Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases

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Article Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases

Silke L. Pittrof 1, Larissa Kaufhold 1, Anja Fischer 1 and Daniel Wefers 1,2,*

1 Department of Food Chemistry and Phytochemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany; [email protected] (S.L.P.); [email protected] (L.K.); anja-93-ﬁ[email protected] (A.F.) 2 Food Chemistry–Functional Food, Institute of Chemistry, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany * Correspondence: [email protected]

Abstract: Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as O3- and O4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from Bacteroides thetaiotaomicron and Streptococcus salivarius were cloned and heterologously expressed in Escherichia coli. The two recombinant enzymes as well as two commercial dextranases from Chaetomium sp. and Penicillium sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from Chaetomium sp., Penicillium sp., and Bacteroides thetaiotaomicron, isomaltose was the end product

of the hydrolysis from linear dextrans, whereas Penicillium sp. dextranase led to isomaltose and isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when

Citation: Pittrof, S.L.; Kaufhold, L.; incubated with isomaltotriose. For O3- and O4-branched dextrans, the fungal dextranases yielded Fischer, A.; Wefers, D. Products significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product Released from Structurally Different patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity. Dextrans by Bacterial and Fungal Dextranases. Foods 2021, 10, 244. Keywords: glucans; structure; HPAEC-PAD; enzymatic fingerprinting; oligosaccharides; chromatog- https://doi.org/10.3390/foods raphy; enzymes; exopolysaccharides; enzymatic cleavage 10020244

Academic Editor: Arun K. Bhunia Received: 29 November 2020 1. Introduction Accepted: 21 January 2021 Published: 26 January 2021 Dextrans are versatile polysaccharides that have a high application potential in foods, cosmetics, or medical products [1]. They are formed by dextransucrases which are pro-

Publisher’s Note: MDPI stays neutral duced by several lactic acid bacteria. These enzymes are able to hydrolyze sucrose, release with regard to jurisdictional claims in fructose, and transfer the intermediately bound glucose units to a growing polysaccha- published maps and institutional afﬁl- ride [2]. Dextrans are characterized by a homogenic backbone of α-1,6-linked glucose units. iations. Depending on the dextransucrase, this backbone may be linear or decorated with mono-, di-, or oligomeric side chains at position O2, O3, or O4, which results in a considerable structural complexity [3,4]. A speciﬁc hydrolysis of dextrans can be achieved by the application of endo-dextranases, which hydrolyze the linear, α-1,6-linked areas of the dextran backbone. This results in the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. conversion of polymeric dextrans to glucose, isomaltose, linear isomalto-oligosaccharides, This article is an open access article and, in case of branched dextrans, branched oligosaccharides. The most important ap- distributed under the terms and plication of dextranases is the removal of dextrans during the sugar production process. conditions of the Creative Commons Furthermore, dextranases have potential for some medical purposes such as the treatment Attribution (CC BY) license (https:// of dental plaque [5]. In addition, endo-dextranase hydrolysis and subsequent analysis of the creativecommons.org/licenses/by/ liberated oligosaccharides can be used for structural analysis of dextrans or mixed-linkage 4.0/). α-glucans [6–13]. Dextranases are expressed by a wide range of microorganisms and have

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different properties with regards to their optimum incubation conditions as well as their product patterns [5]. However, information on the product patterns of dextranases from varying origins, especially with regards to branched oligosaccharides, is scarce. Therefore, the aim of this study was to comparatively investigate the release of linear and branched oligosaccharides from linear, O3-branched, and O4-branched dextrans by bacterial and fungal dextranases. Fungal dextranases are widely applied and commercially available, thus, two commercial fungal dextranases (from Chaetomium sp. and Penicillium sp.) were used in this study. Dextranases from both species were previously investigated with regards to their optimum incubation conditions and, in part, their hydrolysis products [7,8,14–24]. Because bacterial dextranases may have a signiﬁcantly different product spectrum, genes encoding for two dextranases were cloned from Streptococcus (S.) salivarius DSM 20560 (SSAL_1145) and Bacteroides (B.) thetaiotaomicron DSM 2079 (BT_3087). As for the fungal dextranases, some investigations were conducted on the optimum incubation conditions and the hydrolysis products of dextranases from these genera/species [25–29]. For a detailed investigation of the products released by hydrolysis with the bacterial and fungal dextranases, dextrans from Ligilactobacillus animalis, Latilactobacillus curvatus, and Limosilactobacillus reuteri were used (for simplicity, the abbreviation L. will be used for all three genera). These dextrans have completely linear, O3-branched, and O4-branched backbones, respectively [11,30]. Be- cause several oligosaccharides released from these dextrans by Chaetomium sp. dextranase were previously isolated and characterized in detail [11,12], an analysis of the hydrolysates by HPAEC-PAD yields detailed information on the product patterns of the dextranases.

2. Materials and Methods 2.1. General AZCL-dextran and dextranase from Chaetomium sp. (EC 3.2.1.11, 490 U/mg) were purchased from Megazyme (Bray, Ireland). Dextranase from Penicillium sp. (EC 3.2.1.11, 100–250 U/mg) was purchased from Sigma-Aldrich (Schnelldorf, Germany). pLIC-SGC1 was a gift from Nicola Burgess-Brown (Addgene plasmid # 39187). If not stated otherwise, all other chemicals used were of “p.a.” grade or better and were purchased from Sigma Aldrich, VWR (Radnor, PA, USA), or Carl Roth (Karlsruhe, Germany).

2.2. Molecular Cloning and Heterologous Expression Genes encoding for dextranase from S. salivarius DSM 20560 (SSAL_1145; GenBank accession number: AIY20395) and B. thetaiotaomicron DSM 2079 (BT_3087; GenBank accession number: AAO78193) were amplified from the respective genomic DNA by using a Phusion High-Fidelity PCR kit (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). For ligation independent cloning of the genes into the pLIC-SGC1 vector, specific overhangs were added at both ends of the genes [31–33]. Genes encoding for glucansucrases from L. animalis TMW 1.971 and L. curvatus TMW 1.624 (previously described by Rühmkorf et al. [34]) were cloned from genomic DNA, while the gene encoding for glucansucrase from L. reuteri TMW 1.106 was previously cloned into the pLIC-SGC1 vector [13]. The sequences of the inserted genes were confirmed by Sanger sequencing (Eurofins GATC Biotech, Konstanz, Germany). For protein expression, plasmids were transformed into OneShot BL21 Star (DE3) cells by heat shock. A single colony was transferred to 5 mL of LB medium (100 µg ampicillin/mL) and grown at 37 ◦C and 225 rpm. After growing the precultures for 6 h at 37 ◦C and 225 rpm, the suspension was transferred to 200 mL of fresh LB medium (100 µg ampicillin/mL). The culture was further incubated for 3 h at 37 ◦C and 225 rpm, and isopropyl-β-D-thiogalactopyranoside (final concentration: 0.1 mM) was added to induce protein expression. After incubating for 16 h at room temperature, cells were harvested by centrifugation, re-suspended in binding buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.5), and Foods 2021, 10, 244 3 of 12

lysed by sonication (amplitude 50%, 3 × 20 s pulse, 59.9 s pause) with a Soniﬁer W−250 D (Branson, Danbury, CT, USA). Cell debris were removed by centrifugation (30 min, 4 ◦C, 20,000× g) and the clear supernatant was transferred to a HisPur Ni-NTA resin (Thermo Fisher Scientiﬁc; pre-equilibrated with binding buffer). The suspension was incubated for 1 h at 4 ◦C, washed with 4 volumes of binding buffer, and the recombinant proteins were eluted with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 100 mM imidazole, pH 7.5).

2.3. Optimum Incubation Conditions Dextranase activity and optimum incubation conditions were determined by monitor- ing the hydrolysis of AZCL-dextran according to the manufacturer’s instructions. Brieﬂy, 20 µL of enzyme solution was added to 100 µL of AZCL-dextran suspension in different buffers (pH 4.0–5.5: 50 mM sodium acetate buffer; pH 6.0–7.0: 50 mM sodium phosphate buffer; pH 7.5 and 8.0: 50 mM Tris buffer), which were pre-equilibrated at the desired temperature. Subsequently, the suspension was incubated for 15 min (temperature from 20–90 ◦C). The reaction was stopped by adding 1 mL Tris-base (2%, w/v) and insoluble ma- terial was removed by centrifugation. The extent of hydrolysis was analyzed by measuring the absorption at 590 nm in a microplate reader.

2.4. Dextran Production Prior to dextran production, the hydrolytic activity of recombinant glucansucrases was analyzed by hydrolyzing 35 mM para-nitrophenyl-α-glucoside in 50 mM sodium acetate buffer (supplemented with 1 mM CaCl2, pH 5.5). The amount of para-nitrophenol formed was continuously measured in a microplate reader (Inﬁnite M200 PRO, Tecan, Männedorf, Switzerland) at 400 nm. Subsequently, dextrans were produced by adding 1 mU (1 U = 1 µmol of released para-nitrophenol/min) dextransucrase to a 500 mM sucrose solution (50 mM sodium acetate buffer, 1 mM CaCl2, 0.05% ProClin 300, pH 5.5). After 16 h of incubation, dextrans were precipitated by adding two volumes of ethanol (96% v/v). Subsequently, the precipitated polysaccharides were recovered by centrifugation and redissolved/resuspended in water. To remove low molecular weight compounds, the solutions/suspensions were dialyzed for 24 h against water (molecular weight cut-off: 3.5 kDa), and freeze-dried.

2.5. Dextranase-Mediated Hydrolysis For dextran hydrolysis, the partially insoluble L. animalis and L. curvatus dextrans were dissolved in 90:10 DMSO/water (v/v, 10 mg/mL) and diluted with water (final concentration 1 mg/mL). L. reuteri dextrans were completely soluble and thus dissolved in water (1 mg/mL). Hydrolysis was carried out by adding different activities (determined by the above described AZCL dextran assay) to the dextran solutions, followed by 24 h of incubation at 30 ◦C (SSAL_1145) or 40 ◦C (all other dextranases). After inactivation of the enzymes by heating to 95 ◦C for 5 min, the solutions were diluted and analyzed by HPAEC-PAD as described below. To isolate isomaltotriose and isomaltotetraose for incubation with Penicillium sp. dextranase, L. animalis dextrans were hydrolyzed with Penicillium sp. dextranase (for isomaltotetraose) and BT_3087 (for isomaltotriose) in preparative scale. After inactivation of the enzymes and freeze-drying, the resulting oligosaccharides were separated on a Bio- Gel P-2 column (85 × 2.6 cm) with water as eluent at 1 mL/min and 40 ◦C. RI-detection was used, fractions collected at fixed time intervals were pooled according to the chromatogram, and freeze-dried. For dextranase hydrolysis, isomaltotriose and isomaltotetraose were dissolved in water (final concentration: 100 µg/mL) and incubated with 0.1 U, 5 U, 25 U, and 100 U as described above. Foods 2021, 10, x FOR PEER REVIEW 4 of 12

Foods 2021, 10, 244 were dissolved in water (final concentration: 100 μg/mL) and incubated with 0.1 U, 45 of U, 12 25 U, and 100 U as described above.

2.6.2.6. High High Performance Performance Anion Anion Exchange Exchange Chromatography Chromatography HPAEC-PADHPAEC-PAD analysis analysis of of dextranase dextranase hydrolysates hydrolysates was was carried carried out out on on an an ICS-5000 ICS-5000 systemsystem (Thermo (Thermo Scientific Scientific Dionex, Dionex, Sunnyvale, Sunnyvale, CA, CA, USA). USA). A A CarboPac CarboPac PA200 PA200 column column (250(250 mm mm ×× 33 mm mm i.d.,i.d., 5.5 5.5 μmµm particle particle size, size, Thermo Thermo Scientific Scientific Dionex) Dionex) with with water water (A), (A),0.1 M0.1 sodium M sodium hydroxide hydroxide (B), 0.1 (B), M 0.1sodium M sodium hydrox hydroxideide + 0.5 M + sodium 0.5 M sodium acetate (C) acetate as eluents (C) as waseluents used was for separation. used for separation. The column The was column held at was 25 °C held and at the 25 flow◦C and rate the was flow 0.4 mL/min. rate was Before0.4 mL/min. every run, Before the column every run, was thewashed column with was 100% washed C for 10 with min 100% and Cequilibrated for 10 min with and 90%equilibrated A and 10% with B 90%for 20 A andmin. 10% After B for injection, 20 min. the After following injection, gradient the following was applied: gradient 0–10 was min,applied: isocratic0–10 90% min ,A isocratic and 10% 90% B; 10–20 A and min, 10% B;linear 10–20 from min, 90% linear A and from 10% 90% B Ato and100% 10% B; B20– to 45100% min, B; linear20–45 from min, 100% linear B from to 80% 100% B and B to 20% 80% C; B 45–55 and 20% min, C; linear 45–55 to min, 100% linear C; 55–60 to 100% min, C; isocratic55–60 min 100%, isocratic C. Previously 100% C. characterized Previously characterized oligosaccharides oligosaccharides [11,12] were [used11,12 as] were standard used compounds.as standard compounds.

3.3. Results Results and and Discussion Discussion 3.1.3.1. Dextranase Dextranase Production Production and and Characterization Characterization HeterologousHeterologous expression expression of of the the bacter bacterialial dextranases dextranases SSAL_1145 SSAL_1145 and and BT_3087 BT_3087 fol- fol- lowedlowed by by purification purification with with immobilized immobilized metal metal affinity affinity chromatography chromatography yielded yielded sufficient sufficient amounts of pure enzyme (confirmed by SDS-PAGE, Figure S1). As described above, the amounts of pure enzyme (confirmed by SDS-PAGE, Figure S1). As described above, the focus of this study was on investigating the released oligosaccharides by HPAEC-PAD, focus of this study was on investigating the released oligosaccharides by HPAEC-PAD, however, improper incubation conditions may result in a lack of hydrolysis. Therefore, an however, improper incubation conditions may result in a lack of hydrolysis. Therefore, an assay based on AZCL-dextran was used to determine suitable incubation conditions for assay based on AZCL-dextran was used to determine suitable incubation conditions for the purified and purchased enzymes (Figure1). the purified and purchased enzymes (Figure 1).

FigureFigure 1. Relative activityactivity of of bacterial bacterial (SSAL_1145 (SSAL_1145 and and BT_3087) BT_3087) and and fungal fungal (Penicillium (Penicilliumsp. and sp.Chaetomium and Chaetomiumsp.) dextranases sp.) dex- tranasesat different at different pH values pH and values temperatures, and temperatures, determined determin photometricallyed photometrically by using AZCL-dextran. by using AZCL-dextran. The activity at The each activity condition at eachwas analyzedcondition inwas at leastanalyzed two independentin at least two experiments. independent experiments.

TheThe activity ofof thethe enzymes enzymes at at different different temperatures temperatures was was clearly clearly different. different. Dextranase Dex- tranasefrom Chaetomium from Chaetomiumsp. showed sp. showed signiﬁcant significant activity betweenactivity between 20 and 90 20◦ Cand with 90an °C optimum with an optimumbetween between 50–60 ◦C, 50–60 which °C, is which in good is in agreement good agreement with data with from data thefrom literature the literature and fromand fromthe supplier the supplier [15,18 [15,18,19].,19]. In contrast, In contrast,Penicillium Penicilliumsp. sp. dextranase dextranase showed showed a rather a rather narrow nar- rowactivity activity range range between between 20 20 and and 60 60◦C °C with with a a maximum maximum around around40 40 ◦°C.C. Comparable data data werewere also also obtained obtained in in previous previous studies studies on on PenicilliumPenicillium sp.sp. dextranases dextranases [14,24]. [14, 24Just]. as Just pre- as viouslypreviously described described for fordextranases dextranases from from strept streptococciococci [26–28], [26–28 ],SSAL_1145 SSAL_1145 had had its its optimum optimum temperaturetemperature between between 25 25 and and 40 40 °C◦C and and was was not not active active at at higher higher temperatures. temperatures. Notably, Notably, dextranase from B. thetaiotaomicron showed nearly maximum activity up to 60 ◦C, thus, the enzyme is very active above the optimum growth conditions of its organism of origin.

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Foods 2021, 10, 244 5 of 12 dextranase from B. thetaiotaomicron showed nearly maximum activity up to 60 °C, thus, the enzyme is very active above the optimum growth conditions of its organism of origin. Fungal dextranases were most active (>50 (>50%% relative relative activity) activity) between between pH pH 5 5 and and 7, which is in good agreement with previous studies [[14,15,18,19,21,24].14,15,18,19,21,24]. BT_3087 showed a comparably high activity between pH 4.0 and 7.5, which is again notable with regards to the optimum optimum growth growth conditions conditions of of its its organi organismsm of of origin. origin. In In contrast, contrast, dextranase dextranase from from S. salivariusS. salivarius waswas most most active active at weakly at weakly acidic acidic pH (4.0–5.5), pH (4.0–5.5), which which is in isgood in good agreement agreement with previouswith previous results results on dextranases on dextranases from from streptococci streptococci [26–28]. [26– 28Because]. Because all enzymes all enzymes were were ac- tiveactive at atweakly weakly acidic acidic pH pH and and because because buffe buffersrs may may interfere interfere with the chromatographicchromatographic analyses, incubations ofof structurallystructurally differentdifferent dextrans dextrans were were conducted conducted in in bidistilled bidistilled water water(pH (pH 5.5–6.0). 5.5–6.0).

3.2. Hydrolysis Hydrolysis of Linear Dextrans To investigate to which degree α-1,6-linked-1,6-linked glucose units can bebe cleaved,cleaved, dextrandextran from L.L. animalis animalis waswas used. used. It Ithas has been been demonstrated demonstrated in previous in previous studies studies [30,34] [30, 34and] and in our in laboratory,our laboratory, that that this this polysaccharide polysaccharide is completely is completely linear. linear. However, However, this this also also results results in some solubility issues, which can mostly be ov overcomeercome by solvation in DMSO and dilution with water. water. Nevertheless, Nevertheless, incomplete incomplete solvation solvation may may slow slow down down or inhibit or inhibit enzymatic enzymatic hy- drolysishydrolysis to tosome some extent, extent, which which was was also also indicated indicated by by lower lower peak peak intensities intensities compared compared to other dextrans. ◦ In previous studies, we demonstrated that 24 h of incubation at 40 °CC and enzyme activity of 55 UU dextranasedextranase from fromChaetomium Chaetomiumsp./mg sp./mg dextran dextran is is in in most most cases cases sufﬁcient sufficient for for an end point incubation [8,11,12]. Therefore, 5 U and 24 h of incubation at 30 ◦C (SSAL_1145) an end◦ point incubation [8,11,12]. Therefore, 5 U and 24 h of incubation at 30 °C (SSAL_1145)and 40 C (all and other 40 dextranases) °C (all other were dextranases) initially used were for initially the hydrolysis used for of theL. animalishydrolysisdextran of L. animalisin this study. dextran The in HPAEC-PAD this study. The chromatograms HPAEC-PAD resulting chromatograms from these resulting conditions from arethese shown con- ditionsin Figure are2. shown in Figure 2.

Figure 2. 2. HPAEC-PADHPAEC-PAD chromatograms chromatograms of of L.L. animalis animalis dextransdextrans after after 24 24h of h incubation of incubation with with 5 U 5of U dextranasesof dextranases from from ChaetomiumChaetomium sp.,sp., PenicilliumPenicillium sp.,sp., B. thetaiotaomicronB. thetaiotaomicron (BT_3087),(BT_3087), and andS. salivariusS. salivarius (SSAL_1145). The oligosaccharides shown in the chromatograms were identified by comparison (SSAL_1145). The oligosaccharides shown in the chromatograms were identiﬁed by comparison with standard compounds and were not present in the enzyme and dextran solutions. The later with standard compounds and were not present in the enzyme and dextran solutions. The later eluting peaks in the SSAL_1145 hydrolysate result from linear isomalto-oligosaccharides with a highereluting degree peaks of in polymerization. the SSAL_1145 hydrolysate result from linear isomalto-oligosaccharides with a higher degree of polymerization. As expected, enzymatic hydrolysis with Chaetomium sp. dextranase resulted in glu- As expected, enzymatic hydrolysis with Chaetomium sp. dextranase resulted in glucose and isomaltose. Although small amounts of isomaltotriose were detected (probably cose and isomaltose. Although small amounts of isomaltotriose were detected (probably derived from a delayed hydrolysis of only partially dissolved dextran), this oligosaccharide disappeared with higher enzyme amounts (as already described previously [8,11,12,15]).

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Foods 2021, 10, 244derived from a delayed hydrolysis of only partially dissolved dextran), this oligosaccha- 6 of 12 ride disappeared with higher enzyme amounts (as already described previously [8,11,12,15]). Hydrolysis with dextranase from Penicillium sp. resulted in the formation of isomaltose and isomaltotetraose, whereas glucose or isomaltotriose were not detected. Hydrolysis with dextranase from Penicillium sp. resulted in the formation of isomaltose and Thus, these resultsisomaltotetraose, suggest that whereasthe Penicillium glucose sp. or isomaltotriose dextranase used were is not only detected. able to Thus, release these results even-numberedsuggest isomalto-oligosaccharides. that the Penicillium sp. Ho dextranasewever, usedisomaltotriose is only able was to release detected even-numbered in some preliminaryisomalto-oligosaccharides. experiments, therefore, However,isomaltotriose isomaltotriose and isomaltotetraose was detected inwere some puri- preliminary fied and separatelyexperiments, incubated therefore, with different isomaltotriose amounts and of isomaltotetraose Penicillium sp.were dextranase puriﬁed and separately (Figure 3). incubated with different amounts of Penicillium sp. dextranase (Figure3).

Figure 3.FigureHPAEC-PAD 3. HPAEC-PAD chromatograms chromatograms of isomaltotriose of isomaltotriose and isomaltotetraose and isomaltotetraose after 24 h of after incubation 24 h of with incuba- different amountstion of dextranase with different from Penicilliumamounts ofsp. dextranase from Penicillium sp.

Interestingly, isomaltotetraoseInterestingly, isomaltotetraose was not further was nothydrolyzed further hydrolyzed by Penicillium by Penicillium sp. dex-sp. dextranase. In contrast,tranase. 5 U or In more contrast, of the 5 U enzy or moreme converted of the enzyme isomaltotriose converted isomaltotriose to isomaltose to and isomaltose isomaltotetraose,and which isomaltotetraose, demonstrates which that demonstrates the enzyme that catalyzes the enzyme a disproportionation catalyzes a disproportionation re- reaction with even-numbered isomalto-oligosaccharides as end products. In previous action with even-numbered isomalto-oligosaccharides as end products. In previous inves- investigations on Penicillium sp. dextranases, glucose, isomaltose, isomaltotriose, and tigations on Penicilliumisomaltotetraose sp. dextranases, were described glucose, as the isomaltose, main hydrolysis isomaltotriose, products [14 and,20, 22isomalto-,23]. The absence tetraose were describedof glucose as was the only main described hydrolysis for a products dextranase [14,20,22,23]. from Penicillium The aculeatum absence, butof glu- this enzyme cose was only describedyielded isomaltose for a dextranase and isomaltotriose from Penicillium as end products aculeatum [24]. A, but condensation this enzyme reaction was yielded isomaltosedescribed and isomaltotriose for a bacterial as dextranase end products from Arthrobacter[24]. A condensation globiformis [reaction35], while was it was only described for a hypothesizedbacterial dextranase for a Penicillium from Arthrobacter lilalicum dextranase globiformis [22]. [35], However, while the it was latter only enzyme was hypothesized forable a Penicillium to cleave isomaltotriose lilalicum dextranase to glucose and[22]. isomaltose. However, Therefore, the latter the enzyme preferred was production able to cleave isomaltotrioseof even-numbered to glucose isomalto-oligosaccharides and isomaltose. Therefore, and the catalysis the preferred of a disproportionation produc- reaction is a novel property of Penicillium sp. dextranases. tion of even-numbered isomalto-oligosaccharides and the catalysis of a disproportiona- Because the HPAEC-PAD chromatograms obtained from the hydrolysis of L. ani- tion reaction is amalis noveldextran property with of BT_3087 Penicillium and sp. SSAL_1145 dextranases. contained isomaltotriose as well as higher Because theoligosaccharides HPAEC-PAD chromatograms (in case of SSAL_1145), obtained a higher from enzyme the hydrolysis amount (100of L. U) animalis was applied to dextran with BT_3087investigate and ifSSAL_1145 further hydrolysis contained is possible isomaltotriose (Figure4). as well as higher oligosaccharides (in case of SSAL_1145),As it can be seena higher from enzyme the chromatograms, amount (100 higher U) was activities applied of to both investi- BT_3087 and gate if further hydrolysisSSAL_1145 is resultedpossible in (Figure (almost) 4). complete degradation of higher oligosaccharides and isomaltotriose to isomaltose and glucose. Therefore, both enzymes eventually yield the same end products as dextranase from Chaetomium sp. However, different products may be obtained after the hydrolysis of branched dextrans.

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Figure 4.FigureHPAEC-PAD 4. HPAEC-PAD chromatograms chromatograms of L. animalis ofdextrans L. animalis after dextrans 24 h of incubation after 24 h with of incubation 5 U and 100 with U of dextranases5 U from B. thetaiotaomicronand 100 U of dextranases(BT_3087) and fromS. salivarius B. thetaiotaomicron(SSAL_1145). (BT_3087) The oligosaccharides and S. salivarius shown (SSAL_1145). in the chromatograms The were identiﬁedoligosaccharides by comparison with shown standard in the compoundschromatograms and were were not identified present inby the comparison enzyme and with dextran standard solutions. com- The later elutingpounds peaks and in were the SSAL_1145 not present hydrolysates in the enzyme result and from dextran linear solutions. isomalto-oligosaccharides The later eluting with peaks a higher in the degree of polymerization.SSAL_1145 hydrolysates result from linear isomalto-oligosaccharides with a higher degree of polymerization. 3.3. Hydrolysis of O3- and O4-Branched Dextrans As it can be seenThe from enzymatic the chromatograms, hydrolysis of O3-branched higher dextransactivities was of investigated both BT_3087 by using and dextrans SSAL_1145 resultedproduced in (almost) by glucansucrase complete degradation from L. curvatus of higherTMW 1.624.oligosaccharides Oligosaccharides and iso- that result maltotriose to isomaltosefrom the hydrolysis and glucose. of these Theref dextransore, both with enzymesChaetomium eventuallysp. dextranase yield the were same previously end products asisolated dextranase and characterizedfrom Chaetomium in detail sp. [ However,11]. To achieve different an end-point products hydrolysis may be ofob- the oligo- tained after the andhydrolysis polysaccharides, of branched theenzyme dextrans. amounts needed for complete hydrolysis of L. animalis dextrans were used (bacterial dextranases: 100 U, fungal dextranases: 5 U, Figure5). 3.3. Hydrolysis of O3-Dextranase and O4-Branched from Chaetomium Dextranssp. yielded the expected oligosaccharide pattern (except for the minor amounts of isomaltotriose). With regards to the branched oligosaccharides, The enzymaticPenicillium hydrolysissp. dextranase of O3-branched yielded dextrans a similar productwas investigated pattern and by only using slightly dex- varying trans producedpeak by glucansucrase intensities were from observed. L. curvatus In addition, TMW this1.624. enzyme Oligosaccharides only liberated that isomaltose re- and sult from the hydrolysisisomaltotetraose, of these which dextrans is in good with agreement Chaetomium with the sp. results dextranase described were above. previ- As expected, ously isolated andthe bacterialcharacterized dextranases in detail yielded [11]. glucose To achieve and isomaltose an end-point as the hydrolysis main products, of the however, oligo- and polysaccharides,clearly different the intensities enzyme were amounts detected needed for the for branched complete oligosaccharides hydrolysis of compared L. to Chaetomium Penicillium animalis dextransthe were dextranases used (bacterial from dextranases:sp. and 100 U, fungalsp. dextranases: While D3-Va 5 andU, D3-VI were of comparable abundance, D3-IV showed a lower peak intensity and D3-Vb showed a clearly Figure 5). higher peak intensity compared to the hydrolysates obtained with fungal dextranases. Both oligosaccharides represent monomeric, O3-bound side chains and only differ in the attachment of an additional non-reducing glucose unit bound to the O3-branched backbone unit. Therefore, it is likely that the two oligosaccharides represent the same portion of monomeric side chains, but different modes of actions of the enzymes yield varying abundances of the corresponding oligosaccharides. This could be the result from the attack of different sites within the α-1,6-linked areas of the dextrans, which would lead to different intermediate products and thus in an increased formation of D3-Vb, which cannot be hydrolyzed any further. However, the oligosaccharide proﬁles obtained with bacterial dextranases also clearly indicate the predominant abundance of monomeric side chains. The O4-branched dextrans formed by glucansucrase from L. reuteri TMW 1.106 were also investigated because they represent a rather rare dextran type and because several of the enzymatically released oligosaccharides were previously characterized [11,12]. The resulting HPAEC-PAD chromatograms are shown in Figure6.

Figure 5. HPAEC-PAD chromatograms of L. curvatus dextrans after 24 h of incubation with 5 U of dextranases from Chaetomium sp., Penicillium sp. and 100 U of dextranases from B. thetaiotaomicron

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Figure 4. HPAEC-PAD chromatograms of L. animalis dextrans after 24 h of incubation with 5 U and 100 U of dextranases from B. thetaiotaomicron (BT_3087) and S. salivarius (SSAL_1145). The oligosaccharides shown in the chromatograms were identified by comparison with standard compounds and were not present in the enzyme and dextran solutions. The later eluting peaks in the SSAL_1145 hydrolysates result from linear isomalto-oligosaccharides with a higher degree of polymerization.

As it can be seen from the chromatograms, higher activities of both BT_3087 and SSAL_1145 resulted in (almost) complete degradation of higher oligosaccharides and isomaltotriose to isomaltose and glucose. Therefore, both enzymes eventually yield the same end products as dextranase from Chaetomium sp. However, different products may be obtained after the hydrolysis of branched dextrans.

3.3. Hydrolysis of O3- and O4-Branched Dextrans Foods 2021, 10, x FOR PEER REVIEW 8 of 12 The enzymatic hydrolysis of O3-branched dextrans was investigated by using dextrans produced by glucansucrase from L. curvatus TMW 1.624. Oligosaccharides that result from the hydrolysis of these dextrans with Chaetomium sp. dextranase were previ- (BT_3087), and S. salivarius (SSAL_1145). The oligosaccharides shown in the chromatograms were identifiedously isolated by comparison and characterizedwith previously charin detailacterized [11]. standard To achieve compounds an end-point [11] and were hydrolysis not of the presentoligo- in andthe enzyme polysaccharides, and dextran solutions. the enzyme amounts needed for complete hydrolysis of L. Foods 2021 10 , , 244animalis dextrans were used (bacterial dextranases: 100 U, fungal dextranases: 5 U, 8 of 12 FigureDextranase 5). from Chaetomium sp. yielded the expected oligosaccharide pattern (except for the minor amounts of isomaltotriose). With regards to the branched oligosaccharides, Penicillium sp. dextranase yielded a similar product pattern and only slightly varying peak intensities were observed. In addition, this enzyme only liberated isomaltose and isomaltotetraose, which is in good agreement with the results described above. As expected, the bacterial dextranases yielded glucose and isomaltose as the main products, however, clearly different intensities were detected for the branched oligosaccharides compared to the dextranases from Chaetomium sp. and Penicillium sp. While D3-Va and D3-VI were of comparable abundance, D3-IV showed a lower peak intensity and D3-Vb showed a clearly higher peak intensity compared to the hydrolysates obtained with fungal dextranases. Both oligosaccharides represent monomeric, O3-bound side chains and only differ in the attachment of an additional non-reducing glucose unit bound to the O3-branched backbone unit. Therefore, it is likely that the two oligosaccharides represent the same portion of monomeric side chains, but different modes of actions of the enzymes yield varying abundances of the corresponding oligosaccharides. This could be the result from the attack of different sites within the α-1,6-linked areas of the dextrans, which would lead to different intermediate products and thus in an increased formation of D3-Vb, which cannot be hydrolyzed any further. However, the oligosaccharide profiles obtained with bacterial dextranases also clearly indicate the predominant abundance of monomeric side chains. Figure 5. HPAEC-PADFigureThe O5. 4-branchedHPAEC-PAD chromatograms dextrans chromatograms of L. curvatusformeddextrans by of glucansucrase L. curvatus after 24 h dextrans of from incubation L. after reuteri with 24 h 5TMW Uof ofincubation dextranases1.106 were with from 5 U of Chaetomiumalsodextranasessp., investigatedPenicillium fromsp. because andChaetomium 100 Uthey of dextranases represent sp., Penicillium froma ratherB. thetaiotaomicronsp. rare and dextran 100 U(BT_3087), of type dextranases and and becauseS. salivarius from several B.(SSAL_1145). thetaiotaomicron of The oligosaccharidesthe enzymatically shown in thereleased chromatograms oligosaccharides were identiﬁed were by comparisonpreviously with characterized previously characterized [11,12]. The standard compoundsresulting [11] and HPAEC-PAD were not present chromatograms in the enzyme and are dextran shown solutions. in Figure 6.

Figure 6.FigureHPAEC-PAD 6. HPAEC-PAD chromatograms chromatograms of L. reuteri of dextransL. reuteri afterdextrans 24 h after of incubation 24 h of incubation with 5 U of with dextranases 5 U of from Chaetomiumdextranasessp., Penicillium from sp.Chaetomium and 100 U sp., of dextranases Penicillium from sp. andB. thetaiotaomicron 100 U of dextranases(BT_3087), from and B.S. salivariusthetaiotaomicron(SSAL_1145). The oligosaccharides(BT_3087), and shown S. salivarius in the chromatograms (SSAL_1145). were The identiﬁed oligosaccharides by comparison shown with in previouslythe chromatograms characterized were standard compoundsidentified [11,12] by and comparison were not present with in previously the enzyme charac and dextranterized solutions. standard compounds [11,12] and were not present in the enzyme and dextran solutions.

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The chromatogram obtained from the hydrolysis of L. reuteri dextrans with 5 U of Chaetomium sp. dextranaseThe chromatogram yielded the obtainedpreviously from described the hydrolysis product of L. pattern reuteri dextrans [11]. A withcom- 5 U of parable pattern wasChaetomium detectedsp. in dextranase the hydrolysate yielded the obtained previously with described Penicillium product sp. pattern dextranase, [11]. A com- however, D4-IV andparable D4-Va pattern had was a higher detected inte in thensity, hydrolysate whereas obtained the other with oligomersPenicillium showedsp. dextranase, a lower intensity. Becausehowever, the D4-IV hexa- and D4-Vaand heptameric had a higher oligosaccharides intensity, whereas thecontain other oligomerstwo or three showed a 1,6-linked glucoselower units intensity. on the Because non-reducing the hexa- andend, heptameric the higher oligosaccharides abundance of contain D4-IV two and or three 1,6-linked glucose units on the non-reducing end, the higher abundance of D4-IV and D4-Va may result from an elevated and possibly ongoing hydrolysis of high molecular D4-Va may result from an elevated and possibly ongoing hydrolysis of high molecular weight compounds.weight To compounds.evaluate this, To L. evaluate reuteri this,dextransL. reuteri weredextrans incubated were with incubated a higher with en- a higher zyme amount (100enzyme U) of amountPenicillium (100 sp. U) ofdextranasePenicillium and,sp. dextranase as a comparison, and, as a comparison,dextranase from dextranase Chaetomium sp. (Figurefrom Chaetomium 7). sp. (Figure7).

FigureFigure 7. HPAEC-PAD 7. HPAEC-PAD chromatograms chromatograms of L. reuteri dextrans of L. reuteri after 24dextrans h of incubation after 24 with h of different incubation amounts with of different dextranase from Chaetomiumamounts ofsp. dextranase and Penicillium fromsp. Chaetomium The oligosaccharides sp. and shown Penicillium in the chromatograms sp. The oligosaccharides were identiﬁed shown by comparison in the with previouslychromatograms characterized were standard identified compounds by comparison [11,12] and with were previously not present characterized in the enzyme andstandard dextran com- solutions. pounds [11,12] and were not present in the enzyme and dextran solutions. In case of Penicillium sp. dextranase, an activity dependent degradation of higher oligosaccharides can be observed. While D4-VI and D4-VIIa are the clearly predominating In case of Penicillium sp. dextranase, an activity dependent degradation of higher ol- products at low enzyme activities, they are completely degraded to D4-IV and D4-Va at igosaccharides canhigher be observed. activities. Therefore, While D4-V isomaltoseI and D4-VIIa units are liberatedare the clearly from the predominating non-reducing ends of products at low theenzyme higher activities, oligosaccharides. they ar Interestingly,e completely higher degraded activities to ofD4-IVChaetomium and D4-Vasp. dextranase at higher activities.also Therefore, led to some isomaltose further hydrolysis units are of D4-VIIaliberated (although from the linear non-reducing oligosaccharides ends are of usually the higher oligosaccharides.completely hydrolyzed Interestingly, at this higher activity level).activities As it of can Chaetomium be seen in the sp. chromatograms, dextranase this also led to some compoundfurther hydrolysis (and most likelyof D4-V otherIIa later (although eluting branchedlinear oligosaccharides oligosaccharides) wereare usu- degraded to D4-IV and D4-Va. However, the activity on D4-VIIa seems to be rather low, as only a ally completely hydrolyzedslight improvement at this isactivity observed level). from 5As U overit can 25 be U toseen 100 in U. the Nonetheless, chromatograms,Chaetomium sp. this compound (anddextranase most seemslikely toother be able later to removeeluting isomaltosyl branched or oligosaccharides) isomaltotriosyl units were from de- the non- graded to D4-IV reducingand D4-Va. end ofHowever, branched oligosaccharides.the activity on D4-VIIa Notably, theseems portion to be of rather D4-Vb alsolow, increased as only a slight improvementwith increasing is observed enzyme activity. from 5 ThisU over is most 25 U likely to 100 a result U. Nonetheless, of the hydrolysis Chaeto- of higher mium sp. dextranaseoligosaccharides seems to be with able dimeric to remove side chains isomaltosyl (such as D4-VIIb,or isomaltotriosyl which may beunits present from in trace the non-reducingamounts). end of branched Therefore, oligosacch informationarides. on dimeric, Notably, 1,6-linked the sideportion chains of can D4-Vb also bealso derived from the hydrolysates obtained with higher enzyme activities. increased with increasingThe two enzyme bacterial activity. dextranases This showed is most clearly likely different a result product of the patternshydrolysis compared of to higher oligosaccharidesdextranases with from dimericChaetomium side chainssp. and (suchPenicillium as D4-VIIb,sp. Notably, which D4-IV may and be D4-Vb present (no termi- in trace amounts). Therefore, information on dimeric, 1,6-linked side chains can also be derived from the hydrolysates obtained with higher enzyme activities. The two bacterial dextranases showed clearly different product patterns compared to dextranases from Chaetomium sp. and Penicillium sp. Notably, D4-IV and D4-Vb (no terminal, O6-bound glucose unit) were not detected in significant amounts in the hydrolysates obtained with both enzymes; thus, they are (unlike the fungal dextranases) not able to cleave the 1,6-linkage in a 1,4,6-linked glucose unit. The main products obtained from

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nal, O6-bound glucose unit) were not detected in signiﬁcant amounts in the hydrolysates obtained with both enzymes; thus, they are (unlike the fungal dextranases) not able to cleave the 1,6-linkage in a 1,4,6-linked glucose unit. The main products obtained from the hydrolysis with BT_3087 were D4-Va, D4-VI, and D4-VIIa and the peak intensity decreased from D4-Va to D4-VIIa. SSAL_1145 also yielded these oligosaccharides, but the peak intensity decreased from D4-VIIa to D4-Va. In addition, an unknown oligosaccharide which eluted shortly after D4-VIIa was detected. Therefore, SSAL_1145 showed a clearly lower activity on the O4-branched dextrans than the other enzymes, which is also indicated by the generally low peak intensities and the low abundance of linear isomalto-oligosaccharides.

4. Conclusions Overall, the results of this study demonstrated the diversity of dextranases from different origins. With regards to the incubation conditions, the high activity of all enzymes around 40 ◦C and the weakly acidic pH is in good agreement with previous studies and in most cases the preferred growth conditions of the organisms of origin. Hydrolysis of linear dextrans showed that dextranases from B. thetaiotaomicron, S. salivarius, and Chaetomium sp. eventually lead to the formation of isomaltose. However, isomaltose and isomaltotetraose were detected as the end products of the hydrolysis with Penicillium sp. dextranase. This unique product spectrum is the result of a disproportionation reaction, in which isomaltotriose is converted to isomaltose and isomaltotetraose. For O3-branched dextrans, the product patterns obtained with fungal and bacterial dextranases were comparable within the two groups but different between them. Namely, dextranases from Chaetomium sp. and Penicillium sp. were able to hydrolyze O3-branched dextrans to a higher extent. However, the same conclusions on the dextran structures could be drawn from the oligosaccharide proﬁles. In addition, bacterial dextranases also hydrolyzed O4-branched dextrans to a minor extent compared to fungal dextranases, which resulted in higher portions of longer branched oligosaccharides. Notably, Penicillium sp. dextranase was able to hydrolyze this dextran type to a clearly higher degree than Chaetomium sp. dextranase. Therefore, the enzyme applied for structural analysis or technological purposes has be to chosen carefully. However, the differences between the enzymes can also be used to selectively produce certain oligosaccharides in higher yields.

Supplementary Materials: The following are available online at https://www.mdpi.com/2304-8 158/10/2/244/s1, Figure S1: SDS-PAGE analysis of the two recombinant bacterial dextranases SSAL_1145 and BT_3087. Author Contributions: Conceptualization, D.W.; methodology, S.L.P., L.K., A.F., D.W.; data curation, S.L.P., D.W.; writing, D.W.; supervision, project administration, funding acquisition, D.W. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Carl-Zeiss Foundation. Acknowledgments: The authors are very grateful to Frank Jakob and Rudi Vogel from the Chair of Technical Microbiology (TU Munich) for providing L. animalis TMW 1.971 and L. curvatus TMW 1.624 genomic DNA. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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