Detection of industrially potential enzymes of moderately halophilic bacteria on salted goat skins Tuzlanmış Keçi Derileri Üzerindeki Ilımlı Halofil Bakterilerin Endüstriyel Potansiyele Sahip Enzimlerinin Belirlenmesi

Aim: This study aimed to isolate moderately halophilic bacteria from salted goat skins, to characterize these microorganisms and to determine their industrially important enzymes such as amylase, catalase, oxidase, caseinase, cellulase, DNase, lipase, lecithinase, protease, pullulanase, urease, phospholipase, xylanase and β -galactosidase. Methods: Enzymes of these bacteria, isolated from skin samples belonging to eight countries and identified using phenotypic and genotypic methods, were examined in agar media. Results: Thirty-nine isolates were fairly similar to species of genera Staphylococcus , Bacillus , Salinicoccus , Gracilibacillus , Chromohalobacter and Halomonas . Various carbon sources were utilized, and all isolates produced enzyme. Enzyme-producing species were Staphylococcus saprophyticus subsp. saprophyticus , Staphylococcus arlettae , Bacillus pumilus , Gracilibacillus dipsosauri , Salinicoccus roseus , Bacillus licheniformis , Chromohalobacter beijerinckii Staphylococcus xylosus , Halomonas eurihalina Staphylococcus equorum Fairly high percentage of isolates produced protease (87%) and catalase (100%). While more than 50% of isolates produced lipase (64%), β -galactosidase (59%) and oxidase (56%), less than 50% of isolates produced urease (46%), caseinase (28%), amylase (26%), lecithinase (8%) and cellulase (5%). Conclusion: We detected that moderately halophilic bacteria on skins produced important enzymes, which may be used in diverse industrial applications in leather, feed, detergent, paper, food, chemical, medical, pharmaceutical, textile industries.


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].
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.

Sample collection
Twenty-three salted goat skin samples imported from Australia, Bulgaria, Israel, South Africa, Russia, China, France, and four salted goat skin samples preserved in Turkey were collected from different tanneries in the Leather Organized Tannery Region, Tuzla and Corlu, Turkey. The samples were then placed into sterile prelabeled translucent ziplock bags and transported in a sterile and cold container. The samples were named according to its origin: Australia (AL, AVT), Turkey (E, KE, T, YE), Bulgaria (B, BLG, BT), Israel (IRL), South Africa (KT, K, NE, GK), Russia (RU, RA), China (CCN, CN, CC), France (F, FR, FN, FS) ( Table 1).

Isolation of the moderately halophilic bacteria
To isolate moderately halophilic bacteria, 20 g skin samples were separately soaked in flasks containing 180 mL of 10% NaCl. The flasks were placed into orbital shaker at 100 rpm for 4 h at 25°C. Sterile physiological saline solution containing 10% NaCl was used to dilute skin solutions. An aliquot of 0.1 mL of each direct and serial dilutions (from 10 −1 to 10 −6 ) of skin solutions was spread onto the surface of the agar plates containing Complex Medium I (CMI) supplemented with 0.5% (w/v) yeast extract with 10% final salt concentration (SW10, saline water) consisting of (w/v): 8.1% NaCl, 0.2% KCl, 0.7% MgCl 2 , 0.006% NaHCO 3 , 0.96% MgSO 4 , 0.0026% NaBr and 0.036% CaCl 2 [17]. Yeast extract and all chemicals used in SW10 were from the same company (Merck, Darmstadt, Germany). The pH of the media was adjusted to 7.5 prior to autoclaving. The plates were incubated at 37°C during 24 h. After incubation period, bacterial cultures were selected by their different colony morphologies and re-streaked on CMI agar to obtain pure isolates. Since the selections of isolates were made according to their different colony morphologies, a few different species having similar colony morphologies might have been missed. Then, the phenotypic and genotypic analysis of the pure cultures were performed.

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 H 2 S and N 2 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 NH 3 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).
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. 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.