Introduction

Microbial proteases may be divided into three groups (Keay, 1971) referred to as acid, neutral and alkaline proteases on the basis of their pH optima (Table 1). An alternative method of classification is based on the mechanism of protease action (Hartley ,1960). According to this method there are four main groups:(i)serine proteases;(ii) thiol proteases,(iii) methal proteases and (iv) acid proteases .

Among the various proteases , bacterial proteases are the most significant , compared with animal and fungal proteases. And among bacteria, Bacillus species are specific producers of extracellular protease

Global requirements of thermostable biocatalysts are for greater than those of the mesophiles of which proteases contribute two thirds (Beg et al., 2003).

Thermostable proteases are advantageous in some applications because higher processing temperatures can be employed , resulting in faster reaction rates , increase in solubility of nongaseous reactants and products , and reduced incidence of microbial contamination by mesophilic organisms. Thermophilic bacteria from hot springs produced unique thermostable enzymes (Rao et al., 1998).

In this paper were report isolation and characterization of protease producing Bacillus sp, from soil which might have useful industrial application .

Materials and Methods

Screening ,Isolation and Identification of Microorganisms

Soil samples from different parts of north of Iran (including soils, cattle and birds composts manure , plants composts and hot spring ) were collected. Suspensions were prepared by mixing 10 gram of each samples with 90 ml of sterile distilled water and were shaken vigorously for 2 min. the samples were heated at 60°C for 60 min in water bath. The liquid was serially diluted in sterile saline, and the dilution from 10^-1 to 10^-7 was poured and spread on to nutrient agar using spread plate and pour plate methods. Nutrient Agar containing : (g/L Agar,15; Yeast extract, 2; Peptone, 5; Sodium chloride, 5.

These plates were sealed in plastic bags to reduced evaporation and incubated at 55°C for 24h. (Chilcott and Wigley, 1993). Some of the colonies that grew on the plates were purified by successive streaking on nutrient agar plates.

Pure culture were maintained on nutrient agar slant stored in a refrigerator and subcultured at 3 month interval, all colonies isolated by this procedure proved to be thermophilic and did not grow in nutrient agar medium at or below 44°C for as long as 7 days.

To screening for protease producing bacteria; the plates were incubated for up to skimmilk agar at 55^oC for 24h. A clear zone of skim hydrolysis gave an indication of producing organisms. The colonies with clear zone were picked up isolated for further investigation these were sub-cultured to obtain pure isolated bacteria species using Asilm et al., (2002) methods.

Evaluation of clear zones of each colony was estimated as radius (mm) of the clear zone minus the radius of the colony (Fig. -1).

The diameter of the clear zone represented the protease activity of strains (Table-3). The most active strains was used for former characterization, properties of the isolated bacteria were identified based on cellular and colonial morphology growth condition , gram stain , motility and biochemical tests (Sneeth and Halt, 1986; Watanabe and Hayana, 1993). In the identification of isolated bacteria species , standard taxonomic description from (Sneath, 1986) were used and Bacillus species were identified. (Table 1).

Protease Production

The culture medium used this work for protease production contained(w/v) 0.5% glucose, 0.75%peptone,0.5%MgSO₄.7H₂O, 0.5% KH₂PO₄, and 0.01%FeSO₄.7H₂O maintained at 50^°C for 24 to 72 h in a shaking incubator (140rpm). At the end of each fermentation period , the whole fermentation broth was centrifuged at 10.000rpm at 4°C for 15 min and the clear supernatant was used as crude enzyme preparation.

Determination of Protease Activity

Protease activity was determined in triplicate by incubating 500µl of 0.5% azocasein in Tris-HCl buffer with 100µl enzyme solution for 60 minutes at 50^oC, reaction was stopped by adding 500µl of 15% Trichloracetic acid (TCA) with shaking. This was left for 15 min and centrifuged at 4°C for 15 min at 3000 rpm. 1 ml of supernatant was added to 1 ml of 1µl NaoH and absorbance was read at 440nm. one unit(u) of protease activity was defined as micromole of substrate converted per minute under standard assay conditions (Table -3).

Results and Discussion

Seventy-five thermophilic bacteria have been isolated from soil samples according to the method (Chilcott and Wigley ,1993). The isolated bacteria were gram- positive and gram-negative and of which fourty of isolated were identified as Bacillus species with central spores and predominantly unswollen cylindrical sporangia.

Thirty of isolated of hydrolyse both starch and skim and twenty of which hydrolysis only starch and twenty-five of them hydrolysis skim, all of isolated were catalase positive, Indole was not formed and acetoin formation was positive. No growth was obtained in nutrient broth containing more than 7% NaCl.

All of them grew in nutrient broth at 30-60°C with an optimal at 50°C, on the basis of these morphological and Biochemical characteristic, isolated strain as (Table -2) were classified in the genus Bacillus. (Sneath,1985) and denominated Bacillus sp. They were B.brevis, (5 strain) B.licheniformis, (4strain), B.subtilis (4 strain), B.polymyxa (2strain), B.mycoides (5 strain), B.cereus (6 strain), B.megaterium (8 strain), B. alvei (3 strain), B.coagulans (3 strain) (Table -2). Watanaba and Hayano (1993) identified B.subtilis, B.Licheniformis, B.cereus and B.megaterium in soil isolations.

Table 1: Properties of microbial extracellular proteases

Table 1b: Biochemical and biophysical tests for identification of thermophilic isolate. Click here to View Figure

Figure 1: Some samples of proteas-producing bacteria in skimmed milk agar plates Click here to View Figure

In another investigation (Najafi, 2003) isolated some thermostable alpha-amylase produing bacteria from different soil samples collected from various part of Iran and identified as B.coagulans, B.licheniformis and B.brevis, also Waksman (1991) identified 29 isolated as B.megaterium and 24 isolates as B.subtilis out of 306 soil samples. These agree with the results of this study that Bacillus genera are wide spread among bacteria in soil.

Few publications are devoted to the study of the Bacillus species isolated from various environment. Due to their ubiquity and capability to survive under adverse condition, heterotrophic Bacillus strains and hardly considered to be species of certain habitats (Clus and Berkeley;1986).

The Bacillus genus is an extensive heterogeneous group encompassing 83 validly described species to date. Many species in this taxon one of major clinical importance, such as the B. cereus, B. anthracis, B. thuringiensis, B. mycoides and B. weihenstephanensis), but unfortunately, members of the group shane a great deal of morphological and biochemical similarities (Harrel;L.J, et al., 1995; Priest, et al., 1998). Since the biochemical approach for species identification can be tedious, expensive, and inaccurate, a rapid definitive method is greatly needed. Molecular procedures are increasingly being used for rapid species identification (Jung and Cote; 2002). In contrast, the environmental and nonpathogenic species of this genus exhibit a wide range of physiology DNA base content, and nutritional requirements (Priest, 1993).

Screening for Proteolytic Activity

The proteolytic activity was assayed using skimmilk agar medium and expressed as diameter of clear  zone in mm. Strain Jin E,L,P,S exhibited the highest proteolytic activity with a clear zone diameter of 60-65-mm (Figure ,2 , Table 3 ). Table – 3, showed the quantitative assessment of protease productivity by the isolates using fermentation process strains Jin E,L,P,S also produced the highest enzyme activity (806-820 U/ml). Hence it was selected for subsequent investigation .

Table 2a:  The morphology and characteristics of some thermophilic isolate

Figure 2: Proteolytic activity of isolated bacteria strains of 24h at 55oc Click here to View Figure

Table 3: Protease producing ability of isolated strains

Other  workers  have  also  isolated thermophilic bacteria in the Thai hot spring (Thailand) , which were reported to produce thermostable proteases  (Sookkheo et al., 2000).This might be an indication that the Bacillus species would produce thermostable neutral and alkaline proteases which could find applications in industry and biotechnology .proteases from the Bacillus species are presently being characterized .

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