Discovery and Characterization of a Novel Chitosanase from

Subscriber access provided by UNIV OF DURHAM

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 Downloaded from http://pubs.acs.org on April 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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

3 4

1

5

Sciences, Qingdao 266071, China

6

2

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

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

7 8 9 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: [email protected]

16

ACS Paragon Plus Environment


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


Page 2 of 24

Page 3 of 24

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,



Page 4 of 24

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 70

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

program


Page 5 of 24


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.



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


Page 6 of 24

Page 7 of 24


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



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 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


Page 8 of 24

Page 9 of 24


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



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.


Page 10 of 24

Page 11 of 24


197

■ REFERENCES

198

(1) Shinya, S.; Fukamizo, T. Interaction between chitosan and its related enzymes: A review. Int. J. Biol. Macromol.

199

2017. http://dx.doi.org/10.1016/j.ijbiomac.2017.02.040.

200

(2) Younes, I.; Rinaudo, M. Chitin and chitosan preparation from marine sources. Structure, properties and applications.

201

Mar. Drugs. 2015, 13, 1133-1174.

202

(3) Su, C. X.; Wang, D. M.; Yao, L. M.; Yu, Z. L. Purification, characterization, and gene cloning of a chitosanase from

203

Bacillus species strain S65. J. Agric. Food Chem. 2006, 54(12), 4208-14.

204

(4) Alishahi, A.; Aïder, M. Applications of chitosan in the seafood industry and aquaculture: A review. Food Bioprocess

205

Technol. 2011, 5, 817-830.

206

(5) Jayakumar, R.; Menon, D.; Manzoor, K.; Nair, S. V.; Tamura, H. Biomedical applications of chitin and chitosan

207

based nanomaterials-a short review. Carbohydr. Polym. 2010, 82, 227-232.

208

(6) Antonia, M.; Ana I, R. M.; Nieves, C.; Cecilia, G.; Gabriela, I. Enzymatic generation of chitooligosaccharides from

209

chitosan using soluble and immobilized glycosyltransferase (branchzyme). J. Agric. Food Chem. 2013, 61, 10360-10367.

210

(7) Aranaz, I.; Mengíbar, M.; Harris, R.; Paños, I.; Miralles, B.; Acosta, N.; Galed, G.; Heras, A. Functional

211

characterization of chitin and chitosan. Curr. Chem. Biol. 2009, 3, 203-230.

212

(8) Xia, W. S.; Liu, P.; Zhang, J. L.; Chen, J. Biological activities of chitosan and chitooligosaccharides. Food

213

Hydrocolloid. 2011, 25, 170-179.

214

(9) Park, P. J.; Je, J. Y.; Kim, S. K. Free radical scavenging activity of chitooligosaccharides by electron spin resonance

215

spectrometry. J. Agric. Food Chem. 2003, 51(16), 4624-4627.

216

(10) Maria I, Q. V.; Berit B, A.; John, R.; Morten, S.; Vincent G H, E.; Robert W, H. Adherence

217

inhibition of enteropathogenic Escherichia coli by chitooligosaccharides with specific degrees of acetylation and

218

polymerization. J. Agric. Food Chem. 2013, 61, 2748-2754.



219

(11) Aam, B. B.; Heggset, E. B.; Norberg, A. L.; Sørlie, M.; Varum, K. M.; Eijsink, V. G. H. Production of

220

chitooligosaccharides and their potential applications in medicine. Mar. Drugs. 2010, 8, 1482-1517.

221

(12) Benhabiles, M. S.; Salah, R.; Lounici, H.; Drouiche, N.; Goosen, M. F. A.; Mameri, N. Antibacterial activity of

222

chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrocolloids. 2012, 29, 48-56.

223

(13) Wu, H.; Aam, B. B.; Wang, W.; Norberg, A. L.; Sørlie, M.; Eijsink, V. G. H.; Du, Y. Inhibition of angiogenesis by

224

chitooligosaccharides with specific degrees of acetylation and polymerization. Carbohydr. Polym. 2012, 89, 511-518.

225

(14) Yan, Y. L.; Hu, Y.; Simpson, D. J.; Michael G, G. Enzymatic synthesis and purification of galactosylated chitosan

226

oligosaccharides reducing adhesion of enterotoxigenic Escherichia coli K88. J. Agric. Food Chem. 2017, 65(25).

227

(15) Hrynets, Y.; Ndagijimana, M.; Betti, M. Studies on the formation of Maillard and caramelization products from

228

glucosamine incubated at 37 °C. J. Agric. Food Chem.2015, 63, 6249-6261.

229

(16) Mourya, V. K.; Inamdar, N. N.; Choudhari, Y. M. Chitooligosaccharides: synthesis, characterization and

230

applications. Polym. Sci. Ser. A. 2011, 53, 583-612.

231

(17) Cabrera, J. C.; Van Cutsem, P. Preparation of chitooligosaccharides with degree of polymerization higher than 6 by

232

acid or enzymatic degradation of chitosan. Biochem. Eng. J. 2005, 25, 165-172.

233

(18) Zhu, X. F.; Tan, H. Q.; Zhu, C.; Liao, L.; Zhang, X. Q.; Wu, M. Cloning and overexpression of a new chitosanase

234

gene from Penicillium sp. D-1. AMB Express, 2012, 2(1), 13.

235

(19) Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The carbohydrate-active

236

enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 2009, 37, D233-D238.

237

(20) Bueren, A. L. V.; Ghinet, M. G.; Gregg, K.; Fleury, A.; Brzezinski, R.; Boraston1, A. B. The structural basis of

238

substrate recognition in an exo-β-D-glucosaminidase involved in chitosan hydrolysis. J. Mol. Biol. 2009, 385, 131-139.

239

(21) Thadathil, N.; Velappan, S. P. Recent developments in chitosanase research and its biotechnological applications: A

240

review. Food Chem. 2014, 150(1), 392-399.


Page 12 of 24

Page 13 of 24


241

(22) Yao, Y. Y.; Shrestha, K. L.; Wu, Y. J.; Tasi, H. J.; Chen, C. C.; Yang, J. M.; Ando, A.; Cheng, C. Y.; Li, Y. K.

242

Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase. Protein Eng., Des.

243

Sel. 2008, 21(9), 561-566.

244

(23) Le, D. F.; Bazinet, L.; Furtos, A.; Venne, K.; Brunet, S.; Alexandru, M. M. Separation of chitosan oligomers by

245

immobilized metal affinity chromatography. J. Chromatogr. A. 2008, 1194(2), 165-171.

246

(24) Shee, F. L. T.; Arul, J.; Brunet, S.; Bazinet, L. Chitosan solubilization by bipolar membrane electroacidification:

247

reduction of membrane fouling. J. Membr. Sci. 2007, 290(1), 29-35.

248

(25) Naqvi, S.; Moerschbacher, B. M. The cell factory approach toward biotechnological production of high-value

249

chitosan oligomers and their derivatives: an update. Crit. Rev. Biotechnol. 2017, 37(1), 11-25.

250

(26) Das, S. N.; Madhuprakash, J.; Sarma, P. V.; Purushotham, P.; Suma, K.; Manjeet, K.; Rambabu, S.; Gueddari, N. E.;

251

Moerschbacher, M. M.; Podile, A. R. Biotechnological approaches for field applications of chitooligosaccharides (COS)

252

to induce innate immunity in plants. Crit. Rev. Biotechnol. 2015, 35(1), 29-43.

253

(27) Kohlhoff, M.; Niehues, A.; Wattjes, J.; Bénéteau, J.; Cord-Landwehr, S.; Gueddari, N. E. E.; Bernard, F.; Rivera-

254

Rodriguez, G. R.; Moerschbacher, B. M. Chitinosanase: A fungal chitosan hydrolyzing enzyme with a new and unusually

255

specific cleavage pattern. Carbohydr. Polym. 2017, 174, 1121-1128.

256

(28) Cote, N.; Fleury, A.; Dumont-Blanchette, E.; Fukamizo, T.; Mitsutomi, M.; Brzezinski, R. Two exo-β-D-

257

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

258

Biochem. J. 2006, 394, 675-686.

259

(29) Nanjo, F.; Katsumi, R.; Sakai, K. Purification and characterization of an exo-β-D-glucosaminidase, a novel type of

260

enzyme, from Nocardia orientalis. J. Biol. Chem. 1990, 265, 10088-10094.



261

(30) Nogawa, M.; Takahashi, H.; Kashiwagi, A.; Ohshima, K.; Okada, H.; Morikawa, Y. Purification and

262

characterization of exo-β-D-glucosaminidase from a cellulolytic fungus, Trichoderma reesei PC-3-7. Appl. Environ.

263

Microbiol. 1998, 64, 890-895.

264

(31) Fukamizo, T.; Fleury, A.; Côté, N.; Mitsutomi, M.; Brzezinski, R. Exo-β-D-glucosaminidase from Amycolatopsis

265

orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism. Glycobiology. 2006, 16, 1064-1072.

266

(32) Ike, M.; Isami, K.; Tanabe, Y.; Nogawa, M.; Ogasawara, W.; Okada, H.; Morikawa, Y. Cloning and heterologous

267

expression of the exo-β-D-glucosaminidase-encoding gene (gls93) from a filamentous fungus, Trichoderma reesei PC-3-

268

7. Appl. Microbiol. Biotechnol. 2006, 72, 687-695.

269

(33) Ando, A.; Noguchi, K.; Yanagi, M.; Shinoyama, H.; Kagawa, Y.; Hirata, H.; Yabuki, M.; Fujii, T. Primary structure

270

of chitosanase produced by Bacillus circulans MH-K1. J. Gen. Appl. Microbiol. 1992, 38(2), 135-144.

271

(34) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.;

272

Wallace, I. M.; Wilm, A.; Lopez, R.; et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23, 2947-2948.

273

(35) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis

274

version 6.0. Mol. Biol. Evol. 2013, 30, 2725-2729.

275

(36) Miller, F. M.; Gamson, R. M.; Kramer, D. N. A study of the physical and chemical properties of the esters of

276

indophenols I. preparation. J. Org. Chem. 1959, 24(11), 1742-1747.

277

(37) Masson, J. Y.; Boucher, I.; Neugebauer, W. A.; . A new chitosanase gene from a Nocardioides sp. is a third member

278

of glycosyl hydrolase family 46. Microbiology. 1995, 141 (10), 2629-2635.

279

(38) Okajima, S.; Ando, A.; Shinoyama, H.; Fujii, T. Purification and characterization of an extracellular chitosanase

280

produced by Amycolatopsis sp. CsO-2. J. Ferment. Bioeng. 1994, 77(6), 617-620.

281

(39) Boucher, I.; Dupuy, A.; Vidal, P.; Neugebauer, W. A.; Brzezinski, R. Purification and characterization of a

282

chitosanase from Streptomyces N174. Appl. Microbiol. Biotechnol. 1992, 38, 188-193.


Page 14 of 24

Page 15 of 24


283

(40) Takasuka, T. E.; Bianchetti, C. M.; Tobimatsu, Y.; Bergeman, L. F.; Ralph, J.; Fox, B. G. Structure-guided analysis

284

of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E. Proteins:

285

Struct., Funct., Bioinf. 2014, 82(7), 1245-1257.

286

(41) Johnsen, M. G.; Hansen, O. C.; Stougaard, P. Isolation, characterization and heterologous expression of a novel

287

chitosanase from Janthinobacterium sp. strain 4239. Microb. Cell Fact. 2010, 9(1), 5.

288

(42) Lyu, Q.; Wang, S.; Xu, W.; Han, B.; Liu, W.; David, N. M. J.; Liu, W. Structural insights into the substrate-binding

289

mechanism for a novel chitosanase. Biochem. J. 2014, 461(2), 335-345.

290

(43) Lee, Y. S.; Yoo, J. S.; Chung, S. Y.; Lee, Y. C.; Cho, Y. S.; Choi, Y. L. Cloning, purification, and characterization

291

of chitosanase from Bacillus sp. DAU101. Appl. Microbiol. Biotechnol. 2006, 73(1):113-121.

292

(44) Yoon, H. G.; Kim, H. Y.; Lim, Y. H.; Kim, H. K.; Shin, D. H.; Hong, B. S.; Cho, H. Y. Thermostable chitosanase

293

from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme. Appl. Environ.

294

Microbiol. 2000, 66(9), 3727-3734.

295

(45) Li, H.; Fei, Z.; Gong, J. S.; Yang, T.; Xu, Z. H.; Shi, J. S. Screening and characterization of a highly active

296

chitosanase based on metagenomic technology. J. Mol. Catal. B: Enzym. 2015, 111, 29-35.

297

(46) Chiang, C. L.; Chang, C. T.; Sung, H. Y. Purification and properties of chitosanase from a mutant of Bacillus

298

subtilis IMR-NK1. Enzyme Microb. Technol. 2003, 32(2), 260-267.

299

(47) Shimosaka, M.; Fukumori, Y.; Zhang, X. Y.; He, N. J.; Kodaira, R.; Okazaki, M. Molecular cloning and

300

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

301

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

302

(48) Yoon, H. G.; Lee, K. H.; Kim, H. Y.; Kim, H. K.; Shin, D. H.; Hong, B. S.; Cho, H. Y. Gene cloning and

303

biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108. Biosci. Biotechnol. Biochem.

304

2002, 66(5), 986-995.



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


Page 16 of 24

Page 17 of 24


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



323

Figures

324

Figure 1.

325


Page 18 of 24

Page 19 of 24

326


Figure 2.

327



328

Figure 3.

329 330


Page 20 of 24

Page 21 of 24

331


Figure 4.

332



333

Figure 5.

334 335


Page 22 of 24

Page 23 of 24

336


Figure 6.

337



338

TOC Graphic 339


Page 24 of 24

Discovery and Characterization of a Novel Chitosanase from

Recommend Documents