Iranian Journal of Pharmaceutical Sciences Summer 2011: 7(3): 175-180ijps.sums.ac.ir

R

Original Article

Screening and Isolation of Extracellular Protease ProducingBacteria from the Maharloo Salt Lake

Younes Ghasemia,b,*, Sara Rasoul-Aminia,b, Alireza Ebrahiminezhadb, Aboozar Kazemib, Maryam Shahbazib, Najme Talebniab

aDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University ofMedical Sciences, Shiraz, Iran

bPharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

AbstractScreening and identification of moderately halophilic protease producing bacteria

from different regions of Maharloo, a hypersaline lake in the southern area of Iran,were the objectives of this study. In the preliminary screening, 16 isolates exhibitedproteolytic activity on saline skim milk agar. All isolates were able to growcomfortably in the media containing 7-15% of salt. Protease activity was assessedthrough in vitro colorimetric assays for general proteases and the strains wereidentified by 16S rDNA molecular marker. In comparison to Gram-negative bacteria,the Gram-positive rods, displayed higher proteolytic activities, and Bacillus sp. BCCS041 was found as the highest protease producing strain with 0.43 U/ml of supernatantactivity.

Keywords: Bacillus sp; Moderately halophilic; Protease.Received: February 18, 2011; Accepted: April 27, 2011

1. IntroductionProteases (EC.3.4) are a distinct subgroup

of hydrolytic enzymes which catalyze thecleavage of peptide bonds in proteinoussubstrates. Depending on their mode of actionand catalytic mechanism, proteases are dividedinto four major groups including: serineprotease (EC. 3.4.21), cysteine (thiol) protease(EC. 3.4.22), aspartic proteases (EC. 3.4.23)and metallo-protease (EC. 3.4.24) [1]. Inaddition to their pivotal metabolic andphysiological importance, they possess diverse

commercial applications worldwide [1]. Theirapplications are vast in detergents, chemicals,food, pharmaceutical and leather tanningindustries. Most commercial proteases belongto the neutral and alkaline proteases which areproduced by microorganisms in particularBacillus genus. Other promising applicationshave been associated in potential biotechnolog-ical processes and waste water treatment [2, 3].

Since the majority of industrial processesare accomplished under harsh conditions, itwould be of great importance to enjoymicrobial enzymes that demonstrate optimalactivities at wide ranges of pH, temperatureand salt concentration. Microorganismsinhabiting in hypersaline environments are a

*Correspondinh Author: Prof. Younes Ghasemi, Department ofPharmaceutical Biotechnology, Faculty of Pharmacy, ShirazUniversity of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran Tel. (+98)711-2426070; Fax. (+98)711-24242126E-mail: ghasemiy@sums.ac.ir

Y Ghasemi Asl et al / IJPS Summer 2011; 7(3): 175-180

176

remarkable source for producing suchenzymes. They are expected to have specificproteins presenting characters which aredifferent from proteins produced by organismsfrom nonsaline environments. They may havethe capability to produce halophilic enzymeswhich have potential application in situationsthat require tolerance of high saltconcentration. Moderately halophilic bacteriaare a group of halophilic microorganismswhich can grow optimally in media containinga wide range of salt concentrations (3-15%NaCl) [4]. They are phylogenetically verydiverse and show important advantages forbeing used as a source of halophilic enzymes[5, 6]. The extensive biochemical variety andease of genetically manipulation of theseenzymes could generate new ones for variousbiotechnological applications [6-8].Hypersaline lakes, with high salt concentrationwhich is near saturation, are extremeenvironments; yet, they are the habitats ofremarkable microbial communications.Hence, they are regarded as productivebiological ecosystems. These microorganismsuse different strategies for preserving their cellstructure and function in highly salineconditions. They may produce compoundsof industrial interest, such as extracellularhydrolytic enzymes with diverse potentialapplications in the industries [7]. Therefore,conducting researches for isolation ofmoderately halophiles able to produceextracellular enzymes are of great importance.In this study, we describe the screening ofextracellular protease producer halophilicbacteria, isolated from Maharloo hypersalinelake located in the south of Shiraz, Iran. Thesalt concentration of this lake fluctuatesperiodically. It shows lower salinity at wetseasons and higher salt concentrations duringdry seasons, therefore, it may containmoderately halophilic and halophilic bacteria.

2. Materials and Methods2.1. Isolation and screening of moderately

halophilic bacteria for proteolytic activitiesSediment and water samples were collected

from different parts of Moharloo salt lake inthe south of Shiraz, Iran. All samples wereserially diluted with sterile distilled waterand the dilutions were streaked on salinenutrient agar plates (containing 7% NaCl).After 24 h of incubation at 37 °C, singlecolonies were screened for proteolytic activityin a saline skim milk agar medium thatcontained 2% (w/v) skim-milk, 1% (w/v)tryptone and 7% (w/v) NaCl [6]. The clearzones of hydrolysis over the next 24 h weretaken as evidence of proteolytic activity,qualitatively [2].

2.2. Protease productionThe protease producing isolates were

inoculated in 250 ml Erlenmeyer flaskscontaining 50 ml saline skim-milk broth andincubated at 37 °C and 150 rpm [9]. Afterincubation for 48 h, the cultures werecentrifuged at 4500×g for 10 min at 4 °C, andthe cell free supernatants used as crudeenzyme for extracellular protease assay.

2.3. Protease assayProtease activity was determined using

casein as a substrate. The assay mixturecontained 1.1 ml of 1% (w/v) casein in 0.1 MTris-HCl buffer (pH 8.0) and 0.1 ml ofenzyme solution. After incubation at 37 °C for30 min, the reaction was terminated by adding1.8 ml of 5% (w/v) trichloroacetic acid. Theneach test tube was centrifuged at 4000×g for20 min and the absorbance of the supernatantwas determined at 280 nm. One internationalunit (IU) of protease activity is the amount ofenzyme which liberates 1 μmol of tyrosine permin [3, 11].

2.4. Identification of bacteria Initial morphological identification of

isolates was done by Gram staining. Furthercharacterization was done on the basis of 16SrDNA gene sequencing. Different

Extracellular protease producing bacteria

morphological and cultural characteristics ofthe protease producing isolates were studiedcompared with standard description ofBergey's Manual of DeterminativeBacteriology [9].

2.5. PCR amplification of the 16S rDNA andsequence determination

The purified protease producing bacterialisolates were grown in saline nutrient brothcontaining 7% (w/v) NaCl. After centrifugationat 4500×g for 10 min at 4 °C, and twicewashing, the pellets were selected forchromosomal DNA extraction and PCRamplification. Bacterial DNA was extractedusing heat extraction method. The 16S rRNAgene was amplified by PCR using the universalprokaryotic primers 5'- ACGGGCGGTGTG-TAC -3' and 5'-CAGCCGCGGTAATAC-3'.PCR was performed in a final volume of 50 μlcontaining PCR amplification buffer (1X), TaqDNA polymerase (2.5 U), dNTPs (4 mM),primers (0.4 µM) and template DNA (4 ng).Amplification conditions were as follows:initial denaturation at 94 °C for 5 min., 10cycles at 94 °C for 30 s, 50 °C for 30s and 72°C for 2 min., 20 cycles at 92 °C for 30 s, 50°C for 30 s, and 72 °C for 2.5 min. with a final

extension of 72 °C for 5 min. Taq polymerasewas added to the reaction after the firstdenaturation step. The lower denaturationtemperature (92 °C) during the 20 cycle stepswould prevent loss of enzyme activity [11].The samples were electrophoresed in a 1%(w/v) agarose gel containing ethidiumbromide (1 μg/ml). A single ~800 bp of DNAfragment was extracted from the gel using aCore Bio Gel Extraction Kit. The sequencewas determined by the CinnaGen Company.The data were submitted at GeneBankdatabase. The DNA partial sequences werealigned and compared using BLASTalgorithm for finding homologous sequencesin the GeneBank database in the NationalCentre for Biotechnology Information(NCBI). A bioinformatic tool, GeneDocsoftware, version 2.6.002 was used for moreinvestigation of sequence alignments in themost protease producing Bacillus sp.

3. Results3.1. Isolation and screening of proteaseproducing bacteria

Among 40 isolated bacteria, 16 isolates(40 %) were protease producer and clear zonearound colonies was considered as the

177

Table 1. Protease activity (U/ml supernatant) and lengths and specific accession numbers of the 16S rRNA regionof the isolated halophilic bacteria.Halophilic bacterai Accession number Length (base pair) Activity

(U/ml)Bacillus thuringiensis BCCS 038 FJ624483 804 0.18Paenibacillus sp. BCCS 042 FJ645733 776 0.19Bacillus sp. BCCS 041 FJ645731 824 0.43Bacillus sp. BCCS 040 FJ645730 787 0.17Aeromonas sp. BCCS 037 FJ619745 802 0.39Bacillus sp. BCCS 043 FJ645732 811 0.20Halobacterium sp. BCCS 039 FJ645729 792 0.18Halobacterium sp. BCCS 030 FJ529204 821 0.09Aeromonas veroni BCCS 025 FJ429320 720 0.07Bacillus sp. BCCS 034 FJ607328 802 0.22Bacillus sp. BCCS 032 FJ605152 880 0.01Bacillus sp. BCCS 036 FJ619744 802 0.10Bacillus subtilis BCCS 027 FJ517538 832 0.07Bacillus subtilis BCCS 033 FJ607327 796 0.01Bacillus sp. BCCS 031 FJ554670 809 0.18Bacillus subtilis BCCS 028 FJ517539 784 0.17

Y Ghasemi Asl et al / IJPS Summer 2011; 7(3): 175-180

178

evidence of protease production. Since thereis not necessarily good correlation betweenzones of clearing around colonies on skim-milk agar plates and levels of protease activity,all 16 isolates were identified and cultivatedin broth medium and the extracellular proteaseactivity was assessed.

3.2. 16S rDNA sequence analysisInitial morphological identification showed

12 isolates were gram-positive, spore formingrod-shaped and four of them were gram-negative. The PCR amplification of 16SrDNA gene revealed a single band ofamplified DNA product of ~800-bp, indicatingefficient amplification. The DNA sequenceswere published in the NCBI databases underthe specific accession numbers. The amount

of protease production (U/ml of supernatant)and the lengths of the 16S rDNA region of thestrains and their specific accession numbersare listed in Table 1. The result of PCR blastedwith other bacterial sequences in NCBI. Editedsequences were used as queries in BLASTNsearches (http://blast.ncbi.nlm.nih.gov/Blast.cgi).From 16 protease positive isolates, 11 strains(68.75 %) were identified as Bacillus species andBacillus sp. BCCS 041 had the highestproteolytic activity (0.43 U/ml supernatant).The result of the alignment by GeneDocsoftware was shown in Figure 1. A total of 824nucleotides of the partial sequence of Bacillussp. BCCS 041 were 99 % similar to the 16SrRNA genes in recorded strains of Bacillussubtilis in NCBI (Figure 1).

Figure 1. 16S rRNA gene sequence investigation of Bacillus sp. BCCS 041 by bioinformatic tool, GeneDoc software version2.6.002.

Extracellular protease producing bacteria

4. Discussion Proteolytic bacteria are widespread in

nature and are able to grow under variousgrowth conditions, such as differenttemperatures, pH and ionic strength. Thepresence of halotolerant protease in thehalophilic bacteria could be applied inindustrial processes where the concentratedsalt solution used would inhibit ordinaryproteases [2, 3, 7]. In this experiment, 16bacterial isolates were able to produceproteolytic enzymes. Of these, 12 (75%)strains were Gram positive, including Bacillusspp. and Paenibacillus spp., 2 (12.5 %) strainswere Halobacterium spp., which were Gramnegative, and 2 (12.5%) were archaea,Aeromonas spp. In a similar study, Rohbanand his associates [7] isolated 231 moderatehalophilic and 49 extremely halophilicbacteria from Howz Soltan Lake. Amongwhich there were 172 (61.4%) Gram-positiverods, 56 (20%) Gram-negative rods and 52(18.6%) Gram-positive cocci. They foundthat 70 strains of Gram-positive rods, 13strains of Gram-positive cocci and 17 strainsof Gram-negative rods were proteaseproducer. These data are in agreement withour findings and revealed that Gram positivebacteria are the dominant proteolytic speciesin these saline environments. Aerialdistribution of the dormant spores probablyexplains the occurrence of Bacillus in mosthabitats. Ghasemi and his collaborates [12]isolated 9 Bacillus strains of 13 halotolerantamylase producing bacteria from soil andwater samples of Maharloo hypersaline lakein the south of Shiraz, Iran. In additionOlajuigbe and Ajele [13] have isolated 25bacteria from soil, of which nine isolateswere identified as Bacillus. They wereBacillus brevis, B. licheniformis, B. subtilis,B. macerans, B. mycoides, B. coagulans, B.polymyxa, B. cereus and B. megateriumspecies. These findings confirm that Bacillusspecies are widespread in the most extremeenvironments. The genus Bacillus is well

known as an enzyme producer and manyindustrial processes use species belonging tothis genus for commercial production ofenzymes [2]. Members of this genus are usedfor the synthesis of a very wide range ofimportant medical, agricultural, pharmaceuticaland other industrial products. These include avariety of antibiotics, enzymes, amino acids andsugars. The results of this work indicate thatthe moderately halophilic bacterium, Bacillussp. BCCS 041, could display a potential rolein protease production in biotechnologicalprocesses.

AcknowledgementsThis work was supported by a grant from

the Research Council of Shiraz Universityof Medical Sciences, Shiraz, Iran.

References[1] Rao MB, Tanksale AM, Ghatge MS, Deshpande

VV. Molecular and biotechnological aspects ofmicrobial proteases. Microbiol Mol Biol Rev1998; 62: 597-635.

[2] Sanchez-Porro C, Martin S, Mellado E, VentosaA. Diversity of moderately halophilic bacteriaproducing extracellular hydrolytic enzymes. JAppl Microbiol 2003; 94: 295-300.

[3] Mohapatra BR, Bapuji M, Sree A. Production ofindustrial enzymes (amylase, carboxymethylcel-lulase and protease) by bacteria isolated frommarine sedentary organisms. Acta Biotechnol2003; 23: 75-84.

[4] Ventosa A, Nieto JJ, Oren A. Biology of moder-atelyhalophilic aerobic bacteria. Microbiol MolBiol Rev 1998; 62: 504-44.

[5] Mellado E, Sanchez-Porro C, Ventosa A. Proteasesproduced by halophilic bacteria and archaea. In:Barredo JL, (editor). Microbial Enzymes and Bio-transformations: Methods in biotechnology,Totowa New Jercy.Humana press, 2005; pp 181-90.

[6] Xiong H, Song L, Xu Y, Tsoi M, Dobretsov S,Qian P. Characterization of proteolytic bacteriafrom the Aleutian deep-sea and their proteases. JInd Microbiol Biotechnol 2007; 34: 63-71.

[7] Rohban R, Amoozegar MA, Ventosa A. Screeningand isolation of halophilic bacteria producingextracellular hydrolyses from Howz Soltan Lake,Iran. J Ind Microbiol Biotechnol 2009; 36: 333-40.

[8] Aftab S, Ahmed S, Saeed S, Rasool SA.

179

Y Ghasemi Asl et al / IJPS Summer 2011; 7(3): 175-180

180

Screening, isolation and characterization ofalkaline protease producing bacteria from soil. PakJ Biol Sci 2006; 9: 2122-6.

[9] Sharmin S, Towhid Hossain MD, Anwar MN.Isolation and characterization of a proteaseproducing bacteria Bacillus amovivorus andoptimization of some factors of culture condition forprotease production. J Biol Sci 2005; 5: 358-62.

[10] Olivera NL, Sequeiros C, Nievas ML. Diversityand enzyme properties of protease-producingbacteria isolated from sub-Antarctic sediments ofIsla de Los Estados, Argentina. Extremophiles2007; 11: 517-26.

[11] Fiore MF, Moon DH, Tsai SM, Lee H, Trevors JT.Miniprep DNA isolation from unicellular andfilamentous cyanobacteria. J Microbiolo Methods2000; 39: 159-69.

[12] Ghasemi Y, Rasoul-Amini S, EbrahiminezhadA, Zarrini G, Kazemi A, Mousavi-Khorshidi S,Ghoshoon MB, Raee MJ. Halotolerant amylaseproduction by a novel bacterial strain,Rheinheimera aquimaris. Res J Microbiol 2010;5: 144-9.

[13] Olajuyigbe FM, Ajele JO. Production dynamicsof extracellular protease from Bacillus species. AfrJ Biotechnol 2005; 4: 776-9.