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Detection of Industrially Potential Enzymes of Moderately Halophilic

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Detection of Industrially Potential Enzymes of Moderately Halophilic Turk J Biochem 2018; 43(3): 312–322 Research Article Pinar Caglayan*, Meral Birbir, Cristina Sánchez-Porro and Antonio Ventosa 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 https://doi.org/10.1515/tjb-2017-0127 Chromohalobacter beijerinckii, Staphylococcus xylosus, Received May 10, 2017; accepted June 13, 2017; previously published Halomonas eurihalina, Staphylococcus equorum subsp. online October 18, 2017 equorum, Halomonas zhanjiangensis, Halomonas venusta Abstract and Chromohalobacter canadensis. Fairly high percentage of isolates produced protease (87%) and catalase (100%). Aim: This study aimed to isolate moderately halophilic While more than 50% of isolates produced lipase (64%), bacteria from salted goat skins, to characterize these micro- β-galactosidase (59%) and oxidase (56%), less than 50% organisms and to determine their industrially important of isolates produced urease (46%), caseinase (28%), enzymes such as amylase, catalase, oxidase, caseinase, cel- amylase (26%), lecithinase (8%) and cellulase (5%). lulase, DNase, lipase, lecithinase, protease, pullulanase, Conclusion: We detected that moderately halophilic bac- urease, phospholipase, xylanase and β-galactosidase. teria on skins produced important enzymes, which may Methods: Enzymes of these bacteria, isolated from skin be used in diverse industrial applications in leather, feed, samples belonging to eight countries and identified using detergent, paper, food, chemical, medical, pharmaceuti- phenotypic and genotypic methods, were examined in cal, textile industries. agar media. Keywords: Moderately halophilic bacteria; Industrially Results: Thirty-nine isolates were fairly similar to important enzymes; Biochemical characteristics; Leather species of genera Staphylococcus, Bacillus, Salinicoc- industry; Molecular identification. cus, Gracilibacillus, Chromohalobacter and Halomonas. Various carbon sources were utilized, and all isolates produced enzyme. Enzyme-producing species were Özet Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus arlettae, Bacillus pumilus, Gracilibacillus Amaç: Bu çalışma, ılımlı halofil bakterileri tuzlanmış keçi dipsosauri, Salinicoccus roseus, Bacillus licheniformis, derilerinden izole etmeyi, bu mikroorganizmaları karak- terize etmeyi ve bunların amilaz, katalaz, oksidaz, kase- *Corresponding author: Pinar Caglayan, Division of Plant Diseases inaz, selülaz, DNaz, lipaz, lesitinaz, proteaz, pullulanaz, and Microbiology, Department of Biology, Faculty of Arts and üreaz, fosfolipaz, ksilinaz ve β-galaktosidaz gibi endüst- Sciences, Marmara University, 34722 Istanbul, Turkey, e-mail: [email protected]. http://orcid.org/0000- riyel olarak önemli enzimlerini saptamayı amaçlamıştır. 0002-0238-1754 Yöntemler: Sekiz ülkeye ait olan deri örneklerinden izole Meral Birbir: Division of Plant Diseases and Microbiology, edilerek fenotipik ve genotipik metodlara göre tanımlanan Department of Biology, Faculty of Arts and Sciences, Marmara bu bakterilerin enzimleri, agar besiyerlerinde incelendi. University, Istanbul, Turkey, e-mail: [email protected] Bulgular: Staphylococcus, Bacillus, Salinicoccus, Gracili- Cristina Sánchez-Porro and Antonio Ventosa: Department of Microbiology and Parasitology, Faculty of Pharmacy, University bacillus, Chromohalobacter ve Halomonas cinslerine ait of Sevilla, Sevilla, Spain, e-mail: [email protected] (C. Sánchez-Porro); 39 izolat bu cinslere ait türlere oldukça benzerdi. Çeşitli [email protected] (A. Ventosa) karbon kaynakları kullanıldı ve izolatların tümü enzim Pinar Caglayan et al.: Detection of industrially potential enzymes of moderately halophilic bacteria 313 üretti. Enzim üreten türler Staphylococcus saprophyticus salinity (optimally 3–15% NaCl), pH, and temperatures [1, subsp. saprophyticus, Staphylococcus arlettae, Bacillus 2, 4–6]. Furthermore, moderate halophiles present oppor- pumilus, Gracilibacillus dipsosauri, Salinicoccus roseus, tunities owing to easily growing on low-cost substrate and Bacillus licheniformis, Chromohalobacter beijerinckii, Sta- low water content [7]. In the previous studies, different phylococcus xylosus, Halomonas eurihalina, Staphylococ- moderately halophilic bacterial genera (Bacillus, Chro- cus equorum subsp. equorum, Halomonas zhanjiangensis, mohalobacter, Halobacillus, Halomonas, Marinococcus, Halomonas venusta ve Chromohalobacter canadensis’di. Oceanobacillus, Salibacillus, Salinicoccus, Salinivibrio İzolatların oldukça yüksek yüzdesi proteaz (87%) ve and Thalassobacillus), isolated from natural saline envi- katalaz (100%) enzimlerini üretti. İzolatların yarısından ronments, produced DNase, lipase, xylanase, amylase, fazlası lipaz (64%), β-galaktosidaz (59%) ve oksidaz inulinase, protease, cellulase, pullulanase, pectinase and (56%) enzimlerini üretirken, yarısından azı üreaz (46%), caseinase [5, 8, 9]. kazeinaz (28%), amilaz (26%), lesitinaz (8%) ve selülaz Moderately halophilic bacteria have been isolated (5%) üretti. from hypersaline environments such as saline lakes, Sonuç: Derilerdeki ılımlı halofil bakterilerin, deri, yem, salterns, solar salt evaporation ponds, saline soils, salt deterjan, kâğıt, gıda, kimya, medikal, ilaç, tekstil gibi mine soil, marine sediments and other saline habitats çeşitli endüstriyel uygulamalarda kullanılabilecek önemli such as salted fish, fermented anchovy sauce, meat and enzimleri ürettiklerini saptadık. hides [10–16]. Although characterization of moderately halophilic bacteria, isolated from different saline environ- Anahtar Kelimeler: Ilımlı halofil bakteriler; Endüstriyel ments, using conventional and molecular techniques and açıdan önemli enzimler; Biyokimyasal karakteristikler; detail experiments on their hydrolytic enzymes have been Deri endüstrisi; Moleküler tanımlama. 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 Introduction examination of their industrial enzymes have not been reported previously. Screening of enzyme producing mod- Enzymatic processes provide many advantages in terms erately halophilic bacteria on goat skins cured with differ- of saving energy, water, raw materials and chemicals ent countries’ salt will be important for determination of that cannot be achieved using conventional chemical industrially potential microorganisms. Hence, the present processes. Due to enzymes’ biodegradable structure, study focused on the screening industrially important mod- obtaining high quality product, low energy consump- erately halophilic bacteria producing amylase, catalase, tion, low-cost material and less environmental pollution, oxidase, caseinase, cellulase, DNase, lipase, lecithinase, enzymes are more preferred as economically and ecologi- protease, pullulanase, urease, phospholipase, xylanase cally alternatives to chemicals in industrial applications and β-galactosidase. In order to characterize moderately [1]. The use of microbial enzymes is widespread in bio- halophilic bacteria isolated from the skins and to under- technological applications as metabolic catalysts for cen- stand their physiological and biochemical characteristics turies. Industrial enzymes have been used in food, feed, for enzyme production, phenotypic characteristics and chemical and pharmaceutical, textile, biofuel, paper and comparative partial 16S rRNA gene sequence analysis of pulp, detergent and leather industries as well as produc- these microorganisms were also examined in this study. tion 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 Materials and methods enzyme market is approximately estimated at 7.1 billion dollars by 2018 [3]. Due to the high demand for industrial Sample collection enzymes, researchers have been focused on finding new industrial microorganisms producing different enzymes Twenty-three salted goat skin samples imported from Aus- that can withstand extreme conditions. Especially moder- tralia, Bulgaria, Israel, South Africa, Russia, China, France, ate halophiles are considered to be potential sources for and four salted goat skin samples preserved in Turkey were industrial enzymes such as cellulases, lipases, xylanases, collected from different tanneries in the Leather Organized proteases, amylases, nucleases, and esterases which are Tannery Region, Tuzla and Corlu, Turkey. The samples able to perform their functions under a wide range of were then placed into sterile prelabeled translucent ziplock 314 Pinar Caglayan et al.: Detection of industrially potential enzymes of moderately halophilic bacteria bags and transported in a sterile and cold container. The 1 min, 50°C for 1 min and 72°C for 2 min, with final 10 min samples were named according to its origin: Australia (AL, extension at 72°C. The PCR products were then checked AVT), Turkey (E, KE, T, YE), Bulgaria (B, BLG, BT), Israel on agarose gel with ethidium bromide staining and puri- (IRL), South Africa (KT, K, NE, GK), Russia (RU, RA), China fied using QIAquick PCR Purification Kit (Qiagen, Hilden, (CCN, CN, CC), France (F, FR, FN, FS) (Table 1). Germany). The 16S rRNA gene sequences were determined by IONTEK Laboratory (Turkey). 16S rRNA gene similari- ties between isolates and closely
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