Purification and Characterization of a New … · activity was determined. Effect of commercial...

Research ArticlePurification and Characterization of a New ThermostableHaloalkaline Solvent Stable and Detergent Compatible SerineProtease from Geobacillus toebii Strain LBT 77

Wajdi Thebti Yosra Riahi and Omrane Belhadj

Laboratory of Biochemistry amp Technobiology Faculty of Science of Tunis Tunis El Manar UniversityFarhat Hached University Campus 2092 El Manar Tunisia

Correspondence should be addressed to Omrane Belhadj omranebelhadjfstrnutn

Received 30 December 2015 Accepted 2 March 2016

Academic Editor Pengjun Shi

Copyright copy 2016 Wajdi Thebti et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A new thermostable haloalkaline solvent stable SDS-induced serine protease was purified and characterized from a thermophilicGeobacillus toebii LBT 77 newly isolated from a Tunisian hot springThis study reveals the potential of the protease fromGeobacillustoebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time The protease was purifiedto homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography It was amonomeric enzyme with molecular weight of 30 kDa The optimum pH temperature and NaCl for maximum protease activitywere 130 95∘C and 30 respectively Activity was stimulated by Ca2+ Mg2+ DTNB 120573-mercaptoethanol and SDS The proteasewas extremely stable even at pH 1325 90∘C and 30 NaCl and in the presence of hydrophilic hydrophobic solvents at highconcentrations The high compatibility with ionic nonionic and commercial detergents confirms the utility as an additive tocleaning products Kinetic and thermodynamic characterization of protease revealed 119870

119898

= 1mgmLminus1 119881max = 2175UmLminus1119870cat119870119898 = 99mgmLminus1 Sminus1 119864

119886

= 515 kJmolminus1 and Δ119866lowast = 565 kJmolminus1

1 Introduction

Thermophilic and hyperthermophilic organisms play a keyrole in the recent research and the enzymes produced by thesemicroorganisms are coveted for applications in many areasThe proteases are some of the most commercialized enzymeswith over 65 of total enzyme market [1] They are used invarious catalytic applications in both food and pharmaceuti-cal industries Nowadays their roles in the synthesis of bioac-tive peptides and as additive in commercial detergents aregaining attention [2] To be incorporated in the formulationof detergents the protease must be active and stable in harshwashing conditions like high temperature alkaline pH metalions and high salt concentrations in addition to stability andcompatibility with surfactants and detergents [3 4] Theyare used as additives instead of other chemicals harmful tothe environment Since the first alkaline protease Carlsbergproduced by Bacillus licheniformis was commercialized as anadditive to detergents in the sixties [5] research has empha-sized trying and characterizing new proteases with better

performance Several proteases have been purified andcharacterized from Bacillus circulansDZ100 [6] Streptomycessp AB1 [7] Bacillus subtilis AP-MSU6 [8] Bacillus sp EMB9[9] and Geobacillus caldoproteolyticus [10] The industrialdemand of highly active and stable proteases continues to riseand various attempts have been made to enhance stability ofalkaline proteases by site directed mutagenesis and proteinengineering but the screening of microorganisms fromextreme habitats seems to be the best approach Also in thisstudy a protease fromGeobacillus toebii LBT 77 isolated froma Tunisian hot spring was purified and characterized

2 Materials and Methods

21 Substrates and Chemicals Unless otherwise specifiedall substrates chemicals and reagents were purchased fromSigma-Aldrich (Saint Louis Missouri USA)

22 Microorganism The strain Geobacillus sp LBT 77 pro-ducing alkaline proteases was isolated from the hot spring

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9178962 8 pageshttpdxdoiorg10115520169178962

2 BioMed Research International

ldquoHammam El Atrousrdquo next to the Ichkeul lagoon in BizerteTunisia (37∘08101584018810158401015840N 9∘41101584024710158401015840E) The strain was identi-fied based on the phenotypic characteristics of the Bacillusgenus and phylogenetic analysis of the 16S rDNA sequenceGenomic DNA was extracted as described by Marmur [11]and 16S rRNA gene sequences were amplified using the bac-terial universal primers 16SF (51015840AGAGTTTGATCCTGG-CTCAG31015840) and 16SR (51015840CTACGGCTACCTTGTTACGA31015840)[12] using the following PCR program 1 cycle of 94∘C for5min 30 cycles of 94∘C for 1min 54∘C for 1min and 72∘Cfor 15min and a cycle of 72∘C for 5min The amplifiedproducts were purified and the sequence of the 16S rRNAgene was determined by Sanger method DNA sequencing(ABI 3730xl DNA analyzer USA) Sequence comparisonwiththe databases was performed using BLAST program throughNCBI website A phylogenetic tree was constructed withMEGA version 606 using the neighbor-joining method

23 Screening and Production of Protease Activity Screeningof protease activity was performed by the method of dissemi-nation throughwells onmilk agarmediumat pH9 containing5 gL tryptone 3 gL yeast extract 15 gL agar and 25mLskimmedmilk 60 120583L of an 18 h bacterial culture was injectedinto each well and the plates were incubated for 18 h at 55∘CProtease activity was confirmed by the appearance of a clearzone around the well-testifying degradation of casein milk[6] Production of protease by Geobacillus toebii LBT 77 wascarried out at pH 8 in amedium containing 5 g bactopeptone5 g yeast extract 5 g NaCl 10 g gelatin and 02 g CaCl

2in 1 L

deionised water [9] Inocula were routinely grown in TrypticSoy Broth (Scharlau Spain) medium Media were autoclavedat 120∘C for 20min Cultures were performed on a rotatorshaker (150 rpmmin) for 96 h at 55∘C in 500mL Erlenmeyerflasks with aworking volume of 100mLGrowthwas followedby measuring the optical density at 600 nm every 6 h Theculture medium was centrifuged at 12000 rpm for 20min at4∘C and the cell-free supernatant was used as a crude extractfor estimation of proteolytic activity

24 Assay of Protease Activity Proteolytic activity was deter-mined by using casein as substrate One mL of casein 10 gLin 50mM glycine-NaOH buffer pH 11 was mixed with 900 120583Lof glycine-NaOH Reaction was initiated by the additionof 100120583L of enzyme and the tubes were placed in a waterbath at 70∘C After 20min of incubation 2mL of 10 TCAwas added to stop the reaction The reaction mixture wascentrifuged at 12000 rpm for 10min and absorbance wasmeasured at 280 nm [13] One unit of proteolytic activity wasdefined as the amount of enzyme required to release 1 120583g oftyrosine per minute under experimental conditions

25 Enzyme Purification

251 Ammonium Sulfate Precipitation Cell-free supernatantwas collected by centrifugation at 12000 rpm for 20min at4∘C after 72 h of cultivation Ammonium sulfate was slowlyadded to the supernatant to 80 saturation and the mixturewas incubated overnight at 4∘CThe precipitate was collected

by centrifugation dissolved in a minimal volume of 25mMTris-HCl (pH 85) and dialyzed against three changes of thesame buffer for 24 h

252 Sephadex G-75 Gel Filtration The dialysate was sub-jected to gel filtration on Sephadex G-75 column (21 cm times50 cm) equilibrated with 25mM Tris-HCl (pH 85) Thecolumn was eluted with the same buffer at a flow rate of20mLh and fractions of 3mL were collected and thenanalyzed for protease activity and protein concentrationTheactive fractions were pooled and subjected to ion exchangechromatography

253 DEAE-Cellulose Ion Exchange Chromatography Thisround was performed by applying active fractions from theprevious step to a DEAE-Cellulose (2 cm times 25 cm) equili-brated with 25mM Tris-HCl (pH 85) Bounded proteinswere eluted with a linear gradient of NaCl from 0 to 1M ata flow rate of 50mLh and analyzed for protease activity

All purification steps were performed at 4∘CThe proteincontent of each chromatographic fraction was determined bymeasuring the absorbance at 280 nm

26 Protein Estimation Protein concentration was deter-mined by the colorimetric method of Bradford [14] usingbovine serum albumin as standard

27 Polyacrylamide Gel Electrophoresis and ZymographySDS-PAGE was carried out to determine the purity andmolecular weight of the enzyme as described by Laemmli etal [15] using 5 (wv) stacking and 12 (wv) separating gelsProtein bands were visualized by staining with CoomassieBrilliant Blue R-250 The molecular weight of the enzymewas estimated using a low-molecular weight calibration kit(Biomatik Co Canada) as markers

Zymography was performed by the method of Garcia-Carreno [16] to confirm the enzyme activity After elec-trophoresis the gel was immersed in 100mMTris-HCl buffer(pH 85) containing 25 Triton X-100 at 4∘C with shakingfor 30min to remove SDS then the gel was washed twicewith 100mM Tis-HCl buffer (pH 85) in order to eliminateresidual Triton X-100 and then incubated with 1 (wv)casein in 100mM Tris-HCl (pH 95) at 75∘C for 60minFinally the gel was stained using Coomassie Brilliant BlueR-250 Appearance of a clear halo zone on the dark-bluebackground indicates the presence of enzyme

28 Biochemical Properties of the Purified Protease

281 Effects of Temperature pH andNaCl on EnzymeActivityand Stability To study the effect of temperature on activityof the purified enzyme the reaction mixture was incubatedat different temperatures ranging from 55 to 120∘C Enzymeactivity was measured as described earlier Thermal stabilitywas evaluated by incubating the purified enzyme at 70 8090 and 95∘C for 180min in the presence and absence of 5mMCaCl2 Residual activity wasmeasured at 95∘C and pH 13The

activity of nonheated enzyme was considered as 100

BioMed Research International 3

Aeribacillus pallidus (HF5584471)

Bacillus subtilis (AJ277905)

Bacillus circulans strain ATCC4513 (NR1045661)

Geobacillus toebii (FN5389922)

Geobacillus toebii LBT 77 (KP338027)

Geobacillus kaustophilus (KC2529841)

E coli ATCC11775T (X807251)

Streptomyces tendae strain ATCC19812 (NR0258711)

005

Figure 1 Phylogenetic tree based on 16S rRNA gene sequences drawn using the neighbor-joining method and showing the relationshipbetween Geobacillus toebii LBT 77 and species from genera Bacillus and Geobacillus E coli and Streptomyces tendae were used as out groups

Effect of pH was determined by varying the pH ofthe reaction mixture using the following buffers (100mM)glycine-HCl pH 20ndash50 potassium phosphate pH 60ndash70Tris-HCl pH 80ndash85 glycine-NaOH pH 90 NaHCO

3-

NaOH pH 95ndash10ndash11 NaH2PO4-NaOH pH 115ndash12 KCl-

NaOH pH 125ndash13ndash135ndash1375 To test pH stability theenzyme was preincubated in various buffer solutions (pH 6ndash13) for 12 h at 60∘C The residual enzyme activity was thendetermined at the optimum conditions of assay

Effect of NaCl was carried out by incubating the reactionmixture at different concentrations ofNaCl (0ndash40) Stabilitywas investigated by preincubating the enzyme for 1 h at differ-ent concentrations of NaCl (0ndash40) and residual activity ()was measured at the optimum conditions of assaywa

282 Effects of Inhibitors and Metallic Ions on Activity ofProtease Effect of protease inhibitors such as phenylmethyl-sulfonyl fluoride (PMSF) ethylenediaminetetraacetic acid(EDTA) dithio-bis-nitrobenzoic acid (DTNB) and 120573-mer-captoethanol was determined at 5 and 10mM by measuringrelative activity after preincubating the enzyme for 30min at55∘C The activity at standard conditions without additiveswas considered as 100 [2] Effect of metal ions on enzymeactivity was investigated at 5mM by adding divalent (Zn2+Fe2+ Mg2+ Mn2+ Ca2+ Cu2+ Hg2+ and Co2+) and mono-valent (Na+ K+ Al+ and Li+) ions to the reaction mixtureRelative activities were estimated versus activity without anymetallic ions after 1 h of incubation at 55∘C

283 Effect of Detergents on Enzyme Activity Anionic (SDS)cationic (CTAB) and nonionic detergent (Triton X100) werepreincubated with enzyme for 60min at 55∘C and residualactivity was determined Effect of commercial detergent(OMO Ariel and Nadhif) on protease activity was studiedwith 1 (wv) solution of detergent The enzyme was prein-cubated with the aforementioned detergents at 50∘C for 1 hand then assayed for protease activity

284 Effect of Organic Solvents on Protease Activity Theenzyme wasmixed with 25 and 50 ofmany organic solvents

such as cyclohexane n-butanol ethanol toluene acetonitrileacetone isopropanol methanol and benzene for 4 h at 55∘C

29 Catalytic andThermodynamic Parameters Kinetic para-meters were determined by assaying protease activity againstvarious casein concentrations under optimized assay condi-tions 119881max 119870119898 119870cat and 119870cat119870119898 were calculated using aLineweaver-Burk plot (1119881 versus 1[119878]) Thermodynamicparameters for casein hydrolysis activation energy enthalpyentropy and free energy of activation were calculated as perAkolkar and Desai [17]

All experiments mentioned above were repeated at leastthree times and each value represents the average of threerepetitions

3 Results and Discussion

31 Microorganism From a collection of 161 bacteria 37caseinolytic strains were isolated Among these strains thestrain LBT 77 exhibited a large clear zone of degradationshowing high protease activity Phylogenetic analysis of its 16SrRNA gene sequence indicated that the strain LBT 77 is affili-ated to Geobacillus and is closest to Geobacillus toebii (99homology) (Figure 1)The sequence has been deposited in theEMBLGenBankDDBJ databases under accession numberKP338027

32 Production of Extracellular Protease Protease produc-tion is proportional to bacterial growth in fact themaximumproduction coincides with the end of the stationary phase(1900UmL) at 42 h of incubation (Figure 2) This result isconsistent with reports in the literature [2 18 19]

33 Purification of Protease The protease present in thecrude extract was purified using ammonium sulfate 80followed by Sephadex G-75 and DEAE-Cellulose (Figure 3)Table 1 indicated that the protease was purified to 5-fold withspecific activity of 7395UmgThe reduction of recovery ()from one step to the other is due to the elimination of somelower specific activity during chromatography as reportedpreviously [20]


Table 1 Purification of protease from Geobacillus toebii LBT 77

Purification step Total activity(U)

Total protein(mg)

Specific activity(Umg) Recovery () Purification fold

Crude enzyme 11357 1715 665 100 1Ammonium sulfate precipitation 8064 45 180 71 15Sephadex G-75 2504 7 348 22 45DEAE-Cellulose 22925 3 7395 20 5

0200400600800100012001400160018002000

002040608

112141618

0 6 12 18 24 30 36 42 48 54 60 66 72 84 96Pr

otea

se ac

tivity

(Um

L)

Time (h)

Activity (UmL)

Abso

rban

ce at

600

nm

Cell growth (OD 600nm)

Figure 2 Growth kinetics and protease production of Geobacillustoebii LBT 77

050010001500200025003000

0

05

1

15

2

25

0 10 20 30 40 50 60 70 80 90 100

Prot

ease

activ

ity (U

mL)

Fractions

Protease activity (UmL)

Abso

rban

ce at

280

nm

Absorbance at 280nm

Figure 3 Elution profile of protease from Geobacillus toebii LBT 77on Sephadex G-75 gel filtration chromatography

34 Polyacrylamide Gel Electrophoresis and Zymography Thepurified protease appeared as a single band on the SDS-PAGEwith a molecular weight of approximately 30 kDa (Figure 4)Mostly the molecular weights of bacterial proteases arebetween 15 and 40 kDaThemolecular weight of the proteaseproduced by LBT 77 was lower than those of the Bacilluspumilus CBS (345 kDa) [21] and Bacillus circulans DZ100(32 kDa) [6] and higher than those of the proteases fromBacillus mojavensis A21 (20 kDa) [22] and Bacillus subtilisPE11 (15 kDa) [23]

The zymogramanalysis showed a clear band against a bluebackground indicating the purity and themonomeric formofthe protease

Figure 4 SDS-PAGE and activity staining of purified G toebii LBT77 protease Lane 1 purified proteases after DEAE-Cellulose Lane 2proteases purified by sephadex G75 Lane 3 zymogram of purifiedprotease

Rela

tive p

rote

ase a

ctiv

ity (

)

120

100

80

60

40

20

055 60 65 70 75 80 85 90 95 100 110 120

Temperature (∘C)

Figure 5 Effect of temperature on protease activity


351 Effects of Temperature pH andNaCl on EnzymeActivityand Stability The enzyme was active between 70 and 100∘Cwith an optimum of 95∘C (Figure 5) Relative activities at85 90 and 100∘C were about 72 94 and 65 respectivelyAn optimum of temperature at 95∘C was reported for theprotease from Bacillus sp MLA64 [24]

BioMed Research International 5Re

sidua

l act

ivity

()

120

100

80

60

40

20

00 30 60 90 120 150 180

Time (min)

70∘C80∘C with CaCl290∘C with CaCl295∘C with CaCl2

80∘C90∘C95∘C

Figure 6 Effect of temperature on protease stability

The protease in this study was also found to be completelystable at temperatures lower than 80∘C after 180min ofincubation (Figure 6) Half-life of the enzyme at 95∘C wasestimated to be 70min This is higher than those of otherproteases which retain a lower amount of their initial activityeven in a shorter period of incubation and also at lowertemperatures Ca2+ increased thermal stability of the enzymesince it retained 67 of the original activity after incubationat 95∘C for 180minThe effects of Ca2+ on thermal stability ofproteases were previously reported [25]

The enzyme showed maximum activity at pH 130 andwas highly active in the pH range of 90 to 1325 Indeed theenzyme shows 62 and 80 of its activity at pH 9 and 1325respectively (Figure 7(a)) These findings indicate that thisenzyme belonged to alkaline proteases groupThe particular-ity of this enzyme is its pH optimum of activity In fact mostalkaline proteases listed in the bibliography have optimumpH that does not exceed pH 12 [26 27]The enzymewas stablebetween pH 8 and 13 and retained about 99 of its activityafter incubation at 50∘C for 12 h (Figure 7(b)) These charac-teristics are important for its use as laundry additive [28]

The protease activity has risen by increasing NaCl con-centration until an optimum of 30 and has decreased athigher concentration The same optimum concentration wasobserved for the protease produced by Bacillus alveayuensisCAS 5 [29]Halotolerant proteases are interesting for biotech-nological applications especially in detergent industry andthis one was completely stable at 20 and retained 80 ofactivity at 30 of NaCl (Figure 8) This was higher than theprotease of Bacillus aquimaris VITP 4 [19] which was stableup to 12 and lower than this of Bacillus alveayuensis CAS 5[29] which was stable up to 25

352 Effect of Inhibitors and Metal Ions on Activity ofProtease The effect of inhibitors and chelators on proteasesactivity showed that PMSF strongly inhibits the activityto 20 and 5 at 5 and 10mM respectively (Figure 9)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Rela

tive p

rote

ase a

ctiv

ity (

)

pH

(a)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Resid

ual p

rote

ase a

ctiv

ity (

)pH

(b)

Figure 7 Effect of pH on protease activity (a) and stability (b)

Activ

ity (

)

120

100

80

60

40

Activity

20

Stability

00 5 10 15 20 25 30 35 40

[NaCl] ()

Figure 8 Effect of [NaCl] on protease activity and stability

This confirms its belonging to the group of serine proteases[25 30] Among the tested inhibitors activity was increasedby DTNB and 120573-mercaptoethanol suggesting that it is athiol-depending serine protease [25] The metalloproteasesinhibitor EDTA inhibited activity by 22 at 10mM similarresult was reported earlier in serine protease produced byBacillus licheniformis [31] Regarding the effect of metal ionsthe activity was enhanced with Mg2+ and Ca2+ (Figure 10)

6 BioMed Research InternationalRe

sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM

Figure 9 Effect of protease inhibitors on enzyme activity

Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+

Figure 10 Effect of monovalent and bivalent ions on proteaseactivity

This was similar to result of protease from Geobacillus spYMTC 1049 [32] It has been reported that serine proteasessuch as subtilisin have Ca2+ binding sites and their stabilityat higher temperatures was explained by the strengthening ofinteractions inside protein molecules and the better stabiliza-tion of active site against thermal denaturation [33]

The enzyme was quite stable with Mn2+ K+ Na+ Zn2+Fe2+ Cu2+ Al+ and Li+ at 5MmThe repressive effect of Zn2+and Cu2+ was reported earlier [2 34] Metals like Hg2+ andCo2+ strongly inhibit the activity compared to control

353 Effect of Detergents on Enzyme Activity All testeddetergents did not inhibit protease activity but rather anenhancement was observed with SDS and CTAB (increasingactivity by 20 and 10 resp) This result is consistent withthose reported for alkaline proteases from Bacillus sp andBacillus clausii [35 36] In addition the protease producedby Geobacillus toebii LBT 77 was also compatible with com-mercial detergents like OMO Nadhif and Ariel (Figure 11)

354 Effect of Organic Solvents on Protease Stability Theeffect of various organic solvents at 25 and 50 (vv) on

Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif

Surfactantdetergent (1)

Figure 11 Effect of surfactants and detergents on protease activity

180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2

Figure 12 Effect of organic solvents on protease activity

protease stability is shown in Figure 12 Protease activity wasincreased in the presence of acetonitrile methanol ethanoland n-butanol at 25 vv A similar result was reported forthe protease produced by Bacillus sp EMB9 [9] The mostinteresting observation is that the activity remains stablewith acetone (93 of activity) unlike protease from Bacillussp EMB9 which retains only 20 of its initial activity [9]Activity was stable with isopropanol acetonitrile and n-butanol at 50 vvThere is a small decrease in activity in thepresence of 50 ethanol (91 of activity)

The enzyme keeps 70 62 and 40 of its activity inthe presence of 50 benzene toluene and cyclohexanerespectively Protease from Geobacillus toebii LBT 77 wasfound stable in solvents concentration 50 even after 4 hat 55∘C This was found to be superior stability comparedto protease characterized from Bacillus sp and Bacillus spEMB9 which retain only about 20 of their activity [2 9]

36 Catalytic and Thermodynamic Parameters Thermody-namic and kinetic parameters shown in Table 2 confirm thehigh interest of the enzyme Actually catalytic efficiency ofthe serine protease from LBT 77 (99mgmLminus1 Sminus1) is better


Table 2 Kinetic and thermodynamic parameters of the purifiedenzyme

Kinetic parameters Thermodynamic parameters119870119898

(mgmL) 1 119864119886

(kJmolminus1) 515119881max (UmL) 2175 Δ119867lowast (kJmolminus1) 5119870cat (S

minus1) 945 Δ119878lowast (Jmolminus1) minus229119870cat119870119898(mgmLminus1 Sminus1) 99 Δ119866lowast (kJmolminus1) 565

than those of Bacillus pumilis CBS [21] and Halobacteriumsp1(1) [17] with 45 and 85mgmLminus1 Sminus1 respectively

Briefly the present study demonstrated that the purifiedserine protease from the thermophilic Geobacillus toebiistrain LBT 77 has a number of properties that make it apromising potential candidate for application in the detergentindustry as a bioadditive in detergent formulation In fact itshowed high levels of thermoactivity and thermostability anda marked stability to detergents The enzyme also exhibitedhigh levels of stability against pH NaCl ions detergents andsolvent which responds to the industrial requirements

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was funded by the Tunisian Ministry of HigherEducation and Scientific Research

References

[1] R M Banik and M Prakash ldquoLaundry detergent compatibilityof the alkaline protease from Bacillus cereusrdquo MicrobiologicalResearch vol 159 no 2 pp 135ndash140 2004

[2] D Jain I Pancha S K Mishra A Shrivastav and S MishraldquoPurification and characterization of haloalkaline thermoac-tive solvent stable and SDS-induced protease from Bacillussp a potential additive for laundry detergentsrdquo BioresourceTechnology vol 115 pp 228ndash236 2012

[3] A Gupta I Roy R K Patel S P Singh S K Khare and MN Gupta ldquoOne-step purification and characterization of analkaline protease from haloalkaliphilic Bacillus sprdquo Journal ofChromatography A vol 1075 no 1-2 pp 103ndash108 2005

[4] A Haddar R Agrebi A Bougatef N Hmidet A Sellami-Kamoun and M Nasri ldquoTwo detergent stable alkaline serine-proteases from Bacillus mojavensis A21 purification charac-terization and potential application as a laundry detergentadditiverdquoBioresource Technology vol 100 no 13 pp 3366ndash33732009

[5] M Jacobs M Eliasson M Uhlen and J-I Flock ldquoCloningsequencing and expression of subtilisin carlsberg from Bacilluslicheniformisrdquo Nucleic Acids Research vol 13 no 24 pp 8913ndash8926 1985

[6] A Benkiar Z J Nadia A Badis et al ldquoBiochemical and molec-ular characterization of a thermo- and detergent-stable alkalineserine keratinolytic protease from Bacillus circulans strainDZ100 for detergent formulations and feather-biodegradation

processrdquo International Biodeterioration and Biodegradation vol83 pp 129ndash138 2013

[7] B Jaouadi B Abdelmalek D Fodil et al ldquoPurification andcharacterization of a thermostable keratinolytic serine alkalineproteinase from Streptomyces sp strain AB1 with high stabilityin organic solventsrdquo Bioresource Technology vol 101 no 21 pp8361ndash8369 2010

[8] T Maruthiah P Esakkiraj G Prabakaran A Palavesam andG Immanuel ldquoPurification and characterization of moderatelyhalophilic alkaline serine protease frommarine Bacillus subtilisAP-MSU 6rdquo Biocatalysis and Agricultural Biotechnology vol 2no 2 pp 116ndash119 2013

[9] R Sinha and S K Khare ldquoCharacterization of detergentcompatible protease of a halophilic Bacillus sp EMB9 differ-ential role of metal ions in stability and activityrdquo BioresourceTechnology vol 145 pp 357ndash361 2013

[10] X-G Chen O Stabnikova J-H Tay J-Y Wang and S T-L Tay ldquoThermoactive extracellular proteases of Geobacilluscaldoproteolyticus sp nov from sewage sludgerdquo Extremophilesvol 8 no 6 pp 489ndash498 2004

[11] J Marmur ldquoA procedure for the isolation of deoxyribonucleicacid from micro-organismsrdquo Journal of Molecular Biology vol3 no 2 pp 208ndash218 1961

[12] AV Piterina J Bartlett and J T Pembroke ldquoMolecular analysisof bacterial communityDNA in sludge undergoing autothermalthermophilic aerobic digestion (ATAD) pitfalls and improvedmethodology to enhance diversity recoveryrdquo Diversity vol 2no 4 pp 505ndash526 2010

[13] D Shrinivas and G R Naik ldquoCharacterization of alkalinethermostable keratinolytic protease from thermoalkalophilicBacillus halodurans JB 99 exhibiting dehairing activityrdquo Inter-national Biodeterioration and Biodegradation vol 65 no 1 pp29ndash35 2011

[14] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[15] U K Laemmli F Beguin and G Gujer-Kellenberger ldquoA factorpreventing the major head protein of bacteriophage T4 fromrandom aggregationrdquo Journal of Molecular Biology vol 47 no1 pp 69ndash85 1970

[16] F L Garcia-Carreno L E Dimes and N F Haard ldquoSubstrate-gel electrophoresis for composition and molecular weight ofproteinases or proteinaceous proteinase inhibitorsrdquo AnalyticalBiochemistry vol 214 no 1 pp 65ndash69 1993

[17] A V Akolkar and A J Desai ldquoCatalytic and thermodynamiccharacterization of protease from Halobacterium sp SP1(1)rdquoResearch in Microbiology vol 161 no 5 pp 355ndash362 2010

[18] A Khan K Williams M P Molloy and H NevalainenldquoPurification and characterization of a serine protease andchitinases fromPaecilomyces lilacinus and detection of chitinaseactivity on 2D gelsrdquo Protein Expression and Purification vol 32no 2 pp 210ndash220 2003

[19] P Shivanand and G Jayaraman ldquoProduction of extracellularprotease from halotolerant bacterium Bacillus aquimaris strainVITP4 isolated fromKumta coastrdquoProcess Biochemistry vol 44no 10 pp 1088ndash1094 2009

[20] L Lama I Romano V Calandrelli B Nicolaus and AGambacorta ldquoPurification and characterization of a proteaseproduced by an aerobic haloalkaliphilic species belonging to theSalinivibrio genusrdquo Research in Microbiology vol 156 no 4 pp478ndash484 2005


[21] B Jaouadi S Ellouz-Chaabouni M Rhimi and S Bejar ldquoBio-chemical and molecular characterization of a detergent-stableserine alkaline protease from Bacillus pumilus CBS with highcatalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008

[22] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009

[23] K Adinarayana P Ellaiah and D Prasad ldquoPurification andpartial characterization of thermostable serine alkaline proteasefrom a newly isolated Bacillus subtilis PE-11rdquo AAPS Pharm-SciTech vol 4 pp 440ndash448 2003

[24] M Lagzian and A Asoodeh ldquoAn extremely thermotolerantalkaliphilic subtilisin-like protease from hyperthermophilicBacillus sp MLA64rdquo International Journal of Biological Macro-molecules vol 51 no 5 pp 960ndash967 2012

[25] Q K Beg and R Gupta ldquoPurification and characterization of anoxidation-stable thiol-dependent serine alkaline protease fromBacillus mojavensisrdquo Enzyme and Microbial Technology vol 32no 2 pp 294ndash304 2003

[26] H-S Joo and C-S Chang ldquoProduction of protease from anew alkalophilic Bacillus sp I-312 grown on soybean mealoptimization and some propertiesrdquo Process Biochemistry vol40 no 3-4 pp 1263ndash1270 2005

[27] N Hmidet N El-Hadj Ali A Haddar S Kanoun S-K Alyaand M Nasri ldquoAlkaline proteases and thermostable 120572-amylaseco-produced by Bacillus licheniformis NH1 characterizationand potential application as detergent additiverdquo BiochemicalEngineering Journal vol 47 no 1ndash3 pp 71ndash79 2009

[28] A Deng J Wu Y Zhang G Zhang and T Wen ldquoPurificationand characterization of a surfactant-stable high-alkaline pro-tease from Bacillus sp B001rdquo Bioresource Technology vol 101no 18 pp 7100ndash7106 2010

[29] N Annamalai M V Rajeswari and T BalasubramanianldquoExtraction purification and application of thermostable andhalostable alkaline protease from Bacillus alveayuensis CAS 5using marine wastesrdquo Food and Bioproducts Processing vol 92no 4 pp 335ndash342 2014

[30] A Gessesse ldquoThe use of nug meal as a low-cost substrate forthe production of alkaline protease by the alkaliphilic Bacillussp AR-009 and some properties of the enzymerdquo BioresourceTechnology vol 62 no 1-2 pp 59ndash61 1997

[31] K Jellouli O Ghorbel-Bellaaj H B Ayed L Manni R Agrebiand M Nasri ldquoAlkaline-protease from Bacillus licheniformisMP1 purification characterization and potential application asa detergent additive and for shrimp waste deproteinizationrdquoProcess Biochemistry vol 46 no 6 pp 1248ndash1256 2011

[32] W Zhu D Cha G Cheng Q Peng and P Shen ldquoPurificationand characterization of a thermostable protease from a newlyisolated Geobacillus sp YMTC 1049rdquo Enzyme and MicrobialTechnology vol 40 no 6 pp 1592ndash1597 2007

[33] J J Hyeung C K Byoung R P Yu and S K Yu ldquoA novelsubtilisin-like serine protease from Thermoanaerobacter yon-seiensis KB-1 its cloning expression and biochemical proper-tiesrdquo Extremophiles vol 6 no 3 pp 233ndash243 2002

[34] B Sana D GhoshM Saha and J Mukherjee ldquoPurification andcharacterization of a salt solvent detergent and bleach tolerantprotease from a new gamma-Proteobacterium isolated from themarine environment of the Sundarbansrdquo Process Biochemistryvol 41 no 1 pp 208ndash215 2006

[35] RK PatelM SDodia RH Joshi and S P Singh ldquoPurificationand characterization of alkaline protease from a newly isolatedhaloalkaliphilic Bacillus sprdquo Process Biochemistry vol 41 no 9pp 2002ndash2009 2006

[36] H-S Joo C G Kumar G-C Park S R Paik and C-SChang ldquoOxidant and SDS-stable alkaline protease fromBacillusclausii I-52 production and some propertiesrdquo Journal of AppliedMicrobiology vol 95 no 2 pp 267ndash272 2003


ldquoHammam El Atrousrdquo next to the Ichkeul lagoon in BizerteTunisia (37∘08101584018810158401015840N 9∘41101584024710158401015840E) The strain was identi-fied based on the phenotypic characteristics of the Bacillusgenus and phylogenetic analysis of the 16S rDNA sequenceGenomic DNA was extracted as described by Marmur [11]and 16S rRNA gene sequences were amplified using the bac-terial universal primers 16SF (51015840AGAGTTTGATCCTGG-CTCAG31015840) and 16SR (51015840CTACGGCTACCTTGTTACGA31015840)[12] using the following PCR program 1 cycle of 94∘C for5min 30 cycles of 94∘C for 1min 54∘C for 1min and 72∘Cfor 15min and a cycle of 72∘C for 5min The amplifiedproducts were purified and the sequence of the 16S rRNAgene was determined by Sanger method DNA sequencing(ABI 3730xl DNA analyzer USA) Sequence comparisonwiththe databases was performed using BLAST program throughNCBI website A phylogenetic tree was constructed withMEGA version 606 using the neighbor-joining method

23 Screening and Production of Protease Activity Screeningof protease activity was performed by the method of dissemi-nation throughwells onmilk agarmediumat pH9 containing5 gL tryptone 3 gL yeast extract 15 gL agar and 25mLskimmedmilk 60 120583L of an 18 h bacterial culture was injectedinto each well and the plates were incubated for 18 h at 55∘CProtease activity was confirmed by the appearance of a clearzone around the well-testifying degradation of casein milk[6] Production of protease by Geobacillus toebii LBT 77 wascarried out at pH 8 in amedium containing 5 g bactopeptone5 g yeast extract 5 g NaCl 10 g gelatin and 02 g CaCl

2in 1 L

deionised water [9] Inocula were routinely grown in TrypticSoy Broth (Scharlau Spain) medium Media were autoclavedat 120∘C for 20min Cultures were performed on a rotatorshaker (150 rpmmin) for 96 h at 55∘C in 500mL Erlenmeyerflasks with aworking volume of 100mLGrowthwas followedby measuring the optical density at 600 nm every 6 h Theculture medium was centrifuged at 12000 rpm for 20min at4∘C and the cell-free supernatant was used as a crude extractfor estimation of proteolytic activity

24 Assay of Protease Activity Proteolytic activity was deter-mined by using casein as substrate One mL of casein 10 gLin 50mM glycine-NaOH buffer pH 11 was mixed with 900 120583Lof glycine-NaOH Reaction was initiated by the additionof 100120583L of enzyme and the tubes were placed in a waterbath at 70∘C After 20min of incubation 2mL of 10 TCAwas added to stop the reaction The reaction mixture wascentrifuged at 12000 rpm for 10min and absorbance wasmeasured at 280 nm [13] One unit of proteolytic activity wasdefined as the amount of enzyme required to release 1 120583g oftyrosine per minute under experimental conditions

25 Enzyme Purification

251 Ammonium Sulfate Precipitation Cell-free supernatantwas collected by centrifugation at 12000 rpm for 20min at4∘C after 72 h of cultivation Ammonium sulfate was slowlyadded to the supernatant to 80 saturation and the mixturewas incubated overnight at 4∘CThe precipitate was collected

by centrifugation dissolved in a minimal volume of 25mMTris-HCl (pH 85) and dialyzed against three changes of thesame buffer for 24 h

252 Sephadex G-75 Gel Filtration The dialysate was sub-jected to gel filtration on Sephadex G-75 column (21 cm times50 cm) equilibrated with 25mM Tris-HCl (pH 85) Thecolumn was eluted with the same buffer at a flow rate of20mLh and fractions of 3mL were collected and thenanalyzed for protease activity and protein concentrationTheactive fractions were pooled and subjected to ion exchangechromatography

253 DEAE-Cellulose Ion Exchange Chromatography Thisround was performed by applying active fractions from theprevious step to a DEAE-Cellulose (2 cm times 25 cm) equili-brated with 25mM Tris-HCl (pH 85) Bounded proteinswere eluted with a linear gradient of NaCl from 0 to 1M ata flow rate of 50mLh and analyzed for protease activity

All purification steps were performed at 4∘CThe proteincontent of each chromatographic fraction was determined bymeasuring the absorbance at 280 nm

26 Protein Estimation Protein concentration was deter-mined by the colorimetric method of Bradford [14] usingbovine serum albumin as standard

27 Polyacrylamide Gel Electrophoresis and ZymographySDS-PAGE was carried out to determine the purity andmolecular weight of the enzyme as described by Laemmli etal [15] using 5 (wv) stacking and 12 (wv) separating gelsProtein bands were visualized by staining with CoomassieBrilliant Blue R-250 The molecular weight of the enzymewas estimated using a low-molecular weight calibration kit(Biomatik Co Canada) as markers

Zymography was performed by the method of Garcia-Carreno [16] to confirm the enzyme activity After elec-trophoresis the gel was immersed in 100mMTris-HCl buffer(pH 85) containing 25 Triton X-100 at 4∘C with shakingfor 30min to remove SDS then the gel was washed twicewith 100mM Tis-HCl buffer (pH 85) in order to eliminateresidual Triton X-100 and then incubated with 1 (wv)casein in 100mM Tris-HCl (pH 95) at 75∘C for 60minFinally the gel was stained using Coomassie Brilliant BlueR-250 Appearance of a clear halo zone on the dark-bluebackground indicates the presence of enzyme


281 Effects of Temperature pH andNaCl on EnzymeActivityand Stability To study the effect of temperature on activityof the purified enzyme the reaction mixture was incubatedat different temperatures ranging from 55 to 120∘C Enzymeactivity was measured as described earlier Thermal stabilitywas evaluated by incubating the purified enzyme at 70 8090 and 95∘C for 180min in the presence and absence of 5mMCaCl2 Residual activity wasmeasured at 95∘C and pH 13The

activity of nonheated enzyme was considered as 100










005



3-

















Total protein(mg)



0200400600800100012001400160018002000

002040608

112141618

0 6 12 18 24 30 36 42 48 54 60 66 72 84 96Pr

otea

se ac

tivity

(Um

L)

Time (h)

Activity (UmL)

Abso

rban

ce at

600

nm



050010001500200025003000

0

05

1

15

2

25

0 10 20 30 40 50 60 70 80 90 100

Prot

ease

activ

ity (U

mL)

Fractions


Abso

rban

ce at

280

nm

Absorbance at 280nm





Rela

tive p

rote

ase a

ctiv

ity (

)

120

100

80

60

40

20

055 60 65 70 75 80 85 90 95 100 110 120

Temperature (∘C)





sidua

l act

ivity

()

120

100

80

60

40

20

00 30 60 90 120 150 180

Time (min)


80∘C90∘C95∘C






0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Rela

tive p

rote

ase a

ctiv

ity (

)

pH

(a)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Resid

ual p

rote

ase a

ctiv

ity (

)pH

(b)


Activ

ity (

)

120

100

80

60

40

Activity

20

Stability

00 5 10 15 20 25 30 35 40

[NaCl] ()




sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM


Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+






Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif



180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2








(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References
















































005



3-

















Total protein(mg)



0200400600800100012001400160018002000

002040608

112141618

0 6 12 18 24 30 36 42 48 54 60 66 72 84 96Pr

otea

se ac

tivity

(Um

L)

Time (h)

Activity (UmL)

Abso

rban

ce at

600

nm



050010001500200025003000

0

05

1

15

2

25

0 10 20 30 40 50 60 70 80 90 100

Prot

ease

activ

ity (U

mL)

Fractions


Abso

rban

ce at

280

nm

Absorbance at 280nm





Rela

tive p

rote

ase a

ctiv

ity (

)

120

100

80

60

40

20

055 60 65 70 75 80 85 90 95 100 110 120

Temperature (∘C)





sidua

l act

ivity

()

120

100

80

60

40

20

00 30 60 90 120 150 180

Time (min)


80∘C90∘C95∘C






0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Rela

tive p

rote

ase a

ctiv

ity (

)

pH

(a)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Resid

ual p

rote

ase a

ctiv

ity (

)pH

(b)


Activ

ity (

)

120

100

80

60

40

Activity

20

Stability

00 5 10 15 20 25 30 35 40

[NaCl] ()




sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM


Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+






Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif



180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2








(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References










































Total protein(mg)



0200400600800100012001400160018002000

002040608

112141618

0 6 12 18 24 30 36 42 48 54 60 66 72 84 96Pr

otea

se ac

tivity

(Um

L)

Time (h)

Activity (UmL)

Abso

rban

ce at

600

nm



050010001500200025003000

0

05

1

15

2

25

0 10 20 30 40 50 60 70 80 90 100

Prot

ease

activ

ity (U

mL)

Fractions


Abso

rban

ce at

280

nm

Absorbance at 280nm





Rela

tive p

rote

ase a

ctiv

ity (

)

120

100

80

60

40

20

055 60 65 70 75 80 85 90 95 100 110 120

Temperature (∘C)





sidua

l act

ivity

()

120

100

80

60

40

20

00 30 60 90 120 150 180

Time (min)


80∘C90∘C95∘C






0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Rela

tive p

rote

ase a

ctiv

ity (

)

pH

(a)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Resid

ual p

rote

ase a

ctiv

ity (

)pH

(b)


Activ

ity (

)

120

100

80

60

40

Activity

20

Stability

00 5 10 15 20 25 30 35 40

[NaCl] ()




sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM


Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+






Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif



180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2








(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References








































sidua

l act

ivity

()

120

100

80

60

40

20

00 30 60 90 120 150 180

Time (min)


80∘C90∘C95∘C






0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Rela

tive p

rote

ase a

ctiv

ity (

)

pH

(a)

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 1212

5 1313

25

135

137

5Resid

ual p

rote

ase a

ctiv

ity (

)pH

(b)


Activ

ity (

)

120

100

80

60

40

Activity

20

Stability

00 5 10 15 20 25 30 35 40

[NaCl] ()




sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM


Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+






Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif



180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2








(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References








































sidua

l act

ivity

()

140

120

100

80

60

40

20

0

Con

trol

PMSF

5m

M

PMSF

10m

M

EDTA

5m

M

EDTA

10m

M

DTN

B5

mM

DTN

B10

mM

5m

M120573

-mer

capt

oeth

anol

120573-m

erca

ptoe

than

ol10

mM


Resid

ual a

ctiv

ity (

)

180160140120100

80604020

0

Metal ions (5mM)Con

trol

Zn2+

Fe2+

Mg2

+

Mn2

+

Cu2+

Ca2+

Hg2

+

Co2

+

Na+ K+ Al+ Li+






Resid

ual a

ctiv

ity (

)

140

120

100

80

60

40

20

0

Con

trol

SDS

CTA

B

Trito

n X-

100

OM

O

Arie

l

Nad

hif



180160140120100

80604020

0

Organic solvents

Rela

tive p

rote

ase a

ctiv

ity (

)

Con

trol

Cyclo

hexa

ne

n-bu

tano

l

Etha

nol

Met

hano

l

Tolu

ene

Acet

onitr

ile

Acet

one

Isop

ropa

nol

Benz

ene

log p

32 lo

g p08

log p

minus024

log p

minus082

log p

27 log p

minus03

log p

minus02

log p

005

log p

2








(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References










































(mgmL) 1 119864119886





Competing Interests


Acknowledgments


References







































Purification and Characterization of a New … · activity was determined. Effect of commercial...

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Transcript of Purification and Characterization of a New … · activity was determined. Effect of commercial...