**4. Conclusions**

Compared with other native bacteria, the extracellular collagenases of *Ps* sp. SJN2, composed of proteases (e.g., metalloproteases and serine proteases) with different molecular weights, showed higher enzyme activity towards porcine, bovine and marine collagen. Given the increasing economic relevance of marine collagenases, this study was conducted to optimize a variety of fermentation parameters, including medium composition and culture conditions, for maximal collagenase production. The results indicated that the Plackett–Burman design and response surface methodology are effective and efficient approaches to improve collagenase production via optimization of *Ps* sp. SJN2 fermentation conditions. The maximum collagenase yield of 322.58 ± 9.61 U·mL−<sup>1</sup> was achieved under the optimized conditions, which was in agreement with the production value of 306.68 U·mL−<sup>1</sup> predicted by the model. Finally, for optimal fermentation, the cells were cultured at 17.32 ◦C for 3.72 d in a medium containing soybean 34.23 g·L−1, corn meal 30 g·L−<sup>1</sup> and bran liquid 15 mL·L−<sup>1</sup> at an initial pH value of 8.5 and an inoculum volume of 1% (*v*/*v*). Significant improvement (2.2-fold) in the production of collagenases by *Ps* sp. SJN2 was accomplished. In addition, the marine collagen hydrolysed by collagenases from *Ps* sp. SJN2, especially those from Spanish mackerel fish bone and seabream fish scale, showed better prospects for application as antioxidant peptides. Above all, the optimized medium established in this study could provide a basis for further study of large-scale fermentation of *Ps* sp. SJN2 for marine collagenase yield improvement.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/15/12/377/s1: Figure S1: Gelatin immersing zymography of Col SJN2, Figure S2: Crude enzyme zymography of *Ps* sp. SJN2, Table S1: Collagenases activity in purification process. Supporting information about the methods of inoculum preparation for fermentation and preparation of collagen from fishery by-products is provided in the Supplementary Material.

**Acknowledgments:** This work was supported by the National Natural Science Foundation of China (3140002, 31070061, 31370104, 21205142), the National Sparking Plan Project (2013GA770009), the Opening Foundation of the Chinese National Engineering Research Center for Control and Treatment of Heavy Metal Pollution (2015CNERC-CTHMP-07), the Open-End Fund for the Valuable and Precision Instruments of Central South University (CSUZC201640) and the Fundamental Research Funds for the Central Universities of Central South University (2015zzts273).

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