JCC0753

JOURNAL OF CULTURE COLLECTIONS

Volume 5, 2006-2007, pp. 16-24

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Protease activity of some mesophilic streptomycetes
isolated from Egyptian habitats

Hala M. Rifaat¹⁼*, Osama H. El-Said², Sadia M. Hassanein³ and Manal S. M. Selim²

¹Microbial Chemistry Department, National Research Centre, Bohos Street, Cairo, Eg= ypt,

²Microbial Biotechnology Department, National Resea= rch Centre, Cairo, Egypt;

³Microbiology Department, Ein Shams University, Cairo, Egypt

*Corresponding author, e-mail: halamohamed6@hotmail.com

Summary

Different streptomycetes (317 isolates) were obtai= ned from several sources and areas in Egypt and were screened for proteolytic activity. Thirty nine of them produced proteases and were subje= cted to identification. Streptomyces anulatus formed the most abundant portion of the isolates. This species deserves special attention because it is a good candidate for biotechnological applications.

Key words: Egypt, identification, isolation, protease, streptomyce= tes.

Introduction

Proteases are one of= the three largest groups of industrial enzymes. They account for nearly 60 = ;% of the total enzyme sales covering about 20 % of the world market, and they are used mainly in detergents [15, 26, 37]. Proteases possess high catalytic activity and substrate specificity. They can be industrially produced in large quantities and are economically essential for the deterge= nt, protein, brewing, meat, photographic, leather, and dairy industries.

Proteases are common enzymes in plant and animal tissues, fungi, and bacteria. Microorganisms are the preferred proteases producers, as they grow rapidly, require small cultivation space, and can easily be subjected to genetic manipulation. Bacterial proteases are industrially the most significant compared to animal and fungal proteases [37].

The possible use of streptomycetes for enzyme production has recently been investigated, and proteases have been obtained from various species [2, 21]. The streptomycetes comprise Gram-positive bacteria of high G+C content and unus= ual morphological complexity, which develop in their life cycle substrate and aerial mycelia, sporophores and spores [5]. Several proteases were obtained from streptomycetes and were biochemically characterized, such as serine protease produced by Streptomyces p= actum, metallo- and serine proteases from Str. exfoliatus, and aminopeptidase from Str. rimosus [3, 17, 18, 36]. These enzymes= are involved in the assimilation of proteinaceous nitrogen sources, degradation= of aerial mycelium sporulation process, as well as in antibiotic production [16, 19]. In Eg= ypt, high quantities of proteases are imported from abroad and it was found of g= reat interest and of economic importance to produce industrially these enzymes by use of microorganisms.

The aim of the prese= nt investigation was to isolate and characterize mesophilic streptomycetes str= ains and test them for proteolytic activity. The Streptomyces isolates that showed considerable proteolytic activity were taxonomical= ly identified.

Materials and Methods

Sampling and sampling sites. Samples were collected from three different types = of sources from five Eg= ypt governorates. The in-dustrial source was taken from Cairo Tanneries sewage = (T) and soil around it (Ts), the agricultural soil from Plant Island at = Aswan (As) and agricultural field at El-Sharkia (Es). The water sources were obtained from a fish farm at Port Said (Fw), sediments with neutral (Fs) and alka= line pH (Fs*), and Qaroun Lake water (Q) at El Fayoum.

Isolation of streptomycetes. The serial dilution method was applied for isolati= on of streptomycetes, and each sample was diluted to 10^‑6 [12= ]. Eight agar media were used for isolation as follows: starch-nitrate [24], malt-yeast extract [29], Difco actinomycetes isolation, brain-heart infusion (BHI) [22], modified glucose-aspartic acid-ammonium nitrate, modified starch-aspartic acid-ammonium nitrate [8], lactose-peptone [6] and maltose-leucine-lycine [20]. 0.1 ml inoculum of the appropriate dilution was placed on each plate. The plates were incubated at 28 ^oC fo= r 7‑14 days to allow the slow growing forms to develop. Streptomycetes were isolat= ed based on their specific morphological characteristics and then subjected to purification.

Screening for proteolytic activity. The streptomycete isolates were screened for their ability to produce protease. The enzyme activity was determined by three different methods. Extracellular protease production was assess-ed by the s= ize of the clear zone as poor (≤1), fair (≤2), good (≤3) and = very good (>3) on 3 % brain heart infusion supplemented with gelatin [22] and on egg-yolk agar [25]. The ability of the isolates to liquefy gelatin w= as rated as well [10].

Identification of streptomycetes. The active streptomycetes isolates were taxonomica= lly identified through morphological, physiological and chemotaxonomical investigations. The following tests were carried out: colony and micromorphological characteristics, pigment production, lecithinase, lipoly= sis, proteolysis, hydrolysis of pectin, chitin, hipurate, degradation of xanthin= e, elastine, arbutin, utilisation of sucrose, I-inositol, D-fructose, xylose, galactose, glucose, L-arabinose, rhamnose, D-mannitol, raffinose, NO_{3<=
/sub>^-reduction, H₂Sproduction, whole cell sugar pat=
tern
and cell wall chemotype [13, 33, 34, 38].}<= /p>
Statistical analysis. The streptomycetes isolates showing proteolytic activity were subjec= ted to all the tests listed. The obtained data were examined for normality and homogeneity of variance. Analysis was done using the SPSS software package = for the dendrograms generation [31], similarity calculations were based on simp= le matching (S_SM). The UPGMA algorithm was applied for dendrogam generation.

Results and Discussion

A wide range = of methods can be applied to detect proteases using gelatin as a substrate [11= ]. Another approach is based on substrate hydrolysis as an evidence for gelatinase-like proteases production [4]. Egg yolk is routinely used in proteolytic activity detection test systems [1].

A total of 317 streptomycete isolates were obtained on the eight different isolation media from the various collected samples. All of them were screen= ed for protease production on 3 % BHI supplemented with gelatin, gelatin = and egg-yolk agar medium. 39 isolates demonstrated proteolytic activity (Table 1). They originated from all the studied samples except those f= rom Cairo Tanneries sewage (T). The activity was the best on BHI with gelatin, = and it was moderate on the other two media what conformed to the results of Vermelho et al. [35] who reported that streptomycetes hydrolyzed preferenti= ally gelatin incorporated in BHI. As actinomycetes are known to be good protease producers [9, 27], the protein substrate and the composition of the me= dium could markedly influence the extracellular protease production [35]. Differences in the ability to utilize various protein substrates may be due= to substrate specificity of the produced enzyme [28]. Gelatin, being a type&nb= sp;1 collagen, is the most effective protease inducing substrate. It is possible that the gelatin, as a high molecular weight protein, increases the protease production to degrade the substrate to a suitable form for the microorganis= ms.

The methods described above are quite relevant for detecting extracellular protease directly in the culture medium. The use of gelatin in the culture medium provides a qualitative ass= ay, which is a simple, inexpensive, straight-forward method to assess the proteolytic activity of a given microbial colony. Thus, in addition, substr= ate selection is comfortable since low molecular weight (egg yolk) or high molecular weight (gelatin) proteins could be used. Consequently, simplicity= is the greatest advantage of these methods, although they cannot be used for quantitative analyses.

T= able 1. Proteolytic activity of some mesophilic streptomycetes isolated on diffe= rent media.

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Medium

Number of strains and activity

Poor

Fair

Good

Very good

3 % BHI + Gelatine

2=

Es 48, 112

10=

Es 4, 24, 37, 127
As 81; Fs 1, 19
Fw 13, 14

Q1

18

Es 5, 81, 119, 130

As 8, 10, 45, 49, 53, 71, 75,= 77

Fs 3, 13, 16

Fw 11; Fs*4

Ts 3

9=

Es 22, 117
As 22, 94
Fs 35; Fs*5
Ts 5, 6, 9

Gelatine medium

9

Es 24, 37, 48, 112, 117

As 8, 81
Fs* 4, 5

23

Es 4, 5, 22, 81, 119, 127, 13= 0

As 10, 22, 49, 53, 71, 77, 94=

Fs16

Fw 11, 13, 14
Ts 3, 5, 6, 9

Q1

7

As 45, 75
Fs 1, 3, 13, 19, 35

None

Egg-yolk agar

11

Es 22, 81, 112, 130

As 8, 22, 45, 75
Fw 11, 13

Ts3

15

Es 4, 117, 119
As 10, 49, 53, 71, 81

Fs 13, 19

Fw14
Ts 5, 6, 9

Q1

13

Es 5, 24, 37, 48, 127

As 77, 94
Fs 1, 3, 16, 35

Fs* 4, 5

None

The isolates = that showed proteolytic activity were characterized and identified. The results = of the morphological, physiological and chemotaxonomical tests presented in Ta= bles 2 and 3 were subjected to statistical analysis (SPSS) and a dendrogram of t= he active streptomycetes isolates was plotted (Fig. 1). Using the determinative keys [13, 33, 34, 38] different Streptomyces species were identifi= ed. The cell walls of all the strains contained the diagnostic amino acid LR= 09;DAP and did not contain diagnostic sugars. This is characteristic for the speci= es of genus Streptomyces.

The most frequently isolated streptomycetes (43.6 %) were identified as Str. anulatus (Fig. 2). = The strains in this group belonged to the yellow and grey colour series, and fo= rmed spores with smooth surface arranged in rectiflexibiles or occasionally spir= al chains. As a rule, they did not produce melanoid pigments, with a few posit= ive exceptions, particularly on tyrosine agar. This phenotypically variable spe= cies is widespread in nature where decaying organic matter is present. Based on = the phenotypic scheme of Williams et al. [38], Str. alboniger, Str. aureofaciens and Str. griseus are nomen species of = Str. anulatus, and all they p= roduce extracellular proteases [20, 23, 32].

It is intrigu= ing that the closest phenotypic relative of Str. anulatus, Str. albidoflavus was also detected (10.3 %). The two clusters were connected at 80 % S_=
SM, and that was in accordance with the results of Williams et al. [38] who reported the level of similarity at 77.5 %. Most strains= in this group had rectiflexibiles spore chains and smooth spore surface. Melan= oid pigments were rarely produced. The spore colour was yellow or sometimes whi= te. Proteases have been detected in cultures of Str. albidoflavus [16, 30]. The genome sequencing of Str. coelicolor, subjective synonym of Str. albidoflavus, reveals a multide of putative protease genes [14].

Five strains (12.8 %) were identified as St= r. microflavus. Their sporophores were rectiflexibiles or spiral, the spore surface was smo= oth and the spore mass was grey. Most strains produced melanoid pigments.<= /o:p>

Another clust= er was identified as Str. exfolia= tus (5.1 %). These strains also had rectiflexibiles spore chains, smooth s= pore surface, and the spore mass was grey. Some strains produced melanoid pigmen= ts. Kim et al. has mentioned that Str.&= nbsp;exfoliatus produces proteases. Single member phenons were also detected as Str. lydicus (5.1 %), Str. chromofuscus (5.1 %= ), and Str. lavendulae (2.6 = %). Two small clusters were identified as Str. atroolivaceus and Str. violaceus (5.1 % each). Only two isolates (5.1 %) could not be identified at species-level and remained to be refferred as Streptomyces sp. All of the Streptomyces species studied possessed rectiflexibiles or spiral spore chains, the spore surface was smooth, spiny or hairy, and the spore m= ass was yellow or grey. Melanoid pigments were produced by all but few of the strains. All the identified Strepto= myces species are well known as active producers of proteases [7, 16].<= /p>
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T= able 2. Morphological characteristics of some streptomycetes isolates.

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Characteristics

Number of positive strains

Spore chain morphology

Closed spiral

Flexible

Flexible, straight=

Straight

Straight, flexible=

Hooks

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=

1

15

12

4

3

4=

<= span lang=3DEN-US style=3D'mso-bidi-font-size:12.0pt;line-height:115%;font-fam= ily: "Times New Roman";letter-spacing:0pt;mso-ansi-language:EN-US'> =

=

Es4

Es 5, 37, 81, 112, 117, 130; As 49, 53, 75; Fs 3, 13, 16, 19, 35; Fw14

Es24; As 8, 71, 77, 81, 94; Fw13; Ts 3, 5, 6, 9;= Q1

Es 22, 48, 127; As45

Es119; As10; Fs*4

As22; Fs1; Fw11; Fs*5

Spore surface ornamentation

Smooth

Spiny

Hairy

34

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4

1

Es 4, 5, 22, 24, 37, 48, 81, 127, 130; As 8, 10,= 22, 45, 49, 71, 77, 81, 94; Fs 1, 3, 13, 16, 19, 35; Fw 11, 13, 14; Fs* 4, 5;= Ts 3, 5, 6, 9; Q1

Es 112, 117, 119; As75

As53

Colour of spore mass

Medium grey

Light grey

Dark grey

Grey

Yellow

Green

White

Blue

Pink

=

4

11

3

5

4

3

4

1

4

Es 4, 5, 48; As81

Es 24, 81, 112, 117; As 10, 45, 77; Fs 1, 13,19; Fw11

As 8, 22, 53

Es37; As71; Fs16; Ts 3, 6

Es 22, 130; Fs 3, 35

Es 119, 127; Fw14

As49; Ts 5, 9; Q1

As 75

As94; Fw13; Fs* 4, 5

Pigmentation of
substrate mycelium

Yellow brown

Yellow

Dark yellow brown

Dark brown

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36

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1

1

1

Es 4, 5, 22, 24, 37, 48, 81, 112, 117, 127, 130;= As 8, 10, 22, 45, 49, 53, 71, 75, 77, 81; Fs 1, 3, 13, 16, 19, 35; Fw 11, 13, 14; Fs* 4, 5; Ts 5, 6, 9; Q1

Es119

As94

Ts3

Diffusible pigments

Yellow

Citrin yellow

Orange=

Citrin orange

Orange citrin

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6

1

1

1

3

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Es22; As 71, 75; Fs 3, 35; Ts9=

Es119

Fw11

Es130

As 49, 94; Fs16

Table 3. Physiological characteristics of streptomycetes isolates.=

Characteristics

Number of strains

Positive

Negative

Utilization of
carbon sources

L-Arabinose

D-Fructose

Sucrose

Rhamnose

20

D-Mannitol

D= -xylose

R= affinose

I= -inositol

Galactose

22

28

27

23

33

28

13

15

37

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Es 4, 24, 37, 48, 81, 112, 117; As 10, 22, 45, 5= 3, 71, 77

Fs 1, 13, 16, 35; Fw13; Ts 3, 5, 6, 9=

Es 4, 5, 22, 24, 37, 81, 112, 127, 130; As 10, 2= 2, 45, 49, 53, 71, 75, 81; Fs 1, 3, 13, 16, 19, 35; Fw 11, 14; Fs*5; Ts3; Q1=

Es 4, 5, 24, 37, 81, 112, 117, 130; As 8, 10, 22, 49, 53, 71, 75, 77, 81; Fs 13, 16, 35; Fw 11, 13; Ts 3, 5, 6, 9; Q1<= /o:p>

Es 4, 5, 24, 37, 81, 117, 119, 127, 130; As 8, 1= 0, 22, 45, 53, 71, 75, 77, 81; Fs 1, 16, 19; Fw13; Ts3

Es 4, 5, 22, 24, 37, 48, 81, 117, 119, 127; As 8, 10, 45, 49, 53, 71, 75, 77, 81, 94; Fs 1, 3, 13, 16, 19, 35; Fw 11, 14

Ts 3, 5, 6, 9; Q1

Es 4, 22, 24, 37, 48, 112, 117, 119, 127; As 8, = 10, 22, 49, 53, 71, 75, 77, 81, 94; Fs 16, 35; Fw 11, 13, 14; Fs* 4, 5; Ts3; = Q1

Es 37, 48, 81, 119; As 8, 10, 22, 71, 75, 77; Fs= 19; Fw11; Ts3

Es 4, 5, 24, 81, 112, 117, 130; As 22, 53, 75, 7= 7, 81

Fs1; Fw11; Ts3

Es 4, 5, 22, 24, 37, 48, 112, = 117, 119, 130; As 8, 10, 22, 45, 49, 53, 71, 75, 77, 81, 94; Fs 1, 3, 13, 16, = 19, 35; Fw 11, 13, 14

Fs* 4, 5; Ts 3, 5, 6, 9; Q1

17

11

12

16

6

11

26

24

2

Es 5, 22,119, 127, 130; As 8, 49, 75, 81, 94; Fs= 3, 19

Fw 11, 14; Fs* 4, 5; Q1

Es 48, 117, 119; As 8, 77, 94; Fw13; Fs*4; Ts 5,= 6, 9

Es 22, 48, 119, 127; As 45, 94; Fs1, 3, 19; Fw14; Fs* 4, 5

Es 22, 48, 112; As 49, 94; Fs 3, 13, 35; Fw 11, = 14; Fs* 4, 5 Ts 5, 6, 9; Q1

Es 112, 130; As22; Fw13; Fs* 4, 5

Es 5, 81, 130; As45; Fs 1, 3, 13, 19; Ts 5, 6, 9=

Es 4, 5, 22, 24, 112, 117, 127, 130; As 45, 49, = 53, 81, 94

Fs 1, 3, 13, 16, 35; Fw 13, 14; Fs* 4, 5; Ts 5, = 6, 9; Q1

Es 22, 37, 48, 119, 127; As 8, 10, 45, 49, 71, 9= 4; Fs 3, 13, 16, 19, 35; Fw 13, 14; Fs* 4, 5; Ts 5, 6, 9; Q1

Es 81, 127

H₂S production=

31

Es 4, 5, 22, 24, 37, 48, 81, 1= 12, 117, 119, 127, 130; As 8, 22, 49, 53, 71, 75, 81, 94; Fs 3, 13, 16, 19, 3= 5; Fw 13, 14

Ts 3, 5, 6, 9

8

<= /span>

As 10, 45, 77; Fs1; Fw11; Fs* = 4, 5; Q1

Nitrate reduction

31

Es 5, 22, 24, 37, 112, 119, 12= 7; As 8, 10, 22, 49, 71, 77, 81, 94; Fs 1, 3, 13, 16, 19, 35; Fw 11, 13, 14; Fs= * 4, 5

Ts 3, 5, 6, 9; Q1

8

<= /span>

Es 4, 48, 81, 117, 130; As 45,= 53, 75

Table 3. Continued.

Characteristics

Number of strains

Positive

Negative

Degradation of

X= anthine

E= lastine

A= rbutin

P= ectine

21

Chitin

16

36

13

Es 4, 5, 81, 119; As 10, 22, 5= 3, 75, 81; Fs1; Fw11

Fs* 4, 5; Ts 5, 6, 9

None

Fs 4,15, 22, 24, 37, 81, 112, 117, 119, 127, 130= ; As 8, 10, 22, 45, 49, 53, 71, 75, 77, 81, 94; Fs 1, 3, 13, 16, 19, 35

Fw 11, 14; Fs* 4, 5; Ts 5, 6, 9; Q1

Es 4, 5, 24, 37, 48; As 8, 49, 53, 71; Fs 1, 13,= 16, 35

None

23

3

26

Es 22, 24, 37, 48, 112, 117, 1= 27, 130; As 8, 45, 49, 71, 77, 94

Fs 3, 13, 16, 19, 35; Fw 13, 14; Ts3; Q1

All

Es48; Fw13; Ts3

Es 22, 81, 112, 117, 119, 127, 130; As 10, 22, 45, 75, 77, 81, 94

Fs 3, 19; Fw 11, 13, 14; Fs* 4, 5; Ts 3, 5, 6, 9= ; Q1

All

Enzyme activity

P= rotease

L= ipase

Lecitinase

38

18

27

Es 4, 5, 22, 24, 37, 48, 81, 1= 12, 117, 119, 127, 130; As 8, 10, 22, 45, 49, 53, 71, 75, 77, 81, 94; Fs 1, 3, 13, 16, 19, 35

Fw 11, 13, 14; Fs* 4, 5; Ts 3,= 5, 6, 9

Es 4, 37, 130; As 10, 22, 45, 49, 71, 81; Fs3, 3= 5

Fw 11, 13, 14; Ts 3, 5, 6; Q1<= /p>
Es 4, 5, 24, 48, 81, 112, 127, 130; As 10, 45, 49, 53, 71, 77, 81

Fs 1, 3, 13, 16, 19, 35; Fw14; Fs* 4, 5; Ts 3, 6= , 9

1

21

12

Q1

Es 5, 22, 24, 48, 112, 117, 119, 127; As 8, 53, = 75, 77, 94

Fs 1, 13, 16, 19; Fs* 4, 5; Ts9

Es 22, 37, 117, 119; As 8, 22, 75, 94; Fw 11, 13; Ts5; Q1

Melanine production on

Iron agar

Tyrosine agar

16

6

Es 22, 48, 112, 119, 130; As 8, 22, 45, 49, 75; Fs 1, 3, 16, 35

Fw 13, 14

Es 22, 112, 119, 130; As75; Fs35

23

33

Es 4, 5, 24, 37, 81, 117, 127;= As 10, 53, 71, 77, 81, 94

Fs 13, 19; Fw11; Fs* 4, 5; Ts 3, 5, 6, 9; Q1

Es 4, 5, 24, 37, 48, 81, 117, = 127; As 8, 10, 22, 45, 49, 53, 71, 77, 81, 94; Fs 1, 3, 13, 16, 19; Fw 11, 13,= 14; Fs* 4, 5; Ts 3, 5, 6, 9; Q1

Fig. 1. Pheno= gram of Streptomyces species, produc= ers of proteolytic enzymes.

Fig. 2. Distribution of Strep= tomyces species, producers of proteolytic enzymes.

Conclusions

The present s= tudy demonstrated the production of multiple extracellular proteolytic enzymes by different Streptomyces species isolated from several sources, which were co-ordinate during growth. More t= han 300 streptomycetes strains were screened for protease activity in three ass= ay media and 39 of them actively produced proteases. Rapid, sensitive detection and qualitative assay of streptomycetes proteases were highly desirable. The production of proteases was much more sensitively detected on 3 % BHI containing gelatin than on gelatin or egg yolk agar media. The streptomycet= es isolates that were active in protease production were subjected to identification. The obtained results displayed Str. anulatus as the major source for proteases. This spec= ies is widely acknowledged as a vast reservoir of natural products with differe= nt activities. The diverse metabolic capacities of Str. anulatus strains and their specific growth characteristics, i. e. mycelium formation and relatively rapid colonization of selective substrates, facilitate their collecting as suited proteases producers. Beside Str.&nb= sp;anulatus, different Streptomyces species contribute to the production of proteases. These organisms grow in various environments. The use of indigenous strains for pro-teases production is an alternative approach, since the organisms have been already adapted to the habitat.

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ПРОТЕА&= #1047;НА АКТИВНОСТ Н = 40; МЕЗОФИЛНИ СТРЕПТОМИЦh= 5;ТИ,

ИЗОЛИР&= #1040;НИ ОТ РАЗЛИЧНИ МЕСТООБИТАi= 3;ИЯ В еГИПЕТ

Хала М. Рифаат¹*, Осама Х. Ел-Саид², Садиа М. Хаса&#= 1085;еин³, Манал С. М. Селим²

Резюме

Триста и седемнадесk= 7;т стрептомицk= 7;тни култури са изолирани о = 90; различни източници и области в Египет и са изпитани за протеолитиm= 5;на активност. О= 090; тях 39 продуцират протеази и т= 077; са определени до вид. Най-много из= 086;лати са отнесени към Streptomyces anulatus. Този вид заслужава особено внимание поради големия си потенциал з = 72; приложение = 74; биотехнолоk= 5;иите.

Medium	Number of strains and activity
Poor	Fair	Good	Very good
3 % BHI + Gelatine	2= Es 48, 112	10= Es 4, 24, 37, 127 As 81; Fs 1, 19 Fw 13, 14 Q1	18 Es 5, 81, 119, 130 As 8, 10, 45, 49, 53, 71, 75,= 77 Fs 3, 13, 16 Fw 11; Fs*4 Ts 3	9= Es 22, 117 As 22, 94 Fs 35; Fs*5 Ts 5, 6, 9
Gelatine medium	9 Es 24, 37, 48, 112, 117 As 8, 81 Fs* 4, 5	23 Es 4, 5, 22, 81, 119, 127, 13= 0 As 10, 22, 49, 53, 71, 77, 94= Fs16 Fw 11, 13, 14 Ts 3, 5, 6, 9 Q1	7 As 45, 75 Fs 1, 3, 13, 19, 35	None
Egg-yolk agar	11 Es 22, 81, 112, 130 As 8, 22, 45, 75 Fw 11, 13 Ts3	15 Es 4, 117, 119 As 10, 49, 53, 71, 81 Fs 13, 19 Fw14 Ts 5, 6, 9 Q1	13 Es 5, 24, 37, 48, 127 As 77, 94 Fs 1, 3, 16, 35 Fs* 4, 5	None