Enzymes are regarded as environmentally friendly chemicals that help to replace or reduce the use of dangerous chemicals in industrial processes, thereby encouraging sustainable production, and manufacturing. Among different industrial enzymes, microbial proteases dominate the world enzyme market due to their potential application in various industries like food (de Souza et al. 2015), pharmaceutical (Monciardini et al. 2014), textile (Jani et al. 2012), photographic (Parpalliwar et al. 2015), leather (Mamun et al. 2016), and detergent (Sarrouh 2014). However, the proteases must be robust enough to meet the process conditions that are usually hostile for successful industrial applications (Olajuyigbe and Falade 2014). Proteases for industrial applications must have activity, and stability over a wide range of temperatures, and pH extremes for prolonged periods, and even in the presence of various potential enzyme inhibitors (Singh et al. 2016). Contact lenses bear tear films, and protein residues on their surface, which affect the optical clarity of the lenses. Mostly, contact lens cleansing solutions have been prepared using plant, and animal proteases, while microbial proteases are also gaining importance (Pawar et al. 2009).
In the present study, protease producing actinomycetes were isolated from the backwaters of Puthuvypeen, Kerala. These sampling sites have high salinity, and alkaline conditions, and are known to be a rich source for various extremophilic isolates (Ballav et al. 2012). A total of 12 halophilic strains were isolated, out of which 4 strains showed protease production. GS-3 exhibited the best results for protease production among the 4 strains. The phenotypic characteristics reported that the strain belongs to the genus Streptomyces. The BLAST result confirmed that the isolate GS-3 belongs to the genus Streptomyces, and hence designated as Streptomyces sp. GS-3. The isolate GS-3 was considered for the maximum protease production at extreme conditions (high salt concentration, and alkaline pH). Thus, the enzymes obtained from this strain were expected to be highly stable under extreme conditions. Streptomyces sp. GS-3 also showed a higher yield of protease (371 U/ml) than previously reported strains, and, hence, might have a bigger role in a variety of industrial practices. Other actinomycetes strains such as S. pseudogrisiolus NRC-15 produce approximately 200 U ml− 1 of protease (El-Sayed et al. 2012), whereas S. griseus SJ_UOM-07-09, Streptosporangium roseum SJ_UOM-18-09, and Streptomyces sp. LD48 exhibits significantly lower enzyme production of 87, 82, and 16 U ml− 1, respectively (Jogaiah et al. 2016; Mehtani et al. 2017).
The growth media for Streptomyces sp. GS-3 was optimized, and wheat bran was found to be the best substrate used for protease production followed by casein, skim milk, and rice bran respectively. Wheat bran was previously proved to be an important substrate to stimulate protease production in different microorganisms (Agrawal et al. 2005; Kalaiarasi and Sunitha 2009). Wheat bran also gave the best yield for protease production by different actinomycetes like Thermoactinomyces thalpophilus PEE 14 (Divakar et al. 2006). The media contained peptone, and yeast extract as the nitrogen source, and glucose as carbon source. These nutrients play an important role in microbial growth, and enzyme production (Al-Askar et al. 2015). RSM by Box–Behnken design was used for further optimization (Fig. 3). The model was used to monitor the optimum values for each variable in an efficient way to maximize the yield. The enzyme production increased when the substrate concentration increased from 1–5.5%. Further increase in substrate concentration decreases the overall production (Fig. 3a). The excess substrate caused growth inhibition, and hindrance in metabolism resulting in low enzyme production (Ait Barka et al. 2016). A sharp decline in enzyme production was noted as the pH was increased beyond 9.5 (Fig. 3b). Though Streptomyces sp. GS-3 can survive in an alkaline environment and sustain enzyme production, excess alkalinity (> 9.5) restricts its growth. Similar findings were earlier observed with the other strains of Streptomyces sp. E-99‐1333 (Kontro et al. 2005). After 9.5 d of inoculation, the yield decreases (Fig. 3c) the bacterium entered the death phase due to the lack of nutrients in media, causing a notable decrease in enzyme production after the 10th day. The degradation of the existing enzyme in the media is also responsible for the lowered enzyme yield (Sharma et al. 2009).
The enzyme extracted from Streptomyces sp. GS-3 was purified, and the molecular weight was calculated to be 25 kD. Previous reports show that low molecular weight proteases (18–35 kD) from other actinobacteria belong to the class of serine proteases (Kim et al. 2006). These low molecular weight proteases have significant applications in various industries, such as food, detergent, textile, and leather industries (Li et al. 2013). The Km and Vmax values of the enzyme were determined to be 5.88%, and 38.46 µmol l− 1 min− 1 mg− 1, respectively, using 1% casein as substrate. The Km value depends on the substrate, and other associated conditions such as temperature, and pH. This value represents the binding affinity of the substrate towards the enzyme. Vmax values represent the number of substrate molecules being catalyzed per minute. The optimum temperature for the maximum enzyme activity was found to be 45 °C, which implies that the enzyme is also thermostable (Briki et al. 2016). Such thermostable proteases are important in biotechnological, and industrial applications due to their stability against denaturing agents, and other chemicals (Barzkar et al. 2018). The enzyme shows maximum activity at pH 9, and hence can be classified as an alkaline protease. Similarly, a novel, thermostable alkaline serine protease with an optimum pH, and temperature of 9, and 60 °C, respectively were obtained from a newly recognized strain, Aeribacillus pallidus C10 (Yildirim et al. 2017). Such enzymes are robust and have a huge impact on different industries like leather, detergent, and food. Enzyme activity was inhibited by PMSF even at the minimum concentration (2.5 mmol l− 1) as it strongly blocks the active site of the enzyme, which leads to complete loss of enzyme activity (Rao et al. 1998). As PMSF is a known inhibitor of serine proteases (Geng et al. 2016), it indicates that the isolated enzyme could be a serine-type protease. The enzyme was mostly stable in the presence of metal ions such as Cu2+, Hg2+, Zn2+, Ca2+, and Mg2+ ions. Reports also support the fact that metal ions do not affect protease activity (El-Sayed et al. 2012; Jani et al. 2012). The enzyme was stable in the presence of different organic solvents. However, these solvents act as a medium for enzymatic reactions but can also be responsible for deactivating these enzymes by changing the structural flexibility of the enzyme (Yildirim et al. 2017). This was consistent with the results observed for the protease produced from Streptomyces sp. AB-1, which was also stable in different organic solvents (Jaouadi et al. 2010). Proteases that are stable in the presence of organic solvents are very useful for synthetic reactions.
Mostly, contact lenses were cleaned with three different types of solutions such as surfactant, oxidative, and enzymes. Surfactants are non-toxic to lenses but do not remove the protein deposits efficiently (Szczotka-Flynn et al. 2010). Oxidative cleaners are effective in removing non-protein deposits from contact lenses, however, are harmful to the lenses (Jung and Rapp 1993). Enzyme cleaners are safe to lenses and efficient in removing the main component of contact lens debris, namely lysozyme. Bacterial protease degrading lysozyme was known for successfully removing the protein debris, and acting as a cleansing agent (Greene et al. 1996). To study the efficiency of the enzyme as a cleansing agent, the lenses were previously coated with an artificial tear solution. The spectroscopic analysis of the contact lens indicated that before coating the contact lenses with lysozyme the percent transmittance was 97%, and by the earlier studies (Jadhav et al. 2014), after deposition of protein, it was reduced to 68%. After enzymatic treatment, the transmittance was again increased to 95% (Table 5). Thus the increased transmittance indicated that enzyme has potential in the removal of protein deposits from the contact lens. Similarly, a post-treatment transmittance of control using phosphate buffer was 70%, indicating no protein removal. Effects of treatment of lenses with enzyme, and phosphate buffer are statistically significant.