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Actinobacteria isolated from termite guts as a source of novel oxidative enzymes
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  ORIGINAL PAPER Actinobacteria isolated from termite guts as a sourceof novel oxidative enzymes Marilize Le Roes-Hill  • Jeffrey Rohland  • Stephanie Burton Received: 3 March 2011/Accepted: 20 June 2011/Published online: 1 July 2011   Springer Science+Business Media B.V. 2011 Abstract  Amulti-facetedscreeningprogrammewasdesignedtosearchfortheoxidases,laccase,peroxidaseand tyrosinase. Actinobacteria were selectively iso-lated from the paunch and colonregion of the hindgutsof the higher termite,  Amitermes hastatus .The isolateswere subjected to solid media assays (dye decolouri-zation, melanin production and the utilization of indulin AT as sole carbon source) and liquid mediaassays. Eleven of the 39 strains had the ability todecolourize the dye RBBR, an indicator for theproduction of peroxidases in actinobacteria. Melaninproduction on ISP6 and ISP7 agar plates served as agoodindicatorforlaccaseand/ortyrosinaseproductionand the ability of the strains to grow in the presence of indulin AT as a sole carbon source served as a goodindicator of lignin peroxidase and/or general peroxi-dase production. Enzyme-producing strains were cul-tivated in liquid media and extracellular enzymeactivities measured. Strains with the ability to produceoxidative enzymes under the conditions tested wereidentified to genus level by 16S rRNA gene analysisand compared to known oxidase producers. A strongrelationship was observed between the environmentsampled (termite guts where lignocellulose degrada-tion occurs) and the dominant type of oxidativeenzyme activity detected (laccases and peroxidases),which suggests the possibility of future targetedscreening protocols linking the physical properties of thetargetenzymeswithspecificoperationalconditionsrequired, such as lignocellulosic degradation in thepreparation of biofuel feedstocks. Keywords  Actinobacteria    Higher order termite   Oxidative enzymes Introduction Actinobacteria are frequently exploited because of their diverse metabolic capabilities (Arensko¨tter et al.2004; Bull et al. 2000). These organisms can be found in a wide range of environments, with top-soil beingthe predominant source (Gottlieb 1973). One of theenvironments that have hardly been exploited for theisolation of actinobacteria, is that of the guts of  Electronic supplementary material  The online version of this article (doi:10.1007/s10482-011-9614-x) containssupplementary material, which is available to authorized users.M. Le Roes-Hill ( & )    S. BurtonBiocatalysis and Technical Biology Research Group,Cape Peninsula University of Technology,PO Box 1906, Bellville 7535, South Africae-mail: LeroesM@cput.ac.zaJ. RohlandDepartment of Molecular and Cell Biology, Universityof Cape Town, Private Bag 1, Rondebosch,Cape Town 7701, South Africa Present Address: J. RohlandMax Planck Institute for Terrestrial Microbiology,Karl-von-Frisch-Str. 1, 35043 Marburg, Germany  1 3 Antonie van Leeuwenhoek (2011) 100:589–605DOI 10.1007/s10482-011-9614-x  termites (Watanabe et al. 2003). Termites belong tothe insect order Isoptera and are broadly divided intothe lower and higher termites. The lower termites arespecifically wood-feeding insects, while the highertermites have evolved a more diverse diet and gutmicrobiota: some feed on wood and well-rotted plantmatter, some are exclusively soil-feeders, while othersgrow and feed on cellulolytic fungi (Ohkuma 2003).Pasti and Belli (1985) isolated various strains of actinobacteria (  Micromonospora  spp. and  Streptomy-ces  spp.) from the hindgut of various higher termites(  Macrotermes, Odontotermes, Amitermes  and  Micr-ocerotermes ), while Watanabe et al. (2003) showedthat actinobacteria are also widespread in both higherand lower termites.Over the past 20 years, there has been increasedinterest in the production of certain oxidative enzymesby actinobacteria, especially in the fields of lignindegradation and detoxification of organic pollutantssuch as polycyclic aromatic hydrocarbons (PAHs),organophosphate pesticides and azo dyes (Pasti et al.1990; Torres et al. 2003). While the biological role of  these oxidative enzymes in actinobacteria is largelyunknown, it has been suggested that such enzymesmay have a similar role to that found in fungi, indegrading phenolic compounds to support a sapro-phytic life cycle (Endo et al. 2003). However, someoxidative enzymes from actinobacteria may play arole in morphogenesis or antibiotic production (Endoet al. 2003; Suzuki et al. 2006). As part of the microbiota found in the hindguts of termites, actino-bacteria that produce oxidative enzymes may beinvolved in the degradation of the lignocellulolyticfood ingested by termites, but the extent of theirinvolvement still needs to be determined (Ohkuma2003; Pasti et al. 1990; Varma et al. 1994). Oxidative enzymes are included in the vast groupof enzymes known as the oxidoreductases. Theseenzymes catalyse biological oxidation/reduction reac-tions and play a major role in many chemical andbiochemical transformations (May 1999). The oxida-tive enzymes, laccase, peroxidase and tyrosinase havebeen extensively researched, particularly those of fungal origin. Laccases (EC 1.10.3.2) catalyze theoxidation of various aromatic compounds, specificallyphenols and amino-phenols, while concomitantlyreducing molecular oxygen to water. In the case of blue laccases (predominant type of laccase describedin literature), non-phenolic oxidation is also possible,but is dependent on the co-presence of primarylaccase substrates which act as mediators (yellowlaccases do not require mediators to oxidize non-phenolic compounds; Bourbonnais and Paice 1990;Pozdnyakova et al. 2004; Pozdnyakova et al. 2006). Peroxidases (EC 1.11.1.X) catalyse the oxidation of alarge variety of aromatic and non-aromatic substratesin the presence of hydrogen peroxide (Banci 1997).They are widespread in nature where their majorfunctions range from polymerization reactions inlignin production in plants to protection againstoxidative stress in bacteria (Atack and Kelly 2009;Marjamaa et al .  2009). Tyrosinases (EC 1.18.14.1,also called polyphenol oxidases) are ubiquitous innature. They are essential for the formation of melaninand are involved in wound healing and in primaryimmune responses in plants and invertebrates (Clausand Decker 2006). They typically catalyze two typesof reaction: 1) a cresolase or monophenolase typereaction, which involves the  ortho -hydroxylation of monophenols to  o -diphenols, and 2) a diphenolase orcatecholase type reaction, which involves the sub-sequent oxidation of the  o -diphenols to  o -quinones(Claus and Decker 2006). Phenoxazinone synthases(PHS; EC 1.10.3.4) have recently been identified ashaving the ability to catalyze similar reactions to thoseof laccases and tyrosinases (and can therefore influ-ence any screening results) and are phylogeneticallyclosely related to these two enzyme groups (Le Roes-Hill et al .  2009). PHSs have been shown to beinvolved in the production of the antibiotic grixazonein  Streptomyces griseus  subsp.  griseus  and arepotentially involved in spore pigmentation in  Strep-tomyces antibioticus . The true distribution of theseenzymes in nature is unknown and their unsuspectedpresence may influence screening for other oxidases(Le Roes-Hill et al .  2009).In this study, a focussed screening program wasused in which the target enzymes of interest were theoxidative enzymes laccase, peroxidase, phenoxazi-none synthase and tyrosinase produced by actinobac-teria. Actinobacteria were selectively isolated fromthe hindguts (paunch and colon) of the higher termite,  Amitermes hastatus , and the isolated, pure cultureswere subjected to a multi-faceted screening programto determine the distribution of the target activities inthe isolated strains. The enzyme producers wereidentified to the genus level by 16S rRNA geneanalysis. 590 Antonie van Leeuwenhoek (2011) 100:589–605  1 3  Materials and methods Collection of termites and the isolationof actinobacteriaTwenty specimens of the higher termite,  A. hastatus ,were collected from within a termite mound byJeffrey Rohland from the Tygerberg Nature Reservein Cape Town, South Africa, on 13 March 2006. Theidentity of the termite was confirmed based onmorphology (entomologist Dr Mike Picker, Depart-ment of Zoology, University of Cape Town) and bythe DNA sequencing of the mitochondrial cyto-chrome oxidase II gene (Katja Meuser, Brune Grouptechnical assistant, Max Planck Institute for Terres-trial Microbiology, Marburg, Germany).Twenty termites were surface sterilized in 70%ethanol for 2 min followed by three washes in steriledistilled water (dH 2 O). The posterior paunch andcolon regions (Fig. 1) were dissected from the termiteabdomen and placed in separate reaction vesselscontaining 2 ml sterile phosphate buffer (10 mMNa 2 HPO 4  2H 2 O, 1.8 mM KH 2 PO 4 ; pH 7.0). Thecontents were vigorously vortexed for 5 min before astandard serial dilution series was performed usingsterile phosphate buffer (same as above). 100  l l of each dilution was plated onto four separate media:Medium 3 (M3 agar, g/l: 5.0 carboxymethylcellulose,4.0 yeast extract, 10.0 malt extract, 12.0 bacteriolog-ical agar; Wenzel et al. 2002), Medium II (MII agar,g/l: 1.9 K  2 HPO 4 , 0.94 KH 2 PO 4 , 1.68 NaHCO 3 , 1.6KCl, 1.43 NaCl, 0.15 NH 4 Cl, 0.037 MgSO 4  7H 2 O,0.017 CaCl 2  2H 2 O, 0.1 yeast extract, 0.16 Na 2 SO 4  9H 2 O, 0.32 cysteine, 0.2 ml trace salts solution;Cazemier et al. 2003) containing glucose as solecarbon source (10.0 g/l; MIIG), MII agar containingcarboxymethylcellulose (sodium, salt, low viscosity)as sole carbon source (10.0 g/l; MIIC) and MII agarcontaining oat spelts xylan as sole carbon source(1.0 g/l; MIIX). Each medium was prepared at pH6.0, 6.5, 7.0 and 7.5, and supplemented with 50  l g/ ml cycloheximide and 10  l g/ml nalidixic acid. ThepH range selected follows the same strategy used byPasti and Belli (1985), which was based on previousreports that the pH of the termite gut ranges from pH6.0 to 7.5. All plates were incubated under aerobicconditions at 30  C and were monitored for 28 days.Actinobacteria were selected on the basis of colonymorphology and were transferred onto fresh agarmedia using sterile toothpicks. Strains were main-tained on agar plates at room temperature and asstocks at  - 20  C in 20% glycerol (v/v).Solid media assaysThe triphenylmethane dye, crystal violet (CV;SIGMA)andtheanthraquinonedye,RemazolBrilliantBlue R (RBBR; SIGMA) were used for the determi-nation of oxidative enzyme production in solid media.ISP2agar (International Streptomyces  Projectmedium2, g/l: 4.0 yeast extract, 4.0 glucose, 10.0 malt extract,pH 7.2; Shirling and Gottlieb 1966) was used as thebasalmediumfortheassay.Stocksolutionsofthedyeswere prepared in dH 2 O (2%, w/v) and filter sterilised.The dye was added to the autoclaved media at a finalconcentration of 0.003% (w/v). The test strains werestab-inoculated in duplicate into the agar media andincubated for 21 days at 30  C. The plates weremonitored for the formation of clear zones around thestab-inoculated colonies and compared to un-inocu-lated plates for dye decolourization. Visual distinctionwasmadebetween decolourizationandbio-absorption(uptake of the dye into the cell mass).Actinobacterial strains were stab-inoculated into thefollowing agar media to test for melanin production:ISP6(International Streptomyces Projectmediumnum-ber 6,g/l: 15.0 peptone,5.0proteose peptone,0.5ferricammonium citrate, 1.0 K  2 HPO 4 , 0.08 Na 2 S 2 O 5 , 1.0yeast extract, 15.0 agar, pH 7.0; Shirling and Gottlieb1966) and ISP7 (International  Streptomyces  Projectmediumnumber7,g/l:15.0glycerol,0.5 L -tyrosine,1.0 L -asparagine monohydrate, 0.5 K  2 HPO 4 , 0.5MgSO 4  7H 2 O, 0.5 NaCl, 0.01 FeSO 4  7H 2 O, 1 ml tracesalts, 20.0 agar, pH 7.2; Shirling and Gottlieb 1966).Tracesaltssolutioncontained0.1 gFeSO 4  7H 2 O,0.1 gMnCl 2  4H 2 O and 0.1 g ZnSO 4  7H 2 O in 100 ml dis-tilled water, filter-sterilized.  S. antibioticus  NRRLB-2770 T was used as a positive, reference control. Fig. 1  Schematic representation of the gut morphology of ahigher order termite.  C   crop,  G  gizzard,  M   mid gut, T   malpighian tubules,  mS   mixed segment,  1  proctodealsegment,  2  enteric valve,  3  paunch,  4  colon,  5  rectum (adaptedfrom Varma et al. 1994)Antonie van Leeuwenhoek (2011) 100:589–605 591  1 3  The plates were incubated at 30  C and inspected at 7and 14 days for the production of a dark, black-brownpigment in comparison to un-inoculated agar plates.The ability of the strains to utilize indulin AT(MeadWestvaco) as a sole carbon source was testedby inoculating the strains on International  Strepto-myces  Project medium number 9 agar plates supple-mented with different concentrations of indulin AT[ISP9; g/l: 2.64 (NH 4 ) 2 SO 4 , 2.38 KH 2 PO 4 , 5.65K  2 HPO 4  3H 2 O, 1.0 MgSO 4  7H 2 O, 1 ml trace salts,15.0 agar, pH 6.8–7.0; Shirling and Gottlieb 1966].Trace salts solution contained 0.64 g CuSO 4  5H 2 O,0.11 g FeSO 4  7H 2 O, 0.79 g MnCl 2  4H 2 O, 0.15 gZnSO 4  7H 2 O in 100 ml dH 2 O, filter-sterilized andstored at 3–5  C. Indulin AT is a Kraft pine lignin thatis insoluble in water, but soluble in alkali (in thisstudy the indulin AT was solubilised in 5 M NaOH).It is free of wood sugars and contains the following(wt%): 64.46% C, 5.43% H, 1.01% N, 24.72% O,120 ppm Cl, 1.85% S, 2.43% ash and 3.77%moisture. Trace amounts of various minerals are alsopresent (Beis et al. 2010; Tansey et al. 1977). The ISP9 agar was supplemented with 200, 50, 20, 10 and5 mg/l indulin AT. The test strains were cultivated in10 ml Yeast extract Malt extract (YEME) broth(International  Streptomyces  Project medium number2, ISP2; g/l: 4.0 yeast extract, 4.0 glucose, 10.0 maltextract, pH 7.2; Shirling and Gottlieb 1966) for5 days at 30  C, shaking at 160 rpm on an orbitalshaker. The cells were harvested by centrifugation at10000 rpm for 5 min, washed twice with steriledistilled water and centrifuged as before. This stepwas repeated twice to ensure that any residual mediacomponents have been removed from around thecells. Two 20  l l volumes were spotted at the top of the agar plate and streaked down with a sterileinoculating loop. All plates were incubated at 30  Cfor 21 days and growth compared to growth on ISP9agar plates that had not been supplemented withindulin AT or any other carbon source.Enzyme activityTo test for extracellular enzyme production in liquidmedia, the strains were inoculated into 10 ml broth of the isolation or maintenance medium. Selected strainswere also cultivated in modified phenoxazinoneproduction medium g/l: 10.0 glucose; 10.0 glycerol;10.0 soya flour; 5.0 yeast extract; 5.0 casamino acids;4.0 CaCO 3 ; 1 ml trace salts; pH 7.1; Graf et al .  2007),modified phenoxazinone production medium supple-mented with 0.2 g/l indulin, modified phenoxazinoneproduction medium supplemented with 1 mMCuSO 4 , an indulin medium (g/l: 1.0 yeast extract,20.0 malt extract, 0.2 indulin; pH 7.0) and modifiedstarch-casein-nitrate medium (g/l: 10.0 solublestarch; 0.3 casein; 2.0 potassium nitrate; 0.3 CaCO 3 ;0.05 MgSO 4  7H 2 O; 0.01 FeSO 4  7H 2 O; 0.5 yeastextract; 1 ml trace salts; pH 7.0; Atlas 2004). Thetrace salts solution contained 1.0 g FeSO 4 , 0.9 gZnSO 4  and 0.2 g MnSO 4  in 100 ml dH 2 O, filter-sterilized.  S. antibioticus  NRRL B-2770 T ,  Strepto-myces coelicolor   A3(2) and  S. griseus  subsp.  griseus NRRL B-2165 were included in the screeningprogram (actinobacterial strains that are known toproduce oxidative enzymes).The flasks were incubated at 30  C on a rotaryshaker (160 rpm) for 10 days. 1 ml samples wereremoved after 3, 5, 7 and 10 days of growth andcentrifuged at 10000 rpm for 5 min. The supernatantwas used in laccase, peroxidase and tyrosinaseassays: 50  l l of the supernatant was mixed with150  l l of the buffer/substrate mix (in duplicate) in a96-well microtitre plate and the rate of substrateoxidation monitored on the Anthos Zenyth 1100multimode detector for 10 cycles of 30 s (total of 5 min) each at specific absorbances (see below).Enzyme assaysLaccase activity was determined using the followingsubstrates: 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfo-nate) (ABTS, Roche), 2,6-dimethoxyphenol (2,6-DMP,SIGMA), 3-hydroxyanthranilic acid (3-HAA, SIGMA),guaiacol (SIGMA) and syringaldazine (SIGMA).The following substrate concentrations and bufferswere used to monitor laccase activity: 0.5 mM ABTSin 0.1 M sodium acetate buffer (pH 5.5 and 3.5)monitored at 420 nm ( e  =  36000 M - 1 cm - 1 ) (Eggertet al. 1995); 1 mM 2,6-DMP in 0.2 M sodium phos-phate buffer (pH 6.0 and 3.5) monitored at 468 nm( e  =  14800 M - 1 cm - 1 ) (Solano et al. 2001); 1 mM3-HAA in 0.2 M sodium phosphate buffer (pH 6.0)monitored at 452 nm ( e  =  18000 M - 1 cm - 1 ) (Eggertet al. 1995); 0.5 mM guaiacol in 0.1 M sodiumacetate buffer (pH 5.5) monitored at 470 nm( e  =  26600 M - 1 cm - 1 ) (Johannes and Majcherczyk 2000); and 0.216 mM syringaldazine (prepared in 592 Antonie van Leeuwenhoek (2011) 100:589–605  1 3  absolute methanol), diluted 1:10 in 0.1 M sodiumacetate buffer (pH 5.5) monitored at 530 nm( e  =  65000 M - 1 cm - 1 ) (Ride 1980).Tyrosinase activity was determined using  L -3,4-dihydroxyphenylalanine (L-DOPA; SIGMA) and  p -cresol (SIGMA) as substrates. 10 mM L-DOPA wasprepared in 50 mM sodium phosphate buffer (pH 6.0;Lerch and Ettlinger 1972) and 2.5 mM  p -cresol wasprepared in 50 mM sodium phosphate buffer (pH 7.0;Orenes-Pin˜ero et al. 2005). The oxidation of L-DOPAwasmonitoredat475 nm( e  =  3600 M - 1 cm - 1 ),whilethe oxidation of   p -cresol was monitored at 400 nm( e  =  1433 M - 1 cm - 1 ).For the detection of peroxidase activity, 170 mMphenol (SIGMA) and 2.5 mM 4-aminoantipyrine(SIGMA) were prepared in distilled water. Asolution of 50 mM H 2 O 2  was prepared in 0.1 Mpotassium phosphate buffer (pH 7.0). An equalamount of the two solutions were used in thereaction mix and oxidation of the substrate wasmonitored at 510 nm ( e  =  7100 M - 1 cm - 1 ; Trinder1966). Peroxidase activity was also monitored using2,4-dichlorophenol (2,4-DCP; SIGMA) as a sub-strate. The reaction contained equal volumes of 0.1 M potassium phosphate buffer (pH 7.0), 25 mM2,4-DCP, 16 mM 4-aminoantipyrine, the enzyme-containing sample and 50 mM H 2 O 2 . The oxidationof the substrate was initiated by the addition of theH 2 O 2  and monitored at 510 nm ( e  =  21647M - 1 cm - 1 ) (Antonopoulous et al. 2001; Winteret al. 1991).Manganese peroxidase activity was determinedusing the phenol red manganese-dependent peroxi-dase assay as described by Mercer et al. (1996) andthe guaiacol/MnSO 4  described by Niladevi andPrema (2005). 0.1 mM MnSO 4  and 0.1 mM phenolred were dissolved in 50 mM potassium phosphatebuffer, pH 7.0 and the reaction initiated by theaddition of 50 mM H 2 O 2 . The reaction was stoppedat 1 min intervals with 0.1 M NaOH and the absor-bance read at 610 nm ( e  =  4460 M - 1 cm - 1 ). Equalamounts of 0.1 mM guaiacol and 0.1 mM MnSO 4 were dissolved in 50 mM potassium phosphatebuffer, pH 7.0 and the reaction initiated by theaddition of 50 mM H 2 O 2 . Absorbance was monitoredat 470 nm for 5 min ( e  =  26000 M - 1 cm - 1 ).One unit of enzyme activity was defined as theamount of enzyme required to oxidize 1  l mol of substrate per minute at 22  C (ambient temperature).Laccase from  Trametes pubescens  CBS 696.94,horseradish peroxidase (SIGMA), manganese perox-idase (FLUKA) and tyrosinase from  Agaricus bisp-orus  (SIGMA) were used as positive controls whileliquid growth media and the buffer/substrate mixtureswere used as negative controls.Identification of the enzyme-producingactinobacteria by 16S rRNA gene analysisStrains were inoculated into 10 ml liquid broth andincubated at 30  C, shaking on an orbital shaker(160 rpm) and grown until maximum cell massproduction. The cells were harvested by centrifuga-tion at 10000 rpm for 5 min. The supernatant wasremoved and cell harvesting was repeated untilapproximately 200  l l of cell mass was collected foreach strain. DNA was isolated according to a methodmodified from Wang et al. (1996) where 25 mg/mllysozyme was used instead of 5 mg/ml in thelysozyme buffer, and incubation with RNase A wasallowed to proceed overnight rather than for 30 min.The 16S rRNA gene was amplified by thepolymerase chain reaction (PCR) using the universalbacterial 16S primers F1 and R5 (Cook and Meyers2003). The PCR products were analysed by electro-phoresis on 1% (w/v) agarose gels containing ethi-dium bromide (10  l g/ml). The PCR cyclingconditions were as follow: 95  C for 5 min; 30 cyclesof 95  C for 30 s, 48–56  C for 1 min and 72  C for1 min; and 72  C for 5 min. Amplicons were purifiedusing the MSB Spin PCRapace  PCR purification kit(Invitek). The concentration of the purified DNA wasdetermined using a nanodrop spectrophotometer andsubmitted for sequencing at the sequencing unit,University of Cape Town.For phylogenetic analysis, the 16S rRNA genesequences for known laccase-, peroxidase- and tyros-inase-producers were downloaded from GenBank (http://www.ncbi.nlm.nih.gov). Known enzyme pro-ducing-strains were selected based on publicationslisted in PubMed, gene sequences in Entrez gene andproteinsequencesintheNCBIproteindatabase(searchcriteria used: laccase, phenoxazinone synthase, o -aminophenol oxidase, CotA, CueO, SilA, EpoA;tyrosinase, monophenol monooxygenase, MelC; per-oxidase, catalase/peroxidase, DyP-type peroxidase,lignin peroxidase and manganese peroxidase). Inaddition, the sequences for the 16S rRNA gene F2 Antonie van Leeuwenhoek (2011) 100:589–605 593  1 3
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