Turk J Biochem 2017; 42(3): 307–315 Research Article Büşra Abanoz, Sezer Okay and Aslıhan Kurt-Kızıldoğan* Highly active and stable protease production by an extreme halophilic archaeon Haloarcula sp. TG1 isolated from Lake Tuz, Turkey Tuz Gölü, Türkiye’den izole edilen ekstrem halofilik arke Haloarcula sp. TG1’in yüksek derecede aktif ve kararlı proteaz üretimi DOI 10.1515/tjb-2016-0191 The TG1 protease was found to possess very high activity Received October 13, 2016; accepted December 26, 2016; previously and stability over a broad pH and temperature ranges. Its published online March 1, 2017 maximum activity was recorded at pH: 4.0, 50°C and 4 M Abstract NaCl. Among inhibitors tested, dimethyl sulfoxide (DMSO) and ethanol caused the highest decrease (ca. 25%) in its Objective: Isolation of halophilic microorganisms from activity. Çankırı salt mine and Lake Tuz in Turkey to explore ver- Conclusion: Due to the high activity and stability over satile protease producers for industry and characteriza- a wide range of extreme conditions, Haloarcula sp. tion of protease enzyme from the best protease producer TG1 protease reported here is a promising candidate in among the isolated strains. biotechnology. Methods: Extreme halophiles were isolated from salt Keywords: Haloarcula sp. TG1; 16S rRNA; protease activity; samples of Çankırı salt mine and Lake Tuz. Their protease Lake Tuz; salt mine. activities were determined. The isolate with the highest protease activity was characterized. Its protease activ- ity was evaluated in different NaCl concentrations, tem- Özet perature and pH ranges, and in the presence of different inhibitors and metals. Thermostability and pH stability Amaç: Türkiye’deki Çankırı kaya tuzu mağarası ve Tuz were also determined. Gölü’nden halofilik mikroorganizmaların izole edilmesi, Results: The highest protease producer strain was iden- endüstriyel anlamda çok yönlü proteaz üreticilerinin keş- tified as Haloarcula sp. on the basis of 16S rRNA analy- fedilmesi ve izole edilen suşlar arasında en iyi proteaz sis. The isolate namely, Haloarcula sp. TG1, was found to üreticisine ait proteaz enziminin karakterizasyonu. be 99% identical to Haloarcula salaria strain HST01-2R. Metod: Çankırı kaya tuzu mağarası ve Tuz Gölü’nden ekstrem halofiller izole edilmiş ve proteaz aktiviteleri belirlenmiştir. En iyi poteaz aktiviteli izolat karakterize *Corresponding author: Aslıhan Kurt-Kızıldoğan, Enzyme and Microbial Biotechnology Section, Department of Agricultural edilmiştir. Farklı NaCl konsantrasyonları, sıcaklık ve pH Biotechnology, Faculty of Agriculture, Ondokuz Mayıs University, aralığı ile farklı inhibitör ve metallerin varlığında proteaz 55139 Samsun, Turkey, Tel.: 00 90 362 312 1919/1131, aktivitesi değerlendirilmiştir. Ayrıca, enzimin termostabi- Fax: 00 90 362 457 6034, e-mail: [email protected] litesi ve pH stabilitesi tespit edilmiştir. Enzyme and Microbial Biotechnology Section, Büşra Abanoz: Bulgular: En yüksek proteaz üreticisi suş 16S rRNA analizi Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz Mayıs University, 55139 Samsun, Turkey ile Haloarcula sp. olarak tanımlanmıştır. Haloarcula sp. Sezer Okay: Department of Biology, Faculty of Science, Çankırı TG1 suşu Haloarcula salaria HST01-2R’e %99 özdeş bulun- Karatekin University, 18100 Çankırı, Turkey muştur. TG1 proteazının oldukça yüksek ve kararlı enzim 308 Büşra Abanoz et al.: Highly active and stable protease production by an extreme halophilic archaeon aktivitesine sahip olduğu belirlenmiştir. Maksimum akti- extreme conditions. Halophilic proteases are widely used vite pH: 4.0, 50°C ve 4 M NaCl koşullarında elde edilmiş- in washing detergent, food, baking, dairy, tannin and tir. Test edilen inhibitörler içerisinde, aktivitede en fazla leather industries, in aspartame production, in pharma- düşüşe (%25) DMSO ve etanol neden olmuştur. ceutical industry and in the manufacture of soy products Sonuç: Çok geniş aralıklı ekstrem koşullarda yüksek akti- [9, 10]. The characterization and high titer production of vite ve kararlılığından dolayı çalışmada rapor edilen Halo- such kind of novel enzymes are of great biotechnological arcula sp. TG1 proteazı endüstriyel anlamda ümit verici bir importance. Therefore, studies still are going on discov- adaydır. ery of novel halophilic hydrolytic enzymes with optimum activity and stability [7, 10, 11]. Anahtar kelimeler: Haloarcula sp. TG1; 16S rRNA; proteaz Turkey possesses extremely saline environments aktivitesi; Tuz Gölü; tuz mağarası. harboring diverse halophilic microorganisms. Different phylogenetic studies have been conducted to explore this biodiversity [12–17]. However, there is limited study on Introduction characterization of industrially important enyzme produc- tion by haloarchaea isolated from saline environments in Hypersaline environments harbor diverse microbial Turkey [18]. Çankırı salt mine and Lake Tuz are among populations from mostly halophilic organisms belong- the most important sources of commercial salt in Turkey. ing to three domains of life [1]. Halophilic organisms are Çankırı salt mine is an ancient 5000 years old cave and categorized as slight halophiles (optimum growth occurs one of the most important rock salt deposit of the country. at 0.2–0.5 M NaCl), moderately halophiles (0.5–2.5 M On the other hand, Lake Tuz, a second largest lake of NaCl) and extremely halophiles (growth over 2.0 M NaCl Turkey with a geologically tectonic origin, is one of the until to saturation) [2] on the basis of their salt require- greatest salty lakes in the world and is distinguished with ments for growth. Although some extremely halophiles attractive flora and fauna. Therefore, in 2000, the lake and are found in Eubacteria domain, major representatives of its surrounding region was announced as Special Environ- this group are the members of domain Archaea [3]. There- ment Protection Area by UNESCO [19]. It has a salt ratio of fore, archaeal species are the subject of many researches 32.4% [20]. In this study, we aimed at isolation and iden- because of capability for living in extreme environments tification of halophilic microorganisms from Çankırı salt with high salt concentrations. Haloarchaea thrive in mine and Lake Tuz in Turkey to explore versatile protease hypersaline environments such as salt lakes or salt rock producers in industrial manner. Next, activity and stabil- deposits having in excess of 2 M NaCl, and the cellular ity of the protease enzyme from Haloarcula sp. TG1, the integrity depends on high molar salt ratio. In general, best protease producer among the isolated strains, were the haloarchaea are named as extremophiles because of determined for the first time. being very well adapted to live even in saturated amounts of sodium chloride. There are 48 different genera of halo- archaea containing 177 species and studies to explore new species are ongoing [4]. Up to now, complete genomes of Materials and methods 10 haloarchaea have been published (http://halo4.umbi. umd.edu). Among them, those of Haloarcula marismor- Sampling site and isolation of halophilic tui and Haloarcula hispanica were released in 2004 [5] microorganisms and 2011 [6], respectively. The survival of haloarchaea under harsh conditions allow them to produce a vast The salt samples were obtained from Çankırı salt mine repertiore of metabolites. Hydrolytic enzymes such as (40°32′39.1″N 33°45′34.7″E) and Lake Tuz (39°04′13.1″N proteases, lipases, cellulases and amylases produced by 33°24′29.5″E) in Turkey. The Çankırı and Lake Tuz Basins haloarchaea are among the invaluable candidates for are located in northern part of central plateu and in the industrial approaches as because they are more stable dry central plateau of Turkey, respectively. The mineral and functional in extreme conditions such as high tem- composition of Lake Tuz is consisted of mainly Na and perature, NaCl and pH [7, 8]. Proteases are one of the Cl, with few quantities of SO4, Mg, Ca and K [20]. The most important hydrolytic enzymes that exist in the first halophilic microorganisms were isolated using sligthly rank of worldwide enzyme market. The protease enzyme modified procedure described by Nagaoka et al. [21] and exerts its effect on protein degradation. The proteases pro- Enache et al. [1]. One gram of samples were inoculated duced by extremophile haloarchaea in particular, attract into 10 mL of Sehgal–Gibbons (SG) medium in the pres- researchers’ attention due to its broad activity range in ence of penicillin (6 μg/mL). All cultures were incubated Büşra Abanoz et al.: Highly active and stable protease production by an extreme halophilic archaeon 309 at 37°C for 2 weeks in an orbital shaker with 200 rpm. and Kimura two-parameter corrections [28]. 16S rRNA The liquid cultures were then used to inoculate SG agar sequence of Halobacillus mangrovi strain MS10 (accession plates in order to obtain pure cultures of halophiles, number DQ888316.1) was used as the outgroup. and were incubated at 37°C for 1 week. SG medium includes the following components in g/L: NaCl, 250; Cultural and phenotypic characterization MgSO ·7H O, 20; KCl, 2; sodium citrate (trisodium salt), 4 2 of Haloarcula sp. TG1 3; casamino acids, 7.5; yeast extract, 1; FeSO4·7H2O, 0.0023; agar, 15 g, pH: 7.55 [22]. Pure culture of extreme halophile Haloarcula sp. TG1 was observed under microscope and its gram staining prop- erty was determined. Colony morphology, color and shape Protease activity of isolated strains were observed by growing on SG agar plate. The salt range that the isolate needs for growth was defined adding A slightly modified procedure of Pathak and Sardar [23] 1–5.5 M NaCl to the SG medium and incubation at 37°C was used to determine protease activity of the isolates. for 1 week and their growth were monitored spectropho- One week cultures (OD ~ 1.9) were centrifuged and 600 tometrically at OD . In addition, different pH (5.0–10.0) the supernatants were used for the assay. One percent 600 and temperature (20, 28, 37 and 50°C) requirements of the of casein dissolved in 50 mM phosphate buffer (pH 7.5) isolate were also tested.