**4. Conclusions**

In this study, an affinity medium more efficient for metalloprotease purification than other currently available techniques was designed, synthesized and experimentally characterized. Testing the adsorption properties of five designed resins, the Sepharose 6B media coupled with 4-APBA via cyanuric chloride spacer was selected for the purification of native metalloproteases from three commercial products. Metalloproteases from these sources were purified in one step with high efficiency and purity (more than 95%). Compared with the previously reported methods, this protocol resulted in several positive features, such as fewer steps, better activity recoveries, and higher purity. Coupled with efficacy, time-saving procedure and accessible reagents, this novel affinity purification protocol represents a potential important tool for industrial application.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/15/1/5/s1. Figure S1: The ESI-MS analysis of the affinity ligand. The possible structures of the chemicals in principal peaks are shown. The ESI-MS cone voltage (170 V) was selected. Scanning was performed from *m*/*z* 100 to 1000 in 10 s, and several scans were summed to obtain the final spectrum, Figure S2: SDS-PAGE analysis of three purified and crude commercial metalloproteases. (A) Analysis of crude (Line 1) and purified (Line 2) MP; (B) Analysis of crude (Line 2) and purified (Line 1) DENIE-B LPS-P; (C) Analysis of crude (Line 2) and purified (Line 1) ViscozymeL.

**Acknowledgments:** This project was funded by National Science Foundation-Shandong province Joint Fund (U1406402-5); International Science and Technology Cooperation and Exchanges (2014DFG30890); Postdoctoral Science Foundation of China (2016M590673 and 2015M582170); Postdoctoral Science Foundation of Shandong; Postdoctoral Researcher Applied Research Project of Qingdao (Q51201601 and Q51201613); National Natural Science Foundation of China (41376175); The Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASKJ02); National Hi-tech R&D Program (2014AA093516); Science and Technique Plan of Qingdao (14-2-4-11-jch). International collaboration (PSR-SIIRI) supported by the Ministry of Science and Technology of China and the Ministry of Economy, Science and Innovation of Quebec, Canada (MESI).

**Author Contributions:** S.L., S.L. and M.S. conceived and designed the experiments. S.L., L.W., J.L., Y.W. and J.H. performed the experiments. S.L., L.W., Y.W. and X.X. analyzed the data. S.L., X.X. and M.S. wrote the main manuscript text. All authors reviewed the manuscript.

**Conflicts of Interest:** We declare that we have no competing financial interests.
