Stefka Antonova-Nikolova*, Nikoleta Tzekova and Ljubomira Yocheva

Sofia University, Biological Faculty, Department= of General and Industrial Microbiology,

8 Dragan Tzankov st., 1164 Sofia, Bulgaria<= /o:p>

Summary

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Strain Streptomyces sp. 3B produces high activity extracellular proteolytic comple= x, in which serine- and metalloproteases are identi= fied. The general and some other significant properties for the taxonomy of the s= train were studied by the methods of the International Streptomycetes Project. The results of the investigation of Streptomyces sp. 3B and the comparative references about Streptomyces species with similar taxonomic characteristics identified it as Streptomyces albovinaceus. The antibiotic activity and sensitivity was also tested. Strain 3B was resistant to the group of the penicillin antibiotics.

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Introduction

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Proteases constitute one of the most important groups of industrial enzymes and find applications in the production of deter-gents, foods, leat= her, silk, chemical and agricultural preparations [3, 12]. In the last years they are used in medicine successfully. This enzyme accounts for 60 % of the world market.

The streptomycetes produce numerous compounds (secondary metabolites), within w= hich antibiotics are of commercial relevance. As corresponds to their habitat, t= hese bacteria are nutritionally quite versatile and the most produce extracellul= ar hydrolytic enzymes that permit the utilization of high molecular weight biopolymers such as proteins, polysaccharides, fats and other substrates.

St= reptomycetes are great producers of proteolytic enz= ymes. The composition of these protein complexes is determined by the taxonomic belonging of the producer. The present paper as a part of a compl= ex investigation of strain Streptomyce= s sp. 3B and the produced proteolytic enzymes describes its taxonomic determination and biological characterization [4].

Materials and methods

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The object of the investigation, Streptomyces strain = 3B from the collection of the Department of General and Industrial Microbiolog= y, was isolated from protein rich samples, collected around Sevlievo. Its proteolytic activity was clearly expressed. Strain Streptomyces sp. 3B produces extra-cellular enzymes from which serine- and metalloproteases <= /span>were established [1, 7].

Ta= xonomic properties of the strain were determined according to the methods and media= of the International Streptomycetes Project (ISP) [9]. The morphology of the strain was studied in yeast-malt extract agar (ISP-2), oatmeal agar (ISP-3), starch agar with inorganic salts (ISP-4) and glycerol-asparagine agar (ISP-= 5). The production of the melanin was tested on peptone-yeast extract iron agar (ISP-6) and tyrosine agar (ISP-7).

Th= e utilization of carbon sources was carried out according to the modified method of Pridh= am and Gottlieb (isp-9) with addition of the following sugars: D-glucose (positive control), L-arabinose, sucrose, D-xylose, I-inositol, D-mannitol, D-fructose, rhamnose, raffinose = and cellulose, and in absence of carbon source (negative control).

The form of the sporoph= ores was studied with light microscope by directly observation and by replica’s method. The morphology of the spores and the spore chains were investigated= by electron microscope.

The= color of the aerial and vege= tative mycelia was determined according to Bondartsev color scale [2]. The morphological determination of the strain w= as made on mature culture.

The production of the m= elanin was observed on 2^d and 4^th day. The results from utilization of the carbon sources were measured on 10^th and 16en day. Species identification of s= train 3B was based on Nonomura’s key [8] and the species description was according to ISP [10, 11].

In addition the following biological characteristics were studied: decompositi= on of gelatin, hydrolysis of starch [5]; growth on milk agar, effect to the mi= lk, antibiotic activity against test-microorganisms Bacillus subtilis and E= scherichia coli according to the method of the agar plates, sensitivity to antibiotics by p= aper disks method [6]. The following antibiotics (“BulBio”) were tested: erythromycin (15 μg/disk), ampicillin (10 μg/disk), streptomycin (10 μg/ disk), gentamycin (10 μg/disk), penicillin (10 Е/ disk) and chlorampheni= col (30 μg/disk).

Results and discussion

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Micromorphological characteristics. Vegetative mycelium of the studied = strain Streptomyces sp. 3B did not fragme= nt. Aerial mycelium formed monopodially si= tuated sporophores with right or lightly curved shape. The sporophores contained more than 10 spores. The spores were elongated with smooth surfac= e (Fig. 1).

Fig. 1. Spore chains and spores of Streptomyces <= span style=3D'mso-bidi-font-style:italic'>sp. 3B: A. Forms of the = spore chains (ISP-2, 300x, 14^en day);
B. Spore number (ISP-3, 6000x, 14^en day); C. Spore surface (ISP-3, 20000x, 21^st day).

Macromorphological characteristics. = Streptomyces sp. 3B was clearly polymorphic.= The strain formed three types co= lonies on the investigated media: round flat colonies with unfledged aerial myceli= um and white concentric lines, smooth surface and edge; completely covered by = the aerial mycelium formless protruding colonies with rugged surface, undulating edge and slightly convex central part; big good formed round colonies with concentric folds around the center of the colony and a fosse or umbilicus in the center, the margin of the colony was entire or fibrillated depending on= its age.

Cultural properties. On more media Streptomyces sp. = 3B showed a good development. The color of= the aerial mycelium was white. It changed from white to gray-white opal pink depending on the medium. The coloring= of the vegetative mycelium varied from pale-yellow to yellow-brown according to the cultural age (Table 1). <= /b>The strain did not form melanoid pigment on = any of the investigated for melanin media. Other soluble pigments were not observe= d.

Physiological and biochemical properties. <= span lang=3DEN-US style=3D'font-size:12.0pt;letter-spacing:.1pt'>The utilization= of carbon sources is shown in Table 2. The strain 3B grew very well on mineral-salt media, containing D-glucose, L-arabinose, D-xylose, D-fructose, rh= amnose and raffinose. On medium with sucrose the growth was weak or absent. The st= rain did not assimilate I-inositol, D-mannitol and cellulose. It did not hydroly= ze starch but decomposed gelatin. The strain 3B caused peptonization of milk b= ut did not its coagulation. It did not attack tyrosine (Table 3).

Table 1. Cultural properties of strain Strep= tomyces sp. 3B.

Medium

Aerial mycelium

Vegetative mycelium

growth

color

growth

color

ISP-2

abundant<= /o:p>

white

abundant<= /o:p>

yellow-brown

ISP-3

moderate

white

moderate

yellow-brown

ISP-4

moderate

pale pink

moderate

yellow-brown

ISP-5

poor

gray-white

poor

yellow-brown

Table 2. Utilization of carbon sour= ces by strain Streptomyces sp. 3B.

Carbon= source

Utilization stage

D-glucose (positive control)=

++

L-arabinose

++

D-fructose

++

D-xylose

+

Rhamnose

+

I-inositol

-

D-mannitol

-

Sucrose

±

Raffinose

++

Cellulose

-

Withou= t carbon sources (negative control)

-

Table 3. Physiological characterist= ics of strain Streptomyces sp. 3B.

Charac= teristics

Activity

Melanin produc= tion

-

Tyrosinase activity

-

Starch hydrolysis

-

Gelatin liquefaction

+

Peptonization of skim milk

+

Coagulation of skim milk

-

Cellulose activity

-

Temperature range of growth<= /p>

18-45 °C

Antagonistic properties and sensitivity to antibiot= ics. Streptomyces <= /i>sp. 3B did not possess antibiotic a= ctivity against B. subtilis and E. coli. It was sensitive to the m= ore antibiotics used in this investigation. The strain was resistant to the act= ion of β-lactame antibiotics (Table 4).

Taxonomic comments. Using references given by = ISP [10, 11], strain Streptomyces s= p. 3B was compared to Streptomyces cu= ltures similar by morphological, cultural, physiological and biochemical properties (Table 5). More Streptomyces strains assigned to the whi= te series show differences as on the degree of the utilization of the sugars, as well= as on the morphological and cultural properties from Streptomyces sp. 3B strain.

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Table 4. Sensitivity of strain Streptomyces sp. 3B to different antibiotics.

Antibiotics

Diameter of sterile zone (mm)

Erythromycin

20

Ampicillin

-

Streptomycin

12

Gentamycin

12

Penicillin

-

Chloramphenicol

8

Table 5. Comparison of general taxonomyc properties of Streptomyces sp. 3B strain with re= lative streptomycetes spesies.

Streptomycetes species

Color of aerial mycelium

Melanoid pigments

Color of vegetative mycelium=

Soluble pigments

Spore chain

Spores in the spore chain

Spore surface

L-arabinose

D-xylose

I-inositol

D-mannitol

D-fructose

Rhamnose

Sucrose

Raffinose

Streptomyces sp. 3В=

white

-

pale yellow yellow-brown

-

Recti-Flexibilis (RF)

> 10

smooth

+

+

-

-

+

+

±

+

Streptomyces&= nbsp; orientalis

white, blue

-

colorless, yellowish

-

Recti-Flexibilis (RF)

> 50

smooth

+

+

+

+

+

+

-

-

Streptomyces albovinaceus

white

-

colorless, yellowish

-

Recti-Flexibilis (RF)

> 10

smooth

+

+

-

+

+

+

-

-

Streptomyces candidus

white

-

colorless, yellowish

-

Recti-Flexibilis (RF)

> 50

smooth

+

+

-

+

nt

+

-

-

Streptomyces sindenensis=

white, red or yellow

-

colorless, yellowish

-

Recti-Flexibilis (RF)

3 > 10

smooth

+

+

-

+

+

-

-

-

The Streptomyces strain 3B is much closed to S. albovinaceus [10]. Similarities: both Streptomyces sp. 3B and S. albovinaceus belong to section Recti-Flexibilis (RF); the mature spore chain contains more than 10 spores with smooth surface; the co= lor of the aerial mycelium is white; the vegetative mycelium is yellowish; the = strains do not produce melanin and other soluble pigments; they grow ver= y well on medium containing L-arabinose, D-xylose, D-fructose and rhamnose; they do not grow on medium with I-inositol and grow weakly on sucrose; Differences:= Streptomyces strain 3B does not sh= ow growth on medium with D-mannitol but on medium containing raffinose it grows very well.

Streptomyces sp. 3B is similar to S. orientalis [10] by morphol= ogical properties and by the utilization of some sugars. In contrast to strain 3B,= S. orientalis grows on media containing I-inositol and D-mannose but does not utilize raffinose. S. orientalis can form blue aerial mycelium on ISP-3 and its spore chain contains more than 50 spores.

Th= e Streptomyces strain 3B also resemb= les to S. candidus [11]. The differences are that S. candid= us has spore chain with more than 50 spores; it grows very well on media with D-mannitol and does not utilize raffinose.

Morphologically, Streptomyces sp. strain 3B is simi= lar to S. sindenesis [11] from wh= ich differs by the length of the spore chain and by the coloring of the aerial mycelium. The mature spore chain of S. sindenesis contains 3 to 10 spo= res. It grows well on media containing D-mannitol but does not utilize rhamnose, sucrose and raffinose.

The results of the inve= stigation of Streptomyces sp. 3B and the comparative references about Strept= omyces species with similar taxonomic characteristics according to ISP [10, 11] ga= ve us the reason to identify it as Str= eptomyces albovinaceus with the name S.&n= bsp;albovinaceus 3B.

= Short characteristic of Streptomyces albovinaceus = 3B. The strain 3B produces monopodially si= tuated right or lightly curved sporophores, fragmenting in spore chains with more than 10 spores. The spore surface is smooth.

The color of the aerial= mycelium is white with different nuances depending on the medium. The color of the vegetative mycelium is pale - yellow to yellow - brown, depending from the age of the culture. The growth temperature range is 18-<= st1:metricconverter ProductID=3D"45 °C" w:st=3D"on">45 °C. Melanoid and other soluble pigments do not produce. The strain liquefies gelatin and peptonizes milk, has not tyrozinase activity and does not hydro= lyze starch. It utilizes D-glucose, L-arabinose, D-xylose, D-fructose, rhamnose = and raffinose but does not utilize I-inositol and D-mannitol. Strain 3B has not antibiotic activity against B. = subtilis and E. coli. It is sensitive to the antibiotics - gentamycin, erhytromycin, streptom= ycin and chloramphenicol. The strain 3B is resistant to the group of the penicil= lin antibiotics.

Acknowledgments. We express our thanks to Vassilis K= apurdov from the National Center of Hygiene for the generous supply of electron microscope.

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References

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1.<= span style=3D'font:7.0pt "Times New Roman"'>    Antonova, S., P. Angelova, P. Ivanova, S. Dimov, D. Petrova, I. Ivanova, 2002= . Tenth congress of the Bulgarian Microbiologists with International Participation,<= span lang=3DEN-US style=3D'font-size:12.0pt'> 9-12 Oct., Plovdiv, Program and Ab= stracts, GAM41.

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4.&n= bsp;   Gauze, G., T. Preobrazhenskaja, M. Sveshnikova, L. Ter= ehova, T. Maksimova, 1983. Guide of Actin= omycetes, Moskva: Nauk= a, 248 (in Russian).

5.<= span style=3D'font:7.0pt "Times New Roman"'>    Gerhardt, F., R. Murry, R. Costilow= , H. Nester, W. Wood, N. Krieg, G. Phillips, 1984= . Manual of Methods for General Bacteriology, v. 3, Moskva:Mir, 263 (in Russian). =

6.<= span style=3D'font:7.0pt "Times New Roman"'>    Gushterov, G.,= P. Andonov, Ts. Todorov, L. Kominkov, M. Gincheva, 1986. Manual of Microbiology and Virology, Sofia: Nauka i izkustvo, 3= 93.

7.<= span style=3D'font:7.0pt "Times New Roman"'>    Petrova, D., P. Angelova, S. Antono= va-Nikolova, 2003. Biotechnol. Biotechnol. Equip= ment, 17 (2), 57-65.

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Medium	Aerial mycelium	Vegetative mycelium
growth	color	growth	color
ISP-2	abundant<= /o:p>	white	abundant<= /o:p>	yellow-brown
ISP-3	moderate	white	moderate	yellow-brown
ISP-4	moderate	pale pink	moderate	yellow-brown
ISP-5	poor	gray-white	poor	yellow-brown

Carbon= source	Utilization stage
D-glucose (positive control)=	++
L-arabinose	++
D-fructose	++
D-xylose	+
Rhamnose	+
I-inositol	-
D-mannitol	-
Sucrose	±
Raffinose	++
Cellulose	-
Withou= t carbon sources (negative control)	-

Charac= teristics	Activity
Melanin produc= tion	-
Tyrosinase activity	-
Starch hydrolysis	-
Gelatin liquefaction	+
Peptonization of skim milk	+
Coagulation of skim milk	-
Cellulose activity	-
Temperature range of growth<= /p>	18-45 °C

Antibiotics	Diameter of sterile zone (mm)
Erythromycin	20
Ampicillin	-
Streptomycin	12
Gentamycin	12
Penicillin	-
Chloramphenicol	8