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Accepted Manuscript Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species Pavel V. Volkov, Alexander V. Gusakov, Ekaterina A. Rubtsova, Alexandra M. Rozhkova, Veronica Yu. Matys, Vitaly A. Nemashkalov, Arkady P. Sinitsyn PII:

S0300-9084(18)30335-3

DOI:

https://doi.org/10.1016/j.biochi.2018.11.010

Reference:

BIOCHI 5548

To appear in:

Biochimie

Received Date: 18 September 2018 Accepted Date: 19 November 2018

Please cite this article as: P.V. Volkov, A.V. Gusakov, E.A. Rubtsova, A.M. Rozhkova, V.Y. Matys, V.A. Nemashkalov, A.P. Sinitsyn, Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species, Biochimie, https://doi.org/10.1016/ j.biochi.2018.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Abstract

The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385) belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude

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enzyme preparations with the recombinant dextranase content of 8–36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were isolated in a homogeneous state using chromatographic techniques. The purified

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enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5–5.0 and 55– 60 oC and a melting temperature of 60.7–60.9 oC. They were characterized by similar specific

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activities (1020–1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500 kDa, as well as similar kinetic parameters in the hydrolysis of 70 kDa dextran (Km = 1.17–1.18 g/L, kcat = 660–700 s-1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different molecular masses according to the data of SDS-PAGE (~63 and ~60

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kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while after the prolonged time (24 h) the reaction system

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contained isomaltose and glucose as the major products and minor amounts of other

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oligosaccharides.

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Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species

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Pavel V. Volkov a,, Alexander V. Gusakov a,b, Ekaterina A. Rubtsova a, Alexandra M. Rozhkova a,b*, Veronica Yu. Matys c, Vitaly A. Nemashkalov c, Arkady P. Sinitsyn a,b

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences,

Leninsky Pr. 33/2, Moscow 119071, Russia

Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11,

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b

Moscow 119991, Russia c

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a

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of

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Sciences, Pushchino 142292, Moscow region, Russia

* Corresponding author. Tel.: +7 495 660 3430; fax: +7 495 954 2732

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E-mail address: [email protected] (A.M.Rojkova)

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Abstract The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385)

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belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude enzyme

preparations with the recombinant dextranase content of 8–36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were

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isolated in a homogeneous state using chromatographic techniques. The purified enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5–5.0 and 55–60 oC and a melting

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temperature of 60.7–60.9 oC. They were characterized by similar specific activities (1020–1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500 kDa, as well as similar kinetic parameters in the hydrolysis of 70 kDa dextran (Km = 1.10–1.11 g/L, kcat = 640–680 s-1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different

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molecular masses according to the data of SDS-PAGE (~63 and ~60 kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while

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after the prolonged time (24 h) the reaction system contained isomaltose and glucose as the major

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products and minor amounts of other oligosaccharides.

Keywords: Dextran; Dextranase; Heterologous expression; Penicillium species; Purification

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1. Introduction Dextranases (6-α-D-glucan 6-glucanohydrolases, EC 3.2.1.11) are inducible enzymes that

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catalyze the hydrolysis of α-1,6-glucosidic linkages in dextrans. Isomaltose, isomaltotriose and glucose with trace amounts of other oligosaccharides are the main products of the enzymatic reaction catalyzed by dextranase [1].

Dextranases are produced by different microorganisms, including bacteria [2–4], yeasts [5]

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and filamentous fungi [1,6,7]. These enzymes are of practical interest due to their ability to produce

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isomaltooligosaccharides (IMO) belonging to the group of prebiotic functional products [8]. One of the most promising applications of dextranases is in a sugar industry [9], in which the enzyme is used for the depolymerization of dextran produced by Leuconostoc bacteria on the surface of raw materials (sugar beet, sugar cane). In this technology, dextran negatively affects the yield and quality of the target product by increasing the viscosity of the sucrose juice and reducing its

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crystallization rate [10,11]. In addition to the food industry, dextranases have found applications in medicine; for example, they are used in production of blood substitutes [1]. Due to the property of depolymerizing dental dextran deposits, dextranases are also used as components of toothpastes

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preventing the development of caries [12,13].

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Fungal dextranases attract more attention of researchers because of their higher activity, and that is why most commercial preparations, such as Dextranase 50L (from Novozymes) or Dextranase2F (from Mitsubishi Chemical), are based on fungal producing strains [14]. According to the CAZy database (http://www.cazy.org/) [15], practically all known

dextranases are classified into families 49 and 66 of glycoside hydrolases (GH49 and GH66). Bacterial dextranases are present in both families mentioned, while those from fungi belong exclusively to the GH49 family. Compared to other glycoside hydrolase families, the GH49 family 3

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is very sparsely populated; there were only 11 entries of eukaryotic enzymes in it by the end of June 2018 (only 7 entries being classified as a dextranase), and just a few of them have been characterized.

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In this paper, we describe studies of a previously unexplored GH49 dextranase from Penicillium funiculosum (anamorph Talaromyces funiculosus), heterologously expressed in two recipient strains of P. canescens RN3-11-7 and P. verruculosum (T. verruculosus) B1-537. Both

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recipient strains have previously been successfully used for production of recombinant fungal enzymes with up to 80% content of the target protein in the culture broth [16–18]. Purification and

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properties of two recombinant forms of Dex49 (rPcDex49 and rPvDex49) are described and discussed.

2. Materials and methods

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2.1. Microbial strains

P. funiculosum BPI 66 basic strain (No. F-977) was from VKPM collection (Russia). P. canescens RN3-11-7 strain [16] and P. verruculosum B1-537 strain [17,18], both deficient in the

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preparation.

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nitrate reductase gene (∆niaD), were used as host strains for transformation and chromosomal DNA

2.2. Construction of the expression plasmids and transformation of fungi Genetic constructions and production of recombinant strains was carried out as described elsewhere [16–18]. Briefly, using the freshly isolated genomic DNA of P. funiculosum fungus and two pairs of primers shown below, the 1827 bp products were synthesized by polymerase chain 4

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reaction (PCR) for further cloning into P. canescens RN3-11-7 and P. verruculosum B1-537 recipient strains, respectively. Dex-UpLIC (RN3)

5’- gcacaggcagcaggagctggagcagtcatgcacccacc - 3’

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Dex-LowLIC (RN3) 5’- agagcaagccgagcaggtcagctaatctgccactgcccccaatag - 3’ 5’- ggcaacagcaggagctggagcagtcatgcacccacc - 3’

Dex-LIC3 (B1)

5’- agaggagggcgacacagtcagctaatctgccactgcccccaatag - 3’

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Dex-LIC5 (B1)

Then, using the method of independent ligation [19], the dexA gene was cloned into the pXEG

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[16] and pCBHI [18] vectors. The modified pUC19 cloning vector pXEG contained the promoter region of the xylA gene and the terminator region of the egl3 gene, while the pCBHI vector contained the promoter and terminator regions of the cbh1 gene (Fig. S1, Supplementary data). Thus, two plasmids, pXylA-DEX and pCBHI-DEX, were obtained. Protoplasts of P. canescens RN3-11-7 and P. verruculosum B1-537 recipient strains were transformed by the pXylA-DEX and

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pCBHI-DEX plasmid, respectively, together with the pSTA10 co-transforming plasmid at the ratio of 6:1 µg using the transformation protocol [20]. The pSTA10 plasmid contained a nitrate reductase

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(niaD) gene, allowing the selection of the resulting transformants on medium with sodium nitrate. An indicator of the effective transformation of the Penicillium fungi was the production of 20–40

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clones per 1 µg of target plasmid DNA [20].

2.3. Screening and cultivation of transformants Screening of transformants was carried out in shake flasks (total volume 500 mL, fermentation volume 100 mL). A medium for cultivation of P. verruculosum transformants contained (in g/L): microcrystalline cellulose – 40; yeast extract – 10; КН2РО4 – 15; (NH4)2SO4 – 5; MgSO4·7H2O – 5

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0.3; СaCl2·2H2O – 0.3. A medium for cultivation of P. canescens transformants contained (in g/L): soybean hulls – 45; corn extract – 50; КН2РО4 – 15. Fermentation was carried out at 30 oC for 144 h. Initial recipient strains of P. verruculosum B1-537 (∆niaD) and P. canescens RN3-11-7 (∆niaD)

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were used as controls. As a result of primary screening, several clones of P. verruculosum (PV2, PV6, PV9 and PV16) and P. canescens (PC3, PC12 and PC27) with a high activity of the culture filtrates against dextran were selected.

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The best selected clones were cultivated in 3-L glass fermenters KF-104/3 (Prointex, Moscow, Russia). The composition of the medium for P. verruculosum fermentation was the following (in

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g/L): microcrystalline cellulose – 60; glucose – 40; wheat bran – 10; yeast extract – 10; КН2РО4 – 7; (NH4)2SO4 – 5; MgSO4·7H2O – 0.3; СaCl2·2H2O – 0.3 (рН 4.5–5.0, 32 ºС, fermentation time 144 h). Fermentation was carried out in fed-batch mode with fractional glucose addition (every 12 h after the first 48 h), three additions of cellulose and one addition of salts. The composition of the medium

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for P. canescens fermentation was identical to that in shake flasks (see above). Fermentation was carried out without additions of medium components for 144 h. After completion of cultivation in fermenters, the culture broth was centrifuged at 4000 rpm

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for 20 min on a centrifuge Avanti JXN-26 (Beckman Coulter, Atlanta, GA, USA) to remove biomass and insoluble components of the nutrient medium. The supernatant was freeze-dried on a

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VirTis BenchTop 2K ES freeze dryer (SP Scientific,Warminster, PA, USA).

2.4. Enzyme purification

Desalting and fractionation of crude freeze-dried enzyme preparations was carried out on a HPLC system ACTA Purifier (GE Healthcare, UK). Proteins were preliminary precipitated with ammonium sulfate (80% saturation) followed by a desalting on a Bio-Gel Р-4column (Bio-Rad 6

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Laboratories, Hercules, CA, USA) with the use of 0.02 М bis-Tris/HCl buffer, pH 6.8. Then, proteins were fractionated by anion-exchange chromatography on a Source 15Q column (Pharmacia, Uppsala, Sweden). The sample was applied to the column in the starting bis-Tris/HCl buffer at pH

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6.8, and the bound proteins were eluted with a gradient of NaCl from 0 to 0.4 M. Protein fractions displaying the dextranase activity were subjected to further purification using hydrophobic

interaction chromatography on a Source 15ISO column (Pharmacia, Uppsala, Sweden). The sample

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was applied to the column at the initial concentration of added ammonium sulfate of 1.4 M in 50 mM Na-acetate buffer, pH 5.0, and the elution of proteins was carried out in a reversed gradient of

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(NH4)2SO4 from 1.4 to 0 M. In the case of rPcDex49, the fraction with a highest activity against dextran was finally purified by gel filtration on a Superose 12 column (GE Healthcare, UK). Elution was performed in the isocratic mode in 0.01 M Na-acetate buffer, pH 5.0. The enzyme purity was characterized by sodium dodecylsulfate polyacrylamide gel

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electrophoresis (SDS-PAGE) and isoelectrofocusing. SDS-PAGE was carried out in 12% gel using a Mini Protean II equipment (Bio-Rad Laboratories, Hercules, CA, USA). Isoelectrofocusing was performed on a Model 111 Mini IEF Cell (Bio-Rad Laboratories, Hercules, CA, USA). Staining of

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protein bands was carried out in Coomassie Blue R-250 (Ferak, Berlin, Germany). Protein concentration in samples was determined by the modified method of Lowry et al.

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[21], using bovine serum albumin as the standard.

2.5. Enzyme identification by MALDI-TOF mass spectrometry Identification of dextranases was carried out by MALDI-TOF mass spectrometry (MS) peptide fingerprinting of trypsin-digested proteins from the bands after isoelectrofocusing as described elsewhere [16,22]. MALDI-TOF MS of peptides was carried out on an UltrafleXtreme 7

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TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Peptides in the mass spectra were indentified using the online service FindPept (https://web.expasy.org/findpept/) with the translated from the dexA gene amino acid sequence of P. funiculosum dextranase. Glycopeptides

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and structures of N-linked glycans were identified using the GlycoMod tool (https://web.expasy.org/glycomod/).

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2.6. Enzyme activity assays

Enzyme activity against polymeric substrates was assayed using the modified Nelson-

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Somogyi method [23] of determination of reducing sugars (RS) released from dextrans with a mean molecular mass of 20, 70, 500 kDa (Sigma, St. Louis, MO, USA), Sephadex G25, G50, G75, G150, G200 (Pharmacia, Uppsala, Sweden) and soluble starch from potato (Reakhim, Russia). A solution of the substrate (0.5%) in 0.1 M Na acetate buffer, pH 5.0, was incubated with the enzyme at 50 °C

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for 10 min (reaction volume 0.2 mL), and the reaction was stopped by adding 0.2 ml of the Somogyi reagent. After incubation of the obtained solution for 40 min in a boiling water bath, 0.2 ml of the Nelson reagent was added. The resulting solution was cooled in cold water for 10 min and 0.4 mL of

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acetone and 1 mL of distilled water were added. After centrifugation of the sample for 2 min at 13,000 rpm, the absorbance of the supernatant at 610 nm was measured against a substrate blank of

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the same concentration (without added enzyme). Isomaltose (Sigma, St. Louis, MO, USA) was used for preparing a calibration plot. Enzyme activities against microcrystalline cellulose (Avicel PH-101, Sigma, St. Louis, MO,

USA), xylan from birch wood and p-nitrophenyl-α-D-glucopyranoside (PNPG, Sigma, St. Louis, MO, USA) were determined as described elsewhere [24,25].

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Enzyme activities were expressed in international units. One unit of activity corresponded to the quantity of enzyme hydrolyzing 1 µmol of substrate or releasing 1 µmol of reducing sugars per minute. All activity assays were performed in triplicates.

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Study of effect of pH on the dextranase activity was performed using dextran (70 kDa) as a substrate. The activity assays were carried out as described above, except 0.1 M citrate–phosphate universal buffer was used for maintaining the necessary pH in the reaction system instead of the

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2.7. Determination of kinetic parameters

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acetate buffer.

The kinetic parameters of purified dextranases were determined at 50 °C and pH 5.0 (0.1 M Na-acetate buffer) using soluble dextran 70 kDa (Sigma, St. Louis, MO, USA) as a substrate. The substrate concentration was varied in the range of 0.1–5.0 g/L. The initial rates of the enzymatic

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reaction were determined by the Nelson-Somogyi method [23] as described above, and then the values of Km and Vmax were calculated by analyzing data obtained by the method of nonlinear

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regression using the OriginPro 8 software. The experiments were conducted in triplicates.

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2.8. Thermostability studies

Solutions of purified recombinant dextranases were incubated at pH 5.0 (0.1 M Na-acetate

buffer) and 40, 50 or 60 oC in a thermostat. After a certain incubation time, aliquots of the solution were taken, immediately cooled in cold water, and then the residual dextranase activity against dextran 70 kDa was determined under standard conditions (see above). The initial activity of the enzyme sample was taken as 100%. The experiments were carried out in triplicates.

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The melting temperature of dextranases (Tm) was determined by differential scanning microcalorimetry on a VP-DSC MicroCalorimeter (MicroCal, LLC, Northampton, MA) using a protein concentration of 1 mg/mL (0.05 M Na-acetate buffer, pH 5.0) in a 0.5 mL cell by applying a

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heating rate of 1 oC/min.

2.9. Progress kinetics of dextran and Sephadex hydrolysis

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Hydrolysis of dextran (70 and 500 kDa) and Sephadex G150 by the purified dextranases was carried out for 48 h at pH 5.0 (0.1 M Na-acetate buffer) and 40 ºC using a substrate concentration of

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50 g/L. The enzyme dosage was 2.5, 5 or 10 units of dextranase activity per 1 mL (U/mL). During the enzymatic reaction, samples were taken from the reaction mixture, boiled in a water bath for 5 min to stop the reaction and centrifuged to remove the denatured protein. The RS concentration was determined in the supernatants [23].

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Analysis of dextran (500 kDa) hydrolysis products was also carried out by HPLC using an Agilent 1100 chromatographic system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Coulochem III electrochemical detector (Thermo Fisher Scientific Inc., Waltham, MA, USA).

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The samples were dissolved in 100 mM NaOH solution and applied on a CarboPac PA-100 column (Thermo Fisher Scientific Inc., Waltham, MA, USA). An isocratic elution mode was used for the

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first 10 min, and then a linear gradient elution up to 450 mM of Na-acetate was used for 30 min. Detection was carried out in the mode of pulsating amperometry (+100 mV for 400 ms, –2000 mV for 20 ms, +600 mV for 30 ms, –100 mV for 50 ms). Calibration was performed using D-glucose and isomaltose (Sigma, St. Louis, MO, USA) as the standards.

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3. Results and discussion 3.1. Expression of recombinant dextranases

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The P. funiculosum GH49 family dextranase was cloned and heterologously expressed in P. canescens RN3-11-7 and P. verruculosum B1-537 recipient strains. The corresponding recombinant enzymes are referred here as rPcDex49 and rPvDex49, respectively. After screening of the

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transformants in shake flasks, several clones of P. verruculosum (PV2, PV6, PV9 and PV16) and P. canescens (PC3, PC12 and PC27) with a highest activity of the culture filtrates against dextran were

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selected. The SDS-PAGE analysis of the crude enzyme preparations (Fig. S2, Supplementary data) showed that new protein bands with a mass of ~63 kDa and ~60 kDa appeared in culture filtrates based of new recombinant strains of P. canescens and P. verruculosum, carrying the dexA gene, compared to the control samples based on the initial Penicillium strains. Specific activities of the mentioned enzyme preparations against a variety of substrates are

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shown in Table 1. In the case of a series of samples derived from P. verruculosum, the highest specific activity against dextran was observed for PV9 and PV2 clones (980–1010 U/mg protein), while the highest activity in a P. canescens series was displayed by PC3 and PC27 samples (90–105

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U/mg protein). It should be noted that in the case of the initial P. canescens recipient strain (PC-

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control) the dextranase activity was absent, while the control P. verruculosum enzyme preparation (PV-control) displayed only a trace level of this activity (0.02 U/mg). It is noteworthy that the specific activity of the PC-control enzyme preparation against xylan was rather high (105 U/mg), while it decreased 3–4-fold in the series of PC samples containing the recombinant rPcDex49. Analogously, the specific activity toward microcrystalline cellulose (Avicel) in the series of PV samples containing the recombinant rPvDex49 decreased relative to the PV-control preparation. These two phenomena maybe related to using the xylA and cbh1 gene promoters of the respective 11

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recipient strains for the heterologous expression of the recombinant dextranase under study, resulting in lower expression of xylanase A and cellobiohydrolase I (Avicelase) by the new recombinant strains of P. canescens and P. verruculosum, respectively.

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PV9 and PC3 transformants were selected for cultivation in 3-L laboratory fermenters, and

rPcDex49, respectively.

3.2. Purification of recombinant dextranases

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the enzyme preparations obtained were used for isolation and purification of rPvDex49 and

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Preliminary ammonium sulfate precipitated and desalted solutions of proteins from the crude enzyme preparations PV9 (P. verruculosum) and PC3 (P. canescens) were subjected to anion exchange chromatography on a Source 15Q column. During the fractionation of the PC3 preparation, the dextranase activity was detected in a fraction containing 0.12–0.15 M NaCl (Fig.

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1A), while in the case of the PV9 sample the activity was detected in a fraction containing 0.06–0.1 M NaCl (Fig. 1B). According to the SDS-PAGE data (not shown), the obtained fractions contained impurity proteins together with a major band of dextranase, so the enzymes were further purified by

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hydrophobic interaction chromatography on a Source 15ISO column. In the case of rPvDex49 additional protein purification was not required since the enzyme was

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homogeneous according to the SDS-PAGE and isoelectrofocusing (Fig. 2). In the second case (rPcDex49), the dextranase fraction after the hydrophobic chromatography was finally purified to a homogeneous state by gel filtration on a Superose 12 column (Fig. 2). The rPvDex49 and rPcDex49 were characterized by a slightly different molecular mass in the SDS-PAGE (~60 and ~63 kDa, respectively), while displaying the same pI 4.55 after isoelectrofocusing. Data on purification of rPvDex49 and rPcDex49 are summarized in Table 2. It should be noted that in an earlier study [26] 12

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two forms of a dextranase were isolated from P. funiculosum, which were characterized by the same molecular mass of 44 kDa but different pI values: 3.98 and 4.19. The difference in the properties of P. funiculosum dextranases isolated previously [26] and studied in this paper apparently results from

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poor systematization of microorganisms and the lack of nucleotide or amino acid sequences of dextranases in the beginning of 1970s.

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3.3. Amino acid sequence and peptide mass fingerprinting of rPvDex49 and rPcDex49

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The amino acid sequence of P. funiculosum dextranase translated from the nucleotide sequence of the amplified dexA gene is shown in Fig. 3. The nucleotide sequence of the gene was deposited in GenBank with an accession number MH581385. According to NCBI BLAST analysis, the enzyme under study displayed high similarity to other known fungal dextranases from the GH49 family. The highest sequence identity (99%) was shared with dextranases from Talaromyces

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minioluteus (P. minioluteum, GenBank accession number AAB47720), T. pinophilus (P. pinophilum, ACC95417), T. cellulolyticus (GAM43713). Slightly lower identity was found with dextranases from T. funiculosus (97%, CAB91097) and T. aculeatus (95%, AHJ14526). Alignment of the amino

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acid sequences of the mentioned dextranases is shown in Fig. S3 (Supplementary data). Although

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the sequence of another dextranase from P. funiculosum (T. funiculosus) is present in the GenBank database (CAB91097), the enzyme itself has not been characterized previously. Compared to the P. funiculosum dextranase studied by us, the CAB91097 dextranase is longer by 8 aa residues at the Cterminus, and the enzymes also differ by a few aa residues in the signal sequence and the mature protein sequence (Fig. S3). In order to identify the isolated rPvDex49 and rPcDex49, pieces of protein bands after isoelectrofocusing (Fig. 2) were cut from the gel, digested with trypsin, and the resulting peptide 13

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mixtures were analyzed by MALDI-TOF MS. Specific tryptic peptides found with the FindPept tool (https://web.expasy.org/findpept/) and matching the dextranase amino acid sequence are shown in bold in Fig. 3.

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Preliminary manual analysis of the mass spectrum of the digested rPcDex49 showed the presence of peaks differing by 162 Da (the mass of anhydrohexose residue) in the higher-molecular region of the spectrum, which indicated a possible N-glycosylation of the enzyme (Fig. S4,

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Supplementary data). There are three potential N-glycosylation sites in the dextranase (Asn39, Asn571 and Asn574) predicted by the MotifScan tool (http://myhits.isb-sib.ch/cgi-bin/motif_scan).

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The MS/MS fragmentation of peaks with m/z of 5282 and 5413 (Fig. S5, Supplementary data) followed by data analysis using the GlycoMod tool (https://web.expasy.org/glycomod/) showed that these peaks represent peptides 36 GTTNNTHCGADF(C-2Da)TWWHDSGEINTQTPVQPGNVR 69 and 35 MGTTNNTHCGADF(C-2Da)TWWHDSGEINTQTPVQPGNVR 69, both of them being

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decorated with N-linked high-mannose glycan (Hex)4 + (Man)3(GlcNAc)2. Both these peptides, differing by one Met residue at the N-terminus, belong to N-terminal region of dextranase (Fig. 3), and they could not be formed after the enzyme digestion with trypsin since the preceding residues

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are not either Arg or Lys (the specific sites for a protein cleavage by trypsin). Thus, these sequences likely represented the N-terminus of two closely related isoforms of rPcDex49, differing by one

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residue (Met) at the N-terminus. Using the SignalP tool (http://www.cbs.dtu.dk/services/SignalP), a signal peptide sequence of 20 aa residues was predicted in the amino acid sequence of P. funiculosum dextranase under study (shown in small letters in Fig. 3). A signal sequence of similar size is present in P. minioluteum (T. minioluteus) dextranase, sharing 99% identity to our enzyme under study (see above); moreover, the presence of a propeptide after the signal peptide has been found in the amino acid sequence of P. minioluteum dextranase [27]. Thus, the N-terminal sequence 14

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of a mature protein from P. minioluteum starts from the 35-th residue (see also UniProtKB entry P48845), which is in a very good agreement with our data (the sequence of a propeptide is underlined in Fig. 3).

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Using the sequences of the mature protein starting from the 35-th and 36-th residues (Fig. 3), the N-glycosylated peptides and glycan structures were discriminated in rPcDex49 and rPvDex49 with the GlycoMod tool (Tables S1 and S2, Supplementary data). In rPcDex49, high-mannose

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glycans, described by the formula (Hex)2-5 + (Man)3(GlcNAc)2, were found at the Asn39 Nglycosylation site. Similar N-glycosylation pattern has previously been observed for other enzymes

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secreted by P. canescens, and such glycoform heterogeneity is the result of sequential enzymatic trimming of a higher N-linked oligosaccharide by α-mannosidases present in the fungal culture broth [28,29]. Rather interesting, the analysis of MS data revealed that both the Asn571 and Asn574 potential N-glycosylation sites, located not far from each other in the same peptide NVA...GQK,

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seem to be decorated by glycans, one of them occupied by (Man)2(GlcNAc)2, while the other by (Man)3(GlcNAc)2 (Table S1). One may think that closely located Asn571 and Asn574 residues may hinder the glycosylation of both sites. However, modeling the 3D glycoprotein structure of

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rPcDex49 showed that the mentioned oligosaccharides do not cause steric hindrances to each other, and thus the N-glycosylation at both Asn residues is quite plausible (Fig. S6, Supplementary data).

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In rPvDex49, only a single N-linked GlcNAc residue was found at the Asn39 N-

glycosylation site (Table S2). Regarding the Asn571 and Asn574 potential N-glycosylation sites, it seems that only one of them is occupied by a single GlcNAc. However, it is quite possible that both mentioned Asn residues are decorated with GlcNAc since it is unclear what the (GlcNAc)2 glycan found in a glycopeptide with m/z of 4464 represents: either a dimer of GlcNAc at one of the Nglycosylation sites (Asn571 or Asn574) or two single GlcNAc residues on both sites mentioned. The 15

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difference in N-glycosylation patterns of rPcDex49 and rPvDex49 explains the difference in their molecular masses displayed in SDS-PAGE (Fig. 2). The presence of a single N-linked GlcNAc and the absence of high-mannose glycans in the case of recombinant dextranase expressed in P.

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verruculosum may be attributed to the secretion of an endogenous Endo-H- or Endo-F-like activity in the cultures, as has previously been reported for enzymes from Trichoderma reesei [30].

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3.4. Substrate specificity and kinetic parameters of purified dextranases

Specific activities of rPvDex49 and rPcDex49 against different natural and synthetic

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substrates are shown in Table 3. The rPvDex49 and rPcDex49 exhibited high activity toward different dextrans, and the dextranase activity of both recombinant enzymes increased with a decrease in the molecular mass of dextran. The highest specific activity (1330–1340 U/mg protein) was observed in the case of 20 kDa dextran. Also, to verify the enzyme specificity toward the α-1,6-

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glucosidic linkage, insoluble Sephadexes G25–G200 (the chromatographic carriers based on crosslinked dextrans) were tested as substrates. As expected, rPvDex49 and rPcDex49 were active against different kinds of Sephadex, except for the most cross-linked G25, and the enzyme specific activity

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increased with decreasing the degree of cross-linking (in a row from G50 to G200). The formation of reducing sugars in the course of Sephadex hydrolysis was accompanied by dissolution of the

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substrates. A few other substrates with other α-glucosidic linkages were also tested (Table 3) but the recombinant dextranases were inactive toward them. Kinetic parameters (Km and kcat) for the rPvDex49 and rPcDex49 in hydrolysis of 70 kDa

dextran were practically the same: 1.10–1.11 g/L and 640–680 s-1, respectively (pH 5.0, 50 oC). The observed values of Km and kcat were comparable to those reported for other GH49 family dextranases

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from other microorganisms [31,32]. For example, the Km values for enzymes from P. minioluteum

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and Hypocrea lixii were 2.05 and 2.35 g/L, respectively.

3.5. Temperature and pH-dependences of activity and thermostability

Effect of pH on the activity of rPvDex49 and rPcDex49 is shown in Fig. 4A. Both enzymes

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were characterized by very similar pH-profiles, exhibiting the maximum activity at pH 4.5–5.0, like other fungal dextranases [32–34]. The temperature optima of both recombinant dextranases were

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also exactly the same (55–60 °C) as shown in Fig. 4B.

Thermostability studies showed that rPcDex49 and rPvDex49 are stable at 40 °C and pH 5.0 at least for 3 h incubation (Fig. 5). At 50 oC, the enzymes retained 88% of the activity after 30 min of incubation, and 44% of the activity was preserved after 3 h of incubation at pH 5.0. At 60 oC, the

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half-life time of both dextranases did not exceed 10 min.

In addition, the stability of rPcDex49 at 40 °C and pH 3.0 was investigated (Fig. 5). The

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enzyme half-life time under these conditions was ~2 h.

3.6. Progress kinetics of dextran and Sephadex hydrolysis

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Proceeding from the fact that dextranases are not very stable at 50 oC, the prolonged

hydrolysis of polymeric substrates was carried out under conditions of the enzyme stability (40 oC, pH 5.0). Dextrans with a mean molecular mass of 70 kDa and 500 kDa as well as Sephadex G150 (all substrates at a concentration of 50 g/L) were hydrolyzed by rPcDex49 at a dosage of 2.5, 5 and 10 U/mL. After 10 min, 3, 6, 24 and 48 h of the reaction, aliquots of the reaction mixture were

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taken, in which the RS concentration was determined. The composition of the hydrolysis products was also determined by HPLC in some samples. As can be seen from Fig. 6, the accumulation of RS during the hydrolysis of 500 kDa dextran

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and Sephadex G150 was nearly completed after 24 h, while in the case of 70 kDa most of the products were formed in the first 3 h of the reaction. The maximum concentration of RS was obtained when the most high-molecular dextran (500 kDa) was used as the substrate (23.4 g/L and

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28.5 g/L of RS were formed after 24 h at 2.5 and 10 U/mL enzyme loading, respectively).

HPLC analysis showed that at the first stage of the reaction (10 min) with the lowest enzyme

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dosage (2.5 U/mL) the reaction products were different isomaltooligosaccharides (having a polymerization degree of up to 16) and relatively minor amounts of isomaltose (Fig. S7A). When the dextranase dosage increased up to 5–10 U/mL, the concentration of isomaltose and higher oligosaccharides increased (data not shown). At the final stage of the reaction (24–48 h), isomaltose

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and glucose were the major products together with minor amounts of other oligosaccharides (an example chromatogram is shown in Fig. S7B). Thus, by varying the dosage of dextranase and the reaction time, either isomaltooligosaccharides or isomaltose can be obtained as the predominant

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4. Conclusions

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products of dextran hydrolysis.

A novel fungal dextranase from P. funiculosum, not characterized previously, was cloned

and successfully expressed in the recipient strains of P. canescens and P. verruculosum fungi under the control of the strong gene promoters (xylA and cbh1, respectively). The expression of the recombinant dextranase was much higher in P. verruculosum than in P. canescens. Both recombinant proteins were isolated in a homogeneous form. The purified dextranases displayed very 18

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similar properties, such as pI, temperature and pH-optima, specific activities toward different dextrans and Sephadexes, and differed only by molecular masses as a result of different patterns of enzyme N-linked glycosylation. Experiments on the progress enzymatic hydrolysis of dextrans

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demonstrated that by varying the reaction conditions it is possible to obtain either

isomaltooligosaccharides or isomaltose as the predominant products, which may find applications as

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prebiotic functional products or in the food industry.

Acknowledgements

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This work was supported by Federal Agency of Scientific Organizations (Registration number 01201351359). Part of the measurements was carried out on the equipment of the SharedAccess Equipment Centre “Industrial Biotechnology” of Federal Research Center “Fundamentals of

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activity against polymeric substrates based of 3D model structures of the intact enzymes, Biochimie 110 (2015) 45–51.

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[19] C. Aslanidis, P.J. de Jong, Ligation-independent cloning of PCR products (LIC-PCR), Nucleic Acids Res. 18 (1990) 6069–6074. 21

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industrial strain of Chrysosporium lucknowense, Enzyme Microb. Technol. 36 (2005) 57–69. [25] O.A. Sinitsyna, F.E. Bukhtoyarov, A.V. Gusakov, O.N. Okunev, A.O. Bekkarevitch, Y.P. Vinetsky, A.P. Sinitsyn, Isolation and properties of major components of Penicillium

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canescens extracellular enzyme complex, Biochemistry (Moscow) 68 (2003) 1200–1209. [26] M. Sugiura, A. Ito, T. Ogiso, K. Kato, H. Asano, Studies on dextranase: purification of

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dextranase from Penicillium funiculosum and its enzymatic properties, Biochim. Biophys. Acta 309 (1973) 357–362. [27] H. Roca, B.M. Garcia, E. Rodriguez, D. Mateu, L. Coroas, J.A. Cremata, R. Garcia, T. Pons, J. Delgado, Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris, Yeast 12 (1996) 1187–1200.

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[28] A.V. Gusakov, O.A. Sinitsyna, A.M. Rozhkova, A.P. Sinitsyn, N-Glycosylation patterns in two α-L-arabinofuranosidases from Penicillium canescens belonging to the glycoside hydrolase families 51 and 54, Carbohyd. Res. 382 (2013) 71–76.

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[29] A.S. Dotsenko, A.V. Gusakov, P.V. Volkov, A.M. Rozhkova, A.P. Sinitsyn, N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity, Biotechnol. Bioeng. 113 (2016) 283–291.

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[30] M.J. Harrison, A.S. Nouwens, D.R. Jardine, N.E. Zachara, A.A. Gooley, H. Nevalainen, N.H. Packer, Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant

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strain of Trichoderma reesei, Eur. J. Biochem. 256 (1998) 119–127.

[31] D. Wu, H. Zhang, L. Huang, X. Hu, Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production, Process Biochem. 46 (2011) 1942–1950.

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[32] United States patent No. 5,637.491, Dextranase enzyme, method for its production and DNA encoding the enzyme (1997).

[33] A. Hattori, K. Ishibashi, S. Minato, The purification and characterization of the dextranase of

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Chaetomium gracile, Agric. Biol. Chem. 45 (1981) 2409–2416. [34] J.J. Virgen-Ortíz, V. Ibarra-Junquera, P. Escalante-Minakata, J. de Ornelas-Paz, J.A. Osuna-

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Castro, A. González-Potes, Kinetics and thermodynamic of the purified dextranase from Chaetomium erraticum, J. Molec. Catal. B: Enzym. 122 (2015) 80–86.

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Figure Captions Fig. 1. Anion exchange chromatography of PC3 (A) and PV9 (B) crude enzyme preparations on a

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Source 15Q column. Fig. 2. SDS-PAGE (A) and isoelectrofocusing (B) of purified recombinant dextranases. 1, rPvDex49; 2, rPcDex49.

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Fig. 3. Amino acid sequence of P. funiculosum dextranase. A signal peptide, predicted by the SignalP tool (www.cbs.dtu.dk/services/SignalP/), is shown in small letters; a propeptide is

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underlined; matching tryptic peptides identified by MALDI-TOF MS are shown in bold; identified glycopeptides are shaded, and the potential N-glycosylation sites are shown in boxes. Fig. 4. Effect of pH (A) and temperature (B) on rPvDex49 and rPcDex49 activity. Fig. 5. rPvDex49 and rPcDex49 thermostability at 40–60 oC (0.1 M Na-acetate buffer, pH 5.0) and

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40 oC (0.1 M citrate-phosphate buffer, pH 3.0 and 5.0). 1, PV2 crude enzyme preparation (50 oC, pH 5.0); 2, rPvDex49 (50 oC, pH 5.0); 3, rPcDex49 (50 oC, pH 5.0); 4, rPcDex49 (60 oC, pH 5.0); 5, rPvDex49 (60 oC, pH 5.0); 6, rPcDex49 (40 oC, pH 3.0); 7, rPvDex49 and rPcDex49 (40 oC, pH 5.0).

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Fig. 6. Progress kinetics of dextran 70 kDa (A), dextran 500 kDa (B) and Sephadex G150 (C)

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hydrolysis by purified rPcDex49. The enzyme was added at the dosage of 10 (1), 5 (2), and 2.5 (3) U/mL of the reaction mixture. Conditions: 0.1 M Na-acetate buffer, pH 5.0, 40 °C, substrate concentration 50 g/L.

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Table 1 Specific activities (U/mg protein) of crude enzyme preparations against different substrates (pH 5.0, 50 oC). Dextran 70 kDa

Avicel a

PV-control

0.020 ± 0.002

0.55 ± 0.04

PV2

1010 ± 50

0.25 ± 0.02

PV6

250 ± 10

0.44 ± 0.04

PV9

980 ± 50

0.47 ± 0.03

12 ± 1

PV16

750 ± 40

0.37 ± 0.03

14 ± 1

PC-control

<0.01

0.10 ± 0.01

105 ± 4

PC3

90 ± 4

0.10 ± 0.01

25 ± 2

PC12

35 ± 2

0.12 ± 0.01

31 ± 3

PC27

105 ± 5

0.11 ± 0.01

25 ± 2

22 ± 2

4.0 ± 0.4

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17 ± 2

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Activity was measured at 40 oC.

Xylan

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a

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Enzyme sample

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Purification of rPcDex49 and rPvDex49. Purification step

Total protein (mg)

Specific activity (U/mg)

Crude enzyme

13

90

Source 15Q

2.1

600

Source 15ISO

1.5

1010

Superose 12

1.3

1210

Crude enzyme

10

980

Source 15Q

4.5

1000

Source 15ISO

3.4

1200

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rPvDex49

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rPcDex49

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Table 2

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Table 3 Specific activities (U/mg protein) of rPvDex49 and rPcDex49 against natural and synthetic substrates (pH 5.0, 50oC). Main linkage

rPvDex49

Dextran 20 kDa

α-1,6

1330 ± 50

Dextran 70 kDa

α-1,6

1200 ± 40

Dextran 500 kDa

α-1,6

1020 ± 30

Potato starch

α-1,4

<0.01

Sucrose

α-1,2

<0.01

PNPGa

α-1,4

<0.01

Sephadex G25

α-1,6

<0.01

Sephadex G50

α-1,6

Sephadex G75

α-1,6

Sephadex G150

α-1,6

Sephadex G200

α-1,6

1210 ± 40

1030 ± 40

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<0.01 <0.01 <0.01 <0.01

41 ± 3

40 ± 3

43 ± 4

41 ± 3

500 ± 20

510 ± 30

770 ± 30

780 ± 30

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Activity was measured at 40oC.

1340 ± 50

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a

rPcDex49

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Substrate

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mpsmvkilalslaisesaigAVMHPPGNSHPGTHMGTTNNTHCGADFCTWWHDSGEINTQ 60 TPVQPGNVRQSHKYSVQVSLAGTNNFHDSFVYESIPRNGNGRIYAPTDPPNSNTLDSSVD 120 DGISIEPSIGLNMAWSQFEYSHDVDVKILATDGSSLGSPSDVVIRPVSISYAISQSDDGG 180 IVIRVPADANGRKFSVEFKTDLYTFLSDGNEYVTSGGSVVGVEPTNALVIFASPFLPSGM 240 IPHMTPDNTQTMTPGPINNGDWGAKSILYFPPGVYWMNQDQSGNSGKLGSNHIRLNSNTY 300 WVYLAPGAYVKGAIEYFTKQNFYATGHGILSGENYVYQANAGDNYVAVKSDSTSLRMWWH 360

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NNLGGGQTWYCVGPTINAPPFNTMDFNGNSGISSQISDYKQVGAFFFQTDGPEIYPNSVV 420 HDVFWHVNDDAIKIYYSGASVSRATIWKCHNDPIIQMGWTSRDISGVTIDTLNVIHTRYI 480 KSETVVPSAIIGASPFYASGMSPDSRKSISMTVSNVVCEGLCPSLFRITPLQNYKNFVVK 540 NVAFPGGLQTNSIGTGESIIPAASGLTMGLNISNWTVGGQKVTMENFQANSLGQFNIDGS 600

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Highlights

P. funiculosum dextranase was heterologously expressed in two Penicillium species.

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The GH49 enzyme was expressed under the control of strong cbh1 and xylA promoters.

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Expression of the dextranase was higher in P. verruculosum than in P. canescens.

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The purified recombinant dextranases displayed very similar properties.

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High specific activity of dextranase makes it a good candidate for the food industry.

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•