 μg/mL kanamycin (Solarbio, Qingdao, China) for 12 h.

76

Amino Acid Sequence Analysis of CaLJ96

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Based on the gene sequence of calj96, the amino acid sequence of CaLJ96 was

78

obtained by DANMAN (Lynnon, USA). Protein was analyzed by the National Center

79

for Biotechnology Information (NCBI, USA) (http://www.ncbi.nlm.nih.gov/). Protein

80

homologous sequences and phylogenetic tree analysis of CaLJ96 were performed


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using ClustalX 2.1, ESPript (http://espript.ibcp.fr/ESPript/ESPript/) and MEGA 6.06,

82

respectively.

83

Cloning and Expression of α-Agarase CaLJ96

84

The primers CaLJ96-F (5'-GGATCCGCGACCCATTTG-3') and CaLJ96-R

85

(5'-AAGCTTGCGGCCGCACTC-3')

were

designed

using

NEBuilder

86

(http://nebuilder.neb.com/) to amplify the calj96 gene without signal peptide sequence.

87

The primers are synthesized by TsingKe. The purified PCR product of calj96 was

88

ligated into pET28a(+) vector containing two 6×His tags, and then the recombinant

89

plasmid was transformed into E. coli DH5α, which was grown in solid LB medium

90

containing 50 μg/mL of kanamycin for 16 h at 37°C. The suitable single colony was

91

picked, and the nucleotide sequence was verified by PCR and sequencing. The final

92

recombinant expression vector was transformed into E. coli BL21(DE3), which was

93

cultured using ZYP-5052 autoinduction medium (0.05% MgSO4, 0.05% glucose,

94

0.2% α-lactose•H2O, 0.3% (NH4)2SO4, 0.5% glycerin, 0.5% yeast extract, 0.7%

95

Na2HPO4, 0.7% KH2PO4, 1% tryptone) for 48 h at 20°C with shaking (220 rpm).

96

Purification of α-Agarase CaLJ96

97

The purification steps in this study were carried out all operated at 4°C. The cells

98

were first collected by centrifugation at 8000 rpm for 10 min at 4°C, and then the cell

99

pellet was resuspended in 20 mM phosphate buffer (Na2HPO4 and NaH2PO4) at pH

100

8.0, and disrupted by sonication treatment (on 3 s, off 3 s) for 30 min at 4°C. After the

101

disruption, the cell debris was removed by centrifugation at 9000 rpm for 20 min at

102

4°C, and the supernatant was defined as the crude enzyme. The supernatant was



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103

filtered and purified using Ni2+-NTA resin, which was preequilibrated with 6 column

104

volumes of buffer 1 (20 mM phosphate buffer, 10 mM imidazole and 500 mM NaCl).

105

The weakly bound impurity was removed by elution with buffer 2 (20 mM phosphate

106

buffer, 20 mM imidazole and 500 mM NaCl). Finally, a fraction with high enzyme

107

activity was eluted with buffer 3 (20 mM phosphate buffer, 50 mM imidazole and 500

108

mM NaCl). After purification, it was concentrated using 30 kDa ultrafiltration device

109

and washed with buffer 4 (pH 7.0, 50 mM Tris-HCl), and the obtained pure enzyme

110

was measured for purity by SDS-PAGE. The protein concentration was determined by

111

using BCA Protein Assay Kit (Thermo Scientific, Waltham, U.S.A.) with bovine

112

serum albumin (BSA) as the standard15.

113

Enzyme Activity Assay

114

Enzyme

activity

was

determined

with

slight

modification

using

115

3,5-dinitrosalicylic acid (DNS) method as previously described16. Each reaction

116

system containing 193 μL of 0.2% low gelling temperature agarose (melting point less

117

than 65°C, Sigma-Aldrich, USA) in Tris-HCl buffer (pH 7.0), 5 μL of pure enzyme

118

and 2 μL of 1 M Ca2+ was incubated at 37°C for 30 min. The reaction was stopped in

119

a boiling water bath for 10 min, then 300 μL of DNS was added to the mixture and

120

subjected to a boiling water bath for 5 min. After cooling, samples were diluted with

121

500 μL of water and the absorbance was measured at 540 nm. The inactivated enzyme

122

was used as a blank control. One α-agarase CaLJ96 activity unit was defined as the

123

amount of enzyme required to produce 1 μmol of reducing sugar per minute by

124

hydrolyzing agarose under the above-mentioned assay conditions.


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125


Biochemical Characterization of α-Agarase CaLJ96

126

50 mM citrate buffer (pH 3.0-6.0), 50 mM phosphate buffer (pH 6.0-8.0), 50 mM

127

Tris-HCl buffer (pH 7.0-9.0), or 50 mM glycine-NaOH buffer (pH 9.0-10.0) were

128

used to determine the optimum pH of α-agarase CaLJ96 by incubating at 37°C for 30

129

min. In order to determine the pH stability of CaLJ96, the enzyme solution at different

130

pH and times was incubated at 4°C, and the residual activity of CaLJ96 was measured

131

according to the above standard method.

132

The optimum temperature of the α-agarase CaLJ96 was determined by

133

incubating CaLJ96 and agarose for 30 min at a temperature range of 20 to 90°C using

134

50 mM Tris-HCl buffer (pH 7.0). The enzyme solution was incubated at the above

135

temperature for 1 h, and the residual activity was measured according to the above

136

standard method to determine the thermal stability of CaLJ96.

137

The effect of chemicals on CaLJ96 activity was investigated by adding metal

138

ions (Ba2+ (BaCl2), Ca2+ (CaCl2), Co2+ (CoSO4), Cu2+ (CuCl2), Fe3+ (FeCl3), K+ (KCl),

139

Mg2+ (MgCl2), Mn2+ (MnCl2), Na+ (NaCl), Zn2+ (ZnSO4)) or chemical reagents

140

(Na2EDTA, SDS) to the reaction system incubating at 37°C for 30 min at a final

141

concentration of 1 mM and 10 mM, respectively.

142

Determination of the Kinetic Parameters of α-Agarase CaLJ96

143

The enzyme was mixed with different concentrations of substrate (substrate

144

concentration range was set as 0 to 8 g/L), which were incubated at 37°C for 10 min,

145

and the kinetic parameters of CaLJ96 were determined under the standard method.

146

The Km, Vmax and kcat values of CaLJ96 were calculated by the Michaelis-Menten



147

equation.

148

Hydrolytic Reactions

149

The reaction mixture which contained 5 μL of purified CaLJ96, 2 μL of 1 M Ca2+

150

and 193 μL of substrates (0.2% low gelling temperature agarose, A3 or A5) was

151

incubated at 37°C. Aliquots were sampled at 0 min, 1 min, 2 min, 5 min, 30 min, 1 h,

152

2 h, 5 h and 12 h. In the case of determing the hydrolytic product of CaLJ96, after the

153

reaction of agarose hydrolysis by CaLJ96 for 24 h, the α-neoagarobiose hydrolase

154

AgaWH11717 or β-galactosidase AgWH2A18 was added into the reaction mixture to

155

incubate at 37°C for another 24 h, then the reaction was terminated by boiling for 10

156

min.

157

Analytical Methods

158

After centrifuging the above reaction mixture, the supernatant was filtered

159

through the 0.22 μm filter, then loaded onto HPLC system with a Sugar Pak I column

160

(Waters, 6.5×300 mm, Milford, MA, USA), and a refractive index detector (RID).

161

HPLC analysis conditions were column temperature of 75°C, mobile phase of 50

162

mg/L ethylenediaminetetraacetic acid disodium calcium salt (EDTA-CaNa2), and

163

flow rate of 0.5 mL/min. The mobile phase was ultrapure water and the negative ion

164

scanning mode was selected in MS analysis.

165

Results and Discussion

166

Discovery of a Novel α-Agarase

167

α-Agarase CaLJ96 contains 1431 amino acids, with the predicted molecular

168

weight of 151.6 kDa by DNAMAN. The coding sequence of α-agarase CaLJ96 is


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169

4296 bp in length (Genbank accession number WP_035015824.1). Based on the

170

sequence alignment of CaLJ96 and three reported α-agarases, CaLJ96 showed

171

sequence identity of 85.55% with AgaA (Genbank accession number AAF26838.1)

172

from A. agarilytica GJ1B, 83.12% with AgaD (Genbank accession number

173

AQN80853.1) from Thalassomonas sp. LD5, and 64.81% with AgaA33 (Genbank

174

accession number BAF44076.1) from Thalassomonas JAMB-A33. The phylogenetic

175

tree analysis indicated that CaLJ96 belongs to family GH96 (Fig. 1). CaLJ96 contains

176

a 78 bp signal peptide sequence in its N-terminus, two lectin-like β-jelly-rolled folded

177

CBM6 domains for substrate binding and three TSP-3 domains which is predicted for

178

Ca2+ binding. Previous studies have shown that calcium binding is beneficial to the

179

thermal stability of CBM on α-agarase structure19. Moreover, CaLJ96 contains a

180

cellulose binding domain type IV (CBD-IV) near the N-terminus (Fig. 2). Based on

181

sequences analysis, it is speculated that CaLJ96 is an extracellularly secreted

182

α-agarase, which can binds agarose and needs Ca2+ for its activity in its natrual host C.

183

agarivorans.

184

The 4218 bp nucleotide sequence of calj96 gene without signal peptide was

185

expressed in E. coli BL21(DE3). After expression, the crude extract of E. coli

186

BL21(DE3) expressing CaLJ96 could hydrolyze agarose to produce reducing sugars

187

(data not shown). This suggested CaLJ96 is indeed an agarase. After purification of

188

the recombinant protein, a band with a molecular weight of approximately 200 kDa

189

was shown by SDS-PAGE (Fig. 3). Both the α-agarases AgaA and AgaD worked as a

190

dimer with 180 kDa on SDS-PAGE and 360 kDa after gel filtration11,


. The

13


191

molecular weight of CaLJ96 was similar with α-agarases AgaA and AgaD11, 13, but

192

was inconsistent with the predicted molecular weight of 151.6 kDa by DANMAN.

193

Perhaps this is because there are three acidic AAs-rich TSP-3 domains in CaLJ96,

194

since the difference between the predicted molecular weight and the real molecular

195

weight could be caused by the content of acidic amino acids (glutamic acid and

196

aspartic acid) 20.

197

Characterization of CaLJ96

198

The specific activity of CaLJ96 was 6.49 U/mg under standard conditions (37°C,

199

pH 7.0, 50 mM Tris-HCl), its Vmax, Km and kcat value were 12.65 μmol/(mg•min),

200

0.135 mg/mL and 2.40 s-1, respectively. The kcat/Km value of CaLJ96 was 17.78

201

mL/(mg•s). AgaA33, the first recombinant α-agarase, was extracellularly produced

202

using Bacillus subtilis, which yielded AgaA33 with the activity up to 6950 U/L21. The

203

specific activity of purified AgaA33 reached 40.7 U/mg. Meanwhile, its optimum

204

temperature and pH, relative molecular mass, specific activity and other parameters

205

were not significantly different from those of natural agarases. AgaD, the latest

206

reported α-agarase, had maximal activity at 35°C and pH 7.4, which was similar with

207

other α-agarases. The specific activity of purified AgaD reached 149 U/mg13. The

208

CaLJ96 showed highest enzyme activity at pH 7.0 in 50 mM Tris-HCl buffer. And

209

more than 80% of the highest enzyme activity was retained at pH 7.0-10.0 (Fig. 4A).

210

However, at pH 3.0-5.0, the activity was less than 10% of that at pH 7.0. This

211

suggests that CaLJ96 could not tolerate an acid environment. On the contrary, CaLJ96

212

exhibited significant stability under neutral or alkaline conditions (pH 7.0-10.0), more


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213

than 70% of initial activity was retained after 28 days at 4°C, which corresponded to

214

the activity spectrum (Fig. 4B). The optimum reaction temperature for CaLJ96 was

215

37°C, more than 80% of the highest enzyme activity was maintained at 30-50°C, and

216

the activity at 20°C was only 12.3% of that at 37°C (Fig. 4C). Staying at temperature

217

higher than 37°C for 1 h, CaLJ96 lost its activity completely, however, staying at

218

20-30°C, its activity maintained well (Fig. 4D). The optimum reaction temperature

219

(37°C) of CaLJ96 is not the optimum stable temperature (30°C). In general, 30°C is

220

lower than the native agarose (1.5% wt) geling temperature at 35-40°C range22,

221

therefore 37°C is still selected as the reaction temperature in the long-term reaction.

222

The above results demonstrated that CaLJ96 is able to hydrolyze agarose under a

223

wide range of pH, and its optimal reaction pH and temperature are similar to those of

224

reported α-agarases11-13.

225

The CaLJ96 activity was obviously inhibited by metal ions and chemicals such

226

as Fe3+, Mg2+, Co2+, Zn2+ and SDS. With higher concentration, stronger inhibition was

227

observed. The CaLJ96 activity was completely inhibited by Na2EDTA and Cu2+, and

228

was slightly inhibited by 10 mM Ba2+ and Mn2+. Maybe metal ions and chemical

229

agents have an affinity interaction with the functional groups of the enzyme, which

230

causes a change in the structure of the catalytic domain, thereby reducing the enzyme

231

activity23-24. Notably, the CaLJ96 activity could be increased by 16% in the presence

232

of 10 mM Ca2+ (Table.1).

233

Agarose Degradation Pattern by CaLJ96

234

After incubation of CaLJ96 with agarose at 37°C for 24 h, three products peaks



235

(P1, P2, P3) were detected by HPLC (Fig. 5A, SI). These three products were then

236

analyzed by MS to obtain the molecular weights of 936 Da (P1), 630 Da (P2) and 324

237

Da (P3), respectively (Fig. 5B, C, D). This suggested that P1, P2 and P3 are agar

238

oligosaccharides with the polymerization degree (PD) of 6, 4 and 2, respectively.

239

Since the even-numbered AOSs have the same molecular weight as the NAOSs,

240

further works were needed to distinguish these products were AOSs or NAOSs. It has

241

been reported that β-galactosidase can cleave the first D-galactose from the

242

non-reducing end of AOS, but cannot act on NAOS25. The α-neoagarobiose hydrolase

243

can cut off the first L-AHG from the non-reducing end of NAOS, but cannot act on

244

AOS26. Based on the hydrolysis mechanism of β-galactosidase and α-neoagarobiose

245

hydrolase, products of agarose degradation by CaLJ96 were re-degraded with

246

β-galactosidase AgWH2A or α-neoagarobiose hydrolase AgaWH117. According to

247

the results of HPLC, β-galactosidase AgWH2A further degraded P1, P2 and P3 (Fig.

248

5A, SII), while no obvious changes were observed in the case of α-neoagarobiose

249

hydrolase AgaWH117. The enzymatic hydrolysis result showed obviously that all of

250

P1, P2 and P3 were AOSs. That is to say, P1 was agarohexaose (A6), P2 was

251

agarotetraose (A4), and P3 was agarobiose (A2). Therefore, our results showed that

252

the α-agarase CaLJ96 could degrade agarose to produce only AOSs but not NAOSs,

253

which was as same as other reported α-agarase such as AgaA, AgaA33 and AgaD11-13.

254

As shown in Fig. 6, during agarose degradation by CaLJ96, A4 was produced

255

within 2 min, A6 started to be detected at 30 min, and A2 emerged at 1 h of reaction.

256

The amount of A6 began to decrease at 1 h, and until 12 h, the amount of A4 and A2


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257

showed a single increasing trend. This indicated that CaLJ96 cannot use A2 and A4 as

258

substrate. The emerged A6 started to decrease after 1 h, this suggested that CaLJ96

259

could degrade A6 into oligosaccharides with PD lower than A6. During the whole

260

degradation process, only 2 oligosaccharides, namely A2 and A4, had the PD lower

261

than A6. Therefore, it is speculated that CaLJ96 could degrade A6 to produce A2 and

262

A4. In the final reaction mixture, the peak area ratio of A2: A4: A6 is 1: 6.91: 3.14, in

263

which A4 is dominant in the products. According to the study of Yukari Ohta, the

264

α-agarase AgaA33 degraded agarose to 9% A6, 77% A4, 9% A2, and 5% other

265

products12. Its product composition is similar to that of CaLJ96. The degraded product

266

of α-agarase AgaD and AgaA was only an even-numbered agarooligosaccharide A4,

267

and no other product contents were mentioned11, 13. Although the degradation of A6

268

and NA6 by α-agarase AgaA33 has been conducted, there is no specifical analysis

269

about the products12. Therefore, the α-agarases degradation products and degradation

270

patterns should be further analyzed.

271

Considering the polymerization degree of products, the reported β-agarases

272

exhibited similar with CaLJ96. β-Agarase Aga50D from Saccharophagus degradans

273

2-40 degraded agarose into NA227-28. Another β-agarase, AgaO, from Flammeovirga

274

sp. strain MY04 has also been shown to produce NA2 by exo-lytic mode10.

275

AgWH50B from Agarivorans gilvus WH0801 could degrade agarose to produce NA2,

276

NA4 and NA6, with NA4 as the main product29.

277

As shown in Fig. 7, it was apparent that the product peak after the reaction of

278

CaLJ96 with agarotriose (A3) is consistent with the substrate peak, indicating that



279

CaLJ96 coulod not act on A3. After the reaction of CaLJ96 with agaropentaose (A5),

280

the emerged two new peaks (P4, P5) were completely different from the substrate

281

peak, and the retention time of P4 and P5 were consistent with that of A3 and A2 (Fig.

282

7A). The molecular weights of the two products P4, P5 were 486 Da and 324 Da,

283

respectively (Fig. 7B, C). These results showed that CaLJ96 could hydrolyze A5 to

284

generate A3 and A2. However, CaLJ96 cannot degrade A2, A3 or A4, but certainly

285

degrades A5 and A6 (Fig. 8), so A5 is the smallest oligosaccharide substrate. Because

286

the sequence similarity between CaLJ96 and other reported α-agarase is high, it was

287

speculated that A5 is also the smallest oligosaccharide substrate of other α-agarases.

288

This is the first time to characterize the substrate degradation mode of α-agarase in

289

detail. In terms of β-agarase, the research of Liang et, al. showed that NA6 acts as the

290

smallest oligosaccharide substrate of AgWH50B, which cannot hydrolyze NA4, the

291

dominant product of agarose degradation by AgWH50B29. This result was similar to

292

our result of CaLJ96, which could not act on A4, the main product of agarose

293

degradation by CaLJ96.

294

In summary, we cloned and characterized a novel α-agarase named CaLJ96,

295

which degraded agarose to A2, A4, A6, in which A4 is the dominant product. CaLJ96

296

could degrade A5 to A2 and A3, A6 to produce A2 and A4, but could not act on A2,

297

A3 or A4. CaLJ96 could be used for saccharification of agarose, and also to produce

298

AOSs which can be effectively applied in cosmetics and medicine.

299

Funding Sources

300

This work was supported by Taishan Scholar Project of Shandong Province


Page 14 of 41

Page 15 of 41


301

(tsqn201812020), Fundamental Research Funds for the Central Universities

302

(201941002), National Key R&D Program of China (2018YFC0311200) and China

303

Postdoctoral Science Foundation (2018T110710).



304

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3. Rees, D. A. Structure, conformation, and mechanism in the formation of

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4. Liu, N.; Mao, X.; Yang, M.; Mu, B.; Wei, D. Gene cloning, expression and

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characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from

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Agarivorans gilvus WH0801. World J. Microbiol. Biotechnol. 2014, 30 (6),

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1691-1698.

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5. Hu, B.; Gong, Q.; Wang, Y.; Ma, Y.; Li, J.; Yu, W. Prebiotic effects of

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neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe.

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2006, 12 (5), 260-266.

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6. Kang, O. L.; Ghani, M.; Hassan, O.; Rahmati, S.; Ramli, N. Novel

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agaro-oligosaccharide production through enzymatic hydrolysis: physicochemical

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properties and antioxidant activities. Food Hydrocolloids. 2014, 42, 304-308.

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7. Wang, J.; Jiang, X.; Mou, H.; Guan, H. Anti-oxidation of agar oligosaccharides

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produced by agarase from a marine bacterium. J. Appl. Phycol. 2004, 16 (5), 333-340.

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8. Yun; Ju, E.; Kim; Taek, H.; Lee; Sun, H.; Kim; Heon, K.; Lee; Saeyoung.


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Enzymatic production of 3,6-anhydro galactose from agarose and its purification and

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in vitro skin whitening and anti-inflammatory activities. Appl. Microbiol. Biotechnol.

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2013, 97 (7), 2961-2970.

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9. Flament, D.; Barbeyron, T., M; Potin, P.; Czjzek, M.; Kloareg, B.; Michel, G.

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Alpha-agarases define a new family of glycoside hydrolases, distinct from

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beta-agarase families. Appl. Environ. Microbiol. 2007, 73 (14), 4691-4694.

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10. Han, W.; Cheng, Y.; Wang, D.; Wang, S.; Liu, H.; Gu, J.; Wu, Z.; Li, F.

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Biochemical characteristics and substrate degradation pattern of a novel exo-type

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β-agarase from the polysaccharide-degrading marine bacterium Flammeovirga sp.

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strain MY04. Appl. Environ. Microbiol. 2016, 82 (16), 4944-4954.

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11. Potin, P.; Richard, C.; Rochas, C.; Kloareg, B. Purification and characterization of

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the α-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur. J.

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Biochem. 1993, 214 (2), 599-607.

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12. Ohta, Y.; Hatada, Y.; Miyazaki, M.; Nogi, Y.; Ito, S.; Horikoshi, K. Purification

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and characterization of a novel α-agarase from a Thalassomonas sp. Curr. Microbiol.

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2005, 50 (4), 212-216.

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13. Zhang, W.; Xu, J.; Liu, D.; Liu, H.; Lu, X.; Yu, W. Characterization of an

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α-agarase from Thalassomonas sp. LD5 and its hydrolysate. Appl. Microbiol.

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Biotechnol. 2018, 102 (5), 2203-2212.

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14. Yan, S.; Yu, M.; Wang, Y.; Shen, C.; Zhang, X. Catenovulum agarivorans gen.

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nov., sp. nov., a peritrichously flagellated, chain-forming, agar-hydrolysing

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gammaproteobacterium from seawater. Int. J. Syst. Evol. Microbiol. 2011, 61,



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15. Bradford, M. M. A rapid method for the quantitation of microgram quantities of

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protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72,

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248–254.

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16. Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing

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sugar. Anal. Chem. 1959, 31 (3), 426-428.

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a new α-neoagarobiose hydrolase from Agarivorans gilvus WH0801 and enzymatic

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production of 3,6-anhydro-l-galactose. Biotechnol. Appl. Biochem. 2016, 63 (2),

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230-237.

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18. Yang, X.; Liu, Z.; Jiang, C.; Sun, J.; Xue, C.; Mao, X. A novel

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agaro-oligosaccharide-lytic β-galactosidase from Agarivorans gilvus WH0801. Appl.

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Microbiol. Biotechnol. 2018, 102, 5165-5172.

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19. Maher, A. H.; Eva Nordberg, K.; Simpson, P. J.; Sara, L.; Peter, S.; Williamson, M.

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P.; Jamieson, S. J.; Gilbert, H. J.; Bolam, D. N.; Olle, H. Calcium binding and

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thermostability of carbohydrate binding module CBM4-2 of Xyn10A from

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Rhodothermus marinus. Biochemistry. 2002, 41 (18), 5720-5729.

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20. Guan, Y.; Zhu, Q.; Huang, D.; Zhao, S.; Li, J. L.; Peng, J. An equation to estimate

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the difference between theoretically predicted and SDS PAGE-displayed molecular

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weights for an acidic peptide. Sci. Rep. 2015, 5 (1), 13370.

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21. Yuji, H.; Yukari, O.; Koki, H. Hyperproduction and application of alpha-agarase to

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enzymatic enhancement of antioxidant activity of porphyran. J. Agric. Food Chem.


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22. Zhang, N.; Wang, J.; Ye, J.; Zhao, P.; Xiao, M. Oxyalkylation modification as a

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promising method for preparing low-melting-point agarose. Int. J. Biol. Macromol.

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2018, 117, 696-703.

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23. Gupta, V.; Trivedi, N.; Kumar, M.; Reddy, C. R. K.; Jha, B. Purification and

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characterization of exo-β-agarase from an endophytic marine bacterium and its

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catalytic potential in bioconversion of red algal cell wall polysaccharides into

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galactans. Biomass Bioenergy. 2013, 49 (3), 290-298.

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24. Yukari, O.; Yuji, H.; Susumu, I.; Koki, H. High-level expression of a

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neoagarobiose-producing beta-agarase gene from Agarivorans sp. JAMB-A11 in

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Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol. Appl.

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Biochem. 2005, 41 (2), 183-191.

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25. Lee, C. H., Kim, H. T., Yun, E. J., Lee, A. R., Kim, S. R., Kim, J. H., Choi, I. G.,

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Kim K. H. A novel agarolytic β-galactosidase acts on agarooligosaccharides for

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complete hydrolysis of agarose into monomers. Appl. Environ. Microbiol. 2014, 80

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(19), 5965-5973.

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26. Day, D. F.; Yaphe, W. Enzymatic hydrolysis of agar: purification and

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characterization of neoagarobiose hydrolase and p-nitrophenyl alpha-galactoside

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hydrolase. Can. J. Microbiol. 1975, 21 (6), 1512-1518.

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27. Pluvinage, B.; Hehemann, J. H.; Boraston, A. B. Substrate recognition and

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hydrolysis by a family 50 exo-beta-agarase, Aga50D, from the marine bacterium

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Saccharophagus degradans. J. Biol. Chem. 2013, 288, 28078-28088.



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28. Kim, H. T.; Lee, S.; Lee, D.; Kim, H. S.; Bang, W. G.; Kim, K. H.; Choi, I. G.

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Overexpression and molecular characterization of Aga50D from Saccharophagus

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degradans 2-40: an exo-type beta-agarase producing neoagarobiose. Appl. Microbiol.

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Biotechnol. 2010, 86, 227-234.

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29. Liang, Y.; Ma, X.; Zhang, L.; Li, F.; Liu, Z.; Mao, X. Biochemical characterization

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gilvus WH0801. J. Agric. Food Chem. 2017, 65, 7982-7988.

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Figure Legend

401

Figure 1. Phylogenetic analysis of CaLJ96 with other agarolytic enzymes. Based

402

on amino acid sequences, it was showed as a neighbor-joining tree on MEGA 6.0. The

403

numbers showed in the branches indicate bootstrap confidence values of 1000

404

repetitions. The scale bar indicates 0.2 substitutions per site.

405

Figure 2. Modular structure of the α-agarase CaLJ96. The protein contains

406

N-terminal signal peptide (residues 1-26, in blue), a CBD IV (residues 26-162, in

407

black), two CBM6 (residues 224-346 and 674-801, in dark blue) and three TSP-3

408

(residues 163-184, 415-446 and 449-479, in gray).

409

Figure 3. SDS-PAGE analysis of purified CaLJ96 expressed in E. coli. M,

410

molecular mass marker of proteins; Lane 1, crude enzyme; Lane 2, purified CaLJ96.

411

Figure 4. Effect of pH and temperature on enzyme activity and stability of

412

CaLJ96. (A) Effects of pH on the activity of CaLJ96: citrate buffer (pH 3.0–6.0)，

413

phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 7.0–9.0), glycine-NaOH buffer

414

(pH 9.0–10.0). (B) Effects of pH on the stability of CaLJ96: citrate buffer (pH

415

3.0–6.0), Tris-HCl buffer (pH 7.0–8.0), glycine-NaOH buffer (pH 9.0–10.0). (C)

416

Effects of temperature on the activity of CaLJ96. (D) Effects of temperature on the

417

thermostability of CaLJ96. All measurements were performed in triplicate; error bars

418

indicate standard deviation of measurement.

419

Figure 5. Analysis of degradation products of agarose hydrolyzed by CaLJ96. (A)

420

HPLC analysis of the different degradation products of agarose hydrolyzed by

421

α-agarase CaLJ96 (SI), CaLJ96 and β-galactosidase AgWH2A (SII) or CaLJ96 and



422

α-neoagarobiose hydrolase AgaWH117 (SIII). (B) Negative ion ESI-MS spectrum of

423

degradation product P1 of CaLJ96. (C) Negative ion ESI-MS spectrum of degradation

424

product P2 of CaLJ96. (D) Negative ion ESI-MS spectrum of degradation product P3

425

of CaLJ96.

426

Figure 6. HPLC analysis of the degradation patten of agarose hydrolyzed by

427

CaLJ96. The retention times of the AOSs are indicated by arrows (A2: 9.219 min, A4:

428

7.249 min, A6: 6.192 min).

429

Figure 7. Hydrolysis of A3 or A5 by CaLJ96. (A) HPLC analysis of degradation

430

products of A3 and A5 by CaLJ96. (B) Negative ion ESI-MS spectrum of degradation

431

product P4 of CaLJ96 incubated with A5. (C) Negative ion ESI-MS spectrum of

432

degradation product P5 of CaLJ96 incubated with A5.

433

Figure 8. Degradation mode of different AOSs with CaLJ96.


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434


Table 1. Effects of various metal ions and chemical agents on CaLJ96 activity. Chemicals

Relative Activity (%) 1 mM

10 mM

Control

100 ± 4.9

100 ± 4.9

Ca2+

103.9 ± 10.3

116.7 ± 8.6

Ba2+

94.2 ± 3.9

96.5 ± 0.5

Mn2+

76.1 ± 4.4

97.8 ± 10.2

K+

57.3 ± 2.7

58.4 ± 8.3

Mg2+

99.3 ± 9.8

46.0 ± 6.3

SDS

92.8 ± 1.9

11.2 ± 4.3

Co2+

86.1 ± 3.3

28.9 ± 1.9

Na+

65.1 ± 2.8

58.1 ± 4.2

Zn2+

61.1 ± 2.0

0 ± 0.1

Fe3+

49.6 ± 2.3

1.2 ± 1.3

Cu2+

3.7 ± 0.6

3.3 ± 0.4

Ni2+

2.6 ± 6.5

0 ± 0.1

Na2EDTA

0.7 ± 2.0

0 ± 0.1

435

The relative activity was expressed as the percentage of CaLJ96 activity under various

436

metal ion and chemical agents treatments to the control without any added reagents.

437

All measurements were performed in triplicate.

438



439

Figure 1.

440


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441


Figure 2.

442



443

Figure 3.

444


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445


Figure 4.

446



447

Figure 5.

448


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449


Figure 6.

450



451

Figure 7.

452


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Figure 8.

454



455

TOC

456


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Figure 1. Phylogenetic analysis of CaLJ96 with other agarolytic enzymes. Based on amino acid sequences, it was showed as a neighbor-joining tree on MEGA 6.0. The numbers showed in the branches indicate bootstrap confidence values of 1000 repetitions. The scale bar indicates 0.2 substitutions per site. 184x176mm (300 x 300 DPI)



Figure 2. Modular structure of the α-agarase CaLJ96. The protein contains N-terminal signal peptide (residues 1-26, in blue), a CBD IV (residues 26-162, in black), two CBM6 (residues 224-346 and 674-801, in dark blue) and three TSP-3 (residues 163-184, 415-446 and 449-479, in gray).


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Figure 3. SDS-PAGE analysis of purified CaLJ96 expressed in E. coli. M, molecular mass marker of proteins; Lane 1, crude enzyme; Lane 2, purified CaLJ96. 40x87mm (300 x 300 DPI)



Figure 4. Effect of pH and temperature on enzyme activity and stability of CaLJ96. (A) Effects of pH on the activity of CaLJ96: citrate buffer (pH 3.0–6.0)，phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 7.0–9.0), glycine-NaOH buffer (pH 9.0–10.0). (B) Effects of pH on the stability of CaLJ96: citrate buffer (pH 3.0–6.0), Tris-HCl buffer (pH 7.0–8.0), glycine-NaOH buffer (pH 9.0–10.0). (C) Effects of temperature on the activity of CaLJ96. (D) Effects of temperature on the thermostability of CaLJ96. All measurements were performed in triplicate; error bars indicate standard deviation of measurement. 256x198mm (300 x 300 DPI)


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Figure 5. Analysis of degradation products of agarose hydrolyzed by CaLJ96. (A) HPLC analysis of the different degradation products of agarose hydrolyzed by α-agarase CaLJ96 (SI), CaLJ96 and β-galactosidase AgWH2A (SII) or CaLJ96 and α-neoagarobiose hydrolase AgaWH117 (SIII). (B) Negative ion ESI-MS spectrum of degradation product P1 of CaLJ96. (C) Negative ion ESI-MS spectrum of degradation product P2 of CaLJ96. (D) Negative ion ESI-MS spectrum of degradation product P3 of CaLJ96.



Figure 6. HPLC analysis of the degradation patten of agarose hydrolyzed by CaLJ96. The retention times of the AOSs are indicated by arrows (A2: 9.219 min, A4: 7.249 min, A6: 6.192 min). 136x104mm (300 x 300 DPI)


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Figure 7. Hydrolysis of A3 or A5 by CaLJ96. (A) HPLC analysis of degradation products of A3 and A5 by CaLJ96. (B) Negative ion ESI-MS spectrum of degradation product P4 of CaLJ96 incubated with A5. (C) Negative ion ESI-MS spectrum of degradation product P5 of CaLJ96 incubated with A5. 289x333mm (300 x 300 DPI)



Figure 8. Degradation mode of different AOSs with CaLJ96.


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TOC 191x140mm (300 x 300 DPI)


Biochemical Characterization and Substrate Degradation Mode of a

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