*2.3. One-Step Affinity Purification of Commercial Metalloprotease Products*

Three commercially available products (MP, DENIE-B LPS-P and ViscozymeL) containing metalloproteases were dissolved in the loading buffer to a final concentration of 10 mg/mL each. Then, the enzymes were purified by a one-step purification protocol using the 4-APBA-modified Sepharose 6B medium. We tested different loading and elution conditions to optimize the yield of metalloproteases. Almost all of the metalloproteases contained seven or eight calcium ions stabilizing their three-dimensional structure [29]. Thus, to obtain properly folded enzymes, both of the loading and elution buffers contained 1 mM CaCl2. The 0.1 M Gly-NaOH buffer, pH 8.6, was chosen as the loading buffer because of the highest affinity of MP to the beads and stability of all three enzymes at this pH. Different acetic acid buffers (pH ranging from 4.0 to 6.0) were tested to select an optimal pH for MP elution, as low acidity favored the disruption of the H-bond interactions between MP and the medium. The highest protein yield was obtained at pH 5.4. Thus, 0.1 M acetic acid (pH 5.4) was chosen as the elution buffer. The SDS-PAGE analysis of the crude and purified metalloproteases is shown in Figure S2. The activity and purity of purified enzymes are shown in tab:marinedrugs-15-00005-t002.


**Table 2.** Comparison of affinity and traditional purification methods for three available metalloprotease products.

<sup>a</sup> In the affinity protocol, enzymes were purified by 4-APBA-modified medium; <sup>b</sup> The traditional purification protocol of MP was composed of five steps, including ultrafiltration, ammonium sulfate precipitation, desalting, anion-exchange and gel-filtration chromatography; <sup>c</sup> The traditional purification protocol of DENIE-B LPS-P was composed of three steps, including ammonium sulfate precipitation, desalting and anion-exchange chromatography; <sup>d</sup> The traditional purification protocol of ViscozymeL was composed of six steps, including ammonium sulfate precipitation, hydrophobic chromatography, desalting, anion-exchange chromatography, and two steps of gel-filtration chromatography.

In our previous work, the five-step purification protocol for the purification of MP was developed. It included ammonium sulfate precipitation, desalting, anion-exchange and gel-filtration chromatography and took more than 48 h of work [3,21]. Here, we report a simple and efficient one-step MP purification procedure that takes less than one hour. Our protocol is based on the 4-APBA-modified Sepharose 6B medium that efficiently bound native MP from natural sources. Here we compared this one-step protocol with the previous reported methods, along with all the different purification steps, activity yields, specific activities and time requirement. According to the measurements of MP activity in the initial sample and purified protein, almost 64.1% of initial MP was purified, whereas only 8.9% of initial MP was recovered using the traditional protocol. The specific activity of MP purified by the APBA-modified protocol (95.6 U/mg) is similar with the value obtained through the traditional purification protocol (96.2 U/mg) and the IMAC protocol (94.8 U/mg). However, the purity of MP is different, being higher with the APBA-modified affinity purification (98.8%) with respect to the traditional and IMAC methods (92.5% and 94.7%, respectively). Even if the IMAC protocol results an activity recovery higher than the 4-APBA protocol, it is longer. Moreover, the APBA-modified affinity protocol avoids the use of toxic imidazole and the loss of metallic ions in the MP active pocket that reduce the enzyme stability. Based on all the positive features of this affinity protocol, such as one-step of chromatography, shorter times, and higher purity, it is clear there is potential in this approach for the industrial production of high-purity MP.

To determine whether our medium has an affinity value to metalloproteases from other sources, two other commercial metalloprotease products, DENIE-B LPS-P and ViscozymeL, were used for enzyme purification. DENIE-B LPS-P was an enzyme concentrate produced from *Bacillus subtilis* that was widely used in leather softening [43]. Based on the activity measurement in our study, only 10.7% of metalloprotease was purified from this commercial product using the traditional three-step purification protocol, including ammonium sulfate precipitation, desalting and anion-exchange chromatography on a Q Sepharose column. ViscozymeL was a cell wall degrading enzyme complex from *Aspergillus* sp., containing a wide range of carbohydrases and metalloprotease [44]. Traditional purification of the metalloprotease from ViscozymeL resulted in only less than 60% pure enzyme, which required six steps, including ammonium sulfate precipitation, hydrophobic chromatography, desalting, anion-exchange chromatography, and two steps of gel-filtration chromatography. Meanwhile, IMAC (Cu-IDA ligand) purification of these two protein products resulted in less than 60% purity of metalloproteases (data not shown). However, the 4-APBA-modified medium could efficiently purify metalloproteases from those two products ( tab:marinedrugs-15-00005-t002). The activity recoveries of DENIE-B LPS-P and ViscozymeL were 45.2% and 37.8%, respectively. Meanwhile, when the purified enzymes were analyzed by HPLC with a TSK3000SW gel filtration column, both of

them were more than 95% pure (Figure 5). To sum, our novel methodology had multiple advantages in comparison with all known techniques of metalloprotease purification.

**Figure 5.** Purity analysis of three purified enzyme products. (**A**) SDS-PAGE (10.0%) analysis showed that the enzymes were purified to an apparent homogeneous population with a molecular mass of 48 kDa and the purity was more than 95%. *Lane M*, molecular mass standard protein marker; *Lane 1*, the purified MP; *Lane 2*, the purified DENIE-B LPS-P; *Lane 3*, the purified ViscozymeL; (**B**) HPLC analysis using the size exclusion by gel filtration of the purified MP (**1**), DENIE-B LPS-P (**2**) and ViscozymeL (**3**) on a TSK 3000SW column.

#### **3. Materials and Methods**
