ECOLOGIA BALKANICA2018, Vol. 10, Issue 2 December 2018 pp. 185-197

Isolation and Purification of Proteolytic Enzymes, Produced byStrains of Genus Bacillus

Yordan M. Stefanov*, Ivan Z. Iliev, Mariana Marhova, Sonya Kostadinova

University of Plovdiv, Faculty of Biology, Department of Biochemistry and Microbiology,24 Tzar Assen Str., 4000, Plovdiv, BULGARIA

Corresponding author: iordanstefanov@gmail.com

Abstract. One hundred sixty-six bacterial strains belonging to genus Bacillus were tested forprotease production. Ninety percent of the studied strains demonstrated protease activity onnutrient gelatin and milk agar. A strain Bacillus thuringiensis was selected as the most promising forenzyme production based on its initial enzyme activity of 9.2 U/ml. The nutrient mediumcomposition and cultivating conditions were optimized aiming better yields. The highest proteaseactivity of 15 U/ml was achieved on the following conditions: inoculation of the medium with 5%inoculum (6.0 McF), followed by 16 hours of cultivation in liquid medium containing 0.5% glucose,0.55% Bacto Peptone, 50mM phosphate buffer and 0.2% magnesium ions. The produced enzymeswere partially purified 5.6 fold by ultrafiltration and size-exclusion chromatography on SephadexG-75 and had a specific activity of 17.7 U/mg. The approximate molecular weights weredetermined by SDS-PAGE to be between 45 and 66 kDa.

Key words: Bacillus, protease, enzyme production, SDS-PAGE, zymography.

IntroductionA vast variety of organisms, such as

plants, animals, and microorganisms produceproteolytic enzymes. It is estimated that morethan 40% of the proteases, which are soldworldwide, are of а microbial origin (GODFREY

& WEST, 1996). Microorganisms are attractivesources of proteases because they can becultivated in artificial conditions and in largequantities for a relatively short time byestablished fermentation methods. Screeningand characterization of proteases fromdifferent sources has many advantages bothfrom an environmental and industrial point ofview (MIENDA et al., 2014). In addition,microbial proteins have a long shelf life andcan be stored for weeks under normal

conditions without losing their activity (GUPTAet al., 2002). Bacteria are the best choice forproducers of enzymes. They are preferred tothese with plant or animal origin because theyhave almost all desirable qualities forapplication in biotechnology (HAMZA, 2017).

The species of genus Bacillus are one ofthe most important groups of proteaseproducers. This is mainly due to their highprotein secretion capacity of over 20 g/lprotein (HARWOOD & CRANENBURGH, 2008)and rapid growth rate with shortfermentation cycles (CONTESINI et al., 2017).This is also the reason for the existence of alarge number of patents and "commercial"proteases from strains of Bacillus species(VETTER et al., 1993; MERKEL et al., 2007). The

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Isolation and Purification of Proteolytic Enzymes, Produced by Strains of Genus Bacillus

secreted proteases from bacilli are threedifferent types from which alkaline, neutralproteases are produced in the highestamounts, and esterases, which have weakproteolytic activity, are secreted in minimalquantity (MÄNTSÄLÄ & ZALKIN, 1980;REHMAN et al., 2017; ANANDHARAJ et al.,2016). Proteases, secreted from Bacillusspecies, have some unique properties, whichexplain their application in differentindustries. They have a broad pH andtemperature activity and stability range. Thismakes them suitable as ingredients for theproduction of detergents. The addition ofenzymes in the detergents lowers the needfor high temperatures of the water, reducesthe need for extensive mixing and prolongsthe life of the clothes. Preparations withproteases are ideal for the removal of bloodstains and stains from food with highprotein content (CONTESINI et al., 2017). SomeBacillus strains, used as enzyme producers,have GRAS status, which means that theyare safe and their use in the food productionis allowed. Their proteolytic enzymes can beused for the ripening of cheese, productionof fermented sauces, and for production ofbioactive peptides (CONTESINI et al., 2017;OZCANE & KURDAL, 2012). Bacillus proteasesare also stable in organic solvents, whichmakes them ideal for use in organicsynthesis reactions (FABIANO et al., 2017).

Proteases are produced from Bacillusspecies mainly during stationary phase andthe synthesis is regulated by the source andquantity of carbon and nitrogen, agitation,dissolved oxygen and other parameters(FABIANO et al., 2017). In Bacillusthuringiensis, the synthesis and secretion ofproteases start during the late exponentialphase and stationary phase of growth. Theysecrete them to hydrolyze the remainingpolypeptides in the medium to satisfy thecell needs for nitrogen (BRAR et al., 2007).Proteases are also a crucial factor for theinsecticidal properties of this species. Theseenzymes hydrolyze the prototoxins to formactive insecticidal crystals (ANDREWS et al.,1985). Bt proteases have also some other

favorable properties. They are stable andremain active in the range of pH 7-10 andtemperatures of 30-80 °С (ZOUARI et al.,1999). After the fermentation process, themedium contains not only proteases but alsoδ-endotoxins. The produced toxins are usedin the agriculture for pest control.Theoretically, proteases and endotoxins canbe produced in the same time and can beseparately purified, giving two products ofgreat importance and making the productionprocess cost-effective (ZOUARI et al., 1999). Inthe field of medicine, proteases are used fordigestive aids, for the treatment of skinproblems and they are even used againstinflammation (CRAIK et al., 2011).

The aim of the current study was toexamine the extracellular protease productioncapabilities of Bacillus strains and to optimizethe cultivation conditions for the enzymesynthesis for achieving higher yields.

Materials and MethodsBacterial strainsStrains of genus Bacillus (166 strains)

from the microbial collection of theDepartment of Biochemistry andmicrobiology, Faculty of Biology, Universityof Plovdiv, Bulgaria were examined forproteolytic activity. Cultures of theorganisms were maintained on nutrient agarmedium at 4°С for routine laboratory use.For long-term use, the strains weremaintained in nutrient agar under paraffinlayer at 4°С. The initial screening was basedon the ability of the strains to hydrolyzegelatin and milk casein.

Hydrolysis of gelatinTest tubes, containing soluble gelatin

medium (peptone – 10 g/l; NaCl – 2.5 g/l;gelatin – 120 g/l; pH 6.8) were inoculatedwith the test strains. After 7 to 14 days ofincubation at 37°С, the test tubes werecooled to 4°С. The liquefaction of themedium is considered as a positive result.

Hydrolysis of caseinEach strain was inoculated on a petri

dish containing solid milk agar medium(skim milk – 30 g/l; Nutrient broth

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(HiMedia, India) – 13 g/l; agar – 15 g/l; pH7.2) and incubated for 24 to 48 hours at 37°С.The casein hydrolysis zone appears as atransparent circle around the colony, whichdiameter is measured.

Fermentation conditions and separation ofculture filtrates

A subculture was prepared prior to thefermentation by inoculation of the strain innutrient broth and incubation for 8h on arotary shaker at 37°С, 120 rpm. The celldensity of the culture was then diluted to 6.0McF units. Erlenmeyer flasks with 300 mlvolume, containing 30 ml growth medium(casein hydrolysate – 5.5 g/l; Bacto Peptone– 5 g/l; glucose – 5 g/l; Na2CO3 – 5 g/l;MgSO4 – 2 g/l; pH 8; sterilized for 15minutes at 121°С) were inoculated with 5%(6.0 McF) subculture. The inoculated flaskswere placed in a rotary shaker at 37°С, 120rpm for 12 hours. For the separation of thecells, the culture medium was centrifuged at4°С, 14 000 rpm for 20 minutes. Thesupernatant was collected for furtheranalysis.

Optimization of the culture conditions forenzyme production

Optimum protease production wasstudied at different incubation periods (0 –24 h), inoculum volume (1 – 10%), bufferingagents, carbon sources (mannose, fructose,maltose and ribose), concentrations of thecarbon source (0.2 – 0.8%), and differentmetal ions (Mg2+, Mn2+, Zn2+, Ca2+).

Total protein and Enzyme activity analysisThe protein concentration was

determined by the Bradford (BRADFORD etal., 1976) and Hartree (HARTREE, 1972)methods using BSA as standard.

Protease assayProteolytic activity was determined by

the modified Anson method using a solutionof 0.65% casein as substrate as described byCUPP-ENYARD (2008). In brief the reactionwas carried out in a reaction mixturecontaining 2 ml of 0.65% casein (Sigma-Aldrich, New Zeland) and 1 ml adequatelydiluted supernatant by incubation at 37°Сfor 30 minutes. The reaction was stopped by

the addition of 5 ml of 110 mMtrichloroacetic acid. The quantity of thehydrolyzed casein was determinedspectrophotometrically at A650 against caseintreated with inactive enzyme as blank. Atyrosine standard curve was used for theexpression of the enzyme activity. One unitof protease activity was defined as theamount of enzyme liberating 1 µg of tyrosineper minute.

Purification of proteaseUltrafiltrationThe bacterial supernatant was subjected

to ultrafiltration by Millipore`s AmiconStirred cell UFSC20001 (200 ml), usingmembranes made of regenerated cellulosewith dimensions of the pores of 10 kDa. Aconstant pressure of 0.3 Mpa was generatedusing a pressurized bottle with argon gas.

Sephadex G-75 chromatographyThe concentrate obtained by

ultrafiltration was loaded onto a SephadexG-75 column (2.5 x 94 cm ), pre-equilibratedwith 50 mM Tris-HCl (pH 7.5), at a flow rateof 20 ml/h. Fractions of 7 ml were collectedand those having proteolytic activity weresubjected to electrophoretic separation bySDS-PAGE.

Electrophoretic separationDenaturating SDS-PAGEThe samples were mixed with reducing

sample buffer (0.5 ml 1M Tris-HCl pH 6.8buffer, 2 g glycerol, 0.4 g SDS, 0,3 mlmercaptoethanol, 4 mg Bromophenol blue,8.5 ml distilled water) in 1.5 ml tube. Thetubes were heat treated at 100°C for 90seconds and cooled in ice. A 40 µl persample was loaded on 10% polyacrylamidegel and separated at 120 V for 4h in SDS-Tris-glycine buffer, pH 8.05. The gel wasstained with Coomassie blue R-250 (Merck,USA) for 1h and distained using a solutionof water, methanol and acetic acid. Themolecular weight of the proteins wasdetermined using protein ladder (AmershamLow Molecular Weight Calibration Kit).

Zymography Ten percent polyacrylamide gels were

co-polymerised with 0.1% gelatin. The

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Isolation and Purification of Proteolytic Enzymes, Produced by Strains of Genus Bacillus

samples were mixed with non-reducingsample buffer (0.5 ml 1M Tris-HCl pH 6.8buffer, 2 g glycerol, 0.4 g SDS, 4 mgBromophenol blue, 8.5 ml distilled water)and incubated for 5 min at 25°C before beingloaded into gels. The followingelectrophoresis at 120 V at 4°C. After theelectrophoretic separation, the gels wereincubated for 30 min in wash buffer (2.5%Triton X-100 in H2O). Then left in developingbuffer at 37 for 16 h (10 mM Tris pH 7.5 with5 mM CaCl2, 1 M ZnCl2). Gels were stainedwith 30% methanol and 10% acetic acid,containing 0.5% Coomassie brilliant blue R-250 (Merck, USA). De-staining wasperformed with 50% methanol and 10%acetic acid. Gelatinase activity wasvisualized as unstained bands on a bluebackground, representing areas ofproteolysis of the substrate protein(MCKENNA et al., 2017).

Statistical analysisData analysis was performed using

Statistica™ (StatSoft). The results werepresented as mean ± 95% confidence interval.

Results and DiscussionIn the present study, 166 strains of genus

Bacillus were screened for proteolytic activityusing milk agar and gelatin medium. Ninetypercent of the tested strains showedproteolytic activity, their taxonomicdistribution is shown on Figure 1 a. Sixteenstrains with hydrolysis zone with radius,larger than 5 mm, were selected for furtherquantitative determination of theirextracellular protease activity. The strainsdemonstrated highly variable enzyme activity,but the highest activity was established for B.cereus №67, (8.04 U/ml), B. cereus №88 (7.74U/ml), B. thuringiensis №14 (9.20 U/ml) andB. thuringiensis №4 (8.13 U/ml) (Fig. 1 b).Further analyses were carried out with B.thuringiensis №14 as the most productivestrain. Other researchers reported forproducers, whose proteolytic activities rangedfrom 2.85 U/ml (NABRDALIK et al., 2010) to20.67 U/ml (BADHE et al., 2016), and evenhigher values (BHUNIA et al., 2011).

Dynamics of the enzyme production of B.thuringiensis №14

For the determination of the optimalduration of the fermentation process aiminghigh enzyme activity, the studied strain wascultivated for 24 hours and on every 2 hours, asample from the culture was taken foranalysis. The pH of the culture and theextracellular proteolytic activity weredetermined (Fig. 2 a).

The protease secretion began at the 4-thhour of the cultivation and graduallyincreased until it reached its maximum activityof 9.79 U/ml at the 16-th hour. The starting pHof the medium was pH 8, and during thecultivation, it slowly alkalized reaching pH8.7. The highest protease activity wasestablished at pH 7.1. The optimal cultivationperiods is a strain-specific property and canvary – 24 h, 48 h, etc. The optimal pH level ofthe medium can also vary from pH 6 to pH 11(CONTESINI et al., 2017; BADHE et al., 2016).

Optimization of the production of

proteolytic enzymesOne of the most important components

of the medium is the buffering agent. Atfirst, it was buffered with 0.5% Na2C03, asdescribed in the literature (PANT et al., 2015).During the experimental work, thecarbonate-buffering system proved to beinsufficient for maintaining a constant pHwhen cultivating B. thuringiensis №14.Phosphate buffer with varyingconcentrations (from 50 mM to 200 mM) wastested as a substitute buffering system. Thehighest activity was achieved when themedium was buffered with 50 mMphosphate buffer - 11.82 ± 0.74 U/ml (Fig. 2b). Higher phosphate concentrations of 100mM reduced the enzyme activity to 4.16 ±0.63 U/ml, 3.22 ± 0.83 U/ml at 150 mM and1.53 ± 0.47 U/ml at 200 mM (Fig. 2 b).

The volume of inoculum is an essentialfactor that creates a balance between biomassand available resources, which ensuresoptimal enzymatic production (SANDHYA etal., 2005). To determine the optimal volume ofinoculum, equal volumes of culture medium

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Yordan M. Stefanov, Ivan Z. Iliev, Mariana Marhova, Sonya Kostadinova

were inoculated with a 1%, 2.5%, 5%, 7.5%and 10% 8-hour subculture with a celldensity of 6.0 McF (1.8 x 109). The highestenzyme production was achieved when 5%inoculum was used - 13.80 ± 2.85 U/ml (Fig.3 a). When inoculum with volume lower andhigher than 5% was used, the examinedstrain produced a reduced quantity of the

proteolytic enzymes (Fig. 3 a). ABUSHAM etal. (2009) and GEORGE-OKAFOR et al. (2012)described the same results. In the first case,this can be explained by the insufficientnumber of active cells of the strain producer,in the second case when the cell density istoo high, the free surface to volume ratio isreduced.

Fig. 1. Protease activity of strains of genus Bacillus – qualitative (a) and quantitative (b) analysis.

Fig. 2. a) Dynamics of the protease production; b) Influence of the buffering system on theprotease production. *Na2CO3 was used as control (K) buffering agent.

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Fig. 3. Influence of a) the volume of the inoculum; b) different carbohydrates; c) differentconcentrations of glucose; d) different metal ions on the protease production.

The production of protease also dependson the specific preference of the carbonsource. To determine the most appropriatecarbon source for the synthesis of proteaseenzymes four carbohydrates with aconcentration of 0.5% were examined assubstitutes of the glucose – fructose, maltose,ribose, and mannose. The highest proteaseproduction was achieved on the initialmedium with glucose as a sole carbon source- 15.32 ± 2.86 U/ml (Fig. 3 b). When fructosewas used the activity was 8.89 ± 2.41 U/ml,when maltose was used - 7.27 ± 1.55 U/mland ribose - 2.57 ± 0.72 U/ml There was noextracellular protease activity on mediumcontaining mannose. The lack of some keyenzymes may prevent the absorption ofmannose, so the strain develops at the

expense of the pepton available in themedium. At the same time, the high-unchanged monosaccharide concentration init inhibits the synthesis of proteases (KOLE etal., 1988). BADHE et al. (2016) studied theinfluence of various monosaccharides andpolysaccharides and found that the higheststimulation of protease activity is observedwith the addition of glucose. Similar findingsare made by other authors (SEN &SATYANARAYANA, 1993; PASTOR et al., 2001;SANTHI, 2014), which are reporting that thehighest production is observed when glucoseor starch is added to the culture medium.

Another crucial factor for the proteaseproduction is the concentration of the carbonsource. Lower concentrations may beinsufficient to sustain a large bacterial

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population, while higher concentrations mayhave an inhibitory effect over the proteasesynthesis (KOLE et al., 2007). The effect of thedifferent glucose concentrations on theenzyme production was determined (Fig. 3c). Our results confirm that high proteaseproduction is achieved when glucose is usedas a carbon source in the medium at aconcentration of 0.5% - 15.76 ± 2.33 U/ml.When glucose is added to the medium atconcentrations lower or higher than 0.5%,the enzyme production is reduced. Our datadiffers from the findings of other authors.SUGUMARAN et al. (2012) have found thattheir strain produces a maximal quantity ofproteases when the carbon source is 0.6%.ASHA et al. (2018) report that the optimalconcentration of the carbon source for theirstrain is above 1% and MHAMDI et al., (2014)report for preference to even higherconcentrations of 6%. NADEEM et al. (2008)report that increasing the concentration ofglucose to above 0.5% results in increasedprotease production, but our results don`tsupport that observation.

The expression of proteases and theregulation of their activity depends on thepresence of some metal ions (ADINARAYANA

et al., 2003). Sources of metal ions (MgSO4,MnSO4, ZnCl2, CaCl2) were added to theculture medium to evaluate their effect on theextracellular protease activity. The addition ofMg2+

enhanced the enzyme activity up to15.02 ± 1.77 U/ml (Fig 3 d). Manganese, zincand calcium ions had rather inhibitory effectsover the synthesis or the activity of theproteolytic enzymes. These findings areconsistent with the work of other authors(SHARMA et al., 2012; VEERAPANDIAN et al.,2016; KHAJURIA et al., 2015; YILMAZ et al.,2016; SAYEM et al., 2006). Different metalcompounds can stimulate the proteaseproduction, and this is strain specificproperty. Some authors describe thatmanganese and calcium ions can separatelystimulate the production (SHARMA et al., 2012;VEERAPANDIAN et al., 2016; KHAJURIA et al.,2015; CHITTOOR et al., 2016). Others haveshown that the stimulation is achieved by the

synergetic influence of calcium andmagnesium ions (NASCIMENTO et al., 2004).The observed inhibition of protease activity inthe present study is demonstrating theabsence of metalloproteases in thesupernatant of B. thuringiensis № 14. Manyauthors had found a similar inhibitory effecton the synthesis and activity of proteases(SHARMA et al., 2012; VEERAPANDIAN et al.,2016; LAILI et al., 2017; NASCIMENTO et al.,2004; SAYEM et al., 2006) with the inclusion ofzinc, cobalt, mercury and copper ions in themedium. In some cases, calcium andmagnesium may also have an inhibitoryeffect (LAILI et al., 2017; LIU et al., 2013).

Partial purification of proteolytic enzymesfrom Bacillus thuringiensis №14

The strain was cultivated at the optimalconditions, after which the liquid culture wascentrifuged at 14 000 rpm, 4°С for 20 minutes.The collected supernatant subjected toultrafiltration using Millipore`s Amicon®Stirred Cell with filter membranes made ofregenerated cellulose with 10 kDa poresunder pressure (0.3 atm) using argon gas.Using this method, the volume was reduced6.66-fold. The final concentrate showedprotease activity of 15.66 U/ml. The enzymepurification was 1.74-fold times, the yieldbased on the activity was 50.7%, and the totalprotein was reduced 3.43-fold compared withthe supernatant (Fig. 4.).

The contained proteolytic enzymeswere partially purified using size-exclusionchromatography. Ten milliliters of theconcentrate were applied to a column (94 x2.5 cm) filled with Sephadex G-75. Forty-onefractions with 7 ml volume were collected.The proteolytic enzymes were eluted in 13fractions from 172 to 263 ml. The totalvolume collected was 91 ml (Fig. 4, Table 1).

After the gel filtration with Sephadex G-75, the achieved purification was 5.62compared with the specific activity of thesupernatant. The amount of the total proteinwas reduced 11.56-fold and the yieldaccording to the total activity was 48.61%compared with the supernatant. Three

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separated peaks of activity were detected onthe chromatogram corresponding tofractions – 19 (2.56 U/ml), 24 (1.36 U/ml)and 27 (3.77 U/ml), which were elutedrespectively at the 186 – 193 ml; 221 – 228 ml;242 – 249 ml. Our results suggest that thestrain can produce more than oneextracellular protease enzyme, whichmolecular weight varies in a broad range.

Using this method of chromatographicseparation AHMETOGLU et al. (2015) achieved13-fold purification of the protease with a yieldof 23%. Other researchers achieved a 23.78-foldpurification and a 40.9% yield of enzymesproduced by a strain of Bacillus megateriumRRM2 using a Sephadex G-100chromatographic column (RAJKUMAR et al.,2011). Some researchers are carrying out adirect purification with ion-exchangechromatography of the resulting preparationafter. For example, BADHE et al. (2016) achieved2.94-fold purification of a Bacillus subtilispreparation by ion exchange chromatographywith DEAE-cellulose. The cited data confirmedthe high susceptibility of the enzymes to theseparation methods used, associated with lossof activity and low-end yields.

Electrophoretic separation and visualizationSamples of the supernatant, the

concentrated supernatant after ultrafiltration andsamples of the most active fractions obtainedafter size-exclusion chromatography were

analyzed using SDS-PAGE electrophoresis forthe determination of the molecular weight. TheSDS-PAGE results clearly showed that thesupernatant and its concentrate had high proteincontent with a broad range of molecular weight(Fig. 5). The protein content in the fractions wasreduced thanks to the chromatographicpurification. In fractions, 27 and 28 weredetected three identical bends with a molecularweight between 45 and 66 kDa. In fraction 19,there was established a fourth band with lowermolecular weight. The size of the isolatedproteases varied between 45 – 66 kDa. Althoughmany researchers are describing proteases withа molecular weight of 40 kDa (JALKUTE et al.,2017), 40 and 60 kDa (SELIM et al., 2015) theirweight can vary in a broad range between 15kDa to 130 kDa (OZTÜRK et al., 2009).

The same samples (from the supernatant,concentrated supernatant, and the mostactive fractions) were analyzed for proteolyticactivity by zymography (Fig. 6). The methodallowed the identification of gelatinaseactivity in the analyzed samples using SDSpolyacrylamide gels, which wereimpregnated with gelatin. The results showedthat Bacillus thuringiensis №14 producesseveral extracellular protease enzymes withdifferent molecular weight. Two clearlyvisible signals were detected in all studiedsamples, while in fraction 19, there wereadditional signals with lower molecularweight.

Fig. 4. Sephadex G-75 chromatography of protease enzymes produced by B. thuringiensis No 14.

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Table 1. Purification of proteolytic enzymes produced by B. thuringiensis No 14.

StepVolume

(ml)Activity

(U)Protein

(mg)Spec. activity (U/

mg)Purification

(fold)Yields

(%)Supernatant 67.00 308.87 97.82 3.16 - 100Ultrafiltrate 10.00 156.60 28.50 5.49 1.74 50.70Sephadex G75 91.00 150.15 8.46 17.74 5.62 48.61

Fig. 5. SDS gel electrophoresis of samples of the supernatant (9), ultrafiltrate (8) and the three mostactive fractions – 19 (6), 27 (5) and 28 (3).

Fig. 6. Zymography of the supernatant (a – 1, 2, 4 and 7), the ultrafiltrate (a – 4, 5, 6 and 8) and thethree most active fractions 19 (b – 1, 4), 27 (b – 2, 5) and 28 (b – 3, 6).

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Conclusions The studied B. thuringiensis strain

showed consistent extracellular proteaseactivity. The highest activity was recorded atthe end of the exponential phase of growth.Changes of the buffering agent and theaddition of Mg2+ to the culture medium leadto enhancement of the activity up to 15 U/ml. The stable protease production makesthe strain B. thuringiensis №14 a promisingproducer. The partial purification showedthat the strain is producing at least twoseparate proteases with a molecular weightbetween 45 and 66 kDa. Their presence wasconfirmed by zymography. Further studiesare needed in other to separate andcharacterize the proteases.

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Received: 26.11.2018Accepted: 21.12.2018

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