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Journal of Chemical Society Arabia Saudita (2015)

19, 12 22
Universidad Rey Saud
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ORIGINALARTICLE
Biochemicalstudiesonantibioticproductionfrom
Streptomyces SP.:Taxonomy,fermentacin,
isolationandbiologicalproperties
HoussamM.Atta *
BotÆnica y Departamento de Microbiologa, Facultad de Ciencias (Boys), Universidad Al-Azhar, el Cairo, Egipto
Recibido el 27 de noviembre de 2011; aceptado el 09 de diciembre de 2011

Disponible en lnea 16 de diciembre de 2011
PALABRASCLAVE Resumen Tunicamicinaesunantibiticodenucletidoquefueaisladodelcaldodefermentacindecepano.

Antimicrobianoantibitico; unStreptomyces T-4.Deacuerdoconlascaractersticasmorfolgicas,culturales,fisiolgicaybioqumica-
Streptomyces SP.; caractersticas de iCal y 16S rDNA anÆlisis, cepa T-4 fue identican como ulosus de la Streptomyces tor-
16S rDNA; secuencia
. Es activo in vitro contra algunos microbios patgenos viz: Estafilococo Æureo , NCTC 7447;
Propiedadesbiolgicas Micrococcus lutea, ATCC 9341; Bacilo subtilis , NCTC 10400; B. pumilus, NCTC; Klebsiella pneu-
Monia, NCIMB 9111; Escherichia coli, NCTC 10416; Pseudomonas aeruginosa, ATCC 10145; SAC-
charomyces cerevisiae ATCC 9763; Candida albicans, IMRU 3669; Aspergillus ffavus, IMI 111023;
Aspergillus niger IMI 31276; Aspergillus fumigatus ATCC 16424; Fusarium oxysporum; Rhizoctonia
Los medios
solani; de produccin
Alternaria alternata; estaban
Botrytis n butanol
fabae y Penicillium
(1:1, v/v) achrysogenium
pH 7,0. El . anÆlisis estructural qumico con
conrmed
optimizadoanÆpara
lisis espectral UV, IR yde
mÆxima produccin MS que el compuesto
metabolitos producido
secundarios. por
Los metabolitos se obtuvieron usando -
Streptomyces torulosus, T-4 es Tunicamicina antibitico.

ª 2011 produccin y hosting por Elsevier B.V. en nombre de la Universidad Rey Saud.
Acceso abierto bajo licencia CC BY-NC-ND.
1Introduccin y el tipo GØnero de el familia Streptomycetaceae

(Kampfer, 2006). MÆs de 500 especies de Streptomyces He estado
Actinomicetos han proporcionadoimportante bioactivos descrito por Euzeby (2008). Como con los otro Actinobacteria,
compuestos de comerciales alto valoran y continœan siendo rou- Streptomycetes Gram-positivas y han genomas con
revisados
proyectadoparaNuevobioactivos sustancias (Olano et al., alto GC-contenido (Madigan y Martinko, 2005). Streptomy-
2009). Streptomyces es el mÆsgrandeGØnero de Actinobacteria CESSP son ampliamente reconocidos como industrialmente importante
rgano-para
SGSI. sus capacidad Paraelaborar diferentes clases de novela
* Presente Direccin: Biotecnologa Departamento, Facultad de Ciencia metabolitos secundarios (Bibb, 2005). Tunicamycins son
nucle-otide
antibiticos producido por varios Streptomyces especies.
y la educacin, rama de Al-Khurmah, Universidad de Taif, Arabia Saudita.
Direccindecorreoelectrnico:houssamatta@yahoo.com. Ellos son potente inhibidoresde dela el UDP-GlcNAc:polyprenol
RevisinbajoresponsabilidaddelaUniversidadReySaud. familia translocasa de fosfato GlcNAc-1-P y son a menudo utilizados
Parabloque protena N-glicosilacin. El estructuras son altamente
inusual pero bueno caracterizado (Tamura, 1982; Tkacz, 1983;
Eckardt, 1983) y se componen de uracilo, mina N-acetylglucosa-
Producción y hosting por Elsevier (GlcNAc), unœnico 11-carbono 2-aminodialdose azœcar
http://DX.Doi.org10.1016/j.JSCs.2011.12.011
1319-6103 ª 2011 produccin y hosting por Elsevier B.V. en nombre de la Universidad Rey Saud. Acceso abierto bajo licencia CC BY-NC-ND.
Estudios bioqumicos sobre la produccin de antibiticos Streptomyces 13
llamadotunicamine, y un Amida-ligado graso cÆ ido. El AB - Reino de ArabiaSaudita

Arabia, y inoculado en unalmidn
1.1 0- glicosdico acoplamiento entre tunicamine y el GlcNAc nitrato agar. Placas fueronseincubaron en 35 C parasiete das.
el sustituto es tambiØn n œ ico a la familia Tunicamicina de com- El aislamientos fueronindividualmentemantenido en almidnnitrato
libras. Tunicamicina estructural variantes seproducen que difieren agar en 4 C y se almacena como una mezcla de hifas y esporas en
Slo en el naturaleza de el N-vinculadas acil cadena. Nos han re- 20% de glicerol en 80 C la cepa seleccionada se le permiti crecer
recientemente introdujo un sistema de nomenclatura basada en la estructura en un
iden-caldo nitrato de almidn en un propsito de obtener una clara sobrenadante
ties cada Tunicamicina por su firma graso cÆ ido, esdecir
Tun actividad antimicrobiana.
13:1tun 18:1 (Tsvetanova y Precio, 2001). Aunque un
#Pages [11]
gran Deal es conocidoacercadeTunicamicina estructura y FUNC- 2.2. los organismos de prueba
Tion, No anterior AnÆlisis de Tunicamicina biosntesisde ha
se ha informado. La clave para entender la biosntesis de 2.2.1. bacterias
Tunicamicinaes el origen de el 11-carbono tunicamine accesotelefnico
dosis azœcar y el cinØtica parael formacin de el, b-100 , I. Gram-positivas bacterias: Estafilococo aureus, NCTC
11 0- glicosdico bono. A grandenœmero de natural productos de 7447; Bacilo subtilis , NCTC 1040; B. pumilus, NCTC
Streptomyces el origen es sintetizado De 2-carbn unidades Via 8214 y Micrococcus luteus, ATCC 9341.
una secuencia de reaccin tipo polyketide (Khosla, 2000). Sin embargo,
#Pages [10]
los demÆs azœcares de cadena larga como los cÆ idos siÆlicos,
II. Bacterias Gram-negativas: Escherichia coli, NCTC 10416;
Klebsiella neumona , NCIMB 9111 y Pseudomonas
ketodeoxyoctulo-sonate
(KDO) y ketodeoxyheptulosonate son sintetizado aeruginosa, ATCC 10145.
De Condensacin aldlica de baja azœcares con phosphoenol-
piruvato (PEP) (Subramaniam et al., 1998). AdemÆs, la
#Pages [11]
biosntesisde de similares nuclesido antibiticos, Polyoxins y
2.2.2. hongos
nikkomycins, seproduce por ligadura de PEP y Uridina-5-alde- i. Unicelulares hongos:Candida albicans, IMRU 3669 y
Hyde, generando octofuranuloseuronic 8-carbono cÆ ido nucleo- Saccharomyces cervisiae, ATCC 9763.
lado como un intermedio (Isono y Suhadolnik, 1976; Isono II.
#Pages [10]
et al., 1978; Schuz et al., 1992). Aqu, radiolabeling metablico
Hongos filamentosos:Aspergillus niger, IMI 31276; A. ffavus,
#Pages [10]
#Pages [10] IMI111023; A. fumigatous, ATCC 16424; Fusarium oxyspo-
#Pages
experimentos [10]
e incorporaciones de istopos estables han sido Ron; Rhizoctonia solani; Alternaria alternativo; Botrytis fabae
ap-IMPLICITAS
Para desenredarel metablico origen de el 11-carbono accesotelefnico y Penicillium chrysogenium.
14
dosis azœcar, tunicamine. El X2N C] Uridina y [1-14C]
la glucosamina son incorporado Tunicamicina por efciently 2.3. deteccin de actividad antimicrobiana
descansandocØlulas de Streptomyces chartreusis y eso el [1-14C]
glucosamina sealimenta en ambosel 11-carbono tunicamine y La actividad antimicrobiana se determin por el mØtodo de copa
la adjunta n u -100 -GlcNAc residuo. Istopos estables incorpora- como-dicesegœnKavanagh(1972).
#Pages [10]
nes utilizando2h- o 13Glucosa C-etiquetados y competitivo meta-
BOLIC experimentos fueronmonitoreadaspor LC-ESI-CID-MS y 2.4. taxonmicos estudios de Actinomiceto aislar
RMN (H-1, C-13, y HSQC) espectroscopia. El isotpica
Etiquetadopatronesdefueronconstante con carbonocarbono bonos Morfolgicas caractersticas de el mayorapotente producir
formacin entre un precursor de 5 carbonos derivado de Uridina cepa cultivada en medio de agar nitrato de almidn a 35 T-4 C para
y una hexosa 6-carbono intermedia, este lœ timo probablemente 5 das fue examinado bajo exploracin Electron Microscopa
derivadoDe UDP-GlcNAc. Heteronuclear C-13/H-1 RMN (JEOL Technics Ltd.).
13
correlaciones mostraron una incorporacin de la igualdad C la etiqueta de
[1-13C] glucosa en ambosel b-110 y n u -100 anomØrico auto- 2.5. fisiolgicas y bioqumicas caractersticas
bons, indicando que ambossepresentan De uncomœn precursor
piscina. Porlotanto,ambos el La capacidad de la cepa para producir diversas enzimas fue
pseudo-aminogalactopyranosyl
examen-ined mediante el uso de mØtodos estÆndar. Lecitinasa se
(pseudoGalN) anillosde tunicamine y el n u -100 -vinculadas
GLC-
llevacaboenmedio
yemadehuevo segœn Parael mØtodo de Nitsh y
LA! residuo son inicialmente derivadosDe el azœcar nucletido
Kutzner (1969); lipasa (Elwan et al., 1977); proteasa (Chapman,
#Pages [11]
UDP-GlcNAc. Basadoen en el resultados #Pages
de Estos experimentos un [11] #Pages [10]
#Pages
camino biosintØtico es propuesto para Tunicamicina por primera [11] segœn
1952); pectinasa Parael mØtodo de Hankin et Access.
#Pages [10]
tiempo (Schuz et al., 1992). (1971); n u -amilasa segœn el mØtodo de Cowan (1974)
#Pages #Pages [10]
[10]
#Pages [11] y catalasa prueba segœn Parael mØtodo de #Pages [10]
Jones (1949).
En el presente trabajo se describe el aislamiento de un suelo #Pages [10]
Pigmento de la melanina segœn el mØtodo de Pridham
actino, con alto potencial los mycete cepa de la ciudad de #Pages
#Pageset [10]Access.
[11]
Taif, KSA (1957). Degradacin de esculina y xantina segœn el mØtodo de
de actividad antimicrobiana. La identificacin de esta cepa, #Pages [11]
basado en la oferta cultural, morfologa, fisiologa y Gordonet otros(1974).Reduccindelnitrato
#Pages [10]
#Pages [10]
bioqumica char-ateristics, as como 16s rDNA anÆlisis, segœnelmØtodo deGordon
produccindehidrgeno
tambiØn se ha informado. La sustancia bioactiva primaria fue #Pages #Pages
(1966).
asulde[10]
[10]deoxidasay
Prueba
segœnelmØtododeCowan
aislada,determinaronpuriedysusactividadesbio-lgica. (1974).Elde #Pages [10] #Pages [10] fue
utilizacin diferentes carbono y #Pages
nitrgeno [10]
fuentes
segœn los mØtodos de Pridham y Gottlieb (1948). Celular
#Pages [11]#Pages [11]
2Materialsandmethods paredserealizporelmØtododeBeckeretal.,(1964) #Pages
#Pages [10][11] [10]
yLechevalier
#Pages
#Pages [11] y #Pages [10]
Lechevalier (1970). Cultural caractersticas
#Pages [11]
2.1. cepa actinomycete #PagesaØreo,
como el color del micelio [11]color del micelio sustrato y la
pigmentacin de los Actinomiceto seleccionado fueron grabadas
ISP
en Agar medio (Shirling y Gottlieb, 1966). Colores
Cepa T-4 fue aislado De unsuspensin de unsuelomuestra
se evaluaron las caractersticas en la
#Pages
escala
[11] por Kenneth
desarrollada
(Williams y Davies, 1965) recogidos De Taif Ciudad,
#Pages [11]
14 H.M. Atta
y Deane (1955). AdemÆs, la sensibilidad de las cepas a los 2.9.2. extraccin

#Pages [10]
#Pages [10] fue determinado por papel disco mØtodo
antibiticos
diferentes
(Capuchino y Sherman, 2004). Las cultura ltrates se extrajeron dos veces con n-Butanol y los
#Pages [10] extractos solventes combinados fueron se evapor a sequedad bajo
vacopararendirunresiduodecrudo.
2.6. manipulacin y extraccin de ADN
La cepa actinomycete localmente aislada fue cultivada por 5 das 2.9.3. precipitacin
en un almidn agar inclinado a 35C. Seinocularondosmililitrosde
unaesporasus-Pensinenelcaldoalmidnnitratoyincu- El proceso de precipitacin del compuesto crudo se llev
ansiedad durante 3 das en una incubadora del agitador a 200 rpm C
y 30T O utilizando tØ er de petrleo (b.p. 60 80 C) seguido por 11bag-
formar una bolita de cØlulas vegetativas (pre esporulacin). La tamiento a 5000 rpm durante 15 minutos.
prepara-cin de ADN genmico total se realiz segœn los
mØtodosdescritosporSambrooketal(1989).
2.9.4. Purication por el TLC
#Pages [11]#Pages [11]
2.7. Amplication y la secuencia del gen 16S rDNA Separacin de los compuestos antimicrobianos en sus componentes
individuales fue llevacabo por capadelgadacromatografa
Amplication PCR del gen 16S rDNA del local actino-los mycete usando cloroformo y metanol (24:1, v/v) como un sistema de solventes.
cepa fue llevacabo usando dos Imprimaciones,StrepF;
50- ACGTGTGCAGCCCAAGACA-3 0 y estreptococos R; 0
5 - ACAA 2.10. Purication por cromatografa en columna
GCCCTGGAAACGGGGT-3,0 en acuerdo con el Me
dto descrito por Edwards et Access.(1989). La polimerizacin
mezclaen cadena
El purication de los antimicrobianos compuesto fue llevado
#Pages [10] #Pages [10]
#Pages
consisti en 30 pmol de cada iniciador, 100 ng[10]
de cromosmica · 50) cromatografa de, chloro-
utilizando columna de gel de slice (2.5
ADN, 200 lDNTPs M y 2.5 unidades de Taq polimerasa,en forma y metanol 10:2 (v/v) fue utilizado como un solvente de elucin. El
50 ll de tampn de polimerasa. Amplication se llev a cabo para columna fue izquierda
durantelanoche
hasta el slice gel (Prolabo)
30 ciclos de 1 min a 94 C, 1 min de recocido en 53 C, y fue completamente seestablecieron.
Uno mililitro de crudo Extractode Paraser
2 min de extensin en 72 C. La mezcla de reaccin PCR fue fraccionado fue agregado en la superficie de la columna de gel de slice y
Luego analizaron mediante gel de agarosa electro-FØresis y el el extracto fue adsorbido en la parte superior del gel de silicona. 50
permanecer-ing
mezcla fue puried usandoQIA rÆpida POLIMERIZACIN purication ENfracciones
CADENA fueron recogidos (cada uno de 5 ml) y probados para su
reactivos (Qiagen, USA). Gen 16S rDNA fue secuenciado en actividadantimicrobiana.
ambosfilamentosVia el dideoxy cadenaterminacin mØtodo,
comodescrito por Sanger et Access. (1977). El 16S rDNA Gene 2.11. caractersticas fisicoqumicas
#Pages [11]#Pages
secuencia (1,5 kb) de la PCR#Pages
[11]
se adquiri[11]
el producto usando un
Terminator Ciclo Secuenciacin Kit (ABI Prisma 310 GenØtica 2.11.1. elemental anÆlisis
Analizador, Applied Biosystems, USA). El anÆlisis elemental de C, H, O, N y S se llev a cabo en el centro
microanalticos,UniversidaddeelCairo,Egipto.
2.8. secuencia similitudes y anÆlisis filogenØtico
2.11.2. anÆlisis espectroscpico
El EXPLOSINprograma (www.ncbi.nlm.nih.gov/blst) fue El IR, UV y espectros de masas se determinaron en el micro centro
empleado en orden Para evaluar el grado de ADN similitud. analticodelaUniversidaddeelCairo,Egipto.
Mœltiples secuencia alineacin y molecular filogenia fueron
evaluado usando BioEdit software (Hall, 1999). El
filogenØtica rÆ bol fue muestra usando el R
#Pages [10]
` BOL VISTA
2.11.3. actividad biolgica
La concentracin mnima inhibitoria (CMI) ha sido disuadir -
programa. minadaporelanÆlisisdelmØtododetaza(Kavanagh,1972).
#Pages [10]
2.9. factores efectuando en la biosntesis del agente
antimicrobiano 2.11.4. Caracterizacin del agente antimicrobiano de
la antibitico producido por Streptomyces torulosus, T-4 fue
stosincluyerontamaæodelinculo,perododeincubacin,los identican segœn los recomendados internacionales se
refieren-Casde(Umezawa,1977;Berdy,1974,1980a,b,c).
valores de pH, temperaturas de incubacin; diferente carbono y
fuentes de nitrgeno han sido determinadas por los mØtodos
#Pages [11]
estÆndar.
3resultados
2.9.1. fermentacin
3.1. deteccin de la actividad antimicrobiana
El Streptomyces torulosus, T-4 inculo fue introducido
vacan en cada estØril asep-ffask quecontiene el siguientes Noventaysieteactinomycetecepasfueronaisladasdemuestrasdesuelo
ingredientes (g/l): glucosa, 20; KNO,3 2.0; HPO 2 K,4 0.8; fty recogidos De el Taif Ciudad,Reino de ArabiaSaudita
MgSO4Æ7H2O, 0.7 y KCl, 0,5. Se ajust el pH a 7.0 ser- Arabia.LaculturaslounActinomicetoT-4fueencontradaexhiblimitada
esterilizacin Fore. DespuØs de 5 das de incubacin a 35 C. Filtracin Paraproducir ampliaespectro de antimicrobiano actividades
fue llevado a cabo a travØs de algodn y seguido por 11bag - (Gram-positivas y Gram-negativas bacterias y unicelulares
tamiento a 5000 rpm durante 15 minutos. y los hongos lamentous) (tabla 1).
#Pages [4]
Biochemical studies on antibiotic production from Streptomyces 15
chrysogenum
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
25.0
23.0
23.0
19.0
25.0
P.
oxysporum
Fusarium
0.0
0.0
0.0
0.0
0.0
0.0
0.0
27.0
25.0
25.0
21.0
26.0
–
111023
flavus,
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
29.0
26.0
26.0
23.0
28.0
IMI
A.
fumigatus
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
28.0
27.0
27.0
22.0
27.0
A.
A. niger,
31276
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
30.0
30.0
30.0
25.0
30.0
IMI
Plate1 Scanning electron micrograph of the actinomycete

isolate T-4 growing on starch–nitrate agar medium showing spore
S. cervicea
chain spiral shape and spore surfaces warty (25,000·).

ATCC
9763
0.0
0.0
0.0
0.0
0.0
0.0
0.0
23.0
18.0
15.0
19.0
20.0
–
albicans,
Candida
IMRU
Fungi
3.2. Identification of the actinomycete isolate: morphological

3669
characteristics
0.0
0.0
0.0
22
18
15
18
19
–
–
–
Pseudomonas
The vegetative mycelia grew abundantly on both synthetic

aeruginosa,
and complex media. The aerial mycelia grew abundantly

Table 1 Antimicrobial potentialities of the antibiotic-producing microorganisms isolated from
ATCC
10145
on starch–nitrate agar medium and oatmeal agar medium

20.0
12.0
16.0
16.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
(ISP-3). The spore chains were spiral, and had a warty sur-
face ( Plate 1 ). Neither both sclerotic granules and sporangia nor
pneumonia,
flagellated spores were observed.

Klebsiella
NCIMB
9111
0.0
0.0
0.0
0.0
0.0
0.0
0.0
21.0
15.0
19.0
17.0
18.0
15.0
3.3. Cell wall hydrolysate

NCTC
E. coli
10416
The cell wall hydrolysate contains LL-diaminopimelic acid

22.0
17.0
20.0
22.0
21.0
18.0
13.0
0.0
0.0
0.0
0.0
0.0
–
(LL-DAP) and sugar pattern not detected.

Micrococcus
3.4. Physiological and biochemical characteristics

ATCC
luteus,
Mean values of inhibition zones (in mm) against
9341
0.0
0.0
22.0
25.5
18.0
22.0
22.0
20.0
27.0
20.0
17.0
12.0
The actinomycete isolate T-4 could hydrolyze starch, pro-

–
tein, and cellulose, whereas lipid, pectin, lecithin and cata-

NCTC 8214
lase are negative. Melanin pigment is positive, degradation

of xanthine, esculine, production of H2S, nitrate reduction,
pumilus,
Bacillus
decomposition of urea and utilization of citrate and KCN

22.5
23.0
18.0
12.0
20.0
21.0
20.0
29.5
21.0
17.0
12.0
14.0
0.0
are positive. The isolate under study utilizes D-xylose, D-

various localities in Taif governorate.
mannose, D-glucose, D-fructose, D-galactose, mannitol,

Bacillus
subtilis,
NCTC
meso-inositol, sucrose, rhamnose, L-arabinose, raffinose,

1040
0.0
22.0
24.0
18.0
12.0
20.0
22.0
21.0
30.0
21.0
17.0
13.0
14.0
starch and trehalose, but do not utilize lactose, maltose,

and ribose. Good growth on L-glycine, L-asparagines, L-leu-
Staphylococcus
cine L-histidine, L-phenyl alanine and L-lysine. No growth on

NCTC 7447
L-valine, and L-methionine. On the other hand, the isolate T-

Bacteria
4 decreased at high NaCl concentration above (5% w/v).

aureus,
14. 0
The growth is not inhibited in the presence of phenol and

24.0
25.0
20.0
13.0
21.0
19.0
23.0
30.0
20.0
18.0
15.0
0.0
45 C. The actinomycete isolate T-4 is not sensitive to Ampi-

cillin (25 lg/ml), Nalidixic acid l g/ml), Cefoperazone
Organism
(75 lg/ml), and Fusidic acid (10 l(3

number
Gentamicin (10 lg/

0g/ml),
T-16
T-18
T-19
T-27
T-30
T-32
T-35
T-72
T-88
ml) and Kanamycin (30 lg/ml) (Table 2).

T-4
T-5
T-6
T-7
16 H.M. Atta
Table2 The morphological, physiological and biochemical characteristics of the actinomycete isolate T-4.
Characteristic Result Characteristic Result
Morphological characteristics Mannitol ++
Spore chains Spiral L-Arabinose +
Spore mass Gray meso-Insitol ++
Spore surface Warty Lactose
Color of substrate mycelium Light brown–deep brown Maltose
Diffusible pigment Yellowish brown Trehalose ++
Motility Non-motile D-Ribose
Cell wall hydrolysate D-Fructose ++
Diaminopimelic acid (DAP) LL-DAP Utilization of amino acids
Sugar pattern Not detected L-Glycine +
Physiological and biochemical properties L-Leucine +
Hydrolysis of L-Histidine +
Starch + L-Phenylalanine +
Protein + L-Asparagine +
Lipid L-Methionine
Pectin and lecithin L-Lysine +
Cellulose + L-Valine
Catalase test Growth with (% w/v)
Production of melanin pigment on Sodium azide (0.01)
Peptone yeast- extract iron agar + Phenol (0.1) +
Tyrosine agar medium + Thallous acetate (0.001)
Tryptone – yeast extract broth Growth at different temperatures (C)
Degradation of 10
Xanthin + 30–45 ++
Esculin + 50 ±
H2S Production + 55
Nitrate reduction + Growth at different pH values
Citrate utilization + 6–8 +
Urea test + 9
KCN test + Growth at different concentration of NaCl (%) 15
Utilization of carbon sources +
D-Xylose + 7
D-Mannose + Resistance to
D-Glucose +++ Ampicillin (25 lg/ml) and +
D-Galactose + Nalidixic acid (30 lg/ml) +
Sucrose ++ Cefoperazone (75 lg/ml) +
L-Rhamnose ++ Gentamicin (10 lg/ml) +
Raffinose ++ Kanamycin (30 lg/ml) +
Starch +++ Fusidic acid (10 lg/ml) +
+ = positive , = negative , ± = doubtful results, , ++ = moderate growth and +++ = good growth.
3.5. Color and culture characteristics 3.6.1. Amplification of the 16S rDNA gene
The16SrDNAgenewasamplifiedbypolymerasechainreac-tion
The isolate T-4 shows that the aerial mycelium is light gray; sub- (PCR)usingtheuniversalprimers.Theprimersthatwasusedto16S
strate mycelium is light brown, and the diffusible pigment is rDNAsequencingwere16F357ofthesequence5
moderateyellowishbrownornotproduceddiffusible(Table3). -ACGTGTGCAGCCCAAGACA-3
strepF; 0 0
and strpR; 50 -
0
ACAAGCCCTGGAAACGGGGT-3 , the product of the
PCR was analyzed on 1.5% ethidium bromide gel.
3.6. Taxonomy of actinomycete isolate, T-4
3.6.2. Molecular phylogeny of the selected isolate
This was performed basically according to the recommended
InternationalKey’sviz.Buchananand The 16S rDNA sequence of the local isolate was compared to the
Gibbons (1974) and sequencesof Streptomyces spp.Inordertodeterminetherelat-
Hensyl
spe- data
(1994)
and in
andview
numerical
of the comparative
taxonomy of
study
cies of the
Streptomyces
recorded proper- tiesthe basis of the previously collected edness of the local isolate to these Streptomyces strains.Thephy-
program. On
logenetictree(asdisplayedbytheTreeViewprogram)revealedthat
thelocallyisolatedstrainiscloselyrelatedto Streptomyces
of T-4 in relation to the most closest reference strain,
sp., rather related to Streptomyces sp., rather than to Streptomy-
viz. Streptomyces torulosus,itcouldbestatedthatactinomy-of
ces torulosus ( Fig. 1 ). Multiple sequence alignment was con-
cetes isolate, T-4 is suggestive being likely belonging to
ducted by the sequences of the 16S rDNA gene of
Streptomyces torulosus, T-4 ( Table 4 ).
Streptomyces torulosus. Computer assisted DNA searches
Table3 Culture characteristics of the actinomycete isolate T-4.

Medium Growth Aerial mycelium Substrate Diffusible pigments
mycelium
1. Starch–nitrate agar medium Good L.gray264 – light 57-l.br – light 77-m.ybr – moderate
gray brown yellowish brown
2. Tryptone yeast extract broth No – – –
(ISP-1) growth
3. Yeast extract malt extract agar No – – –
medium (ISP-2) growth
4. Oatmeal agar medium (ISP-3) Good L.gray264 – light 57-l.br – light –
gray brown
5. Glycero asparagine agar Poor L.gray264 – light 57-l.br – light –
medium (ISP-4) gray brown
6. Inorganic salts starch agar Moderate L.gray264 – light 86-l. yellow – light yellow
medium (ISP-5) gray
–
7. Peptone yeast extract–iron Moderate L.gray264 – light 57-l.br – light brown
agar medium (ISP-6) gray
59-d.br – deep brown
8. Tyrosine agar medium (ISP-7) Moderate L.gray264 – light 57-l.br – light brown
gray
59-d.br – deep brown
The color of the organism under investigation was consulted with the ISCC-NBS color – name charts illustrated with centroid color.
Table4 A comparative study of the characteristic properties 27.0, 25.0 and 23.0 in case of A. niger, IMI 31276; Staph-
of T-4 in relation to reference strain, Streptomyces torulosus ylococcus aureus, NCTC 7447, Bacillus subtilis, NCTC
( Williams et al., 1989 , p. 2448 and Table 29-12). 1040, C. albicans, IMRU 3669 and Klepseilla pneumonia,
NCIMB, 9111, respectively, at an inoculum size of four
Characteristics T-4 Streptomyces
(disks per 100 ml media). The level of antibiotic yield in-
torulosus
creased gradually with increasing the incubation period up
Morphological characteristics to the end of 5 days, after these maximum values 30.0,
Spore mass Gray Gray 28.0, 27.0, 25.0 and 23.0 in case of A. niger, IMI 31276;
Reverse color Light yellow/light brown Light yellow
Staphylococcus aureus, NCTC 7447; B. subtilis, NCTC
Spore chain Spiral Spiral
Spore surface Warty Warty and
1040, C. albicans IMRU 3669 and K. pneumonia, NCIMB
spiny 9111, respectively. The optimum temperature capable of pro-
Motility Non-motile Non-motile moting antimicrobial agents biosynthesis was at 35oC,
whereas, the diameter of inhibition zone resulted from anti-
Cell wall hydrolysate
microbial agent productivity reached up to 31.0, 29.0, 28.0,
Diaminopimelic acid (DAP) LL-DAP LL-DAP
25.5 and 24.0 in case of A. niger, IMI 31276; Staphylococ-
Sugar pattern Not detected Not detected
Melanin pigment + + cus aureus, NCTC 7447; B. subtilis, NCTC 1040; C. albicans,
IMRU 3669 and K. pneumonia, NCIMB 9111, respectively.
Utilization of carbon sources The optimum initial pH value capable of promoting antimi-
L-Arabinose + +
crobial agent was found to be the value of 7.0 since the
D-Fructose + +
diameter of inhibition zone at from antimicrobial
D-Galactose + + resulted
D-Glucose + + agents productivity reached up to 31.0, 29.0, 28.0, 25.5
Meso-Inositol + + and 24.0 in case of A. niger, IMI 31276; Staphylococcus
D-Mannitol + + aureus, NCTC 7447; B. subtilis, NCTC 1040; C. albicans,
Raffinose + + IMRU 3669 and K. pneumonia, NCIMB 9111, respectively.
Sucrose + ND The effect of the used carbon sources in the production of
D-Xylose + + antimicrobial agent could be arranged in the following
ND = no data. descending manner; for Streptomyces torulosus, KH-4, glu-
cose > starch > mannitol > sucrose >
fructose > Arabinose > D-mannose > D-galactose > xylose
against bacterial database similarly revealed that the 16S rDNA > raffinose > Rhamnose.
sequencewas98%identical Streptomyces torulosus ( Fig. 1 ).
3.8. Fermentation, extraction and purification
3.7. Factors effecting on the biosynthesis of the antimicrobial agent
The fermentation process was carried out for five at
35 C. After incubation period, the filtration was days
The maximum inhibition zones of produced antibiotic conducted
followed by centrifugation at 4000 rpm for 15 min. The entire
against tested microorganisms reached up to 30.0, 28.0,
18 H.M. Atta
Figure3 Ultraviolet absorbance of antimicrobial agent pro-

duced by Streptomyces torulosus, T-4.
Figure1 The phylogenetic position of the local Streptomyces sp.

most active fractions against the tested organisms ranged between
strain among neighboring species. The phylogenetic tree was based 14and23.
on the pairwise comparisons of 16S rDNA sequences.
3.9. Physico-chemical characteristics

culture broth (20 l) was centrifuged (4000 rpm, 15 min) to sep- arate
the mycelium and the supernatant. The supernatant was extracted The purified antimicrobial agent produced by Streptomyces
with torulosus, T-4 produces characteristic odor, their melting
n-butanol (1:1, v/v) and the organic layer was
evaporated to give an oily material. The oily material was then points are 235 C. The compound is freely soluble in chloro-
dissolvedin15%aqueousmethanolanddefattedbypartition- form, ethyl acetate, n-butanol, acetone, ethyl alcohol, metha-
ing with petroleum ether (b.p. 60–80 C) to give a solid extract. nol and 10% isopropyl alcohol, but insoluble in petroleum
Its color is yellowish. Separation of antimicrobial agent into ether, hexan and benzene.
individualcomponentswascarriedoutbythin-layerchroma-using
tography a solvent system composed of chloroform 3.10. Elemental analysis
and methanol (24:1, v/v). Only one band at Rf = 0.55 showed
antimicrobial activity. The purification process through col- The elemental analytical data of the antimicrobial agent produced
umn chromatography packed with silica gel revealed that the by Streptomyces torulosus, T-4 showed the following:
Figure2 I.R. spectrum of antimicrobial agent produced by Streptomyces torulosus, T-4.

Figure4 Mass spectrum of antimicrobial agent produced by Streptomyces torulosus, T-4.
absorption spectrum is recorded a maximum absorption peak at

Table5 A comparative study of the characteristic properties 260 nm (Fig. 3). The Mass spectrum revealed that the
of the antimicrobial agent produced by Streptomyces torulosus, molecular weight is 865 ( Fig. 4 ).
T-4 in relation to reference antibiotic (tunicamycin).
Character purified Tunicamycin 3.12. Biological activities of the antimicrobial agent
antimicrobial
agent Data of the antimicrobial agent spectrum indicated that the
1. Melting point 235 C 234–235 C agent is active against Gram-positive and Gram-negative bac-
2. Molecular 865 865.4 terial and unicellular and filamentous fungi ( Table 5 ).
weight
3.13. Identification of the antimicrobial agent
Chemical analysis
C 53.30 53.31
H 6.87 6.86
On the basis of the recommended keys for the identification of
antibiotics and in view of the comparative study of the re-
N 6.61 6.61
O 29.51 29.51 cordedpropertiesoftheantimicrobialagent,itcouldbestatedthat
S 0.0 0.0 the antimicrobial compound is suggestive of being belonging to
tunicamycin antibiotic ( Umezawa, 1977; Berdy, 1974, 1980a,b,c;
3. Ultraviolet 260 205 and 260
Tsvetanovaetal.,2002)(Table6).
4. Formula C38H62N4O16 C38H62N4O16
5. Active against Active against Active against
Gram-positive Gram-positive
4.Discussion
and Gram- and Gram-
negative negative
bacteria and bacteria and The increase in the frequency of multi-resistant pathogenic
unicellular and unicellular bacteria has created an urgent demand in the pharmaceutical
filamentous and industry for more rational approaches and strategies to the
fungi filamentous fungi
screening of new antibiotics with a broad spectrum of activity,
which resist the inactivation processes exploited by the microbial
enzymes(Mottaetal.,2004).Ninety-sevenactinomycetewere
C = 53.30; H = 6.87; N = 6.61; O = 29.51 and S = 0.0. This analysis strains isolated from fifty soil samples collected from
indicates a suggested empirical formula of the Taif City, Kingdom of Saudi Arabia. Only one actinomy-
C38H62N4O16. cete culture T-4 was found exhibited to produce wide spectrum
antimicrobial activities. Identification process has been carried
3.11. Spectroscopic characteristics according
out to (Hensyl, 1994; Numerical Taxonomy Pro-
gram, 1989 ). For the purpose of identification of actinomycete
The spectroscopic analysis of the purified of antimicrobial isolate, the morphological characteristics and microscopic
compound produced by Streptomyces torulosus, T-4, the infra- examination emphasized that the spore chain is spiral. Spore
red (IR) spectrum showed characteristic band corresponding to mass is light gray; while spore surface is warty, substrate mycelium
26 peaks 551.9, 669.2, 770.1, 847.1, 910.8, 956.3, 980.2, is light yellowish brown and no diffusible pigment was produced on
1039.2, 1091.4, 1110.9, 1204.8, 1254.1, 1264.1, 1380.2, 1461.7, ISP-media. The results of physiological, biochemical
characteristics and cell wall hydrolysate of actinomycetes isolate,
1547.7, 1666.2, 1708.4, 2333.8, 2306.0, 2872.5, 2954.2, 3280.8, exhibitedthatthecellwallcontainingLL-diaminopim-
3341.8, 3668.9 and 3731.5 (Fig. 2). The ultraviolet (UV)
20 H.M. Atta
2004); KNO3 best nitrogen source (Hosokawa et al., 1996;

Table6 Antimicrobial spectrum of the antimicrobial agent(s)
Khalifa, 2008; Atta et al., 2011 ).
by adding paper disk diffusion method ( Kavanagh, 1972 ).
Theactivemetaboliteswereextractedbyn-ButanolatpH7.0(
Test organisms MIC (lg/ml) Atta,2010;Attaetal.2011).Theorganicphasewascol-lectedand
concentration of evaporatedunderreducedpressureusingarotaryevaporator.
antimicrobial The extract was concentrated and treated with
agent produced
petroleum ether (b.p. 40–60 C) for precipitation process
by Streptomyces
torulosus, T-4
where only one fraction was obtained in the form of yellowish
ppt. and then tested for their antimicrobial activity. Separation of
A. Bacteria Gram-positive antibiotic into individual components has been tried by
a. cocci Staphylococcus
aureus thin-layer chromatography using a solvent system composed of
, 52.7 chloroform and methanol (24:1, v/v) as developing solvent ( Zhang
NCTC 7447 etal.,2007;Attaetal.,2009).Thebandwithan
Micrococcus luteus, 52.7 Rf va-
ATCC 9341 lue at 0.55 which indicated the presence of one compound
b. Gram-positive bacilli ( Atta, 2010 ). For the purpose of purification process, the anti-
Bacillus subtilis, 73.78 biotics were allowed to pass through a column chromatography
NCTC 10400 packed with silica gel and eluting solvent was composed of
chloroform and methanol (10:2 v/v), fifty fractions were collected
Bacillus pumilus, 73.78 and tested for their activities. The most active fractions against the
NCTC 8214 tested organisms ranged between 14 and 23. Simi- larly, many
c. Gram-negative bacteria workers used a column chromatography packed with silica gel and
Escherichia coli, 73.78 an eluting solvent composed of various ra- tios of chloroform and
NCTC 10416 methanol(Criswelletal.,2006;Sekig-uchietal.,2007).
Klebsiella pneumonia, 100 <
NCIMB 9111
Pseudomonas 100 <
aeruginosa, ATCC 10145 The physico-chemical characteristics of the purified antibi-
otic revealed that, melting point at 235 C. The compound is
B. Fungi
freely soluble in chloroform, ethyl acetate, n-butanol, acetone,
a. Unicellular fungi
Candida albicans, 73.78 ethyl alcohol, methanol and 10% isopropyl alcohol, but insol-
IMRU 3669 uble in petroleum ether, hexan and benzene; similar results
Saccharomyces 46.9 were recorded by Mellouli et al. (2003), El-Tayeb et al.
cervisiae ATCC 9763 (2004) and Atta (2010) .
b. Filamentous fungi Astudyoftheelementalanalysisoftheantibioticshowedthe
Aspergillus niger, IMI 15.73 following C = 53.30; H = 6.87; N = 6.61; O = 29.51
31276 and S= 0.0 leadoftothe
anmaximum
empirical formula of Cpeak
16 the presence absorption 38H62in
N4UV
O .
Aspergillus fumigatus, 31.25 The260 spectroscopic characteristics of antibiotic revealed
at nm, infrared absorption spectrum showed
ATCC 16424 characteristic band corresponding to 26 peaks 551.9,
Aspergillus flavus, IMI 22.32 669.2, 770.1, 847.1, 910.8, 956.3, 980.2, 1039.2, 1091.4,
111023 1110.9, 1204.8, 1254.1, 1264.1, 1380.2, 1461.7, 1547.7,
Fusarium oxyspoum 46.9 1666.2, 1708.4, 2333.8, 2306.0, 2872.5, 2954.2, 3280.8,
Rhizoctonia solani 52.7 3341.8, 3668.9 and 3731.5. Mass spectrum showed that
Alternaria alternata 46.9 the molecular weight is 865 Tsvetanova
( et al., 2002
).
Botrytis fabae 46.9
The MIC of antibiotic under study exhibited fairly
active against both and
Penicillium 52.7
chrysogenium
Gram-positive Gram-negative bacteria and unicellular
and filamentous fungi. The MIC of antibiotic was determined
elic acid (DAP) and sugar pattern of cell wall hydrolysate andtheresultsshowedthattheminimuminhibitoryconcentra-tion
(MIC)oftheantibioticproducedby Streptomyces torulo-
could not be detected. These results emphasized that the acti-
nomycetes isolate is related to a group of Streptomyces. In sus, T-4 against Staphylococcus aureus, NCTC 7447 was
view of all the previously recorded data, the identification of 52.7 lg/ml, Micrococcus lutea, ATCC 9341 was 52.7 lg/ml,
actinomycete isolate T-4 was suggestive of being belonging and B. subtilis, NCTC 10400 was 73.78 lg/ml, B. pumilus,
to Streptomyces torulosus, T-4. The resulted sequence was NCTC 8214 was 73.78 lg/ml, K. pneumonia, NCIMB 9111,
aligned with available almost complete sequence of type of was >100 lg/ml, E. coli, NCTC 10416 was 73.78 lg/ml, and
strains of family streptomycetaeae. The phylogenetic tree (dia- Pseudomonas aeruginosa, ATCC 10145 was >100 lg/ml, for
gram) revealed that the local isolate is closely related to Strep- A. flavus, IMI 111023 was 31.25 lg/ml and Saccharomyces
tomyces sp rather than to Streptomyces torulosus by a cerevisiae, ATCC 9763 was 46.9 lg/ml, C. albicans, IMRU
similarity matrix is 98%. 3669 was 73.78 lg/ml, A. niger, IMI 31276 was 15.73 lg/ml,
Maximumantimicrobialactivitybiosynthesiscouldbere- A. fumigatus, ATCC 16424 was 31.25 lg/ml, A. flavus, IMI
corded that a different inoculum sizes for four disks; 111023 was 22.32 lg/ml, F. oxysporum was 46.9 lg/ml, R.
incubation period for five days (Adinarayana et al., 2002); solani was 52.7 lg/ml, A. alternata was 46.9 lg/ml, B. fabae
pH 7.0 ( Atta, 2010 ); temperature 35 C(Kunnari et al., 1997; was 46.9 lg/ml, Penicillium chrysogenium was 52.7 lg/ml. Sim-
Atta, 1999); glucose best carbon source (Hoshino et al., ilar investigations and results were attained by Imnagaki et al.
(1998),Sekiguchietal.(2007)andAttaetal.(2009).Identifi-
cation of antibiotic according to recommended international keys Edwards,U.,Rogall,T.,Bocker,H.,Emade,M.,Bottger,E.,1989.Isolation

indicated that the antibiotic is suggestive of being belonging to and direct complete nucleotide determination of entire
tunicamycin antibiotic ( Umezawa, 1977; Berdy, 1974, 1980a,b,c; genes. Characterization of a gene coding for 16s ribosomal DNA.
Tsvetanovaetal.,2002). NucleicAcidRes.17,7843–7853.
El-Tayeb, O.M., Salama, A., Hussein, M., El-Sedawy, H.F., 2004.
Optimization of industrial production of rifamycin B by Amyco-
5.Conclusion latopsis mediterranei I. The role of colony morphology and nitrogen
sources in productivity. Afr. J. Biotechnol. 3, 266–272.
Elwan, S.H., El-Nagar, M.R., Ammar, M.S., 1977. Characteristics of
The present study shows the present data focusing on obtain- ing Lipasesinthegrowthfiltratedialystateof
microbial local isolates which have the ability to produce Bacillus stearothermo-
antimicrobial philus grown at 55 C using a tributryin-cup plate assay. Bull. Fac.
agent against pathogenic microorganisms Sci., Riyadh Univ. 8, 105–119.
(Gram-positive and Gram-negative bacteria and unicellular Euzeby, J.P., 2008. Genus streptomyces. List of Prokaryotic names
and filamentous fungi). with Standing in Nomenclature.
Gordon, R.E., 1966. Some criteria for the recognition of Nocardia
madura (Vincent) Blanchord. J. General Microbiol. 45, 355–364.
References Gordon, R.E., Barnett, D.A., Handehan, J.E., Pang, C.H., 1974.
Nocardia coeliaca; Nocardia autotrophica and Nocardin strain. Int.
Adinarayana, K., Ellaiah, P., Srinivasulu, B., Bhavani, R., Adinara- yana, J. Syst. Bacteriol. 24, 54–63.
G., 2002. Response surface methodological approach to Hall, T.A., 1999. BioEdit: a user-friendly biological sequence align-
optimize the nutritional parameters for neomycin production by ment editor and analysis program for Windows 95/98/NT. Nucleic
Streptomyces marinensis under solid-state fermentation. Process Acid Symp. Ser. 41, 95–98.
Biochem. 38, 1565–1572. Hankin, L., Zucker, M., Sands, D.C., 1971. Improved solid medium for
Atta,
for H.M.,
fermentation
1999. conditions.
ApplicationPh.D.
of Thesis,
biotechnology
Faculty ofinScience,
search the detection and enumeralion of proteolytic bacteria. Appl.
Al-Azhar
antibioticsUniversity, Cairo, Egypt.
from environmental pollutants under solid state Microbiol. 22, 205–509.
Hensyl, W.R., 1994. Bergey’s Manual of Systematic Bacteriology,
ninth ed. Williams and Wilkins, Baltimore, Philadelphia, Hong
Atta,H.M.,2010.Production,purification,physico-chemicalcharac- Kong, London, Munich.
teristicsandbiologicalactivitiesofantifungalantibioticproduced Hoshino, Y., Mukai, A., Yazawa, K., Uno, J., Ando, A., 2004.
by Streptomyces antibioticus, AZ-Z710. American-Eurasian J. Sci. Transvalencin A, a thiazolidine zinc complex antibiotic produced
Res. 5 (1), 39–49. by a clinical isolate of Nocardia transvalensis II. Structure elucida-
Atta,H.M.,Abul-Hamd,A.T.,Radwan,H.G.,2009.Productionof tion. J. Antibiot. 57, 803–807.
Destomycin
Streptomyces –aantibiotic
sp.usingrice strawas by Hosokawa, N., Naganawa, H., Hamada, M., Takeuchi, T., Ikeno, S., Hori,
fermentedsubstrate..Commun.Appl.Biol.Sci.,GhentUniversity74 M., 1996. Hydroxymycotrienins A and B, new ansamycin
(3),879–897.
group antibiotics. J. Antibiot. 49 (5), 425–431.
Atta, H.M., El-Sehrawi, M.H., Bahobail, A.S., 2011. Antifungal Imnagaki, T., Kaneda, K., Suzuki, Y., Hirai, H., Nomura, E., 1998. CJ-12,
macrodiode production by Streptomyces albidoflavus-143: fermen- 373, a novel topoisomerase II inhibitor: fermentation,
tation,purificationandbiologicalactivities.J.Am.Sci.7(3),13–22. isolation, structure, elucidation and biological activities. J. Anti-
Becker,B.,Lechevalier,M.P.,Gordon,R.E.,Lechevalier,H.A.,1964. biot. 51 (2), 112–116.
Rapiddifferentiationbetween
Nocardia and Streptomyces by paper Isono, K., Suhadolnik, R.J., 1976. The biosynthesis of natural and
chromatography of whole cell hydrolysates. Appl. Microbiol. 12, unnatural polyoxins by Streptomyces cacaoi. Arch. Biochem.
421–423. Biophys. 173, 141–153.
Berdy, J., 1974. Recent developments of antibiotic research and Isono, K., Sato, T., Hirasawa, K., Funayama, S., Suzuki, S., 1978.
classification of antibiotics according to chemical structure. Adv. Biosynthesis of the nucleoside skeleton of polyoxins. J. Am. Chem.
Appl. Microbiol. 14, 309–406. Soc. 100, 3937–3939.
Berdy, J., 1980a. Recent advances in and prospects of antibiotics Jones, K.L., 1949. Fresh isolates of actinomycetes in which the
research. Process Biochem. 15, 28–35. presence of sporogenous aerial mycelia is a fluctuating character-
Berdy, J., 1980b. CRC Handbook of Antibiotic Compounds, first istics. J. Bacteriol. 57, 141–145.
ed., vol. 1. CRC Press, Boca Raton (ISBN-13: 9780849334511).
Berdy, J., 1980c. CRC Handbook of Antibiotic Compounds, first ed., Kampfer, P., 2006. The family Streptomycetaceae, Part I: Taxonomy.
vol. 2. CRC Press, Boca Raton (ISBN-13: 978-0849334528). In: Dworkin, M. (Ed.), The Prokaryotes: A Handbook on the
Biology of Bacteria. Springer, Berlin, pp. 538–604.
Bibb,M.J.,2005.Regulationofsecondarymetabolisminstreptomy-cetes. Kavanagh, F., 1972. Analytical Microbiology. vol. 2. Academic Press, New
Curr.Opin.Microbiol.8,208–215. York.
Buchanan, R.E., Gibbons, N.E., 1974. Bergey’s Manual of Determi- Kenneth, L.K., Deane, B.J., 1955. Color universal language and
native Bacteriology, eighth ed. The Williams & Wilkins Company,
Baltimore. dictionary of names. United States Department of Commerce.
National Bureau of Standards. Washington, DC, p. 20234.
Cappuccino, J.G., Sherman, N., 2004. Microbiology, A Laboratory Khalifa, M.A., 2008. Bioprocess development for the biosynthesis
Manual. Pearson Education, Inc., New Delhi, pp. 282–283. of bioactive compounds from microbial origin. M.Sc. Thesis,
Faculty of Science, Al-Azhar University, Cairo, Egypt.
Chapman,G.S.,1952.Asimplemethodformakingmultipletestsofa
microorganism.J.Bacteriol.63,147.
Khosla, C., 2000. Natural product biosynthesis: a new interface
Cowan,S.T.,1974.CowanandSteel’sManualfortheIdentificationof between enzymology and medicine. J. Org. Chem. 65, 8127–
MedicalBacteria,seconded.UniversityPress,Cambridge.
8133.
Criswell, D., Tobiason, V.L., Lodmell, J.S., Samuels, D.S., 2006. Kunnari, T., J.,Hautala,A.,Hakala,J.,Ylihonko,K.,
Mutations conferring aminoglycoside and spectinomycin resistance Mantsala, Tuikkanen,
P., 1997. Isolation and characterization of 8-demethoxy
in Borrelia burgdorferi. Antimicrob. Agents Chemother. 50, 445– steffimycins and generation of 2, 8-demethoxy steffimycins in
452. Streptomyces steffisburgensis by the nogalamycin biosynthesis
Eckardt,K.,1983.Tunicamycins,streptovirudinsandcorynetoxins,a genes. J. Antibiot. 50 (6), 496–501.
specialsubclassofnucleosideantibiotics.J.Nat.Prod.46,544–550.
22 H.M. Atta
Lechevalier,M.P.,Lechevalier,H.A.,1970.Chemicalcompositionasain Sekiguchi, M., Shiraish, N., Kobinata, K., Kudo, T., Yamaguchi, I., 2007.
criterion the classification of aerobic actinomycetes. J. Syst. RS-22A,BandC:newmacrolideantibioticsfrom Strepto-
Bacteriol. 20, 435–443. myces violaceusniger. I. Taxonomy, fermentation, isolation and
Madigan,M.,Martinko,J.,2005.BrockBiologyofMicroorganisms.11th biological activities. J. Antibiot. 48 (4), 289–292.
ed.Prentice-Hall,NewJersey,USA.. Shirling, E.B., Gottlieb, D., 1966. Methods for characterization of
Mellouli, L., Ameur-Mehdi, R.B., Sioud, S., M., Bejar, S., Streptomyces species. Int. J. Syst. Bacteriol. 16, 313–340.
2003. Isolation, purification and partial Salem, of anti- Subramaniam, P.S., Xie, G., Xia, T., Jensen, R.A., 1998. Substrate
characterization
bacterial activities produced by a newly isolated Streptomyces sp. ambiguity of 3-deoxy-D-manno-octulosonate 8-phosphate synthase
US24 strain. Res. Microbiol. 154, 345–352. from Neisseria gonorrhoeae in the context of its membership in a
Motta, A.S., Cladera-Olivera, F., Brandelli, A., 2004. Screening for protein family containing a subset of 3-deoxy-D-arabino-heptulo-
antimicrobial activity among bacteria isolated from the Amazon sonate 7-phosphate synthases. J. Bacteriol. 180, 119–127.
Basin. Braz. J. Microbiol. 35, 307–310. Tamura, G., 1982. Tunicamycin. Japan Scientific Press, Tokyo, pp.
Nitsh,B.,Kutzner,H.J.,1969.Egg-Yolkagarasdiagnosticmediumfor 10–31, ISBN-13: 9784762253096.
Streptomycetes.Cell.Mol.LifeSci.25,220–221. Tkacz, J.S., 1983. Tunicamycin and Related Antibiotics. Springer-
Numerical Taxonomy Program, 1989. Numerical taxonomy of Strep- Verlag, Berlin, pp. 255–278.
tomyces species program (PIB WIN) Streptomyces species. J. Gen. Tsvetanova, B.C., Price, N.P.J., 2001. Liquid chromatography-elec-
Microbiol. 1989, 13, 512-533. trospray mass spectrometry of tunicamycin-type antibiotics. Anal.
Olano,C.,Mendez,C.,Salas,J.A.,2009.Antitumorcompoundsfrom Biochem. 289, 147–156.
marineactinomycetes.Mar.Drugs7,210–248. Tsvetanova, B.C., Kiemle, D.J., Price, N.P.J., 2002. Biosynthesis of
Pridham, T.G., Gottlieb, D., 1948. The utilization of carbon tunicamycin and metabolic origin of the 11-carbon dialdose sugar,
compounds by some Actinomycetales as an aid for species tunicamine. J. Biol. Chem. 277 (38), 35289–35296.
determination. J. Bacteriol. 56 (1), 107–114. Umezawa, H., 1977. Recent advances in bioactive microbial secondary
Pridham,T.G.,Anderson,P.,Foley,C.,Lindenfelser,L.A.,Hessel-tine, metabolites.Jpn.J.Antibiot.Suppl.30,138–163.
C.W., Benedict, R.G., 1957. A selection of media for Williams, S.T., Davies, F.L., 1965. Use of antibiotics for selective
maintenance and taxonomic study of Streptomyces. Antibiot. isolation and enumeration of actinomycetes in soil. J. Gen.
Ann., 947–953. Microbiol. 38, 251–262.
Sambrook,J.,Fritsch,E.F.,Maniaties,T.,1989.MolecularCloning.A Williams, S.T., Sharpe, M.E., Holt, J.G., 1989. In: Bergey’s Manual of
Laboratory Manual. Cold Spring Harbor Laboratory Press, Systematic Bacteriology, vol. 4. Williams and Wilkins, Baltimore,
Hong Kong, London, Sydney.
Springer, Berlin.
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with Zhang, L., Yan, K., Zhang, Y., Huang, R., Bian, J., 2007. High-
chain terminating inhibitors. Proc. Natl. Acad. Sci. 74, 5463–5467. throughput synergy screening identifies microbial metabolites as
Schuz, T.C., Fiedler, H.P., Zahner, H., Rieck, M., Konig, W.A., 1992.
combination agents for the treatment of fungal infections. Proc.
Metabolicproductsofmicroorganisms.263.NikkomycinsSZ,SX,SoZ Natl. Acad. Sci. USA 104, 4606–4611.
and SoX, new intermediates associated to the nikkomycin
biosynthesis of Streptomyces tendae. J. Antibiot. 45, 199–206.

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Journal of Chemical Society Arabia Saudita (2015)

Universidad Rey Saud

Recibido el 27 de noviembre de 2011; aceptado el 09 de diciembre de 2011

PALABRASCLAVE Resumen Tunicamicinaesunantibiticodenucletidoquefueaisladodelcaldodefermentacindecepano.

Streptomyces torulosus, T-4 es Tunicamicina antibitico.

1Introduccin y el tipo GØnero de el familia Streptomycetaceae

llamadotunicamine, y un Amida-ligado graso cÆ ido. El AB - Reino de ArabiaSaudita

y Deane (1955). AdemÆs, la sensibilidad de las cepas a los 2.9.2. extraccin

Plate1 Scanning electron micrograph of the actinomycete

chain spiral shape and spore surfaces warty (25,000·).

3.2. Identiﬁcation of the actinomycete isolate: morphological

The vegetative mycelia grew abundantly on both synthetic

and complex media. The aerial mycelia grew abundantly

on starch–nitrate agar medium and oatmeal agar medium

ﬂagellated spores were observed.

3.3. Cell wall hydrolysate

The cell wall hydrolysate contains LL-diaminopimelic acid

(LL-DAP) and sugar pattern not detected.

3.4. Physiological and biochemical characteristics

The actinomycete isolate T-4 could hydrolyze starch, pro-

tein, and cellulose, whereas lipid, pectin, lecithin and cata-

lase are negative. Melanin pigment is positive, degradation

decomposition of urea and utilization of citrate and KCN

are positive. The isolate under study utilizes D-xylose, D-

mannose, D-glucose, D-fructose, D-galactose, mannitol,

meso-inositol, sucrose, rhamnose, L-arabinose, rafﬁnose,

starch and trehalose, but do not utilize lactose, maltose,

cine L-histidine, L-phenyl alanine and L-lysine. No growth on

L-valine, and L-methionine. On the other hand, the isolate T-

4 decreased at high NaCl concentration above (5% w/v).

The growth is not inhibited in the presence of phenol and

45 C. The actinomycete isolate T-4 is not sensitive to Ampi-

(75 lg/ml), and Fusidic acid (10 l(3

Gentamicin (10 lg/

ml) and Kanamycin (30 lg/ml) (Table 2).

Table3 Culture characteristics of the actinomycete isolate T-4.

Figure3 Ultraviolet absorbance of antimicrobial agent pro-

Figure1 The phylogenetic position of the local Streptomyces sp.

3.9. Physico-chemical characteristics

Figure2 I.R. spectrum of antimicrobial agent produced by Streptomyces torulosus, T-4.

Figure4 Mass spectrum of antimicrobial agent produced by Streptomyces torulosus, T-4.

absorption spectrum is recorded a maximum absorption peak at

2004); KNO3 best nitrogen source (Hosokawa et al., 1996;

cation of antibiotic according to recommended international keys Edwards,U.,Rogall,T.,Bocker,H.,Emade,M.,Bottger,E.,1989.Isolation

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