Antarctic Rahnella Inusitata: a Producer of Cold-Stable Β-Galactosidase Enzymes

International Journal of Molecular Sciences

Article Antarctic Rahnella inusitata: A Producer of Cold-Stable β-Galactosidase Enzymes

Kattia Núñez-Montero 1,2,†, Rodrigo Salazar 1,3,†, Andrés Santos 1,3,†, Olman Gómez-Espinoza 2,4 , Scandar Farah 1 , Claudia Troncoso 1,3 , Catalina Hoffmann 1, Damaris Melivilu 1, Felipe Scott 5 and Leticia Barrientos Díaz 1,*

1 Laboratory of Molecular Applied Biology, Center of Excellence in Translational Medicine, Universidad de La Frontera, Avenida Alemania 0458, Temuco 4810296, Chile; [email protected] (K.N.-M.); [email protected] (R.S.); [email protected] (A.S.); [email protected] (S.F.); [email protected] (C.T.); [email protected] (C.H.); [email protected] (D.M.) 2 Biotechnology Investigation Center, Department of Biology, Instituto Tecnológico de Costa Rica, Cartago 159-7050, Costa Rica; [email protected] 3 Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile 4 Laboratory of Plant Physiology and Molecular Biology, Institute of Agroindustry, Department of Agronomic Sciences and Natural Resources, Faculty of Agricultural and Forestry Sciences, Universidad de La Frontera, Temuco 4811230, Chile 5 Green Technology Research Group, Facultad de Ingenieria y Ciencias Aplicadas, Universidad de los Andes, Santiago 7620001, Chile; [email protected] * Correspondence: [email protected]; Tel.: +56-45-259-2802 Citation: Núñez-Montero, K.; † These authors contributed equally to this work. Salazar, R.; Santos, A.; Gómez- Espinoza, O.; Farah, S.; Troncoso, C.; Abstract: There has been a recent increase in the exploration of cold-active β-galactosidases, as it Hoffmann, C.; Melivilu, D.; Scott, F.; offers new alternatives for the dairy industry, mainly in response to the current needs of lactose- Barrientos Díaz, L. Antarctic Rahnella intolerant consumers. Since extremophilic microbial compounds might have unique physical and inusitata: A Producer of Cold-Stable chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to pro- β-Galactosidase Enzymes. Int. J. Mol. duce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase Sci. 2021, 22, 4144. https://doi.org/ activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, 10.3390/ijms22084144 and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41–62% of its β-galactosidase activity in the cold Academic Editors: (4 ◦C–15 ◦C). Three β-galactosidases genes were found in the R. inusitata genome, which belong Miguel Gueimonde to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular

Received: 26 January 2021 docking, some of these enzymes exhibited higher lactose predicted afﬁnity than the commercial Accepted: 2 April 2021 control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from Published: 16 April 2021 the Antarctic continent as a prominent cold-active β-galactosidase producer.

Publisher’s Note: MDPI stays neutral Keywords: β-galactosidase; Antarctica; lactose; cold-adapted bacteria; extremozymes with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 1. Introduction Antarctica is possibly one of the most hostile habitats on Earth. It can be deﬁned as a polyextreme environment, given the multiple harsh conditions for life, such as low Copyright: © 2021 by the authors. temperatures, low humidity, limited bioavailability of water, high solar radiation intervals, Licensee MDPI, Basel, Switzerland. extended periods of darkness, and freeze–thaw cycles [1]. However, various organisms, This article is an open access article mainly microorganisms, have adapted to colonize and proliferate under these extreme distributed under the terms and conditions and are of great interest as a source of novel bioactive biotechnological com- conditions of the Creative Commons pounds [2,3]. In recent years, there has been a great interest in discovering new enzymes Attribution (CC BY) license (https:// from extremophilic microorganisms, also called extremozymes, for industrial purposes [4]. creativecommons.org/licenses/by/ Such enzymes belong to microorganisms adapted to multiple adverse conditions that 4.0/).

Int. J. Mol. Sci. 2021, 22, 4144. https://doi.org/10.3390/ijms22084144 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4144 2 of 24

might give them different biochemical and biophysical properties compared to those in mesophilic environments. Cold-adapted enzymes are a prime example, produced by organisms inhabiting low-temperature habitats, which are valuable in several industrial applications (e.g., food, processing, cleaning agents, leather processing, and degradation of xenobiotic agents in the cold.) [5]. During the last decades, Antarctic strains capable of producing lipases, proteases, amylases, cellulases, and β-galactosidases have been described [6]. β-galactosidases (lactases) are used in the food industry to produce probiotic galacto- oligosaccharides and reduce the lactose content in dairy products for lactose-intolerant consumers [7]. Lactose intolerance appears when human adults (and most adult mammals) downregulate intestinal lactase production, a necessary enzyme for the digestion of lactose into glucose and galactose [8]. Hence, lactose-intolerant people suffer from bloating, flatulence, cramps, and nausea after milk consumption. It is estimated that 65% of the world’s population suffers from lactose intolerance [8], ranging from 50 to nearly 100%, depending on the country [9]. Industrial lactose hydrolysis in milk is chiefly carried out using thermostable enzymes at high temperatures (optimum activity at ~50 ◦C) [10]. However, low-temperature processing is preferred to improve the hygienic conditions and the milk properties’ conservation, avoiding spoilage and flavor changes. Then an ideal enzyme for treating milk should retain a significant activity at 4 ◦C–8 ◦C. The activity of the major commercial β-galactosidases (e.g., Lactozym Pure® from Novozymes and Maxilact® from DSM Food Specialties) is severely diminished at 4 ◦C, with optimal activity at nearly 37 ◦C[11]. Thus, optional thermostable enzymes with activity ranging from dairy processing conditions (i.e., 4 ◦C) to moderate temperatures (i.e., 37 ◦C) are needed to increase the process efficiency [11,12]. In recent years, several low-temperature active β-galactosidases have been reported from different psychrophilic bacteria, including Rahnella sp. R3 [13], Halobacterium lacusprofundi, Arthrobacter sp. [7,14], Pseudoalteromonas sp. [15,16], Planococcus sp. [17], Paracoccus sp. [18], Enterobacter ludwigii [19], Halomonas sp. [20], Alkalilactibacillus ikkense [21], and Marinomonas sp. [22]. These enzymes have variable optimum temperatures for their activity, a characteristic that might have been driven by different evolutionary pathways [22]. However, most of the β-galactosidases identified so far display a relatively low activity in cold (~4 ◦C), lost their stability at moderate-high temperatures (>30 ◦C), or are produced in low amounts [21]. Since β-galactosidases are widespread (present in animals, plants, fungi, yeasts, bacteria, and Archaea), a great diversity of newly cold-active β-galactosidases is expected to be discovered in untapped environments. In this work, we aimed to characterize the enzymatic activity of the cold-active β-galactosidases produced by Antarctic bacteria. Our work described the strain Rahnella inusitata Se8.10.12, a species non previously reported in the Antarctic continent, with high β-galactosidase activity at low temperatures (4–15 ◦C).

2. Results and Discussion 2.1. A Rahnella Strain Exhibited the Highest Enzymatic Activity among Multiple Antarctic Strains with β-Galactosidase Activity β-galactosidase enzymes cleave the disaccharide lactose to produce galactose and glucose to enter glycolysis. Microorganisms, vegetables, and animals produce β-galactosidases; however, bacterial sources usually have higher productivity [23]. Here we screened β- galactosidase activity of Antarctic bacteria since new cold-active enzymes need to be explored to process lactose-free products. Our results evidenced 81 (27%) out of 304 Antarc- tic strains with positive enzymatic activity according to the detection by the β-Bluo-Gal indicator on agar (Table S1, Figure S1). Similarly, other authors have explored the potential of polar bacterial strains for the production of β-galactosidase enzymes. Screenings based on cleaved X-Gal in agar plates revealed that 56% of Arctic bacteria were capable of milk degradation, and particularly 40% of the Pseudoalteromonas sp. strains showed β-galactosidase activity [24]. Meanwhile, from a collection of 107 Antarctic bacterial strains, Int. J. Mol. Sci. 2021, 22, 4144 3 of 24 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 25

41showed (38%) β were-galactosidase positive foractivity this enzymatic[24]. Meanwhile, activity from [16 ].a collection Both studies of 107 suggest Antarctic that bacte- Arctic and Antarcticrial strains, cultures 41 (38%) are were a relatively positive richfor this source enzymatic of β-galactosidase activity [16]. producers.Both studies suggest that ArcticOther andβ-galactosidases Antarctic cultures with are potential a relatively for rich the agro-foodsource of β industry-galactosidase have producers. been described in differentOther β bacterial-galactosidases species. with Strains, potential such for as theArthrobacter agro-food psychrolactophilusindustry have been, Thermotoga described mar- itimain different, Lactobacillus bacterial acidophilus species. ,Strains,Sulfolobus such solfataricus as Arthrobacter, Enterobacter psychrolactophilus agglomerans, Thermotoga, Alicyclobacillus acidocaldariusmaritima, Lactobacillus, and Bacillus acidophilus licheniformis, Sulfolobus[25 solfataricus] have been, Enterobacter characterized. agglomerans Among, Alicyclo- other lactic acidbacillus bacteria, acidocaldarius such, as andL. Bacillus acidophilus licheniformis, L. bulgaricus, [25] haveand beenStreptococcus characterized. thermophilus Among other[26 –28], psychrotolerantlactic acid bacteria, bacteria such asof L. theacidophilus genus,Arthrobacter L. bulgaricus, haveand Streptococcus been described thermophilus as a producer [26– of thermally28], psychrotolerant stable and bacteria cold-active of the genusβ-galactosidase Arthrobacter with have biotechnologicalbeen described as potentiala producer [ 7,14]. Likewise,of thermally in stable our work, and cold-active most strains β-galactosidase with positive withβ-galactosidase biotechnological activity potential were [7,14]. identified asLikewise,Arthrobacter in ourspecies work, most (Table strains S1), along with withpositive other β-galactosidase taxa previously activity reported were in identified the Antarctic continent,as Arthrobacter including speciesStenotrophomonas (Table S1), along withsp., Sphingomonas other taxa previouslysp., Sporosarcina reported insp., theSerratia Ant- sp., andarcticStreptomonas continent, includingsp. [29,30 Stenotrophomonas]. sp., Sphingomonas sp., Sporosarcina sp., Serratia sp., andQuantification Streptomonas of sp. enzymatic [29,30]. activity was performed from a set of the screened strains withQuantification higher activity of (revealed enzymatic by activity a more was intense performed color change). from a set The of the strain screened Se8.10.12 strains showed colorwith higher diffusion activity on agar (revealed (Figure by S1), a more which intense might color indicate change). extracellular The strain production Se8.10.12 of β- showed color diffusion on agar (Figure S1), which might indicate extracellular production galactosidases. The potential of this strain (Se8.10.12) as the most significant β-galactosidase of β-galactosidases. The potential of this strain (Se8.10.12) as the most significant β-galac- producer was confirmed since it produced the highest quantified activity in liquid culture tosidase producer was confirmed since it produced the highest quantified activity in liq- (>2000 U/mL) compared to other selected strains (<500 U/mL) (Figure1, Table S1) both in uid culture (>2000 U/mL) compared to other selected strains (<500 U/mL) (Figure 1, Table the presence and absence of the IPTG inducer (isopropyl-β-D-1-thiogalactopyranoside). S1) both in the presence and absence of the IPTG inducer (isopropyl-β-D-1-thiogalactopy- IPTGranoside). triggered IPTG triggered a higher a enzymatic higher enzymatic activity ac intivity all thein all assayed the assayed strains. strains. Moreover, Moreover, a faster responsea faster response was observed was observed on strain on Se8.10.12,strain Se8.10.12, whose whose maximum maximum activity activity was measuredwas meas- after fiveured days after offive culture days (120of culture h), changing (120 h), tochanging the third to daythe third (72 h) day after (72 IPTG h) after was IPTG added. was Based onadded. the resultsBased on described the results above, described Se8.10.12 above, (Rahnella Se8.10.12sp.) (Rahnella was selected sp.) was to furtherselected characterize to fur- itstherβ characterize-galactosidase its β activity.-galactosidase Other activity. studies Other have stud previouslyies have previously described described species spe- from the Rahnellacies fromgenus the asRahnella potential genus cold-active as potentialβ-galactosidase cold-active producers β-galactosidase [11,13,31 ],producers which specific enzymatic[11,13,31],which activity specific is discussed enzymatic and activity compared is discussed in Sections and compared 2.3 and 2.4in Sections. However, 2.3 and none of them2.4. However, were obtained none of from them Antarctic were obtained isolates. from Antarctic isolates.

Figure 1. β-galactosidase activity quantiﬁed from enzyme extract of four Antarctic strains (speciﬁed in each graph title) at 15 ◦C reaction in the presence (red lines) and absence (black lines) of IPTG inducer. Error bars display the standard deviation of the data.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 25

Figure 1. β-galactosidase activity quantified from enzyme extract of four Antarctic strains (speci- Int. J. Mol. Sci. 2021, 22, 4144 4 of 24 fied in each graph title) at 15 °C reaction in the presence (red lines) and absence (black lines) of IPTG inducer. Error bars display the standard deviation of the data.

2.2.2.2. Antarctic Strain Se8.10.12 ShowedShowed Cold-StableCold-Stable β-galactosidase ActivityActivity AmongAmong fourfour promising promisingβ-galactosidase β-galactosidase producer producer Antarctic Antarctic strains, strains,Rahnella sp.Rahnella Se8.10.12 sp. exhibitedSe8.10.12 exhibited higher activity higher in activity the broth. in the Hence, broth. we Hence, tested we the tested stability the of stability its lactose of its degrada- lactose tiondegradation activity atactivity low temperatures. at low temperatures. Using HPLC, Using we HPLC, determined we determined the lactose the concentration lactose con- (g/L)centration after (g/L) 12 h inafter a reaction 12 h in a containing reaction cont milkaining and themilk enzymatic and the enzymatic extract or milkextract and or com-milk mercialand commercialA. oryzae βA.-galactosidase. oryzae β-galactosidase. Relative activitiesRelative wereactivities evaluated were evaluated at 4 ◦C, 15 at◦C, 4 25°C,◦ C,15 and°C, 25 37 °C,◦C. and Higher 37 °C. concentrations Higher concentrations of lactose of were lactose detected were indetected the samples in the assamples the temper- as the aturetemperature decreased. decreased. Consequently, Consequently, the enzymatic the enzymatic activity activity was reduced was reduced for both for commercial both com- enzymemercial enzyme and crude and extract crude atextract temperatures at temperatures below 15 below◦C (Figure 15 °C (Figure2). When 2). comparing When compar- the lactoseing the concentrationlactose concentration for each for reaction, each reaction it was observed, it was observed that the that crude the enzymatic crude enzymatic extract ofextract the strain of the Se8.10.12 strain Se8.10.12 showed showed lower activitylower activity than the than commercial the commercial enzyme enzyme at the at same the temperaturesame temperature and concentration and concentration equivalence equivalence (minimum (minimum concentration concentration reached: reached: 12.82 12.82 vs. 0.52vs. 0.52 g/L, g/L, respectively). respectively). However, However, this this behavior behavior is expected is expected for unpurifiedfor unpurified enzymes. enzymes. On theOn otherthe other hand, hand, the relativethe relative activity activi ofty the of commercial the commercial enzyme enzyme of Aspergillus of Aspergillus oryzae oryzae(maximum (max- atimum 37 ◦ C)at is37 significantly °C) is significantly decreased decreased by temperatures by temperatures lower than lower 15 than◦C, losing 15 °C, up losing to 90% up ofto its90% activity of its activity at 4 ◦C (Figureat 4 °C (Figure2). In contrast, 2). In contrast, the raw the enzymatic raw enzymatic extract ofextract the Se8.10.12 of the Se8.10.12 strain showedstrain showed significantly significantly higher higher relative relative activity activity at lower at temperatureslower temperatures than the than commercial the com- enzyme.mercial enzyme. Hence, Hence, the Antarctic the Antarctic strain extractstrain extract showed showed more enzymaticmore enzymatic efficiency efficiency in cold in reaction,cold reaction, keeping keeping up to up 62% to of 62% its activityof its acti atvity 15 ◦ Cat without15 °C without significant significant differences differences below thisbelow temperature this temperature (i.e., 41% (i.e., at 441%◦C), at but 4 °C), significantly but significantly higher than higher the than commercial the commercial enzyme (Figureenzyme2 ).(Figure 2).

Figure 2. Boxplot representing the β-galactosidase relative activity from Antarctic strain Se8.10.12 crude extract (Rahnella sp.) and commercial enzyme (Aspergillus oryzae) determined at 37 ◦C, 25 ◦C, 15 ◦C, and 4 ◦C after 12 h reaction with milk. TheFigure horizontal 2. Boxplot line representing indicates the the mean β-galactosidase values, and the relative length ac oftivity each from whisker Antarctic indicates strain the Se8.10.12 interquartile crude range extract (IQR); (Rahnellan = 4. Differentsp.) and commercial letters represent enzyme statistically (Aspergillus signiﬁcant oryzae) determined differences (one-wayat 37 °C, 25 ANOVA °C, 15 °C, within and temperatures,4 °C after 12 hp reaction< 0.005; witht-student milk. The horizontal line indicates the mean values, and the length of each whisker indicates the interquartile range (IQR); n = between enzymes, p < 0.01). 4. Different letters represent statistically significant differences (one-way ANOVA within temperatures, p < 0.005; t-student between enzymes, p < 0.01). Cold-adapted galactosidases are a promising alternative for the current commercial enzymes used in the food industry because they hydrolyze lactose at low temperatures, thus preventing the degradation of other nutrients in the milk. Most of the recent studies describing robust cold-active β-galactosidases showed enzymes retaining nearly 20% of Int. J. Mol. Sci. 2021, 22, 4144 5 of 24

its highest activity at 4 ◦C (Antarctic Marinomonas sp. [22], Rahnella aquatilis from Xin- Jiang, China soil [32], Planoccocus sp. [33] from Daqing oil field in China and Enterobacter ludwigii from an Arctic Fjord [19]). Other few enzymes showed higher efficiencies in cold, among them, Rahnella sp. showed up to 27% of the maximum activity with ONPG as the substrate and nearly 80% with lactose at 4 ◦C[13], Halomonas sp. retains up to 25% of its activity at 0 ◦C[20], and for Alkalilactibacillus ikkense 60% of its maximal activity at 0 ◦C, and more than 70% at 10 ◦C was retained [21]. Similar results were reports for Arthrobacter psychrolactophilus strains isolated from Hokkaido, Japan, which showed high activity in cold; however, it significantly decreases above 15 ◦C[14], being less attractive because of its insufficient activity in moderate temperatures. This issue might be due to the optimal temperature activity, which is low (<15 ◦C) for most of the latter cases; thus, the enzymes lost activity in moderate temperatures. Our results suggest a highly efficient enzyme in cold, with optimal activity at moderate temperatures (opt T: 37 ◦C). The wide range of activity of our Antarctic β-galactosidase properly fits the necessities of the agro-food industry. On the other hand, Antarctic bacteria have also been tested for potential cold-active β-galactosidase enzymes. Among them, the Antarctic strain Arthrobacter sp. C2-2 retained 20%, of its maximum activity in cold (~4 ◦C) [7]; Pseudoalteromonas sp. maintain up to 28% of activity [15]; a purified enzyme from bgaS gene of the Antarctic Arthrobacter sp. (strain SB) showed 50% of its activity at 0 ◦ C[34]; the purified enzyme of Pseudoalteromonas sp. isolated from Antarctic krill displayed 11–35% of maximum activity at 0 ◦C–20 ◦C[16], and a Planococcus sp. strain isolated from Ice Shelf in Antarctica keeps less than 20% activity below 10 ◦C[35]. Hence, these results indicate that our Rahnella sp. Se8.10.12 Antarctic strain has high activity in cold (retaining 41% of its maximum activity at 4 ◦C), which might help process liquid milk and generate low lactose products, tolerating moderate temperature (25 ◦C–30 ◦C). Thus, further molecular identification, genome sequencing, and analysis of this strain were performed.

2.3. Characterization of Strain Se8.10.12 Revealed an Antarctic Rahnella inusitata with Unique Metal-Resistance Related Genes among the Rahnella Species Strain Se8.10.12, showing higher potential as a β-galactosidase producer among 304 Antarctic strains, was characterized as a Gram-negative bacterium with a short bacillus or coccobacillus structure (Figure3a). This bacterium lacks flagella and has a size of 1.33 µm approximately (Figure3b, c). According to the biochemical test results, this strain can degrade sugars, as is expected from a lactase-producing strain. This bacterium also showed a lack of enzymes for lysine decarboxylation, ornithine decarboxylation, and urea hydrolysis, and it was negative for Citrate as the exclusive carbon pathway. Regarding the 16S rRNA gene-based identity, this was associated with at least three Rahnella inusitata strains showing 99.93–100% identity by BLASTn comparison and 99.93% identity with Serratia sp. strains. The genus Rahnella belongs to the Enterobacteriaceae family; therefore, we evaluated the phylogenetic distances within the Rahnella genus (R. bruchi, R. woolbedingensis, R. variigena, R. victoriana, R. aquatilis, and R. inusitata), and other close members of the Enterobacteriaceae family (Yersinia similis, Yersinia bercovieri, Pectobacterium polaris, Serratia liquefaciens, Hafnia paralvei, Klebsiella aerogenes, and Rouxiella chamberiensis)[36,37]. The phylogenetic distance between the strain Se8.10.12 and the strain Rahnella inusitata relate them as the most proximate taxonomic group (Figure4a). This taxonomic relatedness was also observed when analyzing the core-proteome-based phylogenetic history, showing a single and distant clade for Rahnella inusitata and our Antarctic strain (Figure4b). This identity was further confirmed by Average Nucleotide Identity (ANI) after genome-wide analysis, where ANI values calculated across other eight Rahnella species reference genomes ranged from 84.09% to 99.04%, with alignment lengths from 3.51 Mb to 4.19 Mb (Figure5 and Table S2). These results indicate that strains Se8.10.12 belong to genus Rahnella, crossing the species cut-off of a >96% ANI with Rahnella inusitata. Rahnella sp. are Gram-negative, bacillus-shaped bacteria described for the first time as Rahnella aquatilis [38]. These data agree with the results obtained by our morphological and Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 25

Int. J. Mol. Sci. 2021, 22, 4144 from 3.51 Mb to 4.19 Mb (Figure 5 and Table S2). These results indicate that strains6 of 24 Se8.10.12 belong to genus Rahnella, crossing the species cut-off of a >96% ANI with Rah- nella inusitata. Rahnella sp. are Gram-negative, bacillus-shaped bacteria described for the first time as Rahnella aquatilis [38]. These data agree with the results obtained by our mor- biochemicalphological and characterization biochemical throughcharacterization Gram staining through and Gram sugars staining degradation and sugars capability, degrada- as β expectedtion capability, for a -galactosidaseas expected for producing a β-galactosidase bacteria. producing Although bacteria. some studies Although have some described stud- theies lactasehave described enzymatic the activity lactase for enzymatic other Rahnella activityspecies for other [11, 13Rahnella,32], no species reports [11,13,32], described no a β reports-galactosidase described activity a β-galactosidase from Rahnella activity inusitata from. Rahnella inusitata.

(a)

(b)

(c)

FigureFigure 3. 3.Microscopic Microscopic visualization visualization of of the the strain strain Se8.10.12. Se8.10.12. ( a(a)) Gram Gram staining staining at at 100 100×× magniﬁcation;magnification; (b,c) are images obtained by scanning electronic microscopy showing details of size and morphol- (b,c) are images obtained by scanning electronic microscopy showing details of size and morphology, ogy, respectively. respectively.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 25 Int.Int. J. J. Mol. Mol. Sci. Sci. 20212021, ,2222, ,x 4144 FOR PEER REVIEW 77 of of 25 24

(a) Tree scale: 0.01 (a) Tree scale: 0.01 Helico b a cte r p ylo ri A T CC-4 3 5 0 4 [N R _ 0 4 4 7 6 1.1] HePlic ectobacteriumo b a cte r p ylo ri polariA T CCs- 4[NR3 5 0 _159255.1]4 [N R _ 0 4 4 7 6 1.1] 82 P ectobacteriumK lebsiella aerogenes polaris [NR[NR _159255.1] _102493.2] 82 K lebsiellaH afnia paralveiaerogenes [NR [NR _116898.1] _102493.2] H afniaS erratia paralvei liquefaciens [NR _116898.1] [NR _122057.1] S erratiaR ouxiella liquefaciens cham beriensis [NR _122057.1] [NR _135871.1] 67 R ouxiellaR ahnella cham variigena beriensis SO [NR T2/10 _135871.1] [KF308405.1] 67 71 R ahnellaR ahnella variigena victoriana SO FR T2/10 B -225 [KF308405.1] [NR _146847.1] 71 68 R ahnellaR ahnella victoriana bruchi FR FR B-226 B -225 [NR [NR _146845.1] _146847.1] 68 97 R ahnellaR ahnella bruchi woolbedingensis FR B-226 [NR FR _146845.1] B-227 [NR _146848 97 R ahnellaYersini awoolbedingensis similis [NR _121742.1] FR B-227 [NR _146848 YersiniR ahnellaa similis sp.GS [NR 15 _121742.1] [MH 884023.1] 92 R ahnellaR ahnella sp.GS aquat 15ilis [MH D S 884023.1] M - 4594 [NR _025337.1] 92 R ahnellaR ahnella aquat inusitatailis D S DS M - 4594 M -30078 [NR _025337.1][NR _146846.1] 100 R ahnellaA ntarctic inusitata strain DS Se8.10.12 M -30078 [NR _146846.1] 100 A ntarctic strain Se8.10.12

(b) Tree scale: 0.01 (b) Tree scale: 0.01 R ahnella victoriana [NZ MA EN 01000001.1] R ahnelR ahnella victorianala a q u a tili s[NZ [NC MA 017047.1] EN 01000001.1] 100 R ahnelR ahnella ala q usp-Y9602 a tilis [NC [NC017047.1] 015061.1] 100 R ahnelR ahnella sp-Y9602la sp ER M [NC R 1-05 015061.1] [NZ CP 019062.1] 100 R ahnelA ntarcticla sp ERstrain M R 1-05Se8.10.12 [NZ CP 019062.1] 100 100 A ntarcticR ahnella strain inusitata Se8.10.12 [NZ RAH G 01000001.1] 100 100 R ahnelR ahnella ilanusitata v a riigena [NZ [NZ RAH NS G D01000001.1] J01000001.1] 100 100 R ahnelR ahnella vla a riiwoolbedingensigena [NZ NS Ds J01000001.1] [NZ RA H H 01000001.1] 100 100 R ahnelR ahnella woolbedingensila bruchi [NZ QZWs [NZ I01000001.1] RA H H 01000001.1] 100 R ahnella bruchi [NZ QZW I01000001.1]

Figure 4. Evolutionary history construction of the Antarctic strain Se.8.10.12 based on phylogeny. (a) Phylogenetic analysis Figure 4. Evolutionary history construction of the Antarctic strain Se.8.10.12 based on phylogeny. (a) Phylogenetic analysis Figureof the 4. 16S Evolutionary rRNA gene history constructed construction with Bio-NJ of the Antamethodrctic base straind onSe.8.10.12 the Kimura based two-parameter on phylogeny. distance(a) Phylogenetic estimation analysis model of the 16S rRNA gene constructed with Bio-NJ method based on the Kimura two-parameter distance estimation model of( Helicobacterthe 16S rRNA pylori gene was constructed used as an with outgroup). Bio-NJ (methodb) Maximum based likelihoodon the Kimura phylogenetic two-parameter analysis distance of core-proteome estimation based model on ((HelicobacterHelicobacterthe concatenated pylori wasamino used ac asidas ansequencesan outgroup).outgroup). of ortholog (b(b)) Maximum Maximumous genes likelihood likelihood present phylogenetic phylogeneticin all Rahnella analysis analysis species of of core-proteomeusing core-proteome RAxML. basedGenBank based on on the ac- theconcatenatedcession concatenated numbers amino amino are acid provided ac sequencesid sequences in brackets. of orthologous of ortholog Branch genesouslengths genes present represent present in all thinRahnellae all number Rahnellaspecies of substitutionsspecies using using RAxML. perRAxML. site GenBank (Bar: GenBank 0.01 accession substi- ac- cessionnumberstutions numbers per are site), provided are and provided bootstrap in brackets. in percentagesbrackets. Branch lengthsBranch of 1000 representlengths replicates represent the are number shown the ofnumber substitutionsin the ofbranches. substitutions per site (Bar: per site 0.01 (Bar: substitutions 0.01 substi- per tutionssite), and per bootstrap site), and percentages bootstrap percentages of 1000 replicates of 1000 are replicates shown inare the shown branches. in the branches.

Rahnella inusitata DSM-30078 (NZ_RAHG01000001.1). Rahnella inusitata DSM-30078 (NZ_RAHG01000001.1). Antarctic strain Se8.10.12 Antarctic strain Se8.10.12 Rahnella variigena CIP-10558 (NZ_NSDI01000001.1) Rahnella variigena CIP-10558 (NZ_NSDI01000001.1) Rahnella woolbedingensis DSM-27399 (NZ_RAHH01000001.1) Rahnella woolbedingensis DSM-27399 (NZ_RAHH01000001.1) Rahnella bruchi DSM-27398 (NZ_QZWI01000001.1) Rahnella bruchi DSM-27398 (NZ_QZWI01000001.1) Rahnella sp. ERMR1-05 (NZ_CP019062.1) Rahnella sp. ERMR1-05 (NZ_CP019062.1) Rahnella victoriana BRK18a (NZ_MAEN01000001.1) Rahnella victoriana BRK18a (NZ_MAEN01000001.1) Rahnella aquatilis HX2 (NC_017047.1) Rahnella aquatilis HX2 (NC_017047.1) Rahnella sp. Y9602 (NC_015061.1) Rahnella sp. Y9602 (NC_015061.1)

Figure 5. Hierarchically clustered heatmap of Average Nucleotide Identity (ANI) calculated using BLASTn method (left), Figure 5. Hierarchically clustered heatmap of Average Nucleotide Identity (ANI) calculated using BLASTn method (left), and alignment length (right) between nine Rahnella species genomes including the Antarctic isolate Se8.10.12. GenBank Figureand alignment 5. Hierarchically length clustered(right) between heatmap nine of RahnellaAverage speciesNucleotide genomes Identity including (ANI) calculated the Antarctic using isolate BLASTn Se8.10.12. method GenBank (left), andaccessionaccession alignment numbers numbers length are are (right provided provided) between in in brackets. brackets. nine Rahnella species genomes including the Antarctic isolate Se8.10.12. GenBank accession numbers are provided in brackets.

Int. J. Mol. Sci. 2021, 22, 4144 8 of 24

Rahnella inusitata is an uncommon species since little is known about its origin. It was recently described in damaged oak tissues in the United Kingdom [37], which contrasts with the Antarctic sediment from which strain Se8.10.12 was isolated. This is the first report of this species on the Antarctic continent to the best of our knowledge. The Se8.10.12 strain de novo genome assembly resulted in a 4.7 Mb chromosome in one complete linear contig with 134× coverage, 52.9 GC content, and showing 100% genome completeness based on 105 bacterial gene markers [39]. A total of 4312 coding sequences (CDS), including 22 rRNA, 78 tRNA, and two circular plasmids, were determined by Prokka annotation. CDS were assigned with eggNOG mapper to 4158 protein families. Comparative analysis of orthologous clusters revealed the presence of 101 protein families on our Antarctic strains that are absent in the reference R. inusitata (Figure6 a), most of them are primary biological functions shared with other Rahnella species, and at least seven clusters were unique of the Antarctic R. inusitata Se8.10.12 (Figure6 b). The functional assigned unique clusters are mostly related to heavy metal resistance, including copper, silver, cadmium, and cobalt (Table1). On the other hand, based on gene ontology, the absent genes on our Antarctic strain showed two enriched biological processes: sporu- lation/formation of a cellular spore and response to zinc ion (p < 0.001). Interestingly, zinc response clusters were enriched in the Antarctic strain (R. inusitata Se8.10.12), R. aquatilis, and Rahnella sp. (Table1), while absent in the reference R. inusitata. These data might suggest a gene loss of R. inusitata species related to zinc resistance, which may have been kept in the Antarctica strain by selective pressure due to heavy metals in the environment. These results might evidence the adaptation of this strain to its Antarctic habitat since it was isolated from sediment near a garbage disposal container at the Antarctic Chilean Base Julio Escudero. This environment could be considered contaminated compared to the environments where other Rahnella species have been found, which might influence the variances on metal-resistance-related genes gain/loss on the Antarctic R. inusitata. Besides, it has been extensively reported that contamination with heavy metals (e.g., cadmium, vanadium, arsenic, mercury, chromium, and gold) is usually found in Antarctic environments [3,40–42]. Moreover, a diversity of metal-resistance genes, including novel genes, has been described in the Antarctic bacteria, which occurrence are likely to be influenced by heavy metals at high concentrations (either by Anthropogenic or natural biogeochemical origin), shaping new taxonomic and functional diversities of soil microbial communities [43].

Table 1. Summary of main unique clusters found in the Antarctic Rahnella inusitata Se8.10.12 strain by gene ontology comparative analysis, including annotated clusters related to metal resistance.

Cluster-ID SwissProt ID: Protein, Function GO Annotation Genome|Protein P0CE53: transposase InsH, involved in Rahnella_inusitata_Se81012|AJMHLFKD_00752 GO:0006313; P:transposition, 5124 the transposition of the insertion Rahnella_inusitata_Se81012|AJMHLFKD_00297 DNA-mediated sequence IS5 Rahnella_inusitata_Se81012|AJMHLFKD_03605 Q47459: protein PcoE, Required for the Rahnella_inusitata_Se81012|AJMHLFKD_00787 6148 copper-inducible expression of copper GO:0046872; F: metal ion binding Rahnella_inusitata_Se81012|AJMHLFKD_00806 resistance Q9ZHD4: sensor kinase SilS, component GO:0000155; F:phospho-relay Rahnella_inusitata_Se81012|AJMHLFKD_00805 6151 of the soil cation-efﬂux system that sensor kinase activity Rahnella_inusitata_Se81012|AJMHLFKD_00788 confers resistance to silver Rahnella_inusitata_Se81012|AJMHLFKD_02409 6149 N/A N/A Rahnella_inusitata_Se81012|AJMHLFKD_03172 Rahnella_inusitata_Se81012|AJMHLFKD_02421 6147 N/A N/A Rahnella_inusitata_Se81012|AJMHLFKD_03161 P76514: uncharacterized protein YfdR, Rahnella_inusitata_Se81012|AJMHLFKD_03184 6146 GO:0050897; F: cobalt ion binding probable cobalt ion binding Rahnella_inusitata_Se81012|AJMHLFKD_02399 Q9XJJ6: Spanin inner membrane GO:0019076; P: viral release from Rahnella_inusitata_Se81012|AJMHLFKD_03641 6150 subunit, participates in cell lysis during the host cell Rahnella_inusitata_Se81012|AJMHLFKD_03102 virus exit Int. J. Mol. Sci. 2021, 22, 4144 9 of 24

Table 1. Cont.

Cluster-ID SwissProt ID: Protein, Function GO Annotation Genome|Protein P41059: uncharacterized protein in gpA, Rahnella_sp_ERMR1|LLFBIOOH_04925 5653 GO:0008270; F: zinc ion binding probable zinc ion binding Rahnella_inusitata_Se81012|AJMHLFKD_02768 P74068: uncharacterized transporter GO:0015341; F: zinc efﬂux active Rahnella_aquatilis|AEJLJCIE_02970 4804 sll1263, cadmium, iron, and zinc transmembrane transporter Rahnella_inusitata_Se81012AJMHLFKD_00295 transmembrane transport activity Rahnella_sp_Y9602|CHBNGHAC_02899

14/11/2020 jVenn_chart-3.svg (a) (b)

Rahnella_inusitata_Se81012 Rahnella_inusitata

101 3765 175

Size of each list

3940 3866 3940 1970

0 Rahnella_inusitata_Se81012 Rahnella_inusitata

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Figure 6. Comparative analysis of orthologous gene cluster across Rahnella species genomes. (a) Venn diagram summarizing the distribution of shared orthologous clusters between two Rahnella inusitata genomes, including the Antarctic isolated strain Se8.10.12. (b) The occurrence table of main orthologous clusters between Rahnella species showing the 16 ﬁrst cluster count in descending and ascending order. The presence or absence of a cluster group in the related species is represented by green or gray cells, respectively. The absent and unique clusters on the Antarctic strain Se8.10.12 are marked inside blue boxes.

2.4. Rahnella inusitata Se8.10.12 Genome Harbors Three β-Galactosidase Genes, In Silico Molecular Docking Showed That Two of Them Have a Higher Predicted Afﬁnity for Lactose Than

ﬁle:///Users/kattianunez/Downloads/jVenn_chart-3.svg the Commercial Enzyme 1/1 The genomic and functional annotation results through Prokka and eggNOG delivered three genes associated with the production of β-galactosidases, including bglY, lacZ, and ebgA. BglY belongs to the family GH42 of β-galactosidases (EC 3.2.1.23) according to the CAZy database [44]. This protein has been reported as a cold-active enzyme in other species, including BglY from Alicyclobacillus acidocaldarius [45], and a β-galactosidases enzyme in the Rahnella genus [22]. LacZ is a GH2 β-galactosidase, which is a widely distributed family in bacteria (more than 22,000 GH2-protein entries in databases) [44]. LacZ has been described to have higher activity, at the optimum temperature, than other cold-active enzymes [46]. However, it is known as a mesophilic enzyme with little to no activity at 0 ◦C, regarding the LacZ of Escherichia coli [46]. Likewise, EbgA is also part of the GH2 family, known as an evolved ineffective LacZ paralog [47], since wild type E.coli was reported to have no lactase activity; however, single point mutations showed a drastically improved activity [48,49]. Int. J. Mol. Sci. 2021, 22, 4144 10 of 24

These bioinformatic analyses complement our experimental results, evidencing the existence of three genes of β-galactosidase in the Se8.10.12 strain genome. Despite its similarity with known β-galactosidases based on BLASP comparison, the phylogenetic analysis showed that these sequences are distant from most of the similar proteins and share a separate clade with enzymes from Rahnella inusitata, Serratia sp., and an Enterobac- teriaceae strain (Figure7), which might be related to their evolutionary history adapting to the speciﬁc environments. Moreover, two well-deﬁned clades can be observed in the Int. J. Mol. Sci. 2021, 22, x FOR PEERphylogenetic REVIEW tree, which groups the proteins into the known glycoside hydrolases11 families of 25

GH2 and GH42 (clade 1: LacZ + EbgA, clade 2: BglY).

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a 6 ._ p u [ 9 s t a t 1 B eta-galactosidase BglY e W e 3 p s n 2 i 6 d R s i e 9 _ S r 3 P 5 a _ _ i . a B u 7 s 0 1 i [ a a _ 6 e l t l m ] l l 6 2 1 - a 4 E S e8.10.12-BglY e e r [ 0 1 5 . Q b r . 1 n n 8 2 e ] g h 0 h L 6 3 A

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a R FigureFigure 7. Maximum7. Maximum likelihood likelihood phylogram phylogram basedbased onon three β-galactosidase-galactosidase amino amino acid acid sequences sequences of ofthe the strain strain Se8.10.12 Se8.10.12 (highlighted color) and proteins on nucleotide database with >85% similarity (light color). Aspergillus oryzae β-galacto- (highlighted color) and proteins on nucleotide database with >85% similarity (light color). Aspergillus oryzae β-galactosidase sidase LacA was used as an outgroup. Branch lengths are shown in each branch and represent the number of nucleotide LacAsubstitutions was used per as site. an outgroup.Bootstrap support Branch based lengths on 1000 are shown replicates in eachultrafast branch method and isrepresent represented the as number branch width of nucleotide (1 px substitutionsline = 47% to per 10 site.px line Bootstrap = 100%). supportGenBank/EMBL/DDB based on 1000J accession replicates numbers ultrafast are method provided is representedin brackets. as branch width (1 px line = 47% to 10 px line = 100%). GenBank/EMBL/DDBJ accession numbers are provided in brackets. On the other hand, it was established that Rahnella sp. R3 exhibited much higher cat- alyticTo activity further (27 study U/mg) the at enzymes’ low temperatures speciﬁcity than regarding other β-galactosidases their Antarctic from nature, Rahnella a presence/absencespecies (Rahnella analysis sp. BS of1, genesRahnella and sp. determination HS-39 [31] and ofRahnella syntenic aquatilis patterns 14-1 were [32]) performed[13]. In- terestingly, Rahnella sp. R3 was isolated from a Glacier (Xinjiang, China), suggesting that, even though all the Rahnella species share orthologous β-galactosidase genes, they might evolve according to their specific niches. Our results support this idea, showing an Ant- arctic Rahnella with higher β-galactosidase activity at 4 °C than other Rahnella species previously reported, except for the R3 cold-adapted isolate. Moreover, the BglY β-galactosidase of our Antarctic R. inusitata might have acquired the same cold adaptability as the enzyme from Rahnella sp. R3, which can be deduced from its structural similarity (99%

Int. J. Mol. Sci. 2021, 22, 4144 11 of 24

using PIRATE pangenome toolbox. Results showed that the genes lacZ and bglY were present in the eight of the available genomes of Rahnella strains (6 species), while gene ebgA was absent in the species R. vitoriana and R. varigena. Moreover, syntenic gene cluster was found for LacZ (including the genes: argO, Arginine exporter protein; mscS, small- conductance mechanosensitive channel; lacY, Lactose permease; and lacI Lactose operon repressor), and for EbgA (including ebgC, Evolved β-galactosidase subunit β; ygjK, Glucosi- dase; melR HTH-type transcriptional repressor; and ygjI inner membrane transporter). No syntenic blocks were found associated with gene bglY. These data suggest the presence of complete putative gene operons in the genome of the Antarctic Rahnella strain, which could effectively produce functional β-galactosidases. Moreover, it suggests that this is a highly conserved function in the Rahnella genus, while the EbgA gene might have been acquired by horizontal gene transference only in some species, as previously described [47]. On the other hand, it was established that Rahnella sp. R3 exhibited much higher catalytic activity (27 U/mg) at low temperatures than other β-galactosidases from Rahnella species (Rahnella sp. BS 1, Rahnella sp. HS-39 [31] and Rahnella aquatilis 14-1 [32]) [13]. Interestingly, Rahnella sp. R3 was isolated from a Glacier (Xinjiang, China), suggesting that, even though all the Rahnella species share orthologous β-galactosidase genes, they might evolve according to their speciﬁc niches. Our results support this idea, showing an Antarctic Rahnella with higher β-galactosidase activity at 4 ◦C than other Rahnella species previously reported, except for the R3 cold-adapted isolate. Moreover, the BglY β-galactosidase of our Antarctic R. inusitata might have acquired the same cold adaptability as the enzyme from Rahnella sp. R3, which can be deduced from its structural similarity (99% similarity to Rahnella sp. R3 protein) (Figure8) and phylogenetic distance being more distant from other BglY Rahnella enzymes (Figure7). The in silico prediction of the biochemical properties of R. inusitata Se8.10.12 β- galactosidases revealed that all sequences have similar characteristics to other β-gal proteins (Table2), with complete sequences between 687 and 1032 amino acids, molecular masses of 77 to 116 kDa and pI between 5.2 and 5.8. The local alignment (BLASTp) against different β-gal from the PDB database showed that Se8.10.12 β-gal exhibited 30–40% of self-identity and 31–99% identity with other β-gal. BLASTp analysis of Se8.10.12 β- galactosidase proteins showed a variable degree of sequence similarity with different X-ray crystal structures from the PDB database. The crystal structure of Rahnella sp. R3 shares 99% sequence similarity with the Se8.10.12-BglY sequence. Likewise, high sequence similarity (68.9%) was found between LacZ from Escherichia coli and Se8.10.12-LacZ. In contrast, the sequence Se8.10.12-EbgA displayed the lowest identity percentage out of the three, only 37.24%, with a β-galactosidase structure from Thermotoga maritima (6S6Z_A). Nevertheless, all identity percentages met the recommended threshold for homology modeling using the PDB structures as template.

Table 2. Biochemical properties of Rahnella inusitata Se.8.10.12 β-galactosidases and other β-gal enzymes from PDB database.

Molecular Glycoside PDB % Identity Organism Protein Sequence pI Mass (Da) Hydrolase Family Number Lac-Z BglY EbgA Rahnella inusitata Se.8.10.12 Lac-Z 1032 116,913.82 5.30 GH 2 - 100 29.79 34.10 Rahnella inusitata Se.8.10.12 BglY 687 77,230.21 5.84 GH 42 - 29.79 100 40.91 Rahnella inusitata Se.8.10.12 EbgA 1023 116,917.20 5.20 GH 2 - 34.10 40.91 100 Rahnella sp. R3 β-gal 712 80,188.46 5.98 GH 42 5E9A_1 31.82 99.13 36.84 Escherichia coli Lac-Z 1023 116,385.64 5.28 GH 2 1jz2.1.D 68.9 36.54 34.51 Thermotoga maritima β-gal 1083 127,476.78 5.52 GH 2 6S6Z_A 36.39 29.27 37.24

Three-dimensional models of Se8.10.12 β-galactosidases (Figure8) were reconstructed to compare the enzymes’ structures using homology modeling. The models got Qualitative Model Energy Analysis (QMEAN) Z-score quality values higher than −4.0 (Supplementary Table S3), indicating a good agreement between the model structure and experimental structures of similar size [50]. The SWISS-MODEL outputs were submitted to the YASARA energy minimization server to improve the quality and the accuracy of the predicted Int. J. Mol. Sci. 2021, 22, 4144 12 of 24

models. The reﬁnement process vastly improved the raw β-galactosidases models’ accuracy, obtaining QMEAN Z-scores around zero, indicators of good quality. Ramachandran plots were also constructed for the three modeled enzymes using the PROCHECK server. Based on Ramachandran plot analysis, all models share a high overall structural quality, with Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 25 more than 87–89% of residues in most favored regions, and only a few residues were in unfavorable conformations.

FigureFigure 8.8. OverallOverall predicted structure of the Rahnella inusitata Se8.10.12 β-galactosidases. The The three- three- dimensional structure reconstructed by homology modeling is presented as a ribbon model. The dimensional structure reconstructed by homology modeling is presented as a ribbon model. The reconstruction shows quaternary structure (left) and monomeric (right) views. In the monomers, reconstruction shows quaternary structure (left) and monomeric (right) views. In the monomers, the the catalytic motifs are colored in red and blue, while asterisks indicate the glutamic acid amino catalyticacid in the motifs active are site. colored in red and blue, while asterisks indicate the glutamic acid amino acid in the active site. Three-dimensional models of the R. inusitata Se8.10.12 β-galactosidases showed note- Three-dimensional models of the R. inusitata Se8.10.12 β-galactosidases showed note- worthy differences in their predicted quaternary structures. While BgIY was recon- worthy differences in their predicted quaternary structures. While BgIY was reconstructed structed as a homo-trimer, LacZ was modeled as a homo-tetramer and a homo-octamer as a homo-trimer, LacZ was modeled as a homo-tetramer and a homo-octamer for EbgA. for EbgA. For BgIY, the predicted homo-trimer structure could be associated with enzy- For BgIY, the predicted homo-trimer structure could be associated with enzymatic activity atmatic low activity temperatures, at low basedtemperatures, on its similarity based on with its thesimilarityβ-galactosidase with the β from-galactosidaseRahnella sp. from R3, Rahnella sp. R3, which showed structural characteristics of cold adaptation [13]. Further- more, a recently characterized β-galactosidase from the Antarctic archaea Halorubrum lacusprofundi, with a homo-trimeric quaternary structure, also showed cold adaptation and tolerance for high concentrations of NaCl [51]. However, no other associations have been made regarding a homo-trimeric conformation and cold tolerance. As these differences in conformation are likely to affect its molecular properties, it is important to consider that these assumptions are made by a prediction based on the highest template similarity with

Int. J. Mol. Sci. 2021, 22, 4144 13 of 24

which showed structural characteristics of cold adaptation [13]. Furthermore, a recently characterized β-galactosidase from the Antarctic archaea Halorubrum lacusprofundi, with a homo-trimeric quaternary structure, also showed cold adaptation and tolerance for high concentrations of NaCl [51]. However, no other associations have been made regarding a homo-trimeric conformation and cold tolerance. As these differences in conformation are likely to affect its molecular properties, it is important to consider that these assumptions are made by a prediction based on the highest template similarity with each homologous protein. However, other quaternary structures could occur in nature. Due to this, we included modeling for both the quaternary and monomeric structures for each enzyme. For Se8.10.12 LacZ, the homo-tetrameric assembly shares structural properties with the quaternary structure of LacZ β-galactosidase from E. coli (comparable subunit mass, amino sequence identity, conservation of amino acid residues involved in catalysis, pI) [52]. The in silico analysis showed that both are tetramer of four identical polypeptide chains, each of around 1000 amino acids. Within each monomer, the amino acids form five well-defined structural domains. The central domain possesses the active site and is called (β/α)8- barrel [53]. Other β-galactosidases from psychrophile bacteria with homo-tetrameric structure have proven to be cold-adapted, including the cold-active β-galactosidases from the Antarctic bacteria Pseudoalteromonas haloplanktis [54], Pseudoalteromonas sp. 22b [55], and Alteromonas sp. ML117 [56]. Then, the predicted structure of Se8.10.12 LacZ could be associated with a cold-adapted enzyme. Unfortunately, the crystallized studies of those enzymes are not available for deeper structural comparisons. Regarding the Se8.10.12 EbgA enzyme, the octameric predicted reconstruction of its three-dimensional structure is unusual. The octamer architecture has only been previously reported for one β-galactosidase from Thermotoga maritima, a hyperthermophilic organism isolated from a geothermal, marine volcano [57]. This enzyme has a characteristic TmLac domain, absent in other GH2 proteins, which determines its oligomeric arrangement. Moreover, despite the high conservation in the catalytic site, the enzyme has different features of the catalytic pocket and the regions surrounding it that might be important for interactions with large substrates and transglycosylation reactions [58]. Despite the marked differences in their quaternary structure’s prediction, which might or might not occur in nature regarding the physicochemical conditions, the monomers of the three Se8.10.12 β-galactosidases share characteristic conserved motifs within their structure. Figure8 shows that the red motif is present in all three enzymes, while the blue motif is only present in LacZ and EbgA. These motifs are also shared by other β -galactosidases, such as LacZ from E. coli, and correspond, by the frequency of amino acids to highly conserved residues in the active site of each monomer. Two highly conserved glutamate residues for lactose hydrolysis were observed among both motifs, which are known to serve as proton donor and nucleophile, respectively, during catalysis [59]. The refined Se8.10.12 β-galactosidases 3D models were used to predict the most likely interaction between the active site and lactose through molecular docking (Figure9 a). Molecular docking was performed to investigate whether the experimental results observed in Figure2 would also be supported by in silico analysis. The docking score was the predicted binding affinity in kcal/mol, and the results are presented in Figure9b. As a comparative parameter, the industrial–commercial Aspergillus oryzae β-galactosidase’s crystal structure was subjected to the same docking protocol. The calculated interaction energies between the lactose and the Se8.10.12 β-galactosidases models varied between −4.8 and −6.5 kcal mol−1, versus the −6.1 kcal mol−1 assigned for the interaction between A. oryzae β-galactosidase with lactose. The predicted binding affinity for lactose in Se8.10.12 LacZ was significantly higher than the binding affinity in A. oryzae for this molecule and comparably to the BglY enzyme. In contrast, the docking score in EbgA was significantly lower compared to the other enzymes. Thus, the in silico findings suggest that some β-galactosidases predicted from the Antarctic R. inusitata Se8.10.12 genome might have an effective lactose binding in the catalytic domain, comparable with the commercial control enzyme. Int. J. Mol. Sci. 2021, 22, 4144 14 of 24 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 25

(a) A. oryzae b-gal

Se8.10.12-LacZ

Se8.10.12-EbgA

Se8.10.12-BglY

(b) 0

−1

−2

−3

−4

Energy (kcal/mol) −5 c

−6 ab a b −7 A. oryzae b-gal Se8.10.12-LacZ Se8.10.12-EbgA Se8.10.12-BglY Rahnella inusitata Se8.10.12 β-galactosidases Figure 9. MolecularMolecular docking docking of of the the RahnellaRahnella inusitata inusitata Se8.10.12Se8.10.12 β-galactosidasesβ-galactosidases and andits substrate its substrate lactose. ( (aa) )Molecular Molecular docking docking simulation simulation of oflactose lactose binding binding to Aspergillus to Aspergillus oryzae oryzae β-galactosidaseβ-galactosidase (4IUG) and Rahnella inusitata Se8.10.12 β-galactosidases LacZ, EbgA, and BglY (homology model- (4IUG) and Rahnella inusitata Se8.10.12 β-galactosidases LacZ, EbgA, and BglY (homology modeling). ing). Schematic representation showing enzyme/ligand interactions (left image) and two-dimen- sionalSchematic (2D) representationligand-protein interaction showing enzyme/liganddiagram (right). The interactions hydrogen (left bonds image) are represented and two-dimensional dotted (2D)green ligand-protein lines (Å). (b) Docking interaction scores diagram (AutoDock (right). Vina) The for lactose hydrogen against bonds the areselected represented β-galactosidases. dotted green Errorlines (Å).bars (bdenote) Docking SE of scores mean; (AutoDock n = 5. Different Vina) letters for lactose represent against statistically the selected significantβ-galactosidases. differences Error betweenbars denote models SE of (Kruskal–Wallis mean; n = 5. Different test; p < 0.05). letters represent statistically signiﬁcant differences between models (Kruskal–Wallis test; p < 0.05).

A. oryzae β-galactosidase, used as a control in our experimental and in silico analysis, belongs to the GH35 galactosidase family. While the β-galactosidases from R. inusitata are

Int. J. Mol. Sci. 2021, 22, 4144 15 of 24

from galactosidase families 2 and 42. However, they all share the catalytic domain (β/α)8- barrel, with conserved motifs among them. It has even been proposed that all these families evolved from a common ancestor and shared a similar catalytic process. These types of enzymes share a common arrangement of two catalytically critical Glu residues at the end of the (β/α)8 barrel [60,61]. Hence, the binding affinities’ differences could be associated with details such as hydrophobic amino acids in the pocket, the number of hydrogen bonds, and the distance between them. Our data showed that the four enzymes’ active sites are highly hydrophilic (Figure S2), facilitating binding with the ligand since lactose is a hydrophilic molecule by itself [62]. However, slight tendencies towards neutrality or hydrophobicity can be seen in the active sites of A. oryzae and EbgA β-galactosidases, which matches with the lower binding affinity observed for those two enzymes in the molecular docking. Docking results also showed that glutamate residues in both predicted active sites (E468/E544) of Se8.10.12 LacZ were in a conformation capable of forming hydrogen bonds with lactose; both close to the site of lactose cleavage (Figure9a). The distance of these H-bonds was less than 3 Å, which agrees with the observed hydrogen bridge distances for LacZ from E. coli and β-gal from the Antarctic bacteria Alteromonas sp. and lactose [62]. As shown in Figure9a, the residues ASN-107, ASN-467, GLU-423, and TRP-575 also form hydrogen bonds with lactose, respectively. In contrast, one glutamate residue in the active site of EbgA, BgIY, and A. oryzae (E-371, E-157, and E-298, respectively) does not appear capable of forming hydrogen bonds with lactose under the simulated tested conditions, which could explain why their affinity energies were lower than LacZ. Nevertheless, these models also form other hydrogen bonds with different amino acids in the active site, including tryptophan (TRP). TRP engages in-ring stacking with the glucosyl moiety of lactose and keeps the substrate inside the active pocket [63]. Besides, those H-bonds also were found at a suitable distance for catalysis. Although hydrogen bonds may vary in an in vivo situation, the in silico analysis allowed the understanding of the potential catalytic efficiency, together with the binding affinity scores predicted for the enzymes [63]. Therefore, based on the docking score and hydrogen bond formation capability, the results suggest that Se8.10.12 LacZ has the highest affinity for lactose. In contrast, EbgA has the lowest apparent affinity, in agreement with previous data describing this as an inefficient enzyme. Accordingly, it could be suggested that the β-galactosidase activity observed in vitro could be given to a greater extent by LacZ and/or BgIY. Meanwhile, activity in cold might be expected from both LacZ and BglY based on the structural and sequence similarity to other cold-active enzymes; however, this must be confirmed for each different purified protein from the Antarctic R. inusitata.

3. Materials and Methods 3.1. Biological Material The Antarctic bacteria included in this work (×304 strains) were previously isolated from soil and sediment samples from the Antarctic Peninsula and belong to the Chilean Type Culture Collection of the University of La Frontera (CCCT). The strains were reconsti- tuted from the bacterial collection (−80 ◦C) both in nutritious broth and in nutritive agar and incubated at room temperature for 1 to 5 days until suitable growth.

3.2. Screening of β-Galactosidase Activity in Antarctic Bacteria Determination of the β-galactosidase activity was performed at 15 ◦C in lactose agar (meat extract 0.3%, peptone 0.5%, lactose 0.5%, agar 1.8% and isopropyl-β-D-1- thiogalactopyranoside (IPTG) at 1 mM (Sigma-Aldrich, Gillingham, UK), using 5-Bromo- 3-indolyl-β-D-galactopyranoside (β-Bluo-Gal) at 300 µg/mL (Thermo Fisher Scientiﬁc, Waltham, MA, USA) as an indicator of the β-galactosidase activity. One stripe of each strain (×304) was cultured in 150 × 15 mm plates in quadrants, allowing the simultaneous assay of 25 strains per plate. The positive β-galactosidase activity was determined after 48, Int. J. Mol. Sci. 2021, 22, 4144 16 of 24

72, and 96 h of incubation when the color of the colonies changed to blue. A more intense color was considered indicative of a more bioactive strain.

3.3. Molecular Identification of β-Galactosidase Producing Antarctic Strains DNA of positive β-galactosidase producers was extracted using the UltraClean® Mi- crobial DNA Isolation Kit (MoBio Laboratories, Carlsbad, USA). The complete 16S rRNA gene was selectively amplified using the universal primers 27f (50-AGAGTTTGATCCTGGC TCAG-30) and 1492r (50-GGTTACCTTGTTACGACTT-30). PCR amplification was performed in a Multigene Optimal Thermal Cycler (Labnet, Edison, NJ, USA) in 50 µL reaction containing 25 µL SapphireAmp Fast PCR Master Mix, (Takara Bio Inc., Shiga, Japan), 22 µL ultra-pure water, 1 µL of each primer (10 µM), and 1 µL of genomic DNA. The temperature and cycling conditions were as follows: preheating at 94 ◦C for 2 min; 30 cycles at 94 ◦C for 1 min; 55 ◦C for 1 min; 72 ◦C for 1.5 min; and incubation at 72 ◦C for 10 min. Sequencing was performed with a Dye Terminator Cycle Sequencing Kit and an ABI 3730XL DNA Sequencer (Applied BiosystemsTM Thermo Fisher Scientific, Waltham, USA) by Macrogen Inc. (Korea). The nearest taxonomic group was identified by 16S rDNA nucleotide sequence BLASTN (http://www.ncbi.nlm.nih.gov/blast, accessed on 7 June 2020) using DNA Data Bank of Japan (DDBJ)/European Molecular Biology Laboratory (EMBL)/GenBank nucleotide sequence databases.

3.4. β-Galactosidase Activity Quantification The most promising strains based on the β-galactosidase activity screening (showing the highest intensity of color change) were selected for enzyme activity quantification. The selected Antarctic strains (×4) were cultivated in lactose broth (meat extract 0.3%, peptone 0.5%, lactose 0.5%) at 15 ◦C for 48 h; then, cells were lysed by sonication. The cell lysate was centrifuged at 4000 rpm for 30 min at 4 ◦C, and the supernatant was used as the enzymatic extract [24]. 20 µL of the enzymatic extract was added to a 22 mM solution of o-nitrophenyl- β-D-galactopyranoside (ONPG) (Sigma-Aldrich, Gillingham, UK). The reaction was carried out for 15 min at 15 ◦C and pH 6.5. The amount of released o-nitrophenol (ONP) after ONPG hydrolysis was measured at 420 nm. One unit of β-galactosidase activity (U) was defined as the amount of enzyme required to release 1 µmol of ONP per minute from ONPG under the experimental conditions described above. Additionally, differences in β- galactosidase activity were evaluated in the presence and absence of IPTG (Sigma-Aldrich, Gillingham, UK). All determinations were performed in triplicates.

3.5. Morphological and Biochemical Characterization of Strain Se8.10.12 Strain Se8.10.12 was morphologically characterized by Gram staining and scanning electron microscopy in a SU-3500 Hitachi-Japan electron microscopy. Gram staining was performed based on the description in Gram (1884). For electronic microscopy characterization, the strain was incubated in nutrient broth for 24 h. Bacterial cells were harvested by centrifugation (5000 rpm, 5 min), followed by consecutive washings with sterile distilled water to eliminate traces of the culture broth media. After three washing-centrifugation steps, the supernatant was eliminated, and the strain was fixed with 500 µL of glutaralde- hyde for 48 h. After fixation and before visualization by electronic microscopy, the sample was washed three times by centrifugation (10,000 rpm, 5 min) to eliminate the fixative excess. Once the supernatant was removed, the sample was resuspended in water and ethanol and visualized in the electronic microscope. Besides, to obtain a biochemical characterization of the strain, a battery of bacterial typing biochemical test tubes was used, composed of Triple Sugar Iron Agar (TSI), Motility Indole Ornithine (MIO), Simmons Citrate, Lysine Iron Agar (LIA), and Urea. The strain was inoculated into each media and incubated at room temperature for 24 h before evaluation. Int. J. Mol. Sci. 2021, 22, 4144 17 of 24

3.6. Determination of the Effect of Temperature on the β-Galactosidase Relative Activity of Strain Se8.10.12 A raw enzymatic extract from Se8.10.12 strain was obtained as described in Section 3.4. The enzymatic reaction was performed with a 15 U/mL of a commercial enzyme for each mL of reaction (β-Galactosidase from Aspergillus oryzae CAS-9031-11-2; Sigma-Aldrich, Gillingham, UK), and the equivalent U/mL of the strain Se8.10.12 raw extract in 10 mL total reaction volume with commercial liquid whole milk (COLUN, Los Rios, Chile) at four different temperatures (4 ◦C, 15 ◦C, 25 ◦C, and 37 ◦C) for both enzymes. All samples were incubated for 12 h under constant agitation at 80 rpm. After 12 h, each sample was filtered with 0.22 µm filters and heated to 100 ◦C for 5 min to stop the enzymatic reaction. The samples were kept cold until the determination of the lactose content by HPLC (Shimadzu Prominence LC-20AT, Japan) using an Aminex HPX-87H ion exclusion column (300 × 7.8 mm, 9 µm particle size, 8% cross-linkage and pH range of 1–3; Bio-Rad, Hercules, CA, USA). Aliquots of 20 µL (injection volume) were separated at 0.5 mL min−1 (flow rate), at a constant 60 ◦C column temperature, with 0.01 N aqueous sulfuric acid elution phase. Detection of lactose was accomplished in a refractive index detector compared to a 40 mg/mL lactose commercial standard (Sigma-Aldrich, Saint Louis, MO, USA). Since a non-purified extract was used (targeting psychrophilic enzymes), the enzymatic activity was expressed as relative activity based on the maximum lactose degradation. All measurements were conducted with four replicates.

3.7. Whole-Genome Sequencing and Assembly Strain Se8.10.12 was sequenced using the Illumina MiSeq (Illumina, San Diego, CA, USA) and Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) platforms. Briefly, 1 mL of bacterial culture (24 h growth in nutrient broth) was used for genomic DNA extraction using the UltraClean Microbial DNA Extraction Kit (MoBio Laboratories, Carlsbad, CA, USA). DNA quality was assessed by fluorometry using a QuantiFluor® ONE dsDNA System (Promega Corporation, Fitchburg, WI, USA) and the determination of purity determining ratios 260/280 and 260/230. A library was prepared with the rapid sequencing kits SQK-RAD004 (Oxford Nanopore Technologies, Littlemore, Oxford, UK), and sequencing was performed in a MinION platform (Oxford Nanopore Technologies (ONT)) using the software MinKNOW v2.3.5. DNA was also sequenced on an Illumina MiSeq X Ten sequencing platform using a paired-end library with an average insert size of 350-bp followed by 2 × 150-bp sequencing. The Illumina paired-end sequencing data were evaluated using FastQC v0.11.9 and then adjusted using the Trimmomatic v0.39 program by filtering out adapters and reads of less than a Phred score of 30. In parallel, the Oxford Nanopore data were evaluated using the NanoPlot v1.32.1 software and then adjusted using the NanoFilt v2.8.0 and Porechop v0.2.4 programs, filtering out reads of less than Q10, a read length fewer than 1000 bases, and removing the adapters, respectively. Finally, the hybrid assembly was performed using the Unicycler v0.4.8 program; additional finalization of the assembly was done using the tool FinisherSC v2.0 [64] and quickmerge v0.3 [65] to close the genome before and after Pilon v1.23 polishing [66]. The quality of the assembly was evaluated using the Quast v5.0.2 program [67], while completeness/contamination was determined with CheckM v1.1.3 [68] and miComplete v1.1.0 [39]. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the bioproject accessions PRJNA605861, BioSample SAMN16814261, which includes the genome CP065024 and plasmids CP065025 and CP065026

3.8. Genome-Wide Analysis for Taxonomic Identiﬁcation and Characterization Taxonomic afﬁliation of the strains Se8.10.12 was performed by 16S rRNA gene comparison using BLASTn [69]. Phylogenetic distances among the nearest taxonomic groups were also determined by a tree construction using the rRNA 16S complete gene sequence and core proteins of reference Rahnella sp. genomes. Core proteins were obtained with M1CR0B1AL1Z3R web server (https://microbializer.tau.ac.il/, accessed on 20 October Int. J. Mol. Sci. 2021, 22, 4144 18 of 24

2020) [70] using the following cut-offs: 0.01 maximal e-value, 80.0% identity minimal percent, and 100.0% minimal percentage for the core. rRNA 16S gene phylogenetic tree construction was performed using Mafft alignment version 737 L-INS-i method [71] and SeaView version 5.0.4. with the Bio-NJ algorithm based on the Kimura-2-parameter model selection on 1000 bootstrapped data sets [72]. Core-proteome phylogenetic construction was conducted with a maximum-likelihood algorithm using RAxML [73] based on the inferred core-proteome alignment. Phylogenetic tree visualization was done using the iTOL v3 tool [74]. A genome-wide analysis was performed to confirm the strain identity by Pairwise Average Nucleotide Identity (ANI) values. Those were calculated among the available reference genomes representative of the genus Rahnella using the software Pyani v0.2.10 in mode ANIb [75]. The output was visualized using the software DiMHepy (available at https://github.com/lucaTriboli/DiMHepy, accessed on 12 August 2020). Genomic annotation was performed using Prokka v1.14.5 [76] to determine the Antarctic strain’s genomic profile and potential genes associated with the production of β-galactosidases. The protein sequences from Prokka generated data were analyzed with the OrthoVenn2 web server (https://orthovenn2.bioinfotoolkits.net, accessed on 15 December 2020) for identification and comparison of orthologous clusters [77]. Briefly, OrthoVenn2 uses the OrthoMCL [78] clustering algorithm to annotate and compare ortholog groups. All-against-all protein similarity was obtained by DIAMOND v0.9.24 alignment [79] (0.05 e-value cut-off) from which putative orthology is identified. Disjoint clusters of closely related proteins are generated with the Markov Clustering Algorithm (MCL) [80] with a 1.5 inflation value. The Gene Ontology (GO) was assigned to the corresponding orthologous cluster by sequence similarity to the UniProt database [81].

3.9. Characterization of β-Galactosidase Enzymes of the Strain Se8.10.12 Based on Genomic Data For the analyses focused on the protein sequences attributed to β-galactosidase genes, multiple alignments were carried out with MAFFT v7 [71] between the sequences obtained from strain Se8.10.12 and sequences deposited in NCBI with sequence similarity above 85% after BLASTp comparison. A phylogram was also constructed to determine the genetic distance between the aminoacidic sequences. The alignment file was used to build a Maximum Likelihood (ML) phylogenetic tree as described above by estimating the most accurate substitution model by Model Finder [82] with the Akaike Information Criterion in the IQ-TREE web server v1.4.2 [83]. The tree visualization was done with the iTOL v3 tool [74]. Gene presence/absence and association with syntenic clusters were studied using the pangenome toolbox PIRATE v1.0.4 (default settings used; 60, 70, 80, 90, 95% amino acid identity threshold) [84]. Moreover, in silico structural analyses of Se.8.10.12 β-galactosidase proteins were performed. Three-dimensional models of the β-galactosidases were reconstructed using a homology modeling approach. NCBI BLASTp against the PDB database was performed to identify β-galactosidase structure templates with the highest sequence similarity to the β-galactosidase sequences of Se.8.10.12. Only PDB structures with sufficient sequence similarity (>30%) were selected as modeling templates. A monomer and quaternary structure of each β-galactosidase, excluding any signal peptide sequence, was constructed using the SWISS-MODEL Workspace platform [85]. The crystal structures of Escherichia coli lacZ (PDB: 1jz2.1.D, 2.1 Å), Rahnella sp. R3 β-galactosidase (PDB: 5e9a.1.A, 2.6 Å), and Thermotoga maritima β-galactosidase (PDB: 6S6Z, 2.0 Å) were used as templates for Se8.10.12 LacZ, BglY, and EbgA model reconstruction, respectively. Based on the highest scores (Global Model Quality Estimation (GMQE) and the Qualitative Model Energy Analysis (QMEAN)) among all possible structural templates, the best models were submitted to refinement in the YASARA (Yet Another Scientific Artificial Reality Application) Energy Minimization Server [86]. The refinement outputs were reanalyzed by QMEAN [50] and SAVES v6.0 [87] servers. All three-dimensional models were visualized using UCSF Chimera software v1.14 [88]. Int. J. Mol. Sci. 2021, 22, 4144 19 of 24

Multiple sequence alignment using MultAlin online platform [89] was used to identify active sites in the β-galactosidases three-dimensional (3D) models. Binding conformations between lactose and the β-galactosidases from R. inusitata were performed using the Dock- ing Algorithm of AutoDock Vina v1.1.2 [90]. The ligand structure was downloaded from the PubChem database (Compound ID (CID): 6134), and the energy minimizations were carried out with the PRODRG2 Server [91]. Gasteiger charges and polar hydrogen atoms were assigned in the protein structures and the ligand using AutoDockTools v4.2.6 [92]. The rotatable bonds were set up, and structure data ﬁles were converted into the Protein Data Bank, Partial Charge (Q), and Atom Type (T) format (PDBQT). Flexible molecular docking was applied to the center of the β-galactosidases catalytic site. An exhaustiveness value = 80 and 500 number modes were used. Conformations were docked, clustered, and ranked according to the binding free energy. Docking was conducted using β-galactosidase monomers since only residues from individual monomers interact directly with a substrate. Since the in vitro analysis was performed compared with an A. oryzae enzyme, a β-galactosidase from A. oryzae (PDB 4IUG) was included in the analysis as a reference from commercial lactase enzyme [93]. Discovery Studio Visualizer v4.5 software [94] was used to analyze, summarize, and cluster the AutoDock Vina results.

3.10. Statistic Data Analysis Mean comparison was made with one-way analysis of variance (ANOVA) and In- dependent samples T-Test at a 95% level of signiﬁcance (p < 0.05) using JASP software v0.14.1 [95]. The assumption of data normality and homoscedasticity were tested with the Shapiro–Wilk and Levene’s test. If data failed the parametric inference, Mann–Whitney U and Kruskal–Wallis H test was used instead. Stander Tukey or Dunn Post Hoc Test was carried out in those cases where ANOVA or Kruskal–Wallis H test was signiﬁcant.

4. Conclusions New β-galactosidase alternatives with activity in cold and wide-range thermostability are currently needed in the glycoside hydrolases industry, particularly for the preparation of lactose-free dairy products. Our work highlights Antarctic microorganisms’ bioprospec- tion for extremozymes discovery, especially cold-active enzymes, since this is still an underexplored source of new molecules. Moreover, further efforts for commercial production of a new diversity of β-galactosidases may promote its regular use in other areas such as dairy whey to produce additional valuable bioproducts (i.e., ethanol and sweet syrup) [23]. Here we report for the first time the potential of an Antarctic Rahnella inusitata strain as a cold-active β-galactosidase producer. This strain produced an enzymatic extract with higher efficiency in cold than commercial enzyme from Aspergillus oryzae, regarding its remaining activity in cold compared with the activity at optimal temperature. The Antarctic strain harbors three β-galactosidase genes and its description will make possible future recombinant production of these potential cold-active enzymes to confirm its activity as a purified product and for commercial industrialization in GRAS platforms (e.g., Bifidobac- terium bifidum and Bacillus subtilis). Despite the sequence and structure similarity of R. inusitata β-galactosidases with known enzymes, it is expected that adaptation to the Antarc- tic conditions shaped evolutive differences directly affecting their activity in cold. However, further studies are needed to understand each specific gene’s structure and functioning and possible evolutive variants at the catalytic domains related to thermostability and activity in cold, which is not entirely understood yet.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22084144/s1, Table S1: Antarctic strains with positive detection of β-galactosidase activity based on β-Bluo-Gal reduction detection, Table S2: Alignment lengths and percentage of identity between nine Rahnella species genomes including the Antarctic isolate Se8.10.12. by Average Nucleotide Identity (ANI) calculation using BLASTn method, Table S3: Quality evaluation of the three-dimensional models of Rahnella inusitata Se8.10.12 β-galactosidases, Figure S1: Example of Int. J. Mol. Sci. 2021, 22, 4144 20 of 24

β-galactosidase producing Antarctic strains determined in Lactose Agar using β-Bluo-Gal as an indicator of the β-galactosidase activity, Figure S2: The hydrophobicity of the active site in docking analysis of β-galactosidases. Author Contributions: Conceptualization, R.S., A.S., S.F., F.S. and L.B.D.; methodology, O.G.-E., K.N.-M., R.S., C.T., S.F., D.M., C.H. and A.S.; validation, K.N.-M., R.S., A.S.; formal analysis, K.N.-M., R.S., A.S. and O.G.-E.; investigation, K.N.-M., R.S., A.S., F.S. and L.B.D.; resources, L.B.D., K.N.-M., F.S.; data curation, O.G.-E. and A.S.; writing—original draft preparation, R.S. and K.N.-M.; writing— review and editing, K.N.-M., R.S., A.S., C.T., D.M., C.H., F.S. and L.B.D.; visualization, O.G.-E., K.N.- M., R.S. and A.S.; supervision, L.B.D.; project administration, L.B.D. and F.S; funding acquisition, L.B.D. and F.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the INSTITUTO ANTÁRTICO CHILENO (INACH), grant INACH RT_14-12, and INACH DG_01-19; UFRO/UANDES, grant INI4 from; UNIVERSIDAD DE LA FRONTERA, grant DI19-0079 and grant DI20-2018, NETWORK FOR EXTREME ENVIRONMENTS RESEARCH (NEXER), grant NXR17-0003; and CONICYT, grants CONICYT-PFCHA/Doctorado Nacional/2017-21170263 for K.N.-M., CONICYT-PFCHA/Doctorado Nacional/2017-21171392 to A.S., and CONICYT-PFCHA/Doctorado Nacional/2017-21170265 to O.G.-E. F. Scott gratefully ac- knowledges financial support by the National Agency for Research and Development (ANID Chile) (Fondecyt Iniciación 11170081). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Publicly available datasets were analyzed in this study. These data can be found at https://www.ncbi.nlm.nih.gov under the accession number provided for each genome or nucleotide sequence. Acknowledgments: We acknowledge Carolina Shene deVidts and Allison Leyton from the Center for Food Biotechnology and Bioseparations for performing the Chromatographic Analyses, as well as Karina Godoy for her help in the microscopic analyses, both from the Scientific and Technological Bioresource Nucleus (BIOREN) of Universidad de La Frontera, Temuco, Chile. 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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