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 process- ing 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 lacuspro- fundi, 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 activ- ity, 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 charac- terize 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 b-Galactosidase Activity β-galactosidase enzymes cleave the disaccharide lactose to produce galactose and glu- cose 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 poten- tial 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