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Biochemical Characterization and Substrate Degradation Mode of a Novel Exo-type #-agarase from Agarivorans gilvus WH0801 Yunxiao Liang, Xiaoqing Ma, Lujia Zhang, Fuli Li, Zhen Liu, and Xiangzhao Mao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01533 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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Journal of Agricultural and Food Chemistry

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Biochemical Characterization and Substrate Degradation Mode of a Novel Exo-type

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β-agarase from Agarivorans gilvus WH0801

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Yunxiao Liang1, #, Xiaoqing Ma2, #, Lujia Zhang1, 3, Fuli Li2, Zhen Liu1, Xiangzhao Mao1, * 1

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

Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China

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School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China

8 9 10

#

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*

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

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Tel.: +86-532-82032660

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Fax: +86-532-82032272

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E-mail: [email protected]

LY and MX contributed equally to this work

Corresponding author: Xiangzhao Mao

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1

ACS Paragon Plus Environment


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Abstract

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Agarases are important hydrolytic enzymes for the biodegradation of agar. Understanding the

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degradation mode and hydrolysis products of agarases is essential for their utilization in oligosaccharide

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preparations. Herein, we cloned and expressed AgWH50B, a novel neoagarotetraose-forming β-agarase

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from Agarivorans gilvus WH0801 that has high specific activity and a fast reaction rate. AgWH50B

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consists of a C-terminal glycoside hydrolase family 50 catalytic domain with two tandem noncatalytic

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carbohydrate-binding modules (CBMs) in the N-terminus (residues 45-214 and 236-442). AgWH50B

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exhibited good enzymatic properties with high specific activity and catalytic efficiency (1523.2 U/mg

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and a Vmax of 1700 µmol/min/mg) under optimal hydrolysis conditions of pH 7.0 and 40 °C. Analysis of

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the hydrolysis products revealed that this enzyme is an exo-type β-agarase and that the dominant product

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of agarose or oligosaccharide degradation was neoagarotetraose. These findings suggest that AgWH50B

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could be utilized to yield abundant neoagarotetraose.

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Keywords: Agarase; GH50 family; Agarivorans gilvus; Degradation mode

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INTRODUCTION

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Agar, a type of hydrophilic polysaccharide, is obtained from the cell walls of red macroalgae,

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including Gelidium, Gracilaria, and Pterocladia, and consists of agarose and agaropectin.1, 2 Agarose is

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a linear chain composed of alternating monosaccharide residues of 3-O-linked β-D-galactose and

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4-O-linked 3,6-anhydro-α-L-galactose (L-AHG).1 As the main component of agar, agarose can be

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biologically degraded into agaro-oligosaccharides, which have been reported to have functional

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activities, such as being anti-inflammatory,3 antioxidative,4, 5 and effective as a prebiotic and as having a

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whitening effect on melanoma cells,6, 7 and have been widely applied in the food and medical industries.

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Agarases, which catalyze the hydrolysis of agarose, can cleave the α-1,3 linkages of agarose to

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produce agaro-oligosaccharides (AOS) (α-agarase; EC 3.2.1.158), or they can hydrolyze the β-1,4

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linkages to neoagaro-oligosaccharides (NAOS) (β-agarase; EC 3.2.1.81).8,

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successfully used in biotechnology applications such as protoplast preparation from seaweed and DNA

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gel recovery.10, 11 Until now, most of the reported agarases have been isolated from marine microbes.12

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Only two α-agarases classified as glycoside hydrolase (GH) 96 have been characterized according to the

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CAZy database (http://www.cazy.org/GH96_characterized.html) and literature,13,

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enzymes are β-agarases that were isolated from marine bacteria such as Agarivorans,15,

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Catenovulum,17 Flammeovirga,18 Streptomyces,19 and Vibrio.11, 20 Based on their amino acid sequence

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similarities, β-agarases are usually distributed into four GH families: GH16, GH50, GH86 and GH118.

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To date, several β-agarases of the GH50 family have been identified with either an endo- or exo-mode of

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action, and the main final products are neoagarotetraoses (NA4)15, 20, 21 or neoagarobioses (NA2),19, 22, 23

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respectively. The Aga50D enzyme from Saccharophagus degradans 2-40, which cleaves agarose 3


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Agarases have been

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while the other 16


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oligomers into NA2, is the first and only β-agarase from the GH50 family to have been characterized

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biochemically and structurally.24, 25 Aga50D has a complex structure with two domains: a β-sandwich

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module at the N-terminus and an (α/β)8-barrel catalytic domain at the C-terminus.24 The catalytic domain

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and part of the β-sandwich CBM-like domain forms an active site tunnel that is plugged at one end,

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suggesting an exo-mode of action, and the substrate-binding site residues are present in both the

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catalytic domain and β-sandwich domain. Two catalytic residues (Glu534 and Glu695) situated in the

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active site channel indicate a retaining catalytic mechanism.24 Another β-agarase, AgaO from

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Flammeovirga sp. strain MY04, has been proven to produce NA2 via an exo-lytic pattern.9 However, the

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substrate degradation pattern from the production of NA4, another main enzymatic hydrolysis product of

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agarose, is still unclear.

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Agarivorans gilvus WH0801 is an agarase-producing and non-endospore-forming bacterium that

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was isolated from the surface of seaweed samples.26 The β-agarase AgWH50C from A. gilvus WH0801,

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which is able to hydrolyze agarose into NA2, was previously cloned and characterized.22 In this study,

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we successfully cloned and expressed the novel NA4-forming-β-agarase AgWH50B, which has two

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tandem β-sandwich domains, using the genome sequence of A. gilvus WH0801 (GenBank accession no.

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CP013021).27 The biochemical characterization and degradation mode of AgWH50B with different

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substrates is discussed. To the best of our knowledge, this is the first time that the degradation pattern of

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an NA4-forming β-agarase in the GH50 family has been investigated.

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MATERIALS AND METHODS

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Bacterial strains and culture conditions. A. gilvus WH0801 was cultivated at 28 °C in 2216E

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medium composed of (w/v) 0.5% tryptone, 0.1% yeast extract and 0.001% ferric citrate with clean 4


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seawater as the solvent.26 The yeast extract and tryptone used for the lysogeny broth medium were

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purchased from Oxoid (Basingstoke, England). The Escherichia coli strains were grown in LB medium

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(1% tryptone, 0.5% yeast extract and 1% NaCl) at 37 °C with 100 µg/mL ampicillin (Solarbio, China)

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when required. Other analytical reagents, unless otherwise indicated, were from Sigma (USA).

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Sequence analysis. Using the gene sequence of agWH50B, the protein sequence of AgWH50B was

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obtained with DNAMAN software (Lynnon, USA). Function prediction and homology analysis of

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AgWH50B were performed using the NCBI (National Center for Biotechnology Information, USA)

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database (http://www.ncbi.nlm.nih.gov/). The structure of the protein and its signal peptide were

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predicted using SWISS-MODEL (http://www.swissmodel.expasy.org/interactive) and SignalP 3.0 Server

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(http://www.cbs.dtu.dk/services/SignalP-3.0/), respectively. Phylogenetic analysis was performed with

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MEGA version 5.0.

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Cloning, expression and purification of recombinant AgWH50B. Genomic DNA was extracted

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from A. gilvus WH0801 using a TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China). The

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primers

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(5’-TTTTTTGTAACGCAGATTATATAGATCACGGTTG-3’) were designed for amplification of the

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agWH50B gene without its signal peptide sequence. All of the primers were synthesized by BGI Tech

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(Beijing, China). The agWH50B PCR product was cloned into the pEASY-E2 expression vector, which

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contains a 6×His tag, according to the instructions of the manufacturer (TransGen Biotech, China). The

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nucleotide sequence of the inserted gene fragment was confirmed by sequencing (BGI, Beijing, China).

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The recombinant expression vector was then transformed into E. coli BL21 (DE3) chemically competent

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cells (Tiangen Biotech, China), which were grown on solid LB medium with 100 µg/mL ampicillin.

AgWH50B-F

(5’-GCCGCCGGTGAACAAGTAG-3’)

5


and

AgWH50B-R


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Transformants were selected and screened by PCR to ensure the correctness of the sequence. The

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transformants were grown in auto-induction medium, ZYP-5052 (1% tryptone, 0.5% yeast extract, 0.2%

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MgSO4, 1.25% glycerin, 0.125% glucose and 10% α-galactose), with shaking (220 rpm) for 48 h at

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20 °C after which the cells were collected by centrifugation at 8000×g for 15 min at 4 °C, resuspended

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in binding buffer (50 mM Tris-HCl and 200 mM NaCl, pH 8.0), and then disrupted by sonication. The

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supernatant was collected by centrifugation at 10,000×g for 20 min at 4 °C. The crude extract was

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filtered and purified with Ni2+-NTA resin in accordance with the manufacturer’s instructions (TransGen

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Biotech, China). Finally, the purified protein was analyzed by SDS-PAGE, and its concentration was

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determined using a BCA Protein Assay Kit (Thermo Scientific, USA) with bovine serum album (BSA)

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as the standard. The purified enzyme was then used for further enzyme activity assay and biochemical

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characterization.

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Enzyme activity assay. Enzyme activity assays were performed as previously described using the

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3,5-dinitrosalicylic acid (DNS) method with some modifications.28 Each reaction of 200 µL contained 20

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mM Tris-HCl buffer (pH 7.0), 0.2% low-melting point agarose and 25 µL purified enzyme solution (91.7

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U). After incubation at the optimum temperature for 30 min, the reaction solution was mixed with 300

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µL of DNS reagent, boiled immediately for 5 min and then cooled in a cold water bath. Samples were

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subsequently diluted with water, and the absorbance was determined at 540 nm. Heat-inactivated

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enzyme was used as a control. One unit of enzymatic activity (U) was defined as the amount of enzyme

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that produced 1 µmol of reducing sugar per min by hydrolyzing agarose under the assay conditions.

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Biochemical characterization of AgWH50B. The optimum pH was determined at different pH

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values using 20 mM sodium citrate buffer (pH 4.0 to 6.0), 20 mM Tris-HCl buffer (pH 7.0 to 8.0) and 20 6


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mM glycine-NaOH buffer (pH 9.0 to 10.0) at 40 °C for 30 min. The optimal temperature was

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determined in 20 mM Tris-HCl (pH 7.0) in a temperature range of 10-70 °C. For the thermal stability

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assay, aliquots of enzyme were incubated at various temperatures for 1 h and renatured for 2 h at 4 °C.

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Then, the residual enzyme activity was determined according to the standard method. To examine the

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effects of metal ions (Fe3+, Ni2+, Ca2+, Zn2+, Co2+, Cu2+, Mg2+) and a series of chemicals (Na2EDTA,

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SDS) on the activity of the enzyme, various chemical reagents were added to the reaction mixture at a

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final concentration of 10 mM.

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Determination of the kinetic parameters of AgWH50B. The kinetic parameters of recombinant

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AgWH50B were determined under standard conditions. The enzyme was mixed with substrates at

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concentrations ranging from 1 mg/mL to 12 mg/mL. The Km, Vmax and kcat were calculated using

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GraphPad Prism 5 (GraphPad Software, Inc. USA).

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Degradation pattern analysis of AgWH50B. The degradation products were analyzed by thin

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layer chromatography (TLC). Purified AgWH50B was incubated with 0.3% agarose in Tris–HCl buffer

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at the optimal temperature for 24 h. Aliquots of the reaction product were loaded on silica gel 60 TLC

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plates (Merck, Germany). The plates were developed in a developing solvent composed of

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n-butanol/acetic acid/water (2:1:1 by volume). Then, the spots were visualized by soaking in an ethanol

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solution containing 10% (v/v) H2SO4, followed by heating at 95 °C for 5 min, and each oligosaccharide

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was preliminarily ascertained by colored spots on the TLC plates. NAOS standards (Qingdao BZ Oligo

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Biotech, China) dissolved into the same buffer were used as oligosaccharide markers. To determine the

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sugar content at certain reaction times, both the samples and standards were analyzed by HPLC using a

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Sugar-Pak I column (Waters, USA) under the following conditions: the mobile phase was EDTA calcium 7



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disodium (50 mg/mL), the column temperature was 75 °C, the flow velocity was 0.5 mL/min, and the

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detector was a refractive index detector (RID).

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To estimate the substrate with the lowest DP, highly purified NAOS solutions including NA4,

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neoagarohexaose (NA6) and neoagarooctaose (NA8), were incubated with the enzyme individually.

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After incubation at 40 °C for 12 h, the reaction was stopped by boiling, and the product was analyzed

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using both TLC and HPLC.

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RESULTS AND DISCUSSION

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Sequence analysis of the β-agarase gene. The opening reading frame (ORF) of agWH50B

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encodes a 955 amino acid protein (GenBank accession no. KY417136), the molecular mass and

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isoelectric point of which were predicted to be 105 kDa and 4.42, respectively. The signal peptide

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predicted by SignalP 3.0 Server includes 32 amino acid residues located in the N-terminus of the

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putative protein. Based on sequence alignment, AgWH50B shares its highest sequence identity of 73%

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with two GH50 β-agarases, NA4-producing enzyme HZ2 from Agarivorans sp. HZ105 (GenBank

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accession no. ADY17919.1) and AgD02 from Agarivorans sp. QM38 (GenBank accession no.

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ABM90422.1). However, phylogenetic analysis indicated that AgWH50B clusters with a different

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branch of the GH50 family (Fig. 1). According to homology modeling and amino acid alignment,

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AgWH50B contains two tandem β-sandwich CBM-like domains in its N-terminus (residues 45-214 and

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236-442) and a catalytic domain (residues 472-955) in its C-terminus (Fig. 2A), different from two

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reported NA2-producing GH50 β-agarases, AgWH50C and Aga50D, both of which consist of one

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N-terminal CBM and a C-terminal catalytic domain.22, 25 These features suggested that AgWH50B is a

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novel β-agarase from Agarivorans sp. 8


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Cloning and expression of AgWH50B. The full length AgWH50B gene was cloned and

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successfully expressed in E. coli BL21 (DE3) with a C-terminal His tag. The recombinant protein was

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purified and detected by SDS-PAGE, which showed one evident band corresponding to 105 kDa (Fig.

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2B). This result indicated that the purified recombinant protein was appropriate for analyzing the

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properties of the enzyme.

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The specific activity of purified AgWH50B under standard conditions (40 °C, pH 7.0, 20 mM

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Tris-HCl) was 1523.2 U/mg, and it had a Vmax of 1700 µmol/min/mg for agarose, which are significantly

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higher than other β-agarases, including AgaA (349.3 U/mg and 901.9 µmol/min/mg) and AgaB34 (242

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U/mg and 50 µmol/min/mg).16, 29 AgWH50B also showed a distinct advantage over the other GH50

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agarases Aga50D (17.9 U/mg), rHZ2 (235 U/mg) and AgaA34 (25.54 U/mg) in terms of its Vmax value,12,

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15, 25

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which suggests that AgWH50B has huge potential for industrial applications. Effects of temperature and pH on the activity of AgWH50B. Using 0.2% of agarose as substrate,

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the optimal temperature and pH of AgWH50B were determined to be 40 °C and 7.0, respectively (Fig.

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3A and B). The enzyme was quite stable at 40 °C, retaining more than 90% of its activity after

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incubation for 1 h (Fig. 3A); however, inactivation of AgWH50B occurred above 50 °C. Meanwhile,

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AgWH50B retained more than 80% of its activity in a pH range from 6.0 to 10.0 (Fig. 3C). These results

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indicate that AgWH50B could degrade agarose in a wide pH range similar to a recently reported GH16

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family thermostable and pH-stable β-agarase from the deep-sea bacterium Flammeovirga sp. OC4.30 In

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terms of the optimal temperature and pH, AgWH50B was similar to the β-agarases HZ2 from

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Agarivorans sp. HZ105 and AgaG1 from Alteromonas sp. GNUM1.15,

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AgWH50B has excellent hydrolytic ability in high temperature environments. 9


31

The results suggest that


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Effects of chemicals on agarase activities. The activity of recombinant AgWH50B was evidently

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disabled by some heavy metal ions such as Fe3+, Zn2+ and Cu2+ and was mildly inhibited by Ni2+,

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Co2+and Mg2+ at a concentration of 10 mM (Fig. 3D). For the other chemicals investigated, the enzyme

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was completely inhibited by Na2EDTA. These inhibitory effects may occur because divalent metal ions

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as well as EDTA have affinity interactions with the SH, CO and NH functional groups of the enzyme,

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which causes structural changes in the catalytic moieties and thus decreases activity.32, 33 SDS strongly

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reduced the enzyme activity of AgWH50B to less than 30%, which has been found with other

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β-agarases,17, 34 indicating that this enzyme is not resistant to such a surfactant.

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Agarose degradation pattern analysis. The hydrolysis products detected by TLC showed that

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NA4 was produced at the beginning of the reaction, and then a small amount of NA2 as well as larger

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NAOS such as NA6 and NA8 were observed gradually after incubation with agarose for 10 min. After

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24 h, the hydrolysis of agarose with AgWH50B led to the production of NA4 and NA2 with a small

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amount of NAOS larger than NA4 (Fig. 4A). These preliminary results suggested that the final products

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of AgWH50B may be both NA4 and NA2.

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To further confirm the final products, the hydrolyzed samples and NAOS standard oligosaccharide

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markers (NA2, NA4, NA6 and NA8) were analyzed by HPLC. As shown in Fig. 4B, during agarose

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degradation by AgWH50B, only one peak with a retention time corresponding to NA4 was observed

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after 1 min. The production of NA6 and NA2 was subsequently detected after 10 min. After 24 h, peaks

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corresponding to NA2, NA4 and NA6 still remained in the degradation products (Fig. 4B). The contents

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of NA2, NA4 and NA6 were also calculated by quantitative analysis. After one minute of reaction, the

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content of the NA4 product was 302.49 mg/L with no trace amounts of NA6 (0.00 mg/L) or NA2 (0.00 10


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mg/L). After hydrolysis for 24 h, the content of NA4 reached 900.98 mg/L, which was much higher than

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those of NA2 (53.26 mg/L) and NA6 (139.74 mg/L). These results suggested that the reaction products

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are NA2, NA6 and NA8, with NA4 being the dominant product and that AgWH50B is an exo-lytic

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NA4-producing β-agarase.

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AgWH50B is one of the few reported GH50 family agarases that can produce NA4. Thus far,

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several agarases have been reported to have this activity, such as HZ2 from Agarivorans sp. HZ105 and

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Aga41A from Vibrio sp. CN41, which shares the sequence identity of 73% and 50% with AgWH50B,

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respectively.15, 20 Both HZ2 and Aga41A are endo-lytic β-agarases that randomly depolymerize agarose

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into product with a logarithmic decrease in its average molecular weight during the reaction.35 However,

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in the AgWH50B reaction system, NA4 was produced initially and continued to increase gradually,

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suggesting that AgWH50B can cleave the four-sugar unit processively from the non-reducing end of

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long-chain agarose. Thus, we infer that AgWH50B is an exo-type NA4-producing β-agarase. To the best

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of our knowledge, AgWH50B is the first exo-lytic β-agarase to be reported, which cleaves one

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four-sugar unit at a time.

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TLC and HPLC analysis of the oligosaccharide degradation products. To further investigate

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the cleavage patterns of AgWH50B and to determine its smallest substrate, NAOS with different lengths

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were used as substrates. The NAOS were dissolved individually in deionized water and incubated with

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AgWH50B at 40 °C for 12 h. The samples were then analyzed by TLC and HPLC (Fig. 5). TLC analysis

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showed that with NA4 as the substrate, no new product was detected even after 12 h (Fig. 5A), which

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indicated that AgWH50B cannot hydrolyze NA4 into NA2. However, NA6 and NA8 were degraded into

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NA4, with NA2 as an accompanying product when NA6 was the substrate (Fig. 5B and 5C). HPLC 11



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quantitative analysis further confirmed the TLC results (Fig. 5D and 5E) that NA2 was produced only

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when NA6 was the substrate. NA4 could not be degraded further, and NA8 could only be hydrolyzed

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into NA4 (Fig. 5F). Combined with the product analysis of agarose hydrolysis, the production of NA2

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from agarose degradation was suggested to result from the further hydrolysis of NA6 but not NA4.

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Therefore, the smallest oligosaccharide substrate of AgWH50B is NA6, and the main hydrolysis product

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is NA4.

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Neoagaro-oligosaccharides have been reported to have potential application values because of their

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high biological activity, which is connected to their degree of polymerization. In our previous research,

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we demonstrated that NA4 has the potential to alleviate intense exercise-induced fatigue by modulating

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the structure and function of the gut microbiome.36 This finding shows that neoagaro-oligosaccharides

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have huge application prospects in the food industry, including as antioxidative functional ingredients in

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food products. In addition, NA4 or NA2 can be further degraded by an α-neoagarobiose hydrolase into

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D-galactose and 3, 6-anhydro-L-galactose (L-AHG).37 L-AHG is also valuable due to its

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anti-inflammatory activity and skin whitening function.7 The TLC and HPLC assays demonstrated that

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AgWH50B is an exo-lytic β-agarase that yields NA4 as its main product during agarose degradation. So

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far, several β-agarases have been reported to produce NA4 by degrading agarose. Except AgWH50A,

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which was reported to show both exo- and endo-lytic features,21 all the other NA4-forming agarases

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were endo-lytic enzymes, including rHZ2 and AgaA41 from GH50 family as well as AgaAc and AgaA

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from GH16 family.15, 16, 20, 38 The glucoside hydrolase exo-mode of action has distinct advantages in

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terms of oligosaccharide purity compared to the endo-mode of action because endo-type agarases

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usually produce two or more main neoagaro-oligosaccharides, which increases the complexity of 12


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oligosaccharide purification and decreases the monomer yield. In addition, as an NA4-producing

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β-agarase, AgWH50B showed a significant advantage in reaction rate. Compared to other

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NA4-producing agarases such as AgaB34,29 AgaA,38 and rHZ2,15 the specific activity of AgWH50B

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exhibited more than 5, 3 and 6 times higher, respectively. Therefore, the GH50 family β-agarase

244

AgWH50B is a key tool for NA4 preparation, and the study of its biochemical characteristics will be of

245

great significance.

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In conclusion, AgWH50B is a novel NA4-producing β-agarase isolated from A. gilvus WH0801

247

that belongs to the GH50 family and has good enzyme characteristics, including a high specific activity

248

and

249

neoagaro-oligosaccharides by an exo-lytic pattern to yield abundant NA4, which can be utilized in

250

functional food applications.

251

ABBREVIATIONS

fast

reaction

rate.

The

enzyme

can

effectively

hydrolyze

agarose

as

well

as

252

GH: glycoside hydrolase; AOS: agaro-oligosaccharides, NAOS: neoagaro-oligosaccharides;

253

L-AHG: 3, 6-anhydro-L-galactose; DP: degree of polymerization; DNA, deoxyribonucleic acid; ORF:

254

open reading frame; MEGA: molecular evolutionary genetics analysis; LB: Luria-Bertani; NTA:

255

nitrilotriacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DNS,

256

3,5-dinitrosalicylic acid; EDTA: ethylene diamine tetraacetic; TLC: thin layer chromatography; HPLC:

257

high-performance liquid chromatography; NA2: neoagarobiose; NA4: neoagarotetraose; NA6:

258

neoagarohexaose; NA8: neoagarooctaose.

259

FUNDING

260

This work was supported by the National Natural Science Foundation of China (31471607 and 13



261

31271923) and the China Postdoctoral Science Foundation (2016M590661).

262

Notes

263

The authors declare that they have no competing interests.

264

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267

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

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(13)Potin, P.; Richard, C.; Rochas, C.; Kloareg, B., Purification and characterization of the α-agarase

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β-Agarase, AgaB34, from Agarivorans albus YKW-34. J. Microbiol. Biotechnol. 2009, 19,

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Thermostable and pH-Stable beta-Agarase from Deep-Sea Bacterium Flammeovirga Sp. OC4. J.

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expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and

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exo-β-agarase from an endophytic marine bacterium and its catalytic potential in bioconversion

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of red algal cell wall polysaccharides into galactans. Biomass Bioenerg. 2013, 49, 290-298.

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β-agarase gene from Agarivorans sp. JAMB-A11 in Bacillus subtilis and enzymic properties of

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enzyme in the agarolytic pathway, alpha-neoagarobiose hydrolase from Saccharophagus

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Figure captions

371

Figure 1 Phylogenetic analysis of AgWH50B with other characterized GH50 agarases. The

372

neighbor-joining tree was obtained using MEGA version 5.0 software. The numbers on the

373

branches indicate the bootstrap confidence values from 1,000 replicates. The scale bar indicates 0.1

374

substitutions per site.

375

Figure 2 Sequence characterization of AgWH50B. A. The module structure of AgWH50B. The

376

protein contains an N-terminal signal peptide (residues 1-32, in black), two tandem CBM-like

377

domains (residues 45-214 and 236-442, in dark gray and light gray, respectively) and a C-terminal

378

catalytic domain (residues 472-955, in pink). B. SDS-PAGE analysis of purified AgWH50B. Lane

379

M, protein molecular mass marker, and Lane 1, purified AgWH50B.

380

Figure 3 Biochemical characteristics of AgWH50B. A. Effects of temperature on the activity and

381

thermostability of rAgWH50B. B. Effects of pH on the activity of AgWH50B; pH 4.0 to 6.0, citric

382

acid-Na buffer; pH 7.0 to 8.0, Tris-HCl buffer; and pH 9.0 to 10.0, glycine-NaOH buffer. C. Effects

383

of pH on the stability of AgWH50B. The protein was treated at different pH values, and the residual

384

activity was analyzed at standard conditions. D. Effects of cations, anions, and other chemicals on

385

the enzyme activity of AgWH50B.

386

Figure 4 Agarose degradation patterns of AgWH50B. A. TLC analysis of the agarose degradation

387

products of AgWH50B. The reactions were performed at 40 °C for 24 h in 20 mM Tris-HCl buffer

388

(pH 7.0) containing 0.3% agarose. M: standard oligosaccharide markers and (-): negative control. B.

389

HPLC analysis of the agarose degradation products of AgWH50B. The retention times of the

390

standard NAOS are indicated by arrows. 20


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391


Figure 5 Oligosaccharide degradation products of AgWH50B. TLC analysis of the final products

392

from NA4 (A), NA6 (B) and NA8 (C) hydrolysis by AgWH50B. M: standard oligosaccharide

393

markers and (-): the negative control in which boiled AgWH50B was used. HPLC analysis of the

394

final degradation products from NA4 (D), NA6 (E) and NA8 (F) hydrolysis by AgWH50B.

21



Figures

Figure 1 Phylogenetic analysis of AgWH50B with other characterized GH50 agarases.

22


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Figure 2 Sequence characterization of AgWH50B.

23



Figure 3 Biochemical characteristics of AgWH50B.

24


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Figure 4 Agarose degradation patterns of AgWH50B.

25



Figure 5 Oligosaccharide degradation products of AgWH50B.

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TOC Graphic

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Biochemical Characterization and Substrate Degradation Mode of a

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