Characterization of a novel bifunctional mannuronan C-5 epimerase and alginate lyase from Pseudomonas mendocina. sp. DICP-70

Journal Pre-proof Characterization of a novel bifunctional mannuronan C-5 epimerase and alginate lyase from Pseudomonas mendocina. sp. DICP-70

Ming Sun, Chu Sun, Tang Li, Kuikui Li, Shenggang Yan, Heng Yin PII:

S0141-8130(19)38952-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.126

Reference:

BIOMAC 14743

To appear in:

International Journal of Biological Macromolecules

Received date:

5 November 2019

Revised date:

10 February 2020

Accepted date:

12 February 2020

Please cite this article as: M. Sun, C. Sun, T. Li, et al., Characterization of a novel bifunctional mannuronan C-5 epimerase and alginate lyase from Pseudomonas mendocina. sp. DICP-70, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.02.126

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© 2018 Published by Elsevier.

Journal Pre-proof Characterization of a Novel Bifunctional Mannuronan C-5 Epimerase and Alginate Lyase from Pseudomonas mendocina. sp. DICP-70 Ming Sun1, 2, Chu Sun 3, Tang Li 1, Kuikui Li 1, Shenggang Yan 2, Heng Yin 1 * 1. Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 2. Dalian Maritime University, Dalian, China

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3. University of California, Berkeley, Berkeley, USA

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*: corresponding author. Tel. (0411) 84379061; E-mail: [email protected]

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Journal Pre-proof

Abstract Alginate is a family of industrially important linear polymers consisting of β-Dmannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The function of alginate is closely related to the ratio of M/G. Mannuronan C-5 epimerase, which converts M to G, is a key enzyme involved in the biosynthesis of alginate. A new mannuronan C-5 epimerase isolated from Pseudomonas mendocina. sp. DICP-70

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named PmC5A was characterized in this study. From the 1H NMR analysis of the products, we have found that PmC5A possesses alginate lyase function in addition to mannuronan C-5-epimerase. The optimal pH and temperature of lyase and epimerase

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activity toward PolyMG and G-blocks.

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were found to be 8.0, 9.0 and 40℃, 30℃, respectively. PmC5A also shows lyase

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Keywords: Alginate; Mannuronan C-5 epimerase; Alginate lyase

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Journal Pre-proof

1. Introduction Alginate is a linear copolymer, widely used for medical purposes, including applied as the oral delivery matrices for microencapsulation of cells [1], and as the nanomaterials and scaffolds for tissue engineering [2-4]. It consists of β-Dmannuronic acid (M) and its C5 epimer α-L-guluronic acid (G). In alginate polymers, these residues are arranged in three possible ways: continuous M residues (M-blocks),

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continuous G residues (G-blocks) and alternate M and G residues (MG-blocks). Different lengths and distributions of the blocks have different characters

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corresponding to different applications. For example, in the presence of divalent metal ions, such as Cu2+, Zn2+, Sr2+, Pb2+, Cd2+ and Ca2+, GG-blocks are easier to form

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“egg-box” junctions [5, 6] by cross linking antiparallel chains and thus producing

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strong hydrogel [7, 8]. In comparison, MM-blocks have lower affinity to these ions.

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In addition, alginate oligosaccharides (AOS) have attracted more attention for theirs diverse biological activities, such as increasing fruit quality and storage life [9],

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because postharvest treatment of seaweed oligosaccharides (AOS) delayed the

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accumulation of ABA and ABA conjugates, and inhibited the expression of ABA signaling genes, thereby prolonging the shelf life of fruits [9]. Since mannuronan C-5 epimerase and alginate lyase could alter the distribution of blocks and the M/G ratio, both essential to designing alginates with desired properties, characterizing the properties of these enzymes and understanding their mechanisms has become a popular field of research consequently.

Mannuronan C-5 epimerases, converting M to its C5 epimer G, have been found in brown algae [10] as well as bacteria, Pseudomonas sp. and Azotobacter vinelandii, however, the structure and function of C5 epimerases are well characterized only in 3 / 31

Journal Pre-proof bacteria [11]. Pseudomonas only contains a periplasmic epimerase (AlgG) [12]. In contrast, Azotobacter vinelandii secrets a family of seven mannuronan C-5 epimerases (AlgE1-E7) which are composed of varying numbers of A-modules (about 385 amino acids) and R-modules (about 150 amino acids) [13, 14]. Moreover, except mannuronan C-5 epimerase, the most studied enzyme in alginate biosynthesis and metabolites is alginate lyase. In the Carbohydrate-Active enzymes (CAZY) database, alginate lyases belong to ten polysaccharide lyase (PL) families (PL5, 6, 7, 14, 15, 17,

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18, 32, 34 and 36) [15, 16]. Alginate lyases catalyze the degradation of alginates by a β-elimination mechanism and produce oligosaccharides with unsaturated uronic acid

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at the nonreducing terminus and saturated uronic acid monomers at reducing end.

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In this study, we characterized a new periplasmic enzyme isolated from Pseudomonas

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mendocina. sp. DICP-70 named PmC5A. PmC5A possesses two functions of mannuronan C-5-epimerase and alginate lyase and we systematically studied the

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bifunctional characteristics of the enzyme, including optimal reaction conditions,

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substrate preference, and the effect of metal ions on activity.

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2. Experimental procedures 2.1 Bacterium strain selection and Identification

The entire digestive tract of a sea urchin caught in east coast of Dalian, China was soaked in 100 ml Luria-Bertani (LB) liquid medium (0.5g Tryptone, 1g NaCl and 0.5g yeast extract per 100 ml aqueous solution) and incubated at room temperature in a shaker incubator at 200 r/min at 28℃ for 12hrs. The seeded culture was diluted 105 times with sterile water and spread onto a solid separating plate (0.5% (g/ml) NaCl, 0.1% (g/ml) KH2PO4, 0.05% (g/ml) MgSO4•7H2O, 0.002% (g/ml) FeSO4•7H2O, 4 / 31

Journal Pre-proof 0.5% (g/ml) (NH4)2SO4, 0.3% (g/ml) sodium alginate and 3% (g/ml) agar). The plate was incubated at 20℃ for two days, from which a colony was picked and separated three more times with the same procedure to obtain a pure strain. The obtained colony was picked and inoculated in LB medium at 28℃ for 12hrs with constant shaking at 200 r/min.

The bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit

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(TIANGEN, Beijing, China), following the instruction provided by the manufacturer. To identify the selected bacterium, its 16S rRNA was amplified through Polymerase Chain

Reaction

(PCR)

using

a

pair

of

universal

primers

27F

(5′-

-p

AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-

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3′). The PCR product was sent to Beijing Genomics Institute (BGI) for sequencing.

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The phylogenetic tree was constructed using MEGA7 based on the sequencing result.

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2.2 Cloning, heterogeneous expression and purification of PmC5A from

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Pseudomonas mendocina. sp. DICP-70

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According to the information from phylogenetic tree, the strain belongs to Pseudomonas mendocina and was named as Pseudomonas mendocina. sp. DICP-70. The whole genome from Pseudomonas medocina (GenBank: CP000680.1) was annotated on RAST website (http://rast.nmpdr.org/). And PmC5A coding sequence was obtained from Pseudomonas medocina (GenBank: CP000680.1), a pair of degenerate primers was designed. A pair of degenerate primers was designed: the sequence

of

the

forward

primer

was

5

’

-

ATACATATGAACCCGMHGSARSACCAGTTC-3’ (the added NdeI restriction site

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Journal Pre-proof is underlined); the reverse primer bearing an XhoI restriction site (underlined) was 5′TATCTCGAGRTTGGTCAKYKKCTCGG-3'. After amplification by PCR and digestion with NdeI and XhoI, the target gene with an added sequence coding for the 6 ⅹ His-tag at the N-terminus of the recombinant enzyme was ligated into the NdeIXhoI sites of the PET21a vector (Novagen, San Diego, CA, USA) with a 6ⅹ His-tag at the N-terminus of the recombinant enzyme. Vectors containing the recombinant

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gene were transformed into E. coli competent DH5α cells (Biomed, Beijing, China). The integrity of the recovered plasmid was confirmed by PCR and sequencing

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(service provided by BGI) with the primers described above and the sequencing

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result. The recombinant plasmid was then transformed into competent E. coli BL21(DE3) cells (Biomed, Beijing, China). The cells were incubated in liquid LB

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medium containing 1mM Ampicillin at 37℃ at 200 r/min in a shaker incubator. After

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the optical density (OD) value of the cell culture reach 0.6, the protein expression was induced by adding 0.1mM isopropyl-β-D-thioga-lactoside (IPTG). The induced cell

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culture was incubated at 16 °C with 180 r/min shaking for 24 hours.

All purification steps were performed at 4℃ unless otherwise stated. The cells were harvested by centrifugation and resuspended in 1/10 of the culture volume in binding buffer (20 mM MOPS, 50 mM sodium acetate, 20 mM EDTA, PH 7.0). Cells were disrupted by sonication at 200W for 10 mins, and the cell debris was removed by centrifugation at 12000 r/min for 30 mins. The supernatant that contained the crude recombinant PmC5A was loaded onto a binding buffer pre-equilibrated NTA-Ni Sepharose column (HisTrap™ HP, GE healthcare, Uppsala, Sweden), and eluted by binding buffer containing a linear gradient of 0-0.5M imidazole. The concentration of 6 / 31

Journal Pre-proof purified PmC5A was measured with a BCA protein assay kit following the instruction given by the manufacturer (Beyotime Biotechnology) and using albumin from bovine serum (BSA) as the standard protein. The molecular weight of PmC5A was analyzed with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Comassie Brilliant Blue R-250 as the staining agent.

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2.3 1H NMR analyses of the functionality of PmC5A

The alginate used as substrate for analysis the function and activity of PmC5A was bought in the Qingdao bright moon seaweed group co., LTD, China. The relative

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content of the G residues was about 38% and DPn is about 1030 measured by Gel

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Permeation Chromatography (GPC). Samples for characterization of PmC5A activity

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were prepared by adding 5 μg PmC5A into 1ml of 20 mM MOPS reaction buffer (pH 7.5) which contains 5 mg of alginate. The reactions were carried out at 30℃ and

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stopped by heating at 100℃ for 10min. All samples were freeze-dried and dissolved in 0.55 ml D2O, then analyzed by 1H NMR spectroscopy using a Bruker AM-400

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(400MHZ) spectrometer at 70℃. Because when the NMR detection is performed at

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normal temperature, the position of the water peak is very close to the peak position of the sample, which will cause great interference. At high temperatures, the water peak can be moved properly away from the target peak. The internal standard was 3(trimethylsilyl)-propionic-2,2,3,3-d4-acid, Na+ salt. The time courses of the epimerase and lyase activities were monitored by collecting samples terminated at different time intervals.

2.4 Characteristics of PmC5A as an alginate lyase—temperature, pH, effect of metal ions and stability

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Journal Pre-proof The

characterization

of

PmC5A

as

an

alginate

lyase

was

performed

spectrophotometrically (540 nm) by a 3,5-dinitrosalicylic acid (DNS) method which was used extensively for evaluating the aqueous concentration of reducing sugar [17, 18]. The mixture containing 96 ul DNS solution and 128 ul sample solution was heated in boiling water for 10 minutes. After cooling down to room temperature, the mixture was diluted with 1.376 ml water, and the amount of reducing sugar, manifested by the change in color, was monitored at 540 nm using a Cary 50

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spectrophotometer (Varian, USA).

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To investigate the effect of temperature on PmC5A lyase, reactions were performed at different temperature (such as 20℃, 30℃, 40℃, 50℃ and 60℃) for 30 mins, and were

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terminated by heating in boiling water for 5 mins. The residue activities were assessed

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by DNS assay. Three parallel experiments were set up for the determination of of the relative activity of PmC5A lyase at each temperature. The average value was selected

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as the activity value, and the STDEVA generated by the three sets of data was used as

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the value of the error bar.

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To investigate the effect of pH on PmC5A lyase, reactions were carried out in buffers with different pH, including 50 mM phosphate buffer (pH 6 and 7), 50 mM Tris-HCl (pH 8 and 9), 50 mM CAPS (pH 10). Reactions were terminated by heating in boiling water for 5 mins. The residue activities were determined by the DNS method. Three parallel experiments were set up for the determination of the relative activity of PmC5A lyase at each pH. The average value was selected as the activity value, and the STDEVA generated by the three sets of data was used as the value of the error bar.

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Journal Pre-proof To investigate the effect of metal ions on PmC5A lyase, the reaction buffer (50 mM Tris-HCl, pH 8.0) was supplied with different metal ions, including Zn2+, Ca2+, Mg2+, Mn2+, Co2+, Cu2+, Fe2+ and Fe3+ ions. The residue activities were determined by the DNS method. The reaction without the presence of metal ions was served as the control. Three parallel experiments were set up for the determination of each effect of metal ions on PmC5A lyase. The average value was selected as the activity value, and the STDEVA generated by the three sets of data was used as the value of the error

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bar.

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The thermal and pH stabilities of PmC5A lyase were also investigated. To study the

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thermal stability, the enzyme solution was pre-incubated at 35℃, 40℃, 45℃, 50℃, 55℃ and 60℃ at pH 8 for 1 hours. For pH stability, the enzyme solution was pre-

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incubated at different pH (2, 3, 4, 5, 6, 7, 8, 9, and 10) at 4℃ for 24 hrs. Buffers used for different pH values included 50 mM glycine buffer (pH 2 and 3), 50 mM sodium

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acetate buffer (pH 4 and 5), 50 mM phosphate buffer (pH 6 and 7), 50 mM Tris-HCl

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(pH 8 and 9), 50 mM CAPS (pH 10). Residue activity was accessed by DNS assay. Three parallel experiments were set up for the determination of the thermal and pH stabilities of PmC5A lyase. The average value was selected as the activity value, and the STDEVA generated by the three sets of data was used as the value of the error bar.

2.5 Characteristics of PmC5A as mannuronan C-5 epimerase—temperature, pH The epimerase activity of PmC5A was detected by 1H NMR as described above. To investigate the temperature effect on epimerase activity, the reactions were carried out 9 / 31

Journal Pre-proof at different temperature (20℃, 30℃, 40℃, 50℃, 60℃ and 70℃). For pH effect, reactions were conducted at different pH (6, 7, 8, 9, and 10), similar as the PmC5A lyase assays.

2.6 Substrate specificity of PmC5A as an alginate lyase—PolyM, PolyMG and Gblocks

All of these alginates were provided by Professor Gudmund Skjåk-Bræk and existed

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in the form of Na+ form. The high-molecular-weight mannuronan (PolyM, DPn=1311), FG=0.00 was isolated from an epimerase-negative strain of Pseudomonas

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fluorescens [19]. Polyalternating alginate (PolyMG, DPn=1045), FG=0.47 was

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produced in vitro from mannuronan by using recombinantly produced AlgE4. The G-

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block alginate (G-blocks, DPn=18), FG=1.00 was isolated from the outer cortex of Laminaria hyperborean stipes. To study the substrate specificity, 0.5% of PolyM,

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PolyMG and G-blocks were dissolved in 40 mM phosphate buffer pH 8. These mixtures were incubated at 40℃ for 30min and then it was terminated by heating in

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boiling water for 5 mins. The relative reactivity was determined with the DNS

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method. Three parallel experiments were set up for the determination of the substrate specificity of PmC5A. The average value was selected as the activity value, and the STDEVA generated by the three sets of data was used as the value of the error bar.

3. Results 3.1 Cloning, expression and purification of PmC5A from Pseudomonas mendocina. sp. DICP-70

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Journal Pre-proof A strain from the digestive tract of sea urchin caught in east coast of Dalian (Liaoning, China) was separated as described above. Its 16S rRNA sequence similarity search performed by basic local alignment search tool (BLAST) indicated that the bacterium was a Pseudomonas medocina. sp (Fig. 1). From the information of genome sequence of Pseudomonas mendocina. sp. DICP-70 in the NCBI database, a pair of degenerate primers was designed and the gene was successfully subcloned into pET21a. The obtained gene was named PmC5A, and was successfully overexpressed

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in E.coil BL21 (DE3). The expressed enzyme was then purified by Ni-column. The eluted fraction was analyzed by SDS-PAGE and the molecular mass was about 52.39

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kDa by calculating the known amino acid sequence (Fig. 2). The amino acid sequence

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of PmC5A shared 61% homology with periplasmic alginate epimerase AlgG from Pseudomonas sp. and Azotobacter vinelandii (Fig. 3) [20]. Compared with these

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epimerase, it showed that the important residues DPHD were conserved, which were

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22].

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proposed to be the active sites and carbohydrate-binding/sugar hydrolysis domain [21,

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3.2 1H NMR analysis of the reaction products of PmC5A

To characterize the function of PmC5A, the recombinant protein incubated with alginate at different time intervals and the reaction products were finally analyzed by 1

H NMR spectroscopy. The molar fraction of the monomers G (FG), M (FM) could be

calculated according to the spectrum [23], because the proton-signal integral could reflect the quantities of the respective trequencies and the intensities of G fulfill the following relationships: FG=FGGG+FMGG+FGGM+FMGM. The results showed that the Gmoieties of alginates increased by 9% from 38% to 47% after incubating for 10 min, which confirmed that PmC5A has epimerase activity. 11 / 31

Journal Pre-proof Interestingly, as the reaction time increased, the G-moieties decreased sharply and the signals from end-groups appeared, such as saturated reducing ends (Mred and Gred) and unsaturated nonreducing ends (Δ) (Fig. 5A). The latter signal is corresponding to the production of unsaturated 4-deoxy-L-erythro-hex-4-enepyranosyluronate residues by a β-elimination reaction [24] , indicating that PmC5A also exhibits lyase activity. From the intensities of the signals of the end groups, the average size of the products can be estimated [25]. Meanwhile, only the β-anomeric reducing end signals could be

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integrated due to the overlap of the α-signals and the unsaturated nonreducing Δ-1-G end signals. However, the intensities of the α-signals were found by the ratios of the

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anomeric protons Mα/Mβ≌2.2 and Gα/Gβ≌0.2[24]. So the total molar fraction of

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M- and G-moieties at the reducing ends could be calculated and the DPn was

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estimated according to the principle of end-based analysis from DPn = [IG-5 + IM-1 + (IGred + IMred) ⅹ 2] / [IGred + IMred] (Fig. 4), where I denoted the intensities of the

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relevant signals in the spectra.

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To comparative analysis of the epimerase and lyase activities of the enzyme, the

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changes of G-moieties and DPn of substrates with reaction time were plotted in figure 4B. The results showed that the G-moieties were firstly increased by 9% from 38% to 47%, then decreased to about 40% and remained at that level with further incubation. On the other hand, the DPn of the alginates dropped sharply in first 30 min (from 1030 to 30) of the reaction and further decreased to the average size of 4 units (Fig. 5B).

3.3 Effect of temperature on PmC5A lyase and epimerase activities

The relative lyase and epimerase activities of PmC5A were measured at different temperatures (Fig. 6). The optimum temperature for PmC5A lyase was measured to 12 / 31

Journal Pre-proof be 40℃, and the enzyme maintained more than 75% of its maximum activity between 30 to 50℃. A drastically drop of activity was observed when the temperature rose above 50℃, and the enzyme became almost inactive when temperature reached 60℃. Investigation on thermal stability revealed that the enzyme maintained more than 60% of its activity after been incubated at 45℃ for 1 hours, but it became almost inactive when the incubation temperature reached 55℃.

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In contrast, the optimum temperature for PmC5A epimerase was 30℃. Interestingly, the enzyme kept 80% of its epimerase activity at 60℃ but exhibited relatively low

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activity at 40 to 50℃.

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3.4 Effect of pH on PmC5A lyase and epimerase activities

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The relative lyase and epimerase activities of PmC5A were measured at different pH

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(Fig.6). The optimum pH for PmC5A lyase was 8. Investigation on pH stability revealed that the enzyme was remarkably stable at pH 4-9, and maintained over 70%

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of its activity at pH 3 and 10.

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The optimum epimerase pH of PmC5A was found to be 9. It has a 70% residue activity at pH of 7, but drastically decreased to 0% in the pH range 9-10.

3.5 Effect of metal ions on PmC5A lyase activities

PmC5A lyase activity was tested in the presence of various metal ions by DNS assay. The results showed that most of the tested ions had certain degree of inhibitory effect on the activity except Zn2+ and Ca2+. Among them, Co2+, Cu2+, and Fe2+ strongly inhibited the enzyme activity, and the activity became almost undetectable in the presence of Fe3+ (Fig. 7). 13 / 31

Journal Pre-proof 3.6 Substrate Specificity of PmC5A lyase

To investigate the substrate specificity of PmC5A lyases, PolyM, PolyMG and Gblocks were used as the substrates. The enzyme showed the highest activity toward PolyMG (relative activity of 100%). PolyM (relative activity of 65.96%) and Gblocks (relative activity of 62.80%) were equally utilized as substrates (Fig.7). Since there are differences between substrates that could affect the activities, for instance,

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the degree of polymerization of polyM, polyMG and G-blocks are about 1311, 1045 and 18 respectively, the results only suggest that PmC5A can cleave MG-blocks and

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preference for certain substrates.

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G-blocks. Still further research and evaluation will be needed to explain the enzyme's

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4. Discussion

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Alginate is a major polysaccharide of the cell wall matrix of brown seaweeds [26]. It is initially produced as polymannuronic acid, within which the guluronic acid residues

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are introduced by the action of mannuronan C-5-epimerases [27]. Terrestrial bacteria

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from the Pseudomonas and Azotobacter genera also biosynthesize alginate as a biofilm component [28-30]. So far, two different types of mannuronan C-5-epimerase are identified in A. Vinelandii and P. aeruginosa, the modular AlgE, which contains a family of seven extracellular enzymes (AlgE1-7) [31], and the non-modular AlgG, which is a periplasmic C-5-epimerase [32]. Unlike epimerase, alginate lyase activity degrades alginate through a β-elimination on the glycosidic bond. Several alginate lyases have been identified in alginate-assimilating organisms such as the Flavobacterium sp. strain UMI-01 [33] and alga-associated bacteria Zobellia galactanivorans [34], within which alginate lyase activity is essential for the

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Journal Pre-proof decomposition of brown algal tissues as a carbon source [35]. However, alginate lyase has also been identified in alginate-producing organisms, such as brown algae [36] and P. aeruginosa [37], where it plays a role in alginate biosynthesis [38-40].

In the present study, we isolate and identify an enzyme, named PmC5A, from Pseudomonas mendocina which inhabited in the digestive tract of Strongylocentrotus nudus. After being heterogeneously expressed in Escherichia coli, PmC5A was

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purified and subjected to a series of enzymatic assays. From the 1H NMR results, it showed a significant increase of G-moieties (9%) of alginates after incubating for

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about 10min. However, with the extension of the reaction time, an obvious signal of unsaturated uronic acid at the nonreducing terminus of the products indicated that

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PmC5A had alginate lyase activity[41]. To further confirm the observed lyase activity

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of the enzyme, the produced reducing sugar from alginate was also investigated by the DNS method [17]. Thus, PmC5A was defined as a bifunctional enzyme with

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mannuronan C-5 epimerase and alginate lyase activities. PmC5A shared high

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homology, like more than 61%, with periplasmic alginate epimerase AlgG from

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Pseudomonas sp. and Azotobacter vinelandii [22]. All of these epimerases have the conserved motif DPHD and other reported important residues, showed in Fig. 3. So it could be difficult to know why PmC5A had two functions. Probably, some other different residues which were not reported a lot pointed to such a change. It would be studied in the future study.

So far, only a few mannuronan C-5 epimerases have been reported as polyfunctional. AlgE7 is one of them, it has mannuronan C-5 epimerase and alginate lyase functions which can both be catalyzed by the same active site in the enzyme [25]. AlgE7 is Ca2+-dependent extracellular enzyme and consists of two types of structural modules, 15 / 31

Journal Pre-proof A and R. Specially, A-modules alone can conduct the whole epimerization process, while the presence of R-modules increases the reaction rate, most possible by making Ca2+ more available to the catalytic sites located on A-modules [42]. Similar to AlgE7, PmC5A possesses both the mannuronan C-5-Epimerase and alginate lyase activities, but it is a non-modular periplasmic enzyme. Both of them have the conserved motif “DPHD(E)”, which were proposed to be the active sites and

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carbohydrate-binding/sugar hydrolysis domain [21, 22].

For gut microflora of marine herbivores, such as sea urchins which feeds on algae but

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doesn’t have related enzymes necessary for digesting algae, the breakdown of alginate by them to produce energy sources for their hosts is essential for their relationship

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[43]. The alginate lyases secreted by these bacterial can degrade alginate [44]. The

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secretion of bifunctional enzyme PmC5A by P. mendocina, which lives in the digestive tract of Strongylocentrotus nudus, will give the bacterium a greater

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advantage in energy efficiency of degrading alginate.

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The enzymatic assays showed that the lyase activity of PmC5A has a maximal

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activity at 40℃, which was different from its epimerase activity which has an optimal temperature at 30℃. Interestingly, with the temperature rising above 30℃, the trend of the lyase and epimerase activities formed a complementary X shape, indicating that the relative performance of the two activities of PmC5A were temperature dependent. Moreover, the lyase activity of the enzyme retained about 71%, 73% and 64% residual activity after incubation at 35℃, 40℃ and 45℃ for 1h, respectively, demonstrating its great temperature stability.

The optimum pH of alginate lyase was 8.0 and it showed an excellent stability in the pH range of 3.0-10.0, maintaining more than 70% residual activity by incubating at 16 / 31

Journal Pre-proof 30℃ for 40 min. Similarly, the epimerase activity of PmC5A exhibited a maximal activity at pH 9.0, indicating that both the lyase and epimerase activities of PmC5A prefer alkaline conditions. This is in accordance with the alkaline condition (~pH 9.5) in the stomach of sea urchin larvae [45] where the enzyme is secreted into. The adaptation of the enzyme activity to such high pH environment is probably associated with catalytic amino acids which are required to produce ionization in the alkaline environment. Similar to AlgG from Pseudomonas aeruginosa, its activity

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dramatically decrease at alkaline pH (pH>7.5), because its pH-dependent epimerase

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activity was calculated to be related with the ionization of 3 residues [46].

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Acknowledgements

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Many thanks to Professor Gudmund Skjåk-Bræk from Norwegian University of Science and Technology for kindly providing us PolyM, PolyMG and G-blocks which

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helped us complete our research work successfully. The research was supported by

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the National Key Research and Development Program of China (2018YFC1105600), (2017YFD0200900) Subject 2 (2017YFD0200902), the Chinese National Nature Science

Foundation

(31670803),

Liaoning

Revitalization

Talents

（XLYC1807041）, DICP ZZBS201704 and DICP BioChE-X201801.

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Figure Captions Figure 1: Unrooted neighbor-joining phylogenetic tree derived from 16S rRNA gene sequence of strain Pseudomonas mendocina. sp. DICP-70. Sequence alignment was performed using ClustalW and the tree was built by MEGA 7.0 software and EvolView. The scale indicates the number of DNA sequences per site. Figure 2: SDS-PAGE of PmC5A. Lane M, protein marker of molecular mass; Lane

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1, crude enzyme solution of PmC5A; Lane 2, purified PmC5A. Figure 3: Multiple sequences alignments of PmC5A and related mannuronan C-5 epimerases. Green are the important residues. ALGG_PSEAE is AlgG (accession

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number: WP_003110465.1) from Pseudomonas aeruginosa; ALGG_AZOVI is AlgG

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(accession number: P70805.1) from Azotobacter vinelandii; ALGG_PSEAM is AlgG

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(accession number: WP_011103483.1) from Pseudomonas syringae pv. tomato. Figure 4: 1H NMR analysis of the reaction products of PmC5A. The left side of the

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figure shows the process of alginate being enzymatically catalyzed. First, the Mblocks are partially epimerized into G residues, and then cleaved by lyases. The

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corresponding NMR spectrum is on the right. According to the information provided

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in the spectrum, the corresponding DPn and G content can be obtained. Figure 5: Epimerization and degradation of alginate by PmC5A. A. 1H NMR spectroscopy demonstrating the interplay between epimerization and degradation of alginate. B. units epimerized (FG) and DPn, plotted as a function of incubation time with PmC5A. Figure 6: Enzymatic characteristics of PmC5A. (A) Effect of temperature on PmC5A activity. (B) pH on PmC5A activity. (C) pH stability of PmC5A. (D) temperature stability of PmC5A lyase.

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Journal Pre-proof Figure 7: Effects of metal ions and substrate specificity of PmC5A lyase. (A) Effects

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of metal ions. (B) Substrate specificity.

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Sample CRediT author statement Ming Sun: Investigation, Writing- Original draft preparation; Chu Sun: Writing- Original draft preparation; Tang Li: Supervision; Kuikui Li: Formal analysis; Shenggang Yan: Formal analysis;

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Heng Yin: Writing- Reviewing and Editing, Project administration, Funding

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Highlights -An alginate utilization enzyme was found in the stomach of sea urchin. -PmC5A is an enzyme which had bifunctional activities of epimerase and lyase.

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-In-depth study of the enzymatic properties of PmC5A.

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