*3.7. Molecular Modeling and Structural Alignment*

The three-dimensional structure of AlyA was constructed using Protein Homology/analogY Recognition Engine V 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index), on the basis of the homologues of the known structure (alginate lyase AlyA of *Corynebacterium* sp. ALY-1 (PDB ID: 1UAI) [52]. PyMOL (http://www.pymol.org) was used to visualize and analyze the modeled structure and to construct graphical presentations and illustrative figures.

#### *3.8. ESI-MS Analysis of the Degradation Products of AlyA*

For investigating the degradation pattern of AlyA, the reaction mixtures (800 μL) with pH 7.5 containing 1 μg purified enzyme and 2 mg substrates (polyG and polyM, the average Dp of the two substrates are about 40) were incubated at 30 ◦C for 0–48 h. After incubation, the mixture solutions were boiled for 10 min and then centrifuged at 12,000 rpm for 10 min to remove the unsolved materials. The hydrolysates were loaded onto a carbograph column (Alltech, Grace Davison Discovery Sciences, United Kingdom) to remove the salts after removing the proteins, and then concentrated, dried, and re-dissolved in 1 mL methanol. The supernatant (2 μL) was loop-injected to an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) after centrifugation, to further determine the composition of the products. The oligosaccharides were detected in a negative-ion mode using the following settings, previously described, with the scanning mass range 150–2000 *m*/*z* [30].

#### **4. Conclusions**

In this work, an alginate lyase-producing bacterium was isolated and identified to be *Isoptericola halotolerans* NJ-05. A novel bifunctional alginate lyase, AlyA, with high activity and broad substrate specificity was cloned and characterized. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 ◦C. Additionally, it possessed broad substrate specificity, showing high activities towards not only polyM but also polyG. Furthermore, the *Km* value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). The TLC and ESI-MS analyses indicated that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of alginate lyases with broad substrate specificity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/16/8/258/s1, The nucleotide and protein sequence of AlyA and Table S1. Purification of the recombinant AlyA.

**Author Contributions:** L.N. and Y.J. conceived and designed the experiments; M.C. and Y.H. performed the experiments; L.N. and Y.J. analyzed the data; L.N. wrote the paper; B.Z. revised the paper. All of the authors reviewed the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (No. 31601410 and 81503463).

**Acknowledgments:** The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 31601410 and 81503463).

**Conflicts of Interest:** The authors declare no conflict of interest.
