Detection of industrially potential enzymes of moderately halophilic bacteria on salted goat skins

Introduction

Enzymatic processes provide many advantages in terms of saving energy, water, raw materials and chemicals that cannot be achieved using conventional chemical processes. Due to enzymes’ biodegradable structure, obtaining high quality product, low energy consumption, low-cost material and less environmental pollution, enzymes are more preferred as economically and ecologically alternatives to chemicals in industrial applications [1]. The use of microbial enzymes is widespread in biotechnological applications as metabolic catalysts for centuries. Industrial enzymes have been used in food, feed, chemical and pharmaceutical, textile, biofuel, paper and pulp, detergent and leather industries as well as production of fish sauce and soy sauce, bioremediation, saline waste water, and oil field waste treatment [2].

Annual spending on enzymes is quite high worlwide. According to report by BBC Research (2014), globally the enzyme market is approximately estimated at 7.1 billion dollars by 2018 [3]. Due to the high demand for industrial enzymes, researchers have been focused on finding new industrial microorganisms producing different enzymes that can withstand extreme conditions. Especially moderate halophiles are considered to be potential sources for industrial enzymes such as cellulases, lipases, xylanases, proteases, amylases, nucleases, and esterases which are able to perform their functions under a wide range of salinity (optimally 3–15% NaCl), pH, and temperatures [1], [2], [4], [5], [6]. Furthermore, moderate halophiles present opportunities owing to easily growing on low-cost substrate and low water content [7]. In the previous studies, different moderately halophilic bacterial genera (Bacillus, Chromohalobacter, Halobacillus, Halomonas, Marinococcus, Oceanobacillus, Salibacillus, Salinicoccus, Salinivibrio and Thalassobacillus), isolated from natural saline environments, produced DNase, lipase, xylanase, amylase, inulinase, protease, cellulase, pullulanase, pectinase and caseinase [5], [8], [9].

Moderately halophilic bacteria have been isolated from hypersaline environments such as saline lakes, salterns, solar salt evaporation ponds, saline soils, salt mine soil, marine sediments and other saline habitats such as salted fish, fermented anchovy sauce, meat and hides [10], [11], [12], [13], [14], [15], [16]. Although characterization of moderately halophilic bacteria, isolated from different saline environments, using conventional and molecular techniques and detail experiments on their hydrolytic enzymes have been carried out by several researchers [5], [8], [9], investigation of moderately halophilic bacteria found on salted goat skins, using both conventional and molecular techniques and examination of their industrial enzymes have not been reported previously. Screening of enzyme producing moderately halophilic bacteria on goat skins cured with different countries’ salt will be important for determination of industrially potential microorganisms. Hence, the present study focused on the screening industrially important moderately halophilic bacteria producing amylase, catalase, oxidase, caseinase, cellulase, DNase, lipase, lecithinase, protease, pullulanase, urease, phospholipase, xylanase and β-galactosidase. In order to characterize moderately halophilic bacteria isolated from the skins and to understand their physiological and biochemical characteristics for enzyme production, phenotypic characteristics and comparative partial 16S rRNA gene sequence analysis of these microorganisms were also examined in this study.

Materials and methods

Enzymatic activities of the moderately halophilic isolates

Catalase activity (hydrogen-peroxide oxidoreductase, EC 1.11.1.6) was determined by adding 3% H₂O₂ to colonies grown on CMI agar medium. The immediate appearance of bubbles was accepted as a positive test result. Oxidase activity (ferrocytochrome-c, EC 1.9.3.1) was examined by transfering colony of the isolate with a sterile loop onto filter paper moistened with oxidase reagent (Merck, Darmstadt, Germany). Occurrence of color change from pink to dark purple in a few seconds indicated positive oxidase activity [9], [20], [21], [22]. Amylase activity (4-α-D-glucan glucanohydrolase, EC 3.2.1.1) was detected using CMI agar medium supplemented with 0.5% (w/v) soluble starch. After incubation, the plate was flooded with 0.3% I₂–0.6% KI solution. Clear halos around the colonies indicated starch hydrolysis. The DNase test agar (Merck, Darmstadt, Germany) was used to determine DNase activity (deoxyribonuclease I, EC 3.1.21.1). After incubation, the plate was flooded with 1N HCl. Clear zones around the colonies showed hydrolysis of DNA [8], [21], [22]. The cellulose medium agar plate containing 0.2% (w/v) carboxymethyl cellulose was used to detect production of cellulase (4-β-D-glucan cellobiohydrolase, EC 3.2.1.91). After incubation, 0.1% congo red test reagent (Merck, Darmstadt, Germany) was flooded on the colonies and left for 30 min. Then, the colonies were washed with 1 M NaCl solution. Clear zones around the colonies showed cellulase activity [21], [22], [23]. Hydrolysis of casein was tested with the Plate Count Agar medium containing 2% skim milk. After incubation, clear zones around the colonies were interpreted as caseinase production (caseinolytic protease, EC 3.4.21.92) [9], [21], [22]. Lipase activity (triacylglycerol acylhydrolase, EC 3.1.1.3) was screened on Tween 80 agar medium containing 1% (w/v) Tween 80 (Merck, Darmstadt, Germany). After incubation, opaque zones around the colonies were accepted as evidence of lipase activity [21], [22], [23]. Due to the presence of fats on the skins, lipase activity of the isolates was also tested in agar medium containing 5% (w/v) butter. After incubation, 20% cupper-sulfate solution was flooded onto the plates. Positive bluish green colonies were interpreted as phospholipase activity (phosphatidylcholine 1-acylhydrolase, EC 3.1.1.32) [21], [22], [24], [25]. Protease activity (gelatinase A, EC 3.4.24.24) was screened on gelatin agar medium containing 2% gelatin (w/v). After incubation, the plate was flooded with Frazier solution. Clear zones around the colonies were interpreted as positive protease activities [8], [21], [22]. Urease activity (urea amidohydrolase, EC 3.5.1.5) was detected on Christensen Urea Agar (Difco, Detroit, USA). After growth was obtained, the tube was examined for pink or red color changes [21], [22], [26]. To detect pullulolytic (pullulan 6-α-glucanohydrolase, EC 3.2.1.41) and xylanolytic (4-β-D-xylan xylanohydrolase, EC 3.2.1.8) activities of the isolates, plates containing the chromogenic substrates such as azurine-cross-linked (AZCL)-pullulan and AZCL-xylan (Megazyme, Wicklow, Finland) were used. Clear zones around the colonies were accepted as positive pullulolytic and xylanolytic activities [8], [21], [22]. Lecithinase activity (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) was investigated on lecithine agar plate containing 5% egg yolk (w/v). Opaque zones around the colonies showed lecithinase activity [21], [22], [24], [25]. Beta-galactosidase activity (β-D-galactoside galactohydrolase, EC 3.2.1.23) was detected in test tubes containing 1 mL of 10% NaCl (w/v) sterile distilled water and ONPG (ortho-nitrophenyl-β-galactoside) discs (Sigma-Aldrich, Buchs, Switzerland). The formation of yellow color was accepted as positive β-galactosidase activity [21], [22], [23]. The pH of all media was adjusted to 7.5. The salt mixture (SW10) was used in all biochemical test media.

Figure 1:

Comparison of enzymatic activities of moderately halophilic bacterial isolates.

Results

The 16S rRNA the pairwise sequence similarities of the isolates were found as 98.9–100% for the isolates belonging to Firmicutes and 99–99.9% for the isolates belonging to Proteobacteria [25]. While four different genera [Staphylococcus (16 isolates), Bacillus (8 isolates), Gracilibacillus (5 isolates) and Salinicoccus (3 isolates)] were determined in Firmicutes, two different genera [Halomonas (4 isolates) and Chromohalobacter (3 isolates)] were found in Proteobacteria (Table 1).

Goat skin samples used in this study were cured with each country’s preservation salt. Different species of moderately halophilic bacteria may be found in these salts. Hence, every country’s goat skin samples may have different moderately halophilic bacterial species. To verify this, we collected salted goat skin samples belonging to eight different countries. Our study demonstrated that the isolate numbers, presence and prevalence of different moderately halophilic bacterial species on the goat skin samples showed differences according to countries. While the goat skins belonging to Africa contained five different species, goat skin samples belonging to Israel and Russia contained only one species. The other skin samples contained a few species (Table 2).

While all isolates were catalase positive, more than half of the isolates were oxidase positive. It has been known that catalase and oxidase enzymes are related with aerobic microorganisms. A few bacteria produced indole from tryptophan; fermented glucose and produced acetoin and 2,3 butanediol in Voges-Proskauer test; formed H₂S and N₂ gases. More than half of the isolates catabolized glucose and produced acidic end products; used nitrate as a terminal electron acceptor and reduced nitrate to nitrite; produced NH₃ from peptone broth. Ammonia odor released from the goat skin samples was related to protein catabolism. While 85%, 92%, 56%, 74%, 90%, 74%, 69%, 72%, 62% and 72% of the isolates respectively produced acid from sucrose, D-glucose, D-galactose, fructose, D-trehalose, D-mannose, D-xylose, lactose, maltose and L-arabinose, acid productions from D-melibiose and D-cellobiose were detected as 44% and 28%, respectively. Thirty one percent of the isolates utilized citrate as a sole carbon source for their energy needs (Table 1).

While all isolates produced catalase, 87%, 64%, 59%, 46%, 28%, 26%, 8%, 5% of the isolates produced protease, lipase, β-galactosidase, urease, caseinase, amylase, lecithinase and cellulase, respectively (Table 1).

Some of the isolates exhibited combined enzymatic activities. While 5%, 36% and 31% of isolates produced eight, six and five different enzymes, respectively, 15% of isolates produced three different enzymes. Furthermore, 10% and 3% of the isolates produced two and one enzymes (Figure 1). In the present study, isolates exhibiting most combined activities were belong to the genera Bacillus, Staphylococcus, Gracilibacillus, Salinicoccus and Halomonas. Among the isolates B. licheniformis produced the highest number of enzymes (Figure 1). In order to characterize these enzymes and determine their biochemical properties, more detailed investigation is currently under way.

Discussion

Production of lipase, pullulanase, amylase, protease, xylanase, inulinases, pectinase, cellulases and DNase enzymes by moderately halophilic bacteria (Salicola, Salinicoccus, Marinococcus, Salinivibrio, Virgibacillus, Oceanobacillus, Thalassobacillus, Halovibrio, Halobacillus, Piscibacillus, Halomonas, Gracilibacillus, Bacillus, Chromohalobacter) which were isolated from various saline environments in Spain and Howz Soltan playa (a hypersaline lake) were stated in the previous studies [5], [8]. The enzymatic activities of our test isolates were similar to the enzymatic activities of the same species previously identified [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44].

Catalase enzymes are used in textile industry as a bleaching agent and elimination of hydrogen peroxide in dairy industry [45], [46]. Our all isolates produced this enzyme. Proteases are used in removing hair from hides, leather processing, laundry, detergent production, cheese production, softening meat, improving wool quality [47]. The species of Bacillus licheniformis, Bacillus pumilus, Gracilibacillus dipsosauri, Halomonas eurihalina, Staphylococcus arlettae, Staphylococcus equorum subsp. equorum, Staphylococcus saprophyticus subsp. saprophyticus, Salinicoccus roseus and Staphylococcus xylosus were able to produce protease in this study. Lipases are used in dairy industry for hydrolysis of milk fat, removal of subcutaneous fat in leather industry, biosysnthesis of drugs in pharmaceutical industry [47]. Bacillus licheniformis,Bacillus pumilus, Gracilibacillus dipsosauri, Halomonas eurihalina,Salinicoccus roseus and Staphylococcus saprophyticus subsp. saprophyticus were able to produce lipase (Table 1). Proteases and lipases are notably important because they are mainly used in bating, soaking, degrasing, tanning and final stages of leather product. Hence, salt-tolerant enzymes produced by moderately halophilic bacteria are good candidates for leather industry [48]. The enzyme β-galactosidase, produced by Bacillus licheniformis, Bacillus pumilus, Gracilibacillus dipsosauri, Staphylococcus equorum subsp. equorum, Staphylococcus saprophyticus subsp. saprophyticus and Staphylococcus xylosus, in this study, may be used in the synthesis of galacto-oligosaccharides from lactose [49]. In the present study, moderately halophilic Halomonas eurihalina, Staphylococcus arlettae, Staphylococcus equorum subsp. equorum, Staphylococcus saprophyticus subsp. saprophyticus and Staphylococcus xylosus showed positive urease activity. Microbial ureases are used for wine production to remove urea [50]. Caseinase plays an essential role in degrading casein found in milk [26]. Caseinase activity was seen in Bacillus licheniformis, Bacillus pumilus and Salinicoccus roseus. Amylases play a vital role in starch hydrolysis, food, textile, paper and pulp industry, bread and baking, detergent, pharmaceutical [47], [51]. In the present study, Bacillus licheniformis, Gracilibacillus dipsosauri and Salinicoccus roseus produced amylase. Staphylococcus equorum subsp. equorum and Staphylococcus xylosus in the present study were capable of producing lecithinase enzyme that hydrolysis lecithine. In our study, only moderately halophilic Bacillus licheniformis produced cellulase enzyme which may be used in food, chemical, textile, feed, paper and detergent industries, biomedical science and agriculture [47]. In accordance with data from previous studies that investigated enzymatic studies, members of genera Bacillus, Gracilibacillus, Halomonas, Salinicoccus, Salinivibrio and Staphylococcus were known to secrete extracellular enzymes such as protease, lipase, amylase, urease [5], [8], [51]. None of our moderately halophilic isolates produced pullulanase, xylanase, phospholipase and DNase in the present study (Table 1).

In the present study, moderately halophilic bacteria were especially isolated from goat skin samples belonging to different countries. Presence of moderately halophilic bacteria on all salted goat skins was closely related with the preservation salt used in the curing of goat skin. It has been known that salted goat skin containing fats, proteins, carbohydrates and blood offers an ideal saline environment for growth of moderately halophilic bacteria and production of hydrolytic enzymes. Our biochemical test results proved that all isolates were able to utilize a wide variety of organic compounds and carbon sources. These isolates produced different enzymes such as protease, catalase, lipase, β-galactosidase, urease, caseinase, amylase, lecithinase and cellulase. These enzymes may have a wide range of potential applications in different industries such as baking, beverage, dairy, dry cleaning, feed, food, laundry, meat, paper, pharmaceutical, starch and textile.