Thermophilic and halophilic βagarase from a halop

Authors: Hiroaki Minegishi Yasuhiro Shimane Akinobu Echigo Yukari Ohta Yuji Hatada Masahiro Kamekura Tadashi Maruyama Ron Usami

Publish Date: 2013/08/15

Volume: 17, Issue: 6, Pages: 931-939

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Abstract

An agardegrading archaeon Halococcus sp 197A was isolated from a solar salt sample The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl650 M The molecular mass of the purified enzyme designated as AgaHC was ~55 kDa on both SDSPAGE and gelfiltration chromatography AgaHC released degradation products in the order of neoagarohexose neoagarotetraose and small quantity of neoagarobiose indicating that AgaHC was a βtype agarase AgaHC showed a salt requirement for both stability and activity being active from 03 M NaCl with maximal activity at 35 M NaCl KCl supported similar activities as NaCl up to 35 M and LiCl up to 25 M These monovalent salts could not be substituted by 35 M divalent cations CaCl2 or MgCl2 The optimal pH was 60 AgaHC was thermophilic with optimum temperature of 70 °C AgaHC retained approximately 90  of the initial activity after incubation for 1 hour at 65–80 °C and retained 50  activity after 1 hour at 95 °C In the presence of additional 10 mM CaCl2 approximately 17  remaining activity was detected after 30 min at 100 °C This is the first report on agarase purified from ArchaeaAgar the main cell wall component of red macroalgae is widely used as a gelling agent of microbiological culture media as well as molecular sieving in DNA electrophoresis and gelfiltration chromatography Agar produced commercially from the species of genera Gelidium and Gracilaria is composed of agarose and agaropectin Aoki et al 1990 Rochas et al 1986 Agarose consists of 3 6anhydrolgalactose and dgalactose alternately linked by α1 3 and β1 4 linkages Kloareg and Quatrano 1988 Agaropectin has the same basic disacchariderepeating units as agarase with some hydroxyl groups of 3 6anhydrolgalactose residues replaced by sulfoxy or methoxy and pyruvate residues Hamer et al 1977Agarases are hydrolytic enzymes that degrade agarose into oligosaccharides They were characterized as either αagarase EC 321158 that cleaves α1 3 linkage to produce a series of agarooligosaccharides related to agarobiose Potin et al 1993 or βagarase EC 32181 that cleaves β1 4 linkage to produce neoagarooligosaccharides of series related to neoagarobiose Kirimura et al 1999 Agarosedegrading enzymes have been attracting keen interest in many fields of biochemistry and enzymology Fu and Kim 2010 The neoagarooligosaccharides have various special biological activities such as inhibition of bacterial growth slowing down of starch degradation thereby reducing the calorific value of food and providing anticancer antivirus and antioxidation activities Giordano et al 2006 The βagarases are also used to recover DNA from agarose gel after electrophoresis Finkelstein and Rownd 1978 Burmeister and Lehrach 1989There have been numerous reports on agarase from isolates belonging to genera of the domain Bacteria including Acinetobacter Agarivorans Alteromonas Bacillus Cytophaga Microbulbifer Pseudoalteromonas Pseudomonas Salegentibacter Thalassomonas Vibrio Zobellia etc Hu et al 2009 Fu and Kim 2010 many of which are of marine origin There have been no reports however on agarases from halophilic Bacterial strains requiring high more than 10  NaCl concentrations for growth On the other hand no reports have been published on agarases from any strains of the domain Archaea although glycoside hydrolases such as αamylases PérezPomares et al 2003 Hutcheon et al 2005 chitinase Zhang et al 2010 cyclodextrin glycosyltransferase Bautista et al 2012 βxylanase and βxylosidase Wainø and Ingvorsen 2003 have been identified from halophilic Archaeal strains as well as from hyperthermophilic Archaea Kim and Ishikawa 2010 Rashid et al 2002 Tanaka et al 2001Agardegrading extreme halophiles were screened on agar plates of modified JCM medium No 169 which contained in per liter 2500 g NaCl 075 g casamino acids 10 g yeast extract 03 g trisodium citrate 20 g KCl 200 g MgSO4·7H2O 005 g FeSO4 4H2O 02 g MnSO4 4H2O 200 g Bactoagar Difco pH adjusted to 72 with 40  KOH Approximately 300 salt samples domestic and imported were dissolved in 4 ml of 5  sterile NaCl solution spread on the agar plates and incubated at 37 °C for 2 weeks Two colonies that formed depression or clearing zones on agar were picked up and purified further by the same plating method

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