EurAsian Journal of BioSciences EurAsia J BioSci 5, 1-9 (2011) DOI:10.5053/ejobios.2011.5.0.

Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15
Fikret Uyar1, Ilknur Porsuk1, Gksel Kizil2, Ebru Ince Yilmaz1*
1Department of Biology, Faculty of Science, Dicle University, 21280 Diyarbakir, Turkey 2Department of Chemistry, Faculty of Science, Dicle University, 21280 Diyarbakir, Turkey

*Corresponding Author: eince@dicle.edu.tr

Abstract An alkaline protease producer Bacillus sp. strain CA15 was isolated from soil. The microorganism was found to be closely related to Bacillus cereus based on 16S ribosomal DNA sequencing. The culture conditions for higher protease production were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% skim milk, 1% starch and 0.6% MgSO4.7H2O, initial pH 8.0 at 35C. The best enzyme production was obtained during the stationary phase in which the cell density reached to 1.8x108 cells/mL. The level of protease was found to be low in the presence of inorganic nitrogen sources. The protease production was diminished in the presence of sucrose and lactose. The extreme stability towards Triton X-100, Tween 20 and SDS was observed by Bacillus sp. CA15 alkaline protease. The enzyme activity was inhibited by PMSF suggested that presence of serine residues at the active sites. Keywords: Alkaline protease, Bacillus sp., optimum culture conditions, 16S rRNA gene. Uyar F, Porsuk I, Kizil G, Ince Yilmaz E (2011) Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15. Eurasia J Biosci 5: 1-9. DOI:10.5053/ejobios.2011.5.0.1

INTRODUCTION Proteases constitute a class of industrial enzymes, which alone form approximately 60% of the total world-wide enzyme production (Chu 2007). Among the various proteases, microbial proteases play an important role in biotechnological processes. Alkaline proteases produced are of special interest as they could be used in manufacture of detergents, food, pharmaceuticals and leather (Saeki et al. 2007, Dias et al. 2008). In recent years a number of studies have been conducted to characterize alkaline protease from different microorganisms. However, many of the alkaline proteases applied to industrial purposes face some limitations such as low stability towards surfactants and production cost of the enzymes arisen from growth medium (Joo and Chang 2005). It is known that the amount of
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enzyme produced greatly depend on strain and growth conditions. Using of costeffective growth medium for the production of alkaline proteases from an alkalophilic Bacillus sp. is especially important (Joo et al. 2002). Therefore, there is a need to the search of new strains of bacteria that produce proteolytic enzymes with novel properties and the development of low cost media. In this respect, in the present study, we report the isolation and selection of a bacterium (Bacillus sp. CA15), which is a potent extracellular alkaline protease producer and the optimization of culture conditions required for enzyme production.

Received: December Received in revised form: January Accepted: February Printed: February

2010 2011 2011 2011 1

EurAsian Journal of BioSciences MATERIAL AND METHODS Sample collection and identification of isolate The soil sample was collected from Middle Anatolia Region (Ayas-Beypazari) of Turkey. The habitat was the soil from the rhizosphere of local endemic plant Achillea ketenoglui. Using a sterile spatula, the first five cm of the surface layer of the soil is separated, and then 100 g of soil was collected in the layer subjacent (between 5 and 15 cm of depth). It was placed into a sterile plastic bag and transported to the laboratory. 1 g of fresh weight of soil sample was aseptically added to 9 mL sterile 1/4 strength Ringer's solution [(g/L): NaCl, 7.2; CaCl2.2H2O, 0.23; KCl, 0.37] and shaken on a rotary shaker at 220 strokes per minute for 30 min. Serial dilutions (up to 10-6) of suspensions were prepared and 0.1 mL of aliquots aseptically spread on skim milk agar plates containing peptone (0.1%), NaCl (0.5%) and skim milk (10%) supplemented with cycloheximide (50 g/mL). The inoculated plates were incubated at 30C for one week. In situ protease production was demonstrated by the clearing of opaque milk proteins in the surrounding of colonies growing on the plates. The high-yield protease strains were selected depending on the zone wide. Representative protease producer CA15 was checked by several basic morphological and biochemical parameters. The isolate was maintained as suspensions in 20% glycerol at -20C for long-term storage. Bacterium was grown in nutrient broth (NB) at 30C on a rotary shaker (150 rpm) overnight. Genomic DNA was isolated (Sambrook and Russell 2000) and 16S rRNA gene was amplified by PCR using two general bacterial 16S rRNA primers (BSF8/20; 5`AGAGTTTGATCCTGGCTCAG-3`, BSR1541/20; 5`-AAGGAGGTGATCCAGCCG CA-3`). The PCR amplification was performed by using Qiagen Proof-Start Taq Polymerase kit (Qiagen, Hilden, Germany). PCR results were analyzed by electrophoresis on 0.9% agarose TBE-gels and DNA of the expected size was purified from gel with the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Purified PCR products were sequenced by the chain termination method with dye-labeled dideoxy terminators of the Thermo Sequenase II Dye Terminator Cycle Sequencing Kit
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(Amersham), using a Perkin Elmer-ABI Prism 377 automated sequencer and DNA fragments corresponding to the whole 16S rRNA gene of strain (approximately 1500 bp) was determined on both strands. DNA sequencing was carried out at Iontek Company (Istanbul, Turkey). To verify specific 16S rDNA signatures, amplified PCR fragments were sequenced by using an internal primer (357F; 5'-TAC GGG AGG CAG CAG-3'). Sequence of the 16S rDNA of isolate was first analyzed using the advanced BLAST search program at the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST/. The entire nucleotides of the 16S rDNA sequences were aligned with the 16S rDNA sequences of Bacillus type strains by using the default setting of CLUSTALW (Mega 5, Beta Version) program (Thompson et al. 1994). Distance matrices were calculated with the Kimura-2parameter algorithm (Kimura 1980) and the tree was constructed with the default setting of MEGA 3.1 using the neighbor-joining method (Saitou and Nei 1987). Halobacillus alkaliphilus FP5 was used as outgroup. The 16S rDNA sequence of the isolate CA15 has been deposited in GenBank database with accession number GQ355961. Growth of bacterium Among the isolates, Bacillus sp. CA15 which exhibited a remarkable protease activity was selected for the study of alkaline protease production. The initial pH of the growth medium was adjusted with 1 M HCl or NaOH before sterilization. The medium (50 mL) was inoculated with 0.1 mL of an overnight seed culture in 250 mL flasks and incubated at 30C with shaking at 180 rpm for 120 h. The cell-free supernatant was recovered by centrifugation (10,000 x g for 10 min at 4C) and used for determination of protease activity. Alkaline protease assay Protease activity was determined using sulphanilamide azocasein as a substrate according to the method of Leighton et al. (1973). The reaction mixture containing 250 L of substrate (1%) in 0.1 M carbonate/ bicarbonate buffer (pH 10.0) and 150 L of enzyme solution was incubated for 30 min at 37C. After incubation, the enzyme was inactivated by addition of 1.2 mL trichloroacetic acid solution (10%) and then
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EurAsian Journal of BioSciences the solution was neutralized using 0.8 mL of 1.8 N NaOH. The absorbance was read at 420 nm. One unit of proteolytic enzyme activity was defined as the amount of azocasein that hydrolyzed during 1 h incubation at 37C for mL of supernatant solution. Optimization of culture medium To test the effect of different nitrogen source on the protease production, the starch (1%) containing liquid medium was supplemented with various complex nitrogen sources such as skim milk, tryptone, casamino acid, yeast extract, peptone, and several inorganic nitrogen sources (ammonium sulphate, ammonium chloride and ammonium nitrate) at 1% concentration. The effect of different carbon sources on protease production was studied by supplementing; a liquid medium of skim milk (1%) with starch, glucose, sucrose, glycerol, dextrose, lactose, maltose at 1% concentration. The flasks were incubated for 120 h and cell free supernatant was analyzed for protease activity at 24 h interval. The obtained best carbon and nitrogen source was further optimized in the range of 0.5-3% for maximum enzyme activity. To determine the effect of metal ions on the protease production, different metal salts; CaCO3, K2HPO4, KH2PO4, MgSO4.7H2O, NaCl, and MnCl2 were individually added with 0.4% strength (except for CaCO3, 0.04%) in starch (1%) and skim milk (1%) in liquid medium. Calcium carbonate solution was sterilized separately, and then added to the medium. The flasks were incubated for 120 h and then cell-free supernatants were analyzed for protease activity at 24 h interval. Determination of optimum temperature and medium pH for enzyme production To optimize the culture conditions for maximum production of protease, incubation temperature (30C, 35C, 40C, 45C and 50C) and medium pH (5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0) were investigated. The effects of temperature and pH on protease production were recorded for 120 h incubation periods at 24 h interval. Time course of protease production Bacillus sp. CA15 cells were incubated at the optimized conditions in order to determine the optimum growth phase and cell density for protease production. Viable cells were enumerated by plating at 24 h intervals during
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120 h. Cell-free supernatants were analyzed for enzyme activity. Effect of surfactants and inhibitors on protease activity The cell free supernatants were preincubated with different surfactants such as Triton- X-100, Tween-20 and SDS. To study inhibition of protease, enzyme was preincubated with phenylmethylsulphonylfluoride (PMSF, 1 and 5 mM) and ethylendiamine tetraacetic acid (EDTA, 1 and 5 mM) for 1 h at 37C. After incubation, the residual activity was determined by enzyme assay. The enzyme activity was assayed in the absence of detergents was taken as 100%. RESULTS AND DISCUSSION Identification of bacterium Rhizospheric soil of the plant A. ketenoglui has alkaline pH (pH 8.3) and isolate CA15 was obtained from this sample. This isolate was gram positive, rod-shaped, aerobic, catalase positive and spore forming. Molecular phylogenetic studies showed that the strain CA15 was a member of Bacillus cereus group. A comparison of the DNA sequence with sequences in the NCBI database with BLAST software showed 98% sequence identity with the published 16S rRNA gene sequence of B. cereus (Fig. 1). Effect of nitrogen and carbon sources on the protease production Among used of organic nitrogen sources, skim milk was found to have significant effect on the production of the extracellular protease (Fig. 2), and high level of the production was achieved when the cells were grown in a medium containing 1% (w/v) skim milk (Fig. 3). Increasing skim milk concentration has an inhibitory effect on protease production. Yeast extract, tryptone and casamino acid exhibited poor effect on the protease production in Bacillus sp. CA15 (Fig. 2). Inorganic nitrogen sources caused significant reduction in the protease yields. In some earlier reports, it was found that different nitrogen sources such as soybean meal, glucose, casamino acid, and peptone were effective medium ingredients for the protease production by Bacillus species (Mehrotra et al. 1999, Joo et al. 2002, Puri et al. 2002, Joo and Chang 2005, Patel et al. 2005, Chu 2007). However, there is no report
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Fig. 1. Rooted phylogenetic tree showing the relationship between isolate CA15 to closely related Bacillus species and H. alkaliphilus FP5 as outgroup.

Fig. 2. Effect of different nitrogen sources on protease production at the different incubation periods.

Fig. 3. Effect of different concentration of skim milk on protease production at the different incubation periods.

on the use of skim milk in liquid medium as a sole nitrogen source to produce protease by Bacillus species in the literature. Vidyasagar et al. (2006) observed that archaeon Halogeometricum sp. TSS101 had maximum protease production in a medium containing 1% of skim milk powder. The negative effect of inorganic nitrogen sources on protease production by Bacillus sp. has been observed in earlier investigations (Patel et al. 2005, Shikha et al. 2007). The repression of protease biosynthesis may be attributed to the
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release of ammonia from these inorganic nitrogen sources. Our results related with the repression of the enzyme production by ammonium salts were consistent with previously published other studies (Johnvesly and Naik 2001, Patel et al. 2005). Results indicate that different carbon sources have different impact on the production of extracellular protease by Bacillus sp. CA15. Among the various carbon sources tested, soluble starch was found to be the best support protease production with a concentration of 1% (Fig. 4). It was observed that the production of extracellular protease was not greatly affected at the different concentrations of starch (data not shown). In addition, good protease activity was also observed in the media supplemented with dextrose, maltose, glucose and glycerol (Fig. 4). However, sucrose and lactose showed decrease in protease yields. Following the carbon and nitrogen optimization, the highest yield (44 U/mL) was achieved in Bacillus sp. CA15. Such starch enhancement of alkaline proteases has been reported in both alkaliphilic B. cereus (Zambare et al. 2007) and B. licheniformis extracellular proteases (Sinha and Satyanarayana 1991). The repression effect of lactose was consistent with the findings of the study on alkaline protease production by Bacillus sp. (Chu 2007). Joo et al. (2002) also found that the extracellular alkaline protease production in B. horikoshii was reduced by addition of 1% glucose and lactose to the TSB medium. Effects of metal ions on the protease production The effect of various metal ions on protease production was evaluated. Among these ions MgCl2.7H2O was found to increase protease production (50 U/mL) (Fig. 5). The optimum Mg2+ salt concentration was found to be 0.6% with a 55 U/mL enzyme production (Fig. 6). The enzyme activity was slightly enhanced of by supplementation of K+, Na+ and Ca2+ ions compared to control. It was reported that supplementation of Mg2+, Ca2+ and K+ salts to the culture medium exhibited slightly better production of protease (Ellaiah et al. 2002, Nadeem et al. 2007). However, the supplementation of metal ions was not necessary for Bacillus sp. CA15 enzyme production in our culture medium when compared to other reports
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Fig. 4. Effect of different carbon sources on protease production at the different incubation periods.

Fig. 5. Effect of metal ions on protease production at the different incubation periods.

Fig. 6. Effect of different concentration of magnesium ions on protease production.

(Ghorbel et al. 2005, Nilegaonkar et al. 2007). Optimal pH and temperature for protease production The production of enzyme was observed between pH 6.0-10.0 (Fig. 7). The highest enzyme activity (60 U/mL) was obtained at pH 8.0. However, increased alkalinity was not favorable up to pH 10.0 on enzyme production. Bacteria growth was not observed
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at pH 12.0. Therefore, the enzyme production was also not observed. It was reported that proteases secreted by Bacillus sp. presented activity at a wide range of pH (7.0 to 11.0) and temperature (30C to 60C) (Horikoshi 1999, Gupta et al. 2002, Joo et al. 2002). The pH values further the range of 7.0-11.0 could alter the three-dimensional structure of enzyme or ionization state of amino acids by disturbing the electrostatic interactions among the charged amino acids, resulting in loss of enzyme activity. Temperature is one of the most important factors affecting the enzyme production. The enzyme was produced at the temperature between 30C and 45C (Fig. 8). The optimum protease production by Bacillus sp. CA15 was observed at 35C. At 50C, some growth was observed but enzyme production was not detected in the culture medium. A comparison of the literature on the characteristics of alkaline Bacillus strains producing alkaline proteases revealed that most of the alkaline Bacillus strains were mesophilic type with temperature optima of 30-37C. With this regard, strain CA15 was in agreement with the literature (Banerjee et al. 1999, Mabrouk et al. 1999, Genckal and Tari 2006). Effect of surfactants and inhibitors on the enzyme activity The protease produced by Bacillus sp. CA15 was stable toward both the non-ionic and anionic surfactants, such as Triton-X100, Tween-20 and SDS, respectively (Table 1). The high stability against SDS was obtained and it retained approx. 75% of activity at 5% SDS. Many of the available alkaline proteases exhibited low activity and stability towards anionic surfactants like SDS. An alkaline protease from B. clausii I-52 retained approximately 72% activity on treatment with 5% SDS (Joo et al. 2003), while B. pumilus alkaline protease lost 22% activity on treatment with 0.1% SDS for 1 h (Kumar 2002). From the viewpoint of stability of surfactants, the alkaline protease from B. cereus CA15 can be considered as a good candidate for industrial applications, especially in detergent industry. In the presence of 5 mM PMSF, the activity of enzyme was inhibited by 90%. The enzyme was not remarkable inhibited by EDTA (15%) indicated that it may not be a metalloprotease
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Table 1. Effect of surfactants and inhibitors on protease activity from Bacillus CA15.

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Fig. 9. Time course of protease production by Bacillus sp. CA15 ( Cell density, Protease production).

Fig. 7. Effect of different initial pH on protease production at the different incubation periods.

Fig. 8. Effect of temperature on protease production at the different incubation periods.

(Table 1). This inhibition profile suggested that the protease produced from Bacillus sp. CA15 belongs to the family of serine proteases (Kumar 2002, Genckal and Tari 2006). Many of the Bacillus-derived alkaline proteases reported so far, belong to the class of serine proteases (Gessesse 1997). The slight inhibition of enzyme by EDTA suggested that the enzyme is a metalactivated enzyme. Furthermore, the stability of the enzyme in presence of EDTA is
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advantageous for its use as a detergent additive. Time course of protease production Under the optimum conditions, the protease activity reached to 60 U/mL within 96 h when the cell growth reached stationary phase (Fig. 9). In an earlier report using azocasein as a substrate, the extracellular protease activity was found to be about 60 U/mL during stationary phase of novel-alkali tolerant bacterium B. patagoniensis (Olivera et al. 2006). Production of extracellular proteases during the stationary phase of growth (concomitantly with the sporulation) has been described as characteristic of many bacterial species (Ferrero et al. 1996, Olivera et al. 2006, Bhaskar et al. 2007). Thus, optimization studies resulted in the following findings: the most suitable nutrient medium starch (1%), skim milk (1%) and MgSO4.7H2O (0.6%) of initial pH 8.0, temperature 35C and period of incubation 96 h at cell density 1.8x108 cells/mL. The protease produced from Bacillus sp. CA15 can be considered as a possible candidate for the cost-effective enzyme due to use of inexpensive substrates such as skim milk and starch. Considering the characteristics of Bacillus sp. CA15, proteolytic activity in a wide range of pH, possibility of setting up processes at low temperatures and compatability to surfactants, this strain could be a potential source of alkaline protease to be used as additive in detergent formulation or in the leather industry. The production process can be commercialized after further optimization for enhanced enzyme production and characterization.
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EurAsian Journal of BioSciences ACKNOWLEGEMENT We thank to Dr. Murat Kizil for his helpful REFERENCES comments and discussions preparation of the manuscript.

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Yeni Izole Edilmis Bacillus cereus CA15 Trnden Ektraseller Proteaz Uretimi Iin Optimum Kltr Kosullari zet Topraktan alkalen proteaz reticisi Bacillus sp. CA15 izole edildi. 16S ribosomal RNA gen dizilimine gore bu mikroorganizmanin Bacillus cereus ile ok yakin akraba oldugu tespit edildi. Proteaz retiminin artisini saglamak iin, kltr kosulari; karbon ve azot kaynaklari, metal iyonlari, pH ve isinin etkisi aisindan optimize edildi. En yksek proteaz retimi, %1 yagsiz st tozu, %1 znr nisasta ve %0.6 MgSO4.7H2O varliginda, baslangi pH'si 8.0 ve 35C'de retilen kltrlerden elde edildi. Bu besiyerinde retilen hcrelerin, canli hcre sayisinin, mL'de 1.8x108'e vardigi duragan fazda en iyi enzim aktivitesine rastlandi. Inorganik azot kaynaklari varliginda proteaz seviyesinin olduka dsk oldugu bulundu. Skroz ve laktozun varliginda da proteaz retiminin azaldigi tespit edildi. Bacillus sp. CA15 alkalen proteazinin Triton X-100, Tween 20 and SDS'ye karsi son derece dayanikli oldugu gzlemlendi. Enzim aktivitesinin PMSF varliginda inhibe olmasi bu enzimin bir serin proteaz oldugunu dsndrd. Anahtar Kelimeler: Alkalen proteaz, Bacillus sp., optimum kltr kosullari, 16S rRNA geni.
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