Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 6, Num. 1, 2009, pp. 28-37

Journal of Culture Collections, Vol. 6, No. 1, 2009, pp. 28-37

Proteolytic actinomycetes from Indian habitats

Richa Jain*, S. C. Agrawal and P. C. Jain

Department of Applied Microbiology and Biotechnology
Dr. Hari Singh Gour Vishwavidyalaya, Sagar, Madhya Pradesh 470 003, India
*Corresponding author, e-mail: richajain_198@hotmail.com

Code Number: cc09004

Summary

Three hundred five actinomycetes isolated from different habitats were screenedfor their proteolytic activity using gelatin as substrate. As many as 153 (i.e., 50.17 %) isolates were found positive for proteolytic activity, out of which 12 isolates showed greater relative enzyme activity (REA ³ 8.5). These isolates have been identified and further tested for production of proteases in liquid cultures. Streptomyces exfoliatus CFS 1068 and Streptomyces sampsoniiGS 1322 were found to possess excellent protease activity i.e., equivalent to 115.2 and 116.0 U/ml.h in the culture filtrates. The significance of these actinomycetes in nature and in management of industrial waste has been discussed in the present paper.

Key words: actinomycetes, gelatin, habitat, proteolytic activity.

Introduction

The habitats with various abiotic characteristics may differ greatly in their microbial community. Microorganisms inhabiting different habitats may show diversity in their biochemical characteristics. Actinomycetes have been re-ported as the most productive organisms and have been exploited for production of antibiotics [2, 4, 5, 8, 10, 15]. Considering their diverse biochemical properties, actinomycetes can also be explored for industrial production of other biologically active molecules such as enzymes [6, 9, 12, 16]. The screening of any available collection of microorganisms may yield new information about the nature of the available gene pool. In the present study a total of 305 isolates of actinomycetes, collected from various habitats, have been screened to identify strains with excellent proteolytic activity.

Materials and Methods

Test organisms. Three hundred five actinomycetes, isolated from different habitats [8, 11], were obtained from the Microbial Culture Collection of the Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, India. These include 13 actinomycetes from animal feed (AF), 71 from cultivated field soil (CFS), 46 from compost (CM), 61 from decaying organic matter (DOM), 69 from garden soils (GS), 30 from stored agricultural products (SAP), and 15 from soils near hair saloons (SHS). All these actinomycetes were studied for their proteolytic activity.

Determination of proteolytic activity.

Method I. Test actinomycetes were grown on Difco-nutrient agar with gelatin as protein substrate (pH 6.0), which contained (g/l): peptone - 5; NaCl - 5; Beef extract - 3 and Agar agar - 20 [3]. Separately sterilized aqueous solution of gelatin (8 % w/v) was added to the medium before pouring to obtain 0.4% gelatin concentration in the medium. An aliquot of 20 ml of the medium was poured in each plate. After solidification, the medium was streaked in a straight line along the diameter of the plate with a spore suspension of the test actinomycete. After 7 days of incubation at 28±1 ºC, the plates were flooded with 1.5 % HgCl₂ in 20 % conc. HCl. The appearance of a transparent zone around the colony in contrast to the opaque whitish background indicated production of protease by the test actinomycete. Relative enzyme activity (REA) of each protease positive actinomycete was calculated using the following formula:

REA = Diameter of zone of enzyme activity in mm / Diameter of the colony in mm.

Based on REA test actinomycetes have been categorized into four groups showing excellent (REA ³ 8.5), good (REA ³ 5.0 to < 8.5), fair (REA ³ 2.0 to < 5.0) and poor (REA < 2.0) protease activity.

Method II. The test actinomycetes were grown in nutrient broth (peptone - 5; NaCl - 5; Beef extract – 3, g/l), supplemented with 0.4 % gelatin for 7 days at 28±1 ºC under shaking at 100 rpm. The cell free filtrate from cultures was obtained by centrifugation at 5000 rpm for 10 min followed by millipore filtration using syringe filters (0.22 m). The culture filtrate obtained in this way was then used as a crude enzyme sample. For the assay of protease activity in the culture filtrate, Difco nutrient agar medium containing 0.4 % gelatin was prepared as above and poured into the plates after sterilization. After solidification of the medium, 8 mm well is bored in the center of the plated medium using sterile cork borer. 150ml of crude enzyme sample was then poured in each well and the plates were incubated at 28±1 ºC for 24 hours. To determine protease activity, the plates were flooded with 1.5 % HgCl₂ in 20% conc. HCl. The appearance of a transparent zone around the well as a result of protease activity was measured.

Method III. In this method, the production of proteolytic enzymes by the test actinomycetes was measured in the culture filtrates in termsof enzyme units by using the reduction in the optical density of a gelatin solution as a criterion [21]. For this purpose, the cell free culture filtrates obtained earlier using method II were used as crude enzyme samples. An amount of 0.5 ml culture filtrate was mixed with 0.5 ml of gelatine solution (0.2 %) in phosphate buffer of pH 7. This reaction mixture was incubated at 37 ºC for 1 hour. After incubation, 0.15 ml of TCA solution (0.44 M TCA, 0.88 M sodium acetate, 1.32 M acetic acid) was added to stop the reaction. Acetonitrile (3 ml) was then mixed and stirred for 10 min at 30 ºC to allow the precipitation of un-hydrolyzed gelatin in the solution and the absorbance was measured at 700 nm. One unit of protease activity was defined as the amount of enzyme that caused 1 % reduction in optical density of the gelatin solution (0.2 %) per hour.

Identification of actinomycetes. Cultural, morphological, physiological and biochemical properties of the selected strains were studied according to the methods of Shirling and Gottlieb [19] and Williams [23], and strains were iden-tified using PIBWin computer programme [14].

Results and Discussion

Proteolytic activity of the actinomycetes

In the present study 153 out of 305 test actinomycetes were found to produce protease activity on gelatin containing agar medium (Method I). The protease positive actinomycetes showed variable levels of enzyme activity in terms of their REA. A total of 13 actinomycetes from animal feed were tested. Among these, twelve isolates (92.3 %) showed proteolytic activity but none of them was an excellent producer of this enzyme (Table 1). Eleven actinomycetes (73.33 %) from soils near hair saloons possessed proteolytic activity, two of which showed excellent protease activity (REA ³ 8.50). 46 isolates of garden soils manifested proteolytic properties. Among these, 5 isolates (7.24 %) showed excellent protease activity while 17 (24.63 %), 19 (27.53 %), and 5 (7.24 %) actinomycetes demonstrated good, fair and poor protease activity, respectively. Among isolates from decaying organic matter, 47.54 % isolates have been found protease positive. Out of these, 3 isolates (i.e., 4.91 %) showed excellent production of this enzyme. 43.66 % isolates of cultivated field soil manifested production of protease but only two of them (i.e., 2.81 %) showed excellent activity. It is point worthy to note that none of the test isolates from stored agricultural products, animal feed and compost have showed excellent activity of protease in the present screening programme.

In all 12 actinomycetes (3.93 %) including 3 of cultivated field soil (i.e., CFS 1068, CFS 1072 and CFS 1131), 2 of decaying organic matter (DOM 1099, DOM 1419), 5 of garden soils (i.e., GS 1242, GS 1320, GS 1322, GS 1323 and GS 1432) and 2 of soils near hair saloons (SHS 1422, SHS 1426) have been found to have excellent proteolytic properties and showed relative enzyme activity equivalent to REA ³ 8.50. Waksman [22] demonstrated a wide distribution of proteolytic enzymes in actinomycetes with variable levels of activity on different protein substrates. In the present study 50.16 % of 305 isolates have been found positive with variable levels of protease activity using gelatin as substrate. Proteolytic enzymes of actinomycetes have also been reported to digest and hydrolyze protein fibers [1, 7, 13, 20] thus playing an important role in decomposition of proteins in nature.

Table 1. Distribution of proteolytic activity in actinomycetes from different habitats.

*The values given in parenthesis indicate the percentage of protease positive actinomycetes. 

Proteolytic activity - Excellent, REA ³ 8.5; Good, REA ³ 5.0 to < 8.5; Fair, REA ³ 2.0 to < 5.0; Poor, REA < 2.0; -, Protease activity nil.

Twelve isolates, which showed excellent gelatinolysis following the method of Hankin and Anagnostakis [3], were identified and further tested for the extracellular production of protease using method II. All of these showed protease production when grown on nutrient broth containing 0.4 % gelatin. Maximum protease activity (34 mm zone of enzyme activity) was recorded in the culture filtrate of Streptomyces exfoliatus CFS 1068, followed by S. sampsonii GS 1322 (33 mm), S. somaliensis GS 1242 (26 mm), Streptomyces sp.CFS 1072 (24mm) and Streptomyces sp. DOM 1131 (24 mm). The culture filtrates of all other test actinomycetes produced 14–20 mm zone of protease activity using this method.

Protease activity in the culture filtrates of these actinomycetes was also determined spectrophotometrically [21] using Method III and the results are presented in terms of enzyme units (Table 2). Crude culture filtrates obtained from S. sampsonii GS 1322 showed maximum activity of protease i.e., 116.0 U/ml.h. Nearly similar activity of this enzyme was recorded in cultures of S. exfoliatus CFS 1068 (115.2 U/ml.h) followed by 102.0 U/ml.h in S. somaliensis GS 1242. Other isolates showed variable levels of protease activity in a range of 21.64 to 83.66 U/ml.h. Although the proteases are wide spread in nature, microbes serve as a preferred source of these enzymes [17] because of their rapid growth, limited space requirement for cultivation and the ease with which they can be genetically manipulated to generate novel enzymes with altered properties. In the present study, three test actinomycetes showed greater proteolytic properties using gelatin as a substrate and thus can be of significance in the bioremediation of gelatinous waste littered by gelatin factories, food and pharmaceutical industries particularly those manufacturing soft capsules. These strains can also find application in silver recovery and degradation of waste X-ray films [17, 18]. The cultures of all 12 test actinomycetes have been deposited in our culture collection i.e., Gour Nodal Centre for Industrially Important Microorganisms (GNCIIM) and have been deposited at Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.

Table 2. Gelatinolytic activity of some promising actinomycetes.

* Including the growth zone.

**Including the 8 mm diameter of the agar in good health.

Table 3: Morphological, physiological and biochemical characteristics of actinomycetes.

Table 3. Continued.

Table 3. Continued.

Table 3. Continued.

Characterization and identification of actinomycetes

Twelve actinomycetes, which showed excellent gelatinolytic activity using Method-I, include S. somaliensis GS1242 (MTCC 6229) and S. sampsonii GS 1322 (MTCC 6231). These were identified based on their 16 S rRNA gene sequencing by Dr. Y. S. Suhouche, National Center for Cell Sciences, University of Pune, India.

The remaining 10 unidentified actinomycetes were characterized for their morphological, physiological and biochemical characteristics (Table 3) and were identified using PIBWin computer kit [14]. The results of our study showed dominance of three clusters of Streptomyces, namely S. exfoliatus, S. halstedii and S. diastaticus. Four isolates, i.e., CFS 1068, GS 1320, DOM 1419, and SHS 1426 have been found closely related to S. exfoliatus showingPIBWin ID scores in the range from 0.99996 to 0.95077. Two isolates showed PIBWin ID score more than 0.99 and hence, these have been identified as S. exfoliatus CFS 1068 (ID score 0.99996) and S. exfoliatus DOM 1419 (ID score 0.992100). Other two isolates of this cluster have been designated and assigned to the genus Streptomyces with no specific epithet. Two isolates i.e., CFS 1072 and DOM 1131 were found very close to the known strains of S. halstedii. PIBWin ID scores of these isolates were found to be 0.97969 and 0.98688, respectively and hence these strains have been assigned to genus Streptomyces with no specific epithet. Two isolates i.e., DOM 1099 and SHS 1422 showed maxi-mum similarity with known strains of S. diastaticus with PIBWin ID score of 0.95631 and 0.95829, respectively and hence these were also identified as Streptomyces sp. DOM 1099 and Streptomyces sp. SHS 1422. Test isolates GS 1323 was found closely related to S. violaceus (ID score 0.97836) but could not be assigned to this species because of PIBWin ID score less than 0.99. Isolate GS 1432 have been found very close to the known strains of S. rochei (ID score 0.99882) and hence its species assignment has also been confirmed.

Acknowledgements

Authors are thankful to Dr Y. S. Shouche, National Centre for Cell Sciences, University of Pune, India for the identification of two of our isolates. The senior author is thankful to the Head of the Department of Applied Microbiology and Biotechnology, Dr. H. S. Gour. Vishwavidyalaya, Sagar, M. P., India for providing valuable collection of actinomycetes and laboratory facilities for the present study.

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