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

Abstract

There has been a recent increase in the exploration of cold-active β-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, 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 (4 °C-15 °C). Three β-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active β-galactosidase producer.

Keywords: Antarctica; cold-adapted bacteria; extremozymes; lactose; β-galactosidase.

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.

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. The horizontal line indicates the mean values, and the length of each whisker indicates the interquartile range (IQR); n = 4. Different letters represent statistically significant differences (one-way ANOVA within temperatures, p < 0.005; t-student between enzymes, p < 0.01).

Figure 3

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

Figure 4

Evolutionary history construction of the Antarctic strain Se.8.10.12 based on phylogeny. (a) Phylogenetic analysis of the 16S rRNA gene constructed with Bio-NJ method based on the Kimura two-parameter distance estimation model (Helicobacter pylori was used as an outgroup). (b) Maximum likelihood phylogenetic analysis of core-proteome based on the concatenated amino acid sequences of orthologous genes present in all Rahnella species using RAxML. GenBank accession numbers are provided in brackets. Branch lengths represent the number of substitutions per site (Bar: 0.01 substitutions per site), and bootstrap percentages of 1000 replicates are shown in the branches.

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 accession numbers are provided in brackets.

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.

Figure 7

Maximum likelihood phylogram based on three β-galactosidase amino acid sequences of the strain Se8.10.12 (highlighted color) and proteins on nucleotide database with >85% similarity (light color). Aspergillus oryzae β-galactosidase LacA was used as an outgroup. Branch lengths are shown in each branch and represent the number of nucleotide substitutions per site. Bootstrap support based on 1000 replicates ultrafast method is represented as branch width (1 px line = 47% to 10 px line = 100%). GenBank/EMBL/DDBJ accession numbers are provided in brackets.

Figure 8

Overall predicted structure of the Rahnella inusitata Se8.10.12 β-galactosidases. The three-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, the catalytic motifs are colored in red and blue, while asterisks indicate the glutamic acid amino acid in the active site.

Figure 9

Molecular docking of the Rahnella inusitata Se8.10.12 β-galactosidases and its substrate lactose. (a) Molecular docking simulation of lactose binding to Aspergillus oryzae β-galactosidase (4IUG) and Rahnella inusitata Se8.10.12 β-galactosidases LacZ, EbgA, and BglY (homology modeling). Schematic representation showing enzyme/ligand interactions (left image) and two-dimensional (2D) ligand-protein interaction diagram (right). The hydrogen bonds are represented dotted green lines (Å). (b) Docking scores (AutoDock Vina) for lactose against the selected β-galactosidases. Error bars denote SE of mean; n = 5. Different letters represent statistically significant differences between models (Kruskal–Wallis test; p < 0.05).