**4. Conclusions**

The genome of the marine bacterium *P. hydrolytica* S66T encodes two putative GH20 β-*N*-acetylhexosaminidase (EC 3.2.1.52) having protein sequences that differed remarkably from earlier characterized β-NAHAs (≤30% identity). *Ph*Nah20A was positioned on a phylogenetic tree between β-NAHAs of water-associated bacteria, i.e., *Vibrio furnissii* and *Aeromonas hydrophila*, and unicellular eukaryotes (amobae). *Ph*Nah20A, produced in *E. coli*, was unstable if diluted, but was stabilized by BSA or Triton X-100. *Ph*Nah20A is a genuine β-NAHA with essentially the same catalytic efficiency for *p*NPGlcNAc and *p*NPGalNAc, and thus differs from most of the previously studied bacterial β-NAHAs, which prefer *p*NPGlcNAc as a substrate while some eukaryotic GH20 prefer *p*NPGalNAc. *Ph*Nah20A also hydrolyzed LNT2, a core structure of human milk oligosaccharides, and showed biosynthetic activity (transglycosylation) which is a poorly studied aspect of GH20 β-NAHAs, especially from eukaryotes and water-living prokaryotes. *Ph*Nah20A was able to form LTN2 by transglycosylation using NAG-oxazoline as a donor and lactose as an acceptor, LNT2, β-Gal-1,4-β-Glc-1,1-β-GlcNAc and β-Gal-1,4-(β-GlcNAc)-1,2/3-Glc being identified by NMR as main transglycosylation products. Several monosaccharides were also recognized as acceptors by *Ph*Nah20A. To date, based on pH and temperature optima, kinetic parameters or stability characteristics alone, no clear distinction can be made between eukaryotic versus prokaryotic or terrestrial versus aquatic GH20 β-NAHAs. However, this may be due to the very limited number of characterized β-NAHAs of salt or fresh water origin. *Ph*Nah20A is the first characterized member of a distinct group of GH20 β-NAHAs located phylogenetically between eukaryotic and prokaryotic enzymes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/2/417/s1. The following materials are available online: Supplementary Information 1 containing Figure S1. TLC analysis of growth media of *P. hydrolytica* (A, B, C) and growth phenotype on marine agar medium with X-GlcNAc (5-bromo-4-chloro-3-indolyl *N*-acetyl-β-d-glucosaminide) (D); Figure S2. Phylogenetic tree with bootstrap test (1000 replicates) of *Ph*Nah20A, *Ph*Nah20B (both marked with red circles) and 41 biochemically characterised GH20 (EC 3.2.1.52) enzymes; Figure S3. Schematic domain architecture of full-length and truncated variants of *Ph*Nah20A and *Ph*Nah20B; Figure S4. IPTG-induced *E. coli* transformants growing in LB analysed by SDS-PAGE; Figure S5. Inactivation of 5 <sup>μ</sup>g·mL−<sup>1</sup> *Ph*Nah20A at 50 ◦C and pH 6.0 in the presence of 0.5% BSA; Figure S6. Time course of transglycosylation by *S. plicatus* Hex (10 U·mL<sup>−</sup>1) with 100 mM NAG-oxazoline as donor and 200 mM lactose as acceptor; Figure S7. TLC of trisaccharide-containing fractions of the *Ph*Nah20A reaction (2 h; Figure 7A) separated by gel-permeation chromatography; Figure S8. HSCQ spectrum of the chromatographic fraction 50 (see Figure S7) containing over 80% of compound **1**; Figure S9. HSCQ spectrum of the chromatographic fraction 51 (see Figure S7) containing over 75% of 2 (LNT2); Figure S10. HSCQ spectrum of the chromatographic fraction 53 (see Figure S7) containing primarily 3; Figure S11. Extraction of HPAEC-PAD analysis of transglycosylation products by *Ph*Nah20A (10 U·mL<sup>−</sup>1) reacting 2 h with 100 mM NAG-oxazoline as donor and 200 mM lactose as acceptor (blue line); Figure S12. Time course of transglycosylation by *Ph*Nah20A (10 U·mL<sup>−</sup>1) with 100 mM NAG-oxazoline as donor and 200 mM D-glucose (A), 2-deoxy-d-glucose (B) or L-fucose (C) as acceptor; Table S1. BLAST analysis of putative β-NAHAs (EC 3.2.1.52) from *P. hydrolytica*. Table S2. Information on proteins flanking identified β-NAHAs (presented in Figure 1B); Table S3. NMR assignment of 1. The methyl of the GlcNAc acetyl group was at 2.090 ppm for 1H and 22.81 ppm for 13C and the carbonyl of the acetyl was at 176.06 ppm 13C; Table S4. 3H-H coupling constants for 1 measured through DQF-COSY; Table S5. PCR primers to isolate full-length β-NAHA encoding genes and indicated truncated variants. Underlined sequences are priming with pURI3-TEV expression vector; Supplementary Information 2 containing multiple sequence alignment.

**Author Contributions:** Conceptualization, P.S., T.V. and B.S.; methodology, T.V., C.K., A.L. and L.H.P.; validation, T.V., D.T., C.K. and L.H.P.; formal analysis, T.V., C.K., A.L. and D.T.; investigation, T.V., C.K., D.T., A.L. and L.H.P.; resources, B.S., J.Ø.D., L.H.P., C.A.-M., D.T. and P.S.; data curation, T.V., B.S.; writing—original draft preparation, T.V., D.T., C.K., P.S. and B.S.; writing—review and editing, T.V., D.T., C.K., P.S. and B.S.; visualization, T.V. and D.T.; supervision, P.S., J.Ø.D., L.H.P. and B.S.; project administration, P.S., T.V., B.S.; funding acquisition, P.S., D.T., T.V. and B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Innovation Fund Denmark to the project "OliGram. Design and gram scale enzymatic synthesis of human milk oligosaccharides", grant number 1308-00014B having P.S. as PI. D.T. is grateful to the Novo Nordisk Foundation for a postdoctoral fellowship (grant NNF17OC0025660). The APC was funded by University of Tartu Feasibility Fund grant PLTMRARENG13 to T.V. and the NNF17OC0025660 grant to D.T.

**Acknowledgments:** Karina Jansen (Technical University of Denmark) is thanked for general technical assistance, Pernille K. Bech and Mikkel Schultz-Johansen (University of Copenhagen) for providing the *P. hydrolytica* strain, Corinna Schiano di Cola for preparing autoinduction medium and Tiina Alamäe (University of Tartu) for fruitful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
