Discovery and Characterization of a Novel Chitosanase from

119 toward chitosan. Among them, 13 chitosanases, belonging to GH46, have been experimentally determined to produce. 120. COSs with different DPs (Fig...
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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

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

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Enzymatic Product Specificity Prediction Huihui Sun1, Xiangzhao Mao2, Na Guo2, Ling Zhao1, Rong Cao*1, Qi Liu1

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Sciences, Qingdao 266071, China

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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

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*Corresponding author: Professor Rong Cao

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

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Fishery Sciences, Qingdao 266071, China

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Tel.: +86-532-85830760

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Email: [email protected]

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

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

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

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

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

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

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

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

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

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■ INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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for the development of chitosan and COS applications.

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

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

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

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

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

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

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

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

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

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■ MATERIALS AND METHODS

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

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

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

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

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

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

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

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

program

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

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

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

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

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

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

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

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

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

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

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

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

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

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stored at −20°C.

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

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

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

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

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

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

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

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purification steps were carried out at 4 °C.

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

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

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

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

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

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

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

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

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

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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

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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

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

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

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

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colloidal chitin, and carboxymethyl cellulose (CMC).

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

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

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

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

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

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

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

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

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

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■ RESULTS AND DISCUSSION

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

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

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

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

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

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

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enzyme family GH46.

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

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

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

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

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

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

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any of the groups.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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decrease in activity after incubation at pH 7.0 (Figure 3D).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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(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.

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

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

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

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

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

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

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

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

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

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

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

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

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DP.

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■ ABBREVIATIONS

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

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

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

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■ FUNDING

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

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

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Notes

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The authors declare that they have no competing interests.

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Supporting Information

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

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

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

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

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

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2002, 66(5), 986-995.

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Figure captions

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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Tables

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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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Figures

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Figure 1.

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Figure 3.

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Figure 5.

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TOC Graphic 339

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