Production and purification of a protease from an alkalophilic Bacillus sp. 2-5 strain isolated from soil

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Production and purification of a protease from an alkalophilic Bacillus sp. 2-5 strain isolated from soil
    A  r  c   h   i   v  e   o   f    S   I   D 110 IRANIAN JOURNALof BIOTECHNOLOGY, Vol. 5, No. 2, April 2007    Abstract  This research has focused on isolation and character-ization of a strain of Bacillus sp . from alkaline soil,which was able to produce extracellular alkaline pro-tease at pH s ranging from 8 to 11 and temperatures of 20 to 50 ºC . Also the impact of different carbon andnitrogen sources were investigated. The yield and foldof enzyme purification was 24 % and 50 times, respec-tively. Molecular weight of purified enzyme was meas-ured by SDS-PAGE as 24.7 kDa. The alkaline pro-tease produced by Bacillus sp. 2 - 5 showed the mostcaseinolytic activity (without any gelatinolytic activity)at pH > 10. Keywords:  Alkalophilic Bacillus ; Protease; Processvariables; Purification. Proteases constitute one of the most important groupsof industrial enzymes and have applications in differentindustries such as detergent, food, feed, pharmaceuti-cal, leather, silk and for recovery of silver from used X-ray films (Anisworth, 1994; Fujiwara, 1993). Thisenzyme accounts for 30%of the total world enzyme production (Horikoshi, 1996). Among bacteria,  Bacillus spp . are specific producers of extracellular alkaline proteases (Godfrey and Reichelt, 1985). Theseenzymes are quite often added to laundry detergents (tofacilitate the release of proteinaceous stains) (Masseand Tilburg, 1983) and in detergent preparations usedin the dairy and food industries (to remove proteinfoulants from ultrafiltration and reverse osmosis mem- brane systems). Given the wide application of thisenzyme, it was reported that in the year 2005, the glob-al proteolytic enzyme demand increased dramaticallyto 1.0-1.2 billion dollars (Godfrey and Reichelt, 1985).Therefore, taking this demand into account and know-ing the geographic richness and biodiversity of theIranian local environment, it is assumed that there is potential for alkalophilic  Bacillus species living inthese environments. In this paper isolation and charac-terization of a new strain of  Bacillus sp . from alkalinesoil, and its ability to produce alkaline protease at pH s ranging from 8 to 11 and temperature of 20 to 50ºChave been reported. Also, purification and certain prop-erties of the alkaline protease as well as the effect of  process variables such as carbon and nitrogen sources,temperature, pH and time on alkaline protease activityhave been investigated.Alkaline salty soil sample with a pH 10 was collect-ed (surrounding regions of Yazd and Tehran, Iran), sus- pended in sterile saline water (100 g/l) and incubatedat 80°C for 20 minutes (Hitomi, et al  ., 1994). After cooling, it was spread on specialized culture mediacontaining (g/l): glucose 11.1, peptone 5.5, yeastextract 5.5, K  2 HPO 4 11.1, MgSO 4 .7H 2 O 0.22, andagar 16.6. The plates were incubated at 37°C for 24 h.Pure colonies were transferred to a new medium con-taining (g/l): peptone 5, beef extract 3 (or yeast extract1), agar 15 to prepare stock culture. By comparing theability of microorganisms to hydrolyze gelatin, caseinand starch at two different pH s (7 and 10), an alkaline protease producer was selected for further experimen-tal studies. Aloopful of the prepared stock culture was Short Communication Production and purification of a protease from an alkalophilic Bacillus sp . 2-5 strain isolated from soil Hamidreza Falahatpishe 1* , Mahmoud Jalali 2 , Naser Badami 2 , Nadia Mardani 2 ,Kianoush Khosravi-Darani 1 1 Department of Food Technology Research, National Nutrition and Food Technology Research Institute,Shahid Beheshti University, M.C., P.O. Box 19395-4147, Tehran, I.R. Iran 2  Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, I.R. Iran * Correspondence to: Hamidreza Falahatpisheh, M.Sc. Tel: +98 21 22376426; Fax: +98 21 22360660 E-mail:  Archive of     A  r  c   h   i   v  e   o   f    S   I   D   transferred into a 100-ml flask containing 9 ml of inoculum medium consisting of (g/l): peptone 5.55, beef extract 3.33, yeast extract 1.11, MgSO 4 .7H 2 O0.55, and adjusted to pH 10.5 with 10% (w/v) steril-ized Na 2 CO 3 . The culture medium was incubated at37°C with agitation at 125 rpm on a rotary shaker, for 24h. Then, 10% (v/v) of the prepared inoculum wasadded to culture medium containing (g/l): starch 50,yeast extract 5, casamino acid 3, K  2 HPO 4 1,MgSO 4 .7H 2 O 0.2, and adjusted to pH 10.5 (as men-tioned above). After 72h of incubation in a shaker incubator (Model G-24, New Brunswick, USA) at40°C and 125 rpm, the culture was harvested, clarifiedusing a refrigerated centrifuge (MSE, UK) for 20 minat 10000 ×  g  and 4°C. Alkaline protease activity and protein content were determined in the supernatantsolution. One alkaline of protease activity (APU) isdefined as the amount of enzyme that produces TCAreagent soluble peptide equivalent to 1 µ g of tyrosine(spectrophotometric analysis of tyrosine absorbance at275 nm) per 1 min at 30°C and pH 10 (0.02 M borate- NaOH buffer), using Hammerstein casein (Merck,Germany) as substrate. Protein was measured by themethod of Lowry et al  . (1951), with bovine serumalbumin as the standard. The absorbance of culture broth at 660 nm was measured for estimation of cellgrowth and biomass production (Kobayashi, et al  .,1996). Some of the process variables which wereexpected to influence enzyme production during fer-mentation were also investigated (Fujiwara, 1993;Gessesse, et al  ., 2003; Kobayashi, 1996; Uyar andBaysal, 2004). Organic or inorganic nitrogen sources(casamino acids, yeast extract, peptone, ammoniumchloride, sodium nitrate, ammonium sulfate, urea andL- glutamate) were added (5 g/l) to a basic culturemedium containing (g/l): glucose 2.5, K  2 HPO 4 0.1,MgSO 4 .7H 2 O 0.02 and adjusted to pH 10.5 (as men-tioned above). Also different carbon sources (glucose,starch, sodium citrate, casein and sodium acetate) wereadded at a concentration of 10 (g/l) to basic culturemedium (containing 3 g/l casamino acids). After 48hof incubation at 37°C, with an agitation rate of 125rpm, the media were analyzed for cell mass, total pro-tein concentration, alkaline protease activity and final pH. The nitrogen source (yeast extract and casaminoacids) and carbon source (starch) concentrations werekept constant at 5 and 3 g/l, respectively. Temperaturerange of 20 to 50°C with different incubation times (0to 72 h) were investigated. Alkaline protease activitywas analyzed at 6h intervals after initiation of fermen-tation. Cultures with different initial pH s ranging from6 to 14 were incubated at 37°C with shaking at 125rpm for 36 h, in order to examine the effects of pH onenzyme production. All the purification steps wereachieved at 4°C. The concentrated enzyme wasapplied to a CM Cellulose column (2.5 × 30 cm), after washing the column with 10mM phosphate buffer, pH7.5. The bound enzyme was eluted using a linear gra-dient of KCl (0.5 M) added to the washing buffer.Fractions (2 ml each) containing alkaline proteaseactivity were pooled (80 ml, 40 tubes) and dialyzedagain. 1 ml of purified and concentrated enzyme solu-tion was analyzed for alkaline protease activity, specif-ic activity and protein content. To the cell free supernatant, 125 ml solid ammoni-um sulphate (55% saturation) was added and cen- Falahatpishe et al  . 111 Nitrogen sources Concentration(g/l)Cell mass (660 nm) APU 2 /mlCasamino acidPeptoneyeast extractL-glutamateUrea Ammmonium chloride  Ammmonium sulphate Sodium nitrateCaseinyeast extract + Casamino acidsyeast extract + peptoneyeast extract + L-glutamate55555555105 + 35 + 35 + 30.1870.1800.7100.2600.084----------------0.1110.4620.2640.7100.959878 ± 41.91022 ± 62.11264 ± 63.81118 ± 58.2684 ± 35.2588 ± 29.3 636 ± 31.3780 ± 39.51794 ± 90.71938 ± 96.51938 ± 96.9 1938 ± 96.8 Table 1. Effect of nitrogen sources on alkaline protease production by Bacillus sp.2-5. in mediumcontaining (g/l): glucose 2.5, K 2 HPO 4 0.1, MgSO 4 .7H 2 O 0.02, after 48h incubation at 37°C 1 . 1 Each value is the average of three replications. 2  Alkaline protease unit.  Archive of     A  r  c   h   i   v  e   o   f    S   I   D   trifuged at 4°C. After resuspension of precipitated phase in phosphate buffer, it was dialyzed under vacu-um (Cut off  < 10 kDa). The concentrated enzyme wasapplied to carboxy methyl cellulose (CMC) column(2.5 × 30 cm). The bound enzyme was eluted using alinear gradient of KCl (0.5 M). Fractions containingalkaline protease were pooled (80 ml, 40 tubes) andredialyzed. 1 ml of purified and concentrated enzymewas analyzed for alkaline protease activity, specificactivity and protein content (Bollag and Edelstein,1991).Casamino acids (878 APU/ml, peptone (1022APU/ml), yeast extract (1264 APU/ml), L-glutamate(1118 APU/ml) and urea (684 APU/ml) individuallyreduced protease activity (Table 1). These results aresimilar to those reported by Joo et al  . (2002) whoobserved decreased protease activity of  Bacillus sp.I-312 after growth on peptone. Casein addition (1g/l)had a significant effect on biomass production andenzyme activity, although it was still less than theeffect of mixed nitrogen sources. This observation is inagreement with the results of reduced alkaline proteaseactivities of  Bacillus horikoshii, Bacillus licheniformis MIR29, Bacillus mojavensis and  B. horikoshii 104 inthe presence of casein (Beg and Gupta, 2003; Joo et al  ., 2002). But it was somewhat different from the  Bacillus spp.I-312 (Glazer and Nikaido, 1995). Itshould be mentioned that application of synthetic andunpurified nitrogen sources influence yield not only asnitrogen sources but also as sources for excess carbonduring protease production. Data in Table 2 shown thatthe highest protease production was achieved by addi-tion of starch at a concentration of 5 g/l. Higher con-centrations did not affect protease production, signifi-cantly. Similar results have been reported with respectto the influence of corn, potato starch and wheat flour as carbon sources on protease production by  Bacillus sp.I-312 (Glazer and Nikaido, 1995). It seems that the“catabolite repression” mechanism, is the best possibleexplanation for the reduce of protease production inthe presence of glucose (Glazer and Nikaido, 1995),therefore, it is preferable to use complex carbonsources. Glucose (1 g/l), sodium citrate and sodiumacetate (1 g/l) decreased protease production yield by34%, 43% and 20%, respectively. Addition of glucose(1 g/l) to basal media reduced alkaline protease pro-duction by  B. horikoshii to 45% (Joo et al  ., 2002).Gessesse and colleagues (2003) also reported that pro-tease production in  Bacillus pseudofirmus AL-89increased in the presence of glucose, whereas in  Nesternkonia sp. AL-20 was suppressed. Table 3shows the effect of purification steps on specific activ- IRANIAN JOURNALof BIOTECHNOLOGY, Vol. 5, No. 2, April 2007 112 Carbon sourceConcentration (g/l)Cell mass (660 nm) APU 1 /mlStarchGlucoseSodium citrateSodium acetate0.112.5571110.250.480.4750.4950.20.6290.6480.4888001200155025001400160015041600 Table 2. The effect of carbon source on alkaline protease production by Bacillus sp.2-5. in amedium containing (g/l) casamino acids 3, yeast extract 5, after 48h incubation at 37°C. Purification stepsVolume(ml)Proteaseactivity(APU/ml)Protein con-tent (mg)Specific activity(APU/mg)PurificationfoldRecovery(%)SupernatantSalting out + UF 1 CMC 2 + UF 1221012379111478575742084.11.80.4282982148143550129501007024 Table 3 . The effect of purification steps on specific activity, purification fold and percentage of purified alkaline protease recovery. 1  Alkaline protease unit (APU).1Ultrafiltration2 Carboxy methyl Cellulose  Archive of     A  r  c   h   i   v  e   o   f    S   I   D ity, purification fold and percent of purified alkaline protease recovery. Final purification yield and foldwas obtained as 24% and 50 times, respectively (Table3).In this study, the alkalophilic  Bacillus sp.2-5 strainshowed higher protease production at pH 10.Therefore, this isolate can be a potential source of alkaline protease for use as an additive in industrialapplications. The objective of the future study is tofind the optimal conditions for Ca-alginate gel immo- bilization of the new isolated bacterium and to deter-mine the operational stability of the resulting biocata-lyst for the production of alkaline protease under semi-continuous cultivation conditions. References Anisworth SJ (1994). Soap and detergents. Chem Eng News .72:34-59.Beg QK, Gupta R (2003). Purification and characterization of anoxidation stable thiol-dependent serine alkaline protease from  Bacillus mojavenesis .  Enzyme Microbial Technol  ., 32, 294-304. Bollag DM, Edelstein SJ (1991).  Protein Methods . John Wiley andSons, New York, PP. 71-160. Fujiwara N (1993). Production of thermopilic alkaline proteasefrom  Bacillus sp . B18.  J Biotechnol  . 30, 245-256.Gessesse A, Kabul RH, Gashe BA, Mattiasson B (2003). Novelalkaline protease from alkalophilic bacteria grown on chickenfeather.  Enz Microb Tech . 32, 519-524. Glazer AG, Nikaido H (1995). Microbial Biotechnology In:  Fundamentals of Applied Microbiology , Freeman andCompany, Washington, PP. 256-259.Godfrey TA, Reichelt J (1985).  Industrial enzymology : the applica-tion of enzymes in industry. The Nature Press. London.Hitomi J, Adachi S, Hakamada Y, Takaiawa M, Yoshimatsu T,Watanabe Y, Kobayashi T, Kawai S, Ito S (1994). Alkaline protease isolation from  Bacillus sp.US patent   No.5,296,367. Horikoshi K (1996). Alkalophils from an industrial point of view.  FEMS Microbial Rev . 18: 259-270.Joo HS, Kuma CG, Park CG, Paik SR, Chang CS (2002).Optimization of the production of an extracellular alkaline protease from  Bacillus horikoshii .  Process Biochem . 38: 155-159. Kalisz HK (1988). Microbial proteinases.  Adv Biochem Eng  Biotechnol  . 36: 1-65. Kobayashi T, Hakamada Y, Hitomi J (1996). Purification of alka-line proteases from Bacillus strain and their possible interre-lationship.  Appl Microbiol Biotechnol  . 45: 63-71. Lowry OH, Rosenberg NI. Farr AL. Randall RJ (1951). Proteinmeasurement by folin phenol reagent.  J Biol Chem . 193: 265-275.Masse FWJL, Tilburg RV(1983). The benefit of detergentenzymes under changing washing conditions.  J Am Oil ChemSoc . 60:1672-1675.Uyar F, Baysal Z (2004). Production and optimization of process parameters for alkaline protease production by a newly isolat-ed  Bacillus sp. under solid state fermentation.  Process Biochem . 39:1893-1898. Falahatpishe et al  . 113  Archive of 
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