JCC0751

JOURNAL OF CULTURE COLLECTI= ONS

Volume 5, 2006-2007, pp. 3-9

TAXONOMIC CHARACTE= RIZATION
OF THE THERMOPHILIC ACTINOMYCETE STRAIN 21E –
PRODUCER OF THERMOSTABLE COLLAGENASE

Detelina Petrova* and Stoyan Vlahov

Sofia University, Faculty of Biology, Department of G= eneral and Industrial Microbiology,
8 Dragan Tzankov str., 1164 Sofia, Bulgaria<= /span>

*Corresponding author, e-mail: detelina@bi= ofac.uni-sofia.bg

Summary=

The thermophilic actinomycete strain 21E isolated from saline Bulgarian soils produced a hig= hly thermostable collagenase. Macro- and micromorphological characteristics of = the strain were tested on fourteen media. Some physiological and biochemical properties, enzyme activities, as well as the antibiotic activity and sensi= tivity were examined. It was concluded that the strain 21E was a typical member of= the genus Thermoactinomyces. The comparative references to Thermoactinomyces species with similar taxonomic characteristics identified the strain as Thermoactinomyces sacchari. T. sacchari strain 21E was resistant to the antibiotic novobiocine, and displayed protease, amylase, collagenase and li= pase activities.

Key words: taxonomy, thermophilic actinomycetes, Thermoactinomyces sacchari, soil, collagenase

Introduction

The= study of microorganisms capable to grow in extreme conditions is an important field = of microbiology and biotechnology. Thermophilic representatives of the family = of actinomycetes are becoming more and more important in solving certain probl= ems of ecology, industrial microbiology, hygiene and other fields. Recently, th= ey have been subjected to intensive investigations in search for producers of = new thermostable enzymes.

It = was already shown that the thermophilic actinomycetes can produce amylases, xylanases and cellulose digesting enzymes which retain their activity at hi= gh temperatures (50‑65 ^oC) [17, 22, 27]. It is commonly accept-ed nowadays that such enzymes will find wider application in the industrial practice. Extracellular proteolytic activity was detected in= the cultural supernatant of a number of thermophilic actinomycete strains [9, 11, 20, 29].

The collagenases are specific proteases capable to hydrolyze the triple helix of the native collagen, which is remarkably resistant to other proteases. It s= hould be noted however that the collagenases described so far are isolated from mesophilic microorganisms and these enzymes are not stable at elevated temperatures [10, 15, 23, 25].

A collagen-degrading thermophilic actinomycete strain, designated as 21E, was obtained while searching for collagenolytic thermophilic microorganisms of = soil origin [15]. These microorganisms are harmless for humans, and develop quic= kly in culture ‑ for about 2‑3 days. The potential applicability for the production of a collagenolytic enzyme preparation requires strain 21E t= o be determined taxonomically.

Materials and Meth= ods

The thermophilic actinomycete strain 21E was afforded for taxonomic study by A. Gushterova from the Institute of= Microbiology at the Bulgarian Academy of Science. It was isolated from saline soil [15] and produced a highly thermostable collagenase [24].

The= general morphological, cultural, physiological, and biochemical properties of the actinomycetes were employed for the taxonomic characterization of strain 21= E, and they were determined by the methods and in the media of the Internation= al Streptomycetes Project (ISP) [26]. Additional nutrient media and characteri= stic substances were also applied in the investigation.

Micromorphological and cultural properties. The following media were used: pep-tone-maze extract agar (PM) [14], yeast-malt extract agar (ISP-2), oatmeal agar (ISP-3), starch agar wi= th mineral salts (ISP-4), glycerol-asparagine agar (ISP-5), tyrosine agar (ISP= -7) [26], nutrient agar with glucose [8], yeast ex-tract agar [6], glucose-yeast extract agar [8], potato-carrot agar (PC) [7], potato-glucose agar [2], milk agar [8], and chitin agar [20].

The morphology of the sporophores was studied by direct light microscope observation. The morphology of the spores was examined by a scanning microscope. The micromorphological and cultural properties of the strain we= re investigated after 24, 48, and 72 hours of cultivation. Color descriptions = were made after the Bondartsev's color scale [5].

Physiological and biochemical properties<= /b>. Melanin production was studied on pep-tone-yeast extract iro= n agar (ISP-6) and on tyrosine agar (ISP-7) [26]. The utilization of various carbon sources was tested on basic mineral salts agar (ISP-9) [26] supplemented wi= th the following sugars (1 %, w/v): D-glucose, L-arabinose, sucrose, D-xylose, I-inositol, D-mannitol, D-fructose, rhamnose, raffinose, D-maltose, cellulo= se, and lactose.

Growth temperature optimum. The optimal growth temperature was determined on PM agar in the range from = 25 to 75 ^oC.

Resistance to heating at 100 ^oC<= /span>. Spores of the thermophilic actinomycete 21E were suspended in distilled water, heated in boiling water for 10, 30, 60, and 120 min, and t= hen transferred onto PM agar.

Resistance to sodium chloride. The tolerance to NaCl was tested on PM agar containing diffe= rent concentrations of NaCl from 1 to 15 % (w/v).

Decomposition of gelatin. PM agar supplemented with 0.4 % (w/v) gelatin was used. A clear zone around the colony indicated the gelatin hydrolysis [19].

Hydrolysis of starch. Ten grams of potato starch suspended in 100 ml of cold distilled water were added as a single carbon source to 900 ml of PM agar. The plates were inoculated and incubated at 55 ^oC, and then were flooded wi= th iodide solution. A clear zone around the colony indicated the starch hydrol= ysis [19].

Decomposition of casein. The capability to decompose casein was determined as described by Gordon and Smith [12].

Hydrolysis of esculin and arbutin. Esculin or arbutin (0.1 %, w/v) were added to PM agar w= ith 0.05 % ferric citrate (w/v). The blackening of the medium around the colony was an evidence for the hydrolysis of each of the substrates [4, 18]= .

Decomposition of tyrosine, xanthine, hypoxanthin= e, and adenine. The media and methods applied were as described by Gordon et al. [11] and Kurup and Schmitt [18], except for that= the basic medium was PM. The clearing of the medium around and underneath the colonies indicated tyrosine, xanthine, hypoxanthine, or adenine decompositi= on.

Sensitivity to antibiotics. The agar dif-fusion method was applied. Disks impregnated with ampicillin, penicillin, chloramphenicol, gentamycin, erythromycin, and streptomycin were placed on the inoculated agar plates. The zones of growth inhibition were measured.

Nov= obiocin was added to PM liquid medium to a final concentration of 25 μg/ml and the resistance the strain to the antibiotic was recognized when apparen= tly similar growth in both the test and the control tubes was observed [19].

Antagonistic properties. The streak test on PM agar plates was performed on Bacillus subtilis and Escherichia coli as test micro-organisms [14].

Hydrolysis of tributirin. Hydrolysis of tributirin was tested by the method described by Kurup and Schmitt [18].

Decomposition of native collagen. The de-composition of native collagen was assessed according= to the size of the hydrolyzed zone [28].

The= genus reference, species descriptions, and species identification strain 21E were determined after Bergey’s manual [3] and Agre’s manual [1].

Results and Discus= sion

Micromorphological= and cultural properties

Str= ain 21E produced well-developed aerial mycelium that displayed a tendency to fragme= nt with aging of the culture. It was white and cream-white, but there were also pale-beige and ash color variants. The aerial mycelium was transient, usual= ly disappearing within 3 to 4 days through autolysis of the hyphae. The hyphae= of the aerial mycelium were short, sparse or tufted (Fig. 1, A).

F= ig. 1. Hyphae structure (A) and form = of the colonies (B) of strain 21E (PM= agar, 48 hour).

The= hyphae of the substrate mycelium were branching, penetrating into the agar medium, forming fast-growing, spreading colonies. Several colors of the vegetative = mycelium were observed depending on the medium and the age of the culture. The young colonies were beige or cream to dark beige or yellow-brown. The old colonies were dark brown in color (Table1).

Table 1. Cultural properties of strain 21E.

N= utrient medium

Growth

Color of the aerial mycelium

Color of the substrate mycelium

Soluble pigment

Y= east-malt extract agar (ISP-2)

abundant

cream-white

beige

no

O= atmeal agar (ISP-3)

moderate

white

cream

no

S= tarch agar with inorganic ions (ISP-4)

moderate

white or cream-white

dark beige

no

G= lycerol-asparagine agar (ISP-5)

scanty

no

yellow-brown

no

P= eptone-yeast extract iron agar (ISP-6)

abundant

cream-white

beige

no

T= yrosine agar (ISP-7)

no

no

no

no

P= M agar

abundant

cream-white

dark beige

no

N= utrient agar with glucose

scanty

white

cream

no

Y= east extract agar

abundant

pale beige

dark beige

no

G= lucose yeast extract agar

moderate

ash

beige

no

M= ilk agar

abundant

white

cream

no

P= C agar

abundant

cream-white

dark beige

no

P= otato-glucose agar

moderate

white

dark beige

no

C= hitin agar

no

no

no

no

The= colonies were in diameter from 5‑6 mm on these media where the growth of = the strain was scanty to 25‑30 mm on the media with moderate to abundant growth. The typical colony was with a regular round form, flat, with smooth surface and gentle radial folds to the periphery (Fig. 1, B). Its cent= ral area was slightly convex. The margin of the colony was undulated. Morpholog= ical variants of colonies were also observed with irregular form, rugged surface= and fibrillated edge. They appeared in certain unfavorable conditions. The colo= nies became slimy and bacterial in appearance as the hyphae autolysed.

Str= ain 21E produced abundant or mode-rate aerial mycelium on organic media and scantly grew on synthetic media (Table 1). It had a short growth cycle and sporulated already after 24 hours of cultivation. The strain did not produce soluble pigments.

Sin= gle spores were observed. They were situated usually on short sporophores or directly = on the hyphae of both the substrate and the aerial mycelia. The spores appeared globular under the light microscope, but they looked angular in scanning electron microscopy because of a pattern of ridges on the spore surface (Fig. 2).

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Fig. 2. Spore surf= ace of strain 21E (PM agar, 48 hour, 10 000x).

The spores of strain 21E resembled in certain features the bac= terial endospores. Being thermostable, they were heat-resistant at 100 ^o=C for 60 min. They also could be colored with stains specific for bacterial endospores, e.g. malachite-green.

Physiological and biochemical properties

Str= ain 21E was capable to grow in the temperature range between 35 and 65 ^{o<=
/sup>C,
and did not grow at 33 ^oC and 67 ^oC. The
optimal growth temperature was from 55 to 60 ^oC. Good growth
occurred in the presence of 3‑7 % and it was weak at 10 % N=
aCl
(Table 2).}

Table 2. Some physiological and biochemical properties of stra= in 21E.

Growth at temperature
(^oC)

Growth in NaCl
(%)

33

-

1

+

35

+

3

+

40

+

5

+

50

+

7

+

55

+

10

±

60

+

12

-

65

+

15

-

67

-

Enzyme production

= Protease

+

= Collagenase

+

= Amylase

+

= Lipase

+

The= carbon metabolism is significant in taxonomy and the differences in the utilizatio= n of various carbon sources serve as additional criteria for species differentia= tion. ISP considers the utilization of nine sugars [25]. The investigation of thermophilic actinomycetes requires additional carbon sources to be tested besides the specific for the rest of the actinomycetes.

The= growth of strain 21E was weak on ISP-9 [25] regardless the tested carbon sources (Tab= le 3). Only glucose, fructose, arabinose, and mannitol weakly stimulated its growth. The strain grew scantly on media with xylose, ramnose, inositol and raffinose. It did not assimilate sucrose, lactose, maltose, and cellulose.<= /span>

Table 3. Comparison of strain 21E with Thermoactinomyces sacchari and T. vulgaris.

C= haracteristics

Strain 21E

T. sacchari

T. vulgaris

Aerial mycelium

Abundant

-

-

+

Transient

+

+

-

Lysis

disappearing within
3 to 4 days

rapid, within 3 days

not seen

Color

white or cream-white

white

white

Substrate mycelium

Color

beige, cream to dark beige

colorless to cartridge buff

colorless to brown

Spores

Single spores on aerial/ substrate mycelium

+

+

+

Spores – endospores, with ridged surface

+

+

+

Spores on sporophores

+

+

+

Soluble pigment

Yellow-brown

-

-

-

Growth at

30 ^oC

-

-

-

55 ^oC

+

+

+

NaCl (3 %)

+

+

+

NaCl (5 %)

+

+

-

Novobiocin (25 µg/ml)

+

+

+

Utilization of carbon sources

D-fructose

+

+

+

L-arabinose

+

+

-

L-rhamnose

±

-

-

D-xylose

±

-

-

D-maltose

-

NT

NT

D-mannitol

+

+

+

D-glucose

+

+

+

D-raffinose

±

-

-

Sucrose

-

-

+

I-Inositol

±

-

-

Lactose

-

NT

NT

Degradation of

Casein

+

+

+

Gelatin

+

+

+

Starch

+

+

-

Cellulose

-

-

-

Chitin

-

-

-

Arbutin

±

+

+

Esculine

±

+

ND

Tyrosine

-

-

+

Xanthine

-

-

-

Hypoxanthine

-

-

±

Adenine

-

-

-

Legend: not tested (NT); not= defined (ND).

&= nbsp;

Str= ain 21E caused intensive hydrolysis of starch and casein, but did not decompose chi-tin. It coagulated and peptonized milk. The strain weakly decomposed esculin and arbutin, and did not hydrolyze xanthine, xypoxanthine and adeni= ne. It did not show tyrosinase activity either. The strain hydrolyzed gelatin on the 24^th hour of cultivation. It displayed collagenase and lipase activities.

Str= ain 21E was sensitive to all the antibiotics used in this investigation except for novobiocin (Table 4). The strain did not exhibit any antagonistic activity against B. subtilis and E. coli.

Table 4. Sensitivity to antibiotics of strain 21E.

A= ntibiotic

Concentration (µg/ml)

Sterile zone (mm)

C= hloramphenicol

10

6

A= mpicillin

25

12

G= entamycin

10

25

E= rythromycin

10

28

P= enicillin

10

14

S= treptomycin

10

16

N= ovobiocin

25

0

The= hyphae growth characterized strain 21E as an actinomycete. The established segment= ation of the both substrate and aerial mycelia, the optimum of growth in the inte= rval 55‑60 ^oC, the formation of spores with the characteristics of bacterial endospores, and the biochemical properties determined strain 21E as a representative of the genus Thermoactinomyces. It was compared with other species of this g= enus (Table 3) and was found to be phenotypically close to T. sacchari and T. = ;vulgaris. The presented data showed that strain 21E was much more similar to T. sacchari from which it differed= by the weak utilization of some sugars.

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References

1. Agre, N. S., 1986. Systematics of the termophilic actinom= ycetes, Pushtino: ONTI NCBI Acad. Sci. USSR (in Russian).

2. Baldacci, E., G. F. Comaschi= , T. Scotti, C. Spalla, 1953. 6th International Congress in Microbiology, 6-12 Sept., Rome, 20‑39.

3. Bergey’s manual of determinative bacteriology, J. Holt, N. Krieg, P. Snea= th, J. Staley, S. Williams (Eds), Ninth Edition, Baltimore-Philadelphia-Hong Kong: Williams & Wilkins, 2344-2347.

4. Blair, J., E. Lennett, J. Tr= uant, 1970. Manual of clinical microbiolo= gy, Baltimore: Williams & Wilkins.

5. Bondartsev, A. 1954. Color S= cale, Moscow-Leningrad, Acad. Sci. USSR, 27 (in Russian).

6. Corbaz, R., P. Gregory, M. L= acey, 1963. J. Gen. Microbiology, 32, 449–455.

7. Cross, T., M. Lechevalier, H. Lechevalier, 1963. J. Gen. Microbio= logy, 31, 421‑429.<= /span>

8. Cross, T., A. Maciver, J. La= cey, 1968. J. Gen. Microbiology, 50, 351–359.

9. Daniel, R., H. Toogood, P. Bergquist, 1995. Biotech. Gen. Eng. Re= v., 13, 51‑100.

10. Demina, N. and S. Lysenko, 1= 996. Mikrobiologia, 65, 661–664 (in Russian).

11. Gordon, R., D. Barnet, J. Ha= nderman, C. Pang, 1974. Int. J. Syst. Bacter= iol., 24, 54‑63.

12. Gordon, R., M. Smith, 1955. = J. Bacteriol., 69, 147-150.

13. Gordon, R., J. Mihm, 1957. J. Bacteriol., 73, 15‑27.

14. Greiner-Mai, E., R. M. Kropp= enstedt, F. Korn-Wendisch, H. J. Kutzer, 1987. Syst. Appl. Microbiol., 9, 97= 209;109.

15. Gusterova, A., R. Zvetkova, = P. Christov, R. Rachev, M. Trenev, G. Spassov, 1997. Compt. Rend. Acad. Bulg. Sci., 50 (9‑10), 75̴= 9;78

16. Kabadjova, P., S. Vlahov, I. Dobrev, 1997. Compt. Rend. Acad. Bulg. Sci., 50 (2), 95–98.

17. Kuo, M., P. Hartman, 1966. J. Bacteriology, 92 (3), 199–202.

18. Kurup, P., J. A. Schmitt, 19= 73. Can. J. Microbiol., 19, 1035-1048.

19. Kurup, V., J. Barboriak, J. = Fink, M. Lechevalier, 1975. Int. J. Syst. Bacteriol., 4, 150‑1= 54.

20. Lee, J., Y.Kim, H. Kim, Y. P= ark, T. Oh, 1996. Biosci. Biotech. Biochem.= , 60 (5), 840–846.

21. Lingappa, Y., J. Lockwood, 1= 962. Phytopathology, 52, 317‑323.

22. Loginova, L., M. Yakovleva, = I. Usayte, 1978. Mikrobiologia, 47 (4), 653–658 (in Russ= ian).

23. Matsushita, O., K. Yoshihara= , S. Katayama, J. Minami, A. Okabe, 1994. J. Bacteriol., 1, 149–1= 56.

24. Petrova, D., S. Vlahov, P. D= alev, 2001. Biotechnol.& Biotechnol. = Eq., 15 (2), 57–64.

25. Sasagava, Y., Y. Kamio, Y. Matsubara, Y. Ma-tsubara, K. Suzuki, H. Kojima, K. Izaki,. 1993. Biosci. Biotech. Biochem., 57 (11), 1894–1898.

26. Shirling, E., D. Gottlieb, 1= 966. Int. J. Syst. Bacteriol., I., 16= (3), 313‑340.

27. Stutzenberger, F., 1987. J. Appl. Bacteriol., 63, 239–244.

28. Todorov, O., S. Vlahov, 1993. Ann. Uni= v. Sofia, 82 (3), 65‑73= .

29. Tshuchiya, K., Y. Nakamura, = H. Sakashita, T. Kimura, 1992. Biosci. Biotech. Biochem., 56 (2), 246–250.

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ТАКС&#= 1054;НОМИЧНА ХАРАКТЕРИСi= 8;ИКА НА ТЕРМОФИЛ = 53;ИЯ АКТИНОМИЦЕi= 8;ЕН ЩАМ 21Е – ПРОДУЦЕНТ Н = 40; ТЕРМОСТАБИi= 1;НА КОЛАГЕНАЗА=

=

Детели&#= 1085;а Петрова*, Стоян Влахо = 74;

= Резюме

&= #1058;ермофилния= ;т актиномицеm= 0;ен щам 21Е, изолир= ан от солени почви в България, продуцира в = 80;соко термостабиl= 3;на колагеназа. Проучени са макро- и микроморфоl= 3;огичните характерисm= 0;ики на щама върх= 091; 14 среди. Изследвани са някои физиологичl= 5;и и биохимичн = 80; свойства, ен= 079;имни активности, както и антибиотичl= 5;ата активност и чувствителl= 5;ост. Установено = 77;, че щам 21Е е типичен представитk= 7;л на рода Thermoactinomyces. Сl= 8;авняването с видове Thermoactinomyces с близки таксономичl= 5;и характерисm= 0;ики определя щама като Thermoactinomyces sacchari. Щам T. s= acchari 21E е устойчив на антибиотикk= 2; новобиоцин = 80; показва протеазна, амилазна, колагеназнk= 2; и липазна активности.

N= utrient medium	Growth	Color of the aerial mycelium	Color of the substrate mycelium	Soluble pigment
Y= east-malt extract agar (ISP-2)	abundant	cream-white	beige	no
O= atmeal agar (ISP-3)	moderate	white	cream	no
S= tarch agar with inorganic ions (ISP-4)	moderate	white or cream-white	dark beige	no
G= lycerol-asparagine agar (ISP-5)	scanty	no	yellow-brown	no
P= eptone-yeast extract iron agar (ISP-6)	abundant	cream-white	beige	no
T= yrosine agar (ISP-7)	no	no	no	no
P= M agar	abundant	cream-white	dark beige	no
N= utrient agar with glucose	scanty	white	cream	no
Y= east extract agar	abundant	pale beige	dark beige	no
G= lucose yeast extract agar	moderate	ash	beige	no
M= ilk agar	abundant	white	cream	no
P= C agar	abundant	cream-white	dark beige	no
P= otato-glucose agar	moderate	white	dark beige	no
C= hitin agar	no	no	no	no

Growth at temperature (^oC)	Growth in NaCl (%)
33	-	1	+
35	+	3	+
40	+	5	+
50	+	7	+
55	+	10	±
60	+	12	-
65	+	15	-
67	-
Enzyme production
= Protease	+
= Collagenase	+
= Amylase	+
= Lipase	+

C= haracteristics	Strain 21E	T. sacchari	T. vulgaris
Aerial mycelium
Abundant	-	-	+
Transient	+	+	-
Lysis	disappearing within 3 to 4 days	rapid, within 3 days	not seen
Color	white or cream-white	white	white
Substrate mycelium
Color	beige, cream to dark beige	colorless to cartridge buff	colorless to brown
Spores
Single spores on aerial/ substrate mycelium	+	+	+
Spores – endospores, with ridged surface	+	+	+
Spores on sporophores	+	+	+
Soluble pigment
Yellow-brown	-	-	-
Growth at
30 ^oC	-	-	-
55 ^oC	+	+	+
NaCl (3 %)	+	+	+
NaCl (5 %)	+	+	-
Novobiocin (25 µg/ml)	+	+	+
Utilization of carbon sources
D-fructose	+	+	+
L-arabinose	+	+	-
L-rhamnose	±	-	-
D-xylose	±	-	-
D-maltose	-	NT	NT
D-mannitol	+	+	+
D-glucose	+	+	+
D-raffinose	±	-	-
Sucrose	-	-	+
I-Inositol	±	-	-
Lactose	-	NT	NT
Degradation of
Casein	+	+	+
Gelatin	+	+	+
Starch	+	+	-
Cellulose	-	-	-
Chitin	-	-	-
Arbutin	±	+	+
Esculine	±	+	ND
Tyrosine	-	-	+
Xanthine	-	-	-
Hypoxanthine	-	-	±
Adenine	-	-	-

A= ntibiotic	Concentration (µg/ml)	Sterile zone (mm)
C= hloramphenicol	10	6
A= mpicillin	25	12
G= entamycin	10	25
E= rythromycin	10	28
P= enicillin	10	14
S= treptomycin	10	16
N= ovobiocin	25	0