research article production and characterization of keratinolytic...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 175012 14 pageshttpdxdoiorg1011552013175012

Research ArticleProduction and Characterization of KeratinolyticProtease from New Wool-Degrading Bacillus SpeciesIsolated from Egyptian Ecosystem

Mohamed A Hassan1 Bakry M Haroun2 Amro A Amara1 and Ehab A Serour13

1 Protein Research Department Genetic Engineering and Biotechnology Research InstituteCity of Scientific Research and Technological Applications New Borg Al-Arab PO Box 21934 Alexandria Egypt

2 Botany and Microbiology Department Faculty of Science (Boys) Al-Azhar University Cairo Egypt3 King Abdulaziz City for Science and Technology Riyadh Saudi Arabia

Correspondence should be addressed to Mohamed A Hassan m adelmicroyahoocom

Received 2 December 2012 Revised 7 March 2013 Accepted 28 March 2013

Academic Editor Periasamy Anbu

Copyright copy 2013 Mohamed A Hassan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate Egypt In this studythe isolates were identified as Bacillus amyloliquefaciensMA20 and Bacillus subtilisMA21 based onmorphological and biochemicalcharacteristics aswell as 16S rRNAgene sequencingB amyloliquefaciensMA20 andB subtilisMA21 produced alkaline keratinolyticserine protease when cultivated in mineral medium containing 1 of wool straight off sheep as sole carbon and nitrogen sourceThe two strains were observed to degrade wool completely to powder at pH 7 and 37∘C within 5 days Under these conditions themaximum activity of proteases produced by B amyloliquefaciens MA20 and B subtilis MA21 was 922 and 814Uml respectivelyThe proteases exhibited optimum temperature and pH at 60∘C and 9 respectively However the keratinolytic proteases were stablein broad range of temperature and pH values towards casein Hammerstein Furthermore the protease inhibitor studies indicatedthat the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially inpresence of EDTA The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting theirpotential use in biotechnological applications such as wool industry

1 Introduction

Keratins are classified as fibrous proteins called scleroproteinsthat occur abundantly in epithelial cells These proteins areinsoluble in water weak acid and alkali and organic solventsand are insensitive to the attack of common proteolyticenzymes such as trypsin or pepsin [1] The animal remainsrich in 120572-keratin such as animal skin hair claws horns andwools

The important property of these proteins is the presenceof high cystine content that differentiates keratins from otherstructural proteins such as collagen and elastin Both a highcystine content as well as a high content of glycine prolineserine and acidic amino acids and a low content of lysinehistidine and methionine (or their lack) as well as the

absence of tryptophan are also characteristic of keratins [23] Numerous disulfide cystine bonds present in keratin tobind peptide chains and packed as 120572-helices as in hair andwool or in 120573-sheet arrangements as in case of feathers Thedisulfide linkage and the tight secondary structure of keratinsmake them difficult to be hydrolysed by common proteolyticenzymes [4]

Themajor problem of 120572-keratin hydrolysis is the presencea high numbers of disulfide bonds that make it insoluble innature and resistant to proteases hydrolysis [5] Keratinolyticprotease enzymes are spread in nature and elaborated bydifferent groups of microorganisms that can be isolated frompolluted area with keratin wastes [6] A vast variety of Gram-positive bacteria including Bacillus Lysobacter Nesternokia

2 BioMed Research International

Kocuria andMicrobacterium as well as a few strains of Gram-negative bacteria such as Xanthomonas Stenotrophomonasand Chryseobacterium are confined as keratin degraders [7ndash10] Most of keratin degrading bacteria belong to the genus ofBacillus [11]

The keratinous substrate such as feather and wool canbe degraded in basal medium by microorganisms which arecapable of utilize keratin as sole carbon and nitrogen source[12] Keratinolytic proteases have broad substrate specificitywhere they have the ability to hydrolyze soluble protein suchas casein gelatin and bovine serum albumin Additionallythey can hydrolyze the insoluble protein including feathersilk and wool [13] Keratinolytic proteases mostly belong toserine or metalloproteases showing sequence similarity withsubtilisin group of proteases [14 15] In recent years moredemands to keratinolytic proteases are increasing due to theirmultitude in industrial applications such as the feed fertilizerdetergent and textile industries The present study describesthe isolation and identification of new Bacillus amylolique-faciens MA20 and B subtilis MA21 strains from Egyptianecosystem that grow well on wool as sole carbon sourceMoreover the two strains are able to degrade wool and theirenzymes can be used to improve the wool quality This paperincludes full characterization of the keratinolytic proteasewhich explains that the best environmental conditions can beused to improve the wool quality in industry

2 Materials and Methods

21 Sample Collection Different types of samples were col-lected from different Governorates of Egyptian ecosystemincluded Alexandria Behera Qaliubia Mania and AsiutThese fresh sampleswere varied such as soil sand soil humuswaste wool and rhizosphere under olive treesThese sampleswere collected in sterile falcon tubes and transported tothe microbiological lab in (City of Scientific Research andTechnological Applications)

22 Strains Isolation The bacterial strains were isolated bysuspending 1 g of soil samples in a 10mL sterile 085 (wv)saline solution and then treated for 20min at 80∘C This willenable the isolation of the spore-forming bacteria Luria-Bertani (LB) agar medium with 1 (wv) skim milk wasused for their cultivation by spreading 01mL of each 10minus5and 10minus6 dilutions The plates then were incubated for 24hours at different temperatures [4 16] The colonies whichgive clear zones formed by hydrolysis of skim milk werepicked Pure bacterial isolates were obtained by reculturingindividual colonies several times on fresh LB agar medium toproduce single colony from each

23 Strains Selection Twelve selected strains isolated accord-ing to the diameter of clear zone were cultured on mediumcontaining 05 g NaCl 03 g K

2HPO4 04 g KH

2PO4 and

10 g wool per liter pH 7 and incubated for 5 days at 37∘CThe wool was used as sole carbon and nitrogen source fordetecting potent strains that have the ability to degrade thewool completely Three strains degraded the wool and the

supernatants of their culture were assayed on plate containing1 gelatin powder which is soluble in phosphate buffer pH 7After determining the existence of the activity (by the clearzone of the supernatants) Bacillus sp MA20 and Bacillus spMA21 were selected and preserved for further investigation

24 Bacterial Identification While the phenotypic charac-teristics and isolation method of the two selected isolatesindicate that they are related to Bacillus group but furtheridentification was conducted

The strains identification are included the spore mor-phology Gram stain and motility The morphological andphysiological characteristics of the bacterial isolates werecompared with the data from Bergeyrsquos Manual of Determi-native Bacteriology [17]

25 Scanning of Bacillus sp MA20 and Bacillus sp MA21by Scanning Electron Microscope The bacterial smear wasprepared by centrifuging the bacterial cultures at 12000 rpmfor 20minThepellets werewashed 2 times by saline solutionThe pellet was suspended in sterilized distilled H

2O The

bacterial film was prepared and fixed on glass slides tillcomplete drying The smear was coated with gold usingsputter coater The golden coated sample was scanned at20KV acceleration voltages at room temperature

26 Genetic Identification and Differentiation

261 DNA Extraction The genomic DNA of Bacillus strainswas isolated usingmodificationmethod from Sambrook et al[18]

262 Identification by 16S Ribosomal RNA (rRNA)

PCR Amplification according to Sambrook et al [18] The PCRamplification reactions were performed in a total volumeof 50120583L Each reaction mixture contained the followingsolutions 2 120583L of DNA (40 ng) 1 120583L of 10 pmol forwarded16S-rRNA primer (51015840-AAATGGAGGAAGGTGGGGAT-31015840) 1 120583L of 10 pmol reverse 16S rRNA primer (51015840-AGGAGGTGATCCAACCGCA-31015840) 08120583L of 125mM(dNTPrsquos) 5 120583L of PCR buffer included MgCl

2 and 02 120583L

Taq polymerase (1 Unit) and water-free DNAse and RNAsewere added up to 50 120583LThePCR apparatus was programmedas follows 3min denaturation at 95∘C followed by 35 cyclesthat consisted of 1min at 95∘C 1min at 58∘C and 1min at72∘C and a final extension was 10min at 72∘C The productsof the PCR amplification were analyzed by agarose gelelectrophoresis (2)

263 PCR Cleanup and 16S rRNA Sequencing The PCRproducts were cleaned up for DNA sequencing following themethod described by Sambrook et al [18] Automated DNAsequencing based on enzymatic chain terminator techniquedeveloped by Sanger et al [19] was carried out using 3130XDNA Sequencer (Genetic Analyzer Applied BiosystemsHitachi Japan)

BioMed Research International 3

264 Phylogenetic Analysis Similarity analysis of thenucleotides was performed by BLAST searches againstsequences available in GenBank For phylogenetic tree con-struction multiple sequences were obtained from GenBankand the alignments were performed using MEGA 5 softwareversion 51 [20]

27 Keratinolytic Protease Production Bacillus sp MA20and Bacillus sp MA21 were first inoculated in liquid LBmedium to produce large amount of cells After 18 hrs thecolony forming units (CFU)mL culture were 3 lowast 106 and2 volume of the liquid medium was transferred to theproductionmedium using 250mL flask containing 100mL ofthe productionmediumThe productionmedium containing(wv)NaCl 05 gL K

2HPO4 03 gL KH

2PO4 04 gL wool

10 gL and the pHwas adjusted 70ndash72 using 2NofNaOHandHCl [16] The cultivated media were incubated at 37∘C and200 rpm for 5 days Culture supernatants were obtained bycentrifugation at 12000 rpmand 4∘C for 30minThedifferentsupernatants which contain the crude enzymes were used inassay and analysis of enzymes

28 Enzymes Assay

281 Detection of the Proteolytic Activity on Plates The crudeenzyme of Bacillus sp MA20 and Bacillus sp MA21 wasscreened for their proteolytic activity using agarwell diffusionplate method described by Amara et al after modification[21] One gram of gelatin powder was suspended in 100mLphosphate buffer pH 7 and autoclaved After sterilizationthe soluble component was added to sterile water containingagar (18 gm agarL) The suspension then stirred gently anddistributed in Petri dishes (25mLplate) After completesolidification of the agar on plates wells were punched outof the agar by using a clean sterile cork borer The base ofeach hole was sealed with a drop of melted sterile water agar(15 g agar per liter H

2O) using sterile Pasteur pipette Fifty

120583L of each bacterial supernatant was added to each well andpreincubated at 4∘C for 2 hrs and then overnight incubated atdifferent temperatures

282 Visualization of the Enzyme Clear Zone Coomassieblue (025 wv) in methanol-acetic acid-water 5 1 4(vvv) was used in plates staining to visualize the gelatinhydrolysis where 10mL was added to each plate and incu-bated in room temperature for 15min followed by removingthe staining solution from the plates surfaces and washinggently by distilled waterThen the plates were destained usingdestining solution (66mL methanol 20mL acetic acid and114mL H

2Obidest) for a suitable time [22] Also extracellular

protease detection was determined according to Vermelho etal [23] with modification Staining was performed with 01amido black in methanol-acetic acid-water 30 10 60 (vvv)for 5ndash20min (according to the quality of amido black stain) atroom temperature Regions of enzyme activity were detectedas clear areas indicating that hydrolysis of the substrates hasbeen occurred

283 Enzyme Assay Spectrophotometrically

Preparation of Casein in Different Buffers with Different pHCasein Hammarstein was weighted in a quantity of 0325 ganddissolved in 50mLof different buffers at different pHThemixture was dissolved by heating gently to 80ndash90∘C withoutboiling in water bath according to Amara and Serour [24] orusing hot plate with stirring for more accurate because caseinis highly stick with the walls of the container The followingbuffers were used

(i) sodium phosphate pH 6-7-8 (01M)(ii) glycineNaOH pH 9-10 (01M)(iii) sodium phosphate dibasicNaOH pH 11 (005M)(iv) KClNaOH pH 12 (02M)

284 Optimum Temperature of the Enzymes Ten 120583L of eachsupernatant which contains the crude enzyme was addedto 490 120583L of the casein Hammarsten soluble in buffer pH7 The enzymes-substrate mixture was incubated at differenttemperatures of 20 30 40 50 60 70 and 80∘C using waterbath for 15min After the incubation period the enzymereaction stopped by adding 500120583L of 10 trichloroacetic acid(TCA)Themixturewas allowed to stand in ice for 10min andthen centrifuged at 13000 rpm for 10min The absorbanceof each sample was determined spectrophotometrically at280 nm and their tyrosine content derived from the tyrosinestandard curve which was carried out according to Amaraand Serour [24] and the enzyme activity was determined asUnitmL

285 Optimum pH of the Enzymes Ten 120583L of each super-natant which contains the crude enzyme was added to the490 120583L of the casein Hammarsten soluble in different pHvaluesThe enzymes-substratemixture was incubated at 60∘Cwhich acts as optimum temperature in a water bath for15min At the end of incubation period the enzyme reactionstopped by adding 500 120583L of 10 trichloroacetic acid (TCA)The mixture was allowed to stand in ice for 10min and thencentrifuged at 13000 rpm for 10min The absorbance of eachsample was determined spectrophotometrically at 280 nmand their tyrosine content derived from the tyrosine standardcurve and the enzyme activity determined as UnitmL

286 Confirmation of Optimum pH and Temperature Theprevious reaction was performed using casein Hammarstensoluble in GlycineNaOH pH 9 The enzyme substrate mix-ture was incubated at various temperature of 20 30 40 5060 70 and 80∘C in water bath for 15minThe enzyme activitywas calculated as mentioned previously

287 The Optimized Enzymes Reaction The optimized reac-tion is a mixing between 10 uL of crude enzyme and 490 120583Lof the casein Hammarsten soluble in GlycineNaOH pH 9The mixture was incubated at 60∘C in water bath for 15minThe reaction was stopped with 500120583L of 10 TCA Themixture was allowed to stand in ice for 10min and then was


centrifuged at 13000 rpm for 10min The enzyme activitywas determined as mentioned above using tyrosine standardcurve

288 Thermal Stability The thermostability was carried outby preincubating the crude enzyme solution at a temperaturerange of 4∘C to 80∘C for 00 to 24 hrs The residual activitywas measured with standard enzyme reaction The control isenzyme reacted at zero time (consider as 100) [16]

289 pH Stability The pH stability was determined bypreincubating the enzyme solution in buffers with differentpH values (3ndash12) at room temperature from 00 to 48 hrsThe residual activity was measured with standard enzymereaction The control is enzyme reacted at zero time andconsidered as 100 [16] The following buffers were used

(i) citrate buffer pH 3-4-5-6 (01M)

(ii) sodium phosphate pH 7-8 (01M)

(iii) glycineNaOH pH 9-10 (01M)

(iv) sodium phosphate dibasicNaOH pH 11 (005M)

(v) KClNaOH pH 12 (02M)

2810 Effect of Inhibitors The inhibitors were added tosupernatants which contain the produced enzyme and wereincubated for 30min at 30∘C before being tested for prote-olytic activity Protease inhibitors phenylmethanesulphonylfluoride (PMSF) ethylenediaminetetraacetic acid (EDTA)120573-mercaptoethanol and the detergent sodium dodecyl sulfate(SDS) were used The inhibitors stocks were prepared indistilled water except that PMSF was prepared by usingisopropanol The final concentrations of PMSF EDTA and120573-Mercaptoethanol are 5mM and 1mM while SDS is 05and 01 (wv) The control was enzyme mixed with distilledwater instead of inhibitors Control activity was considered tobe 100 [25]

2811 Effect ofMetal Ions Theeffect ofmetal ions onproteaseactivity was investigated using two concentrations of 5mMand 10mM (final concentration) The metal stock solutionswere prepared in distilledwater and diluted to the appropriateconcentrations The enzyme solution was mixed with thedifferent metal solutions and incubated for 30min at 30∘Cbefore assay A control was also included where the enzymewas mixed with distilled water instead of metal solutionControl activity was considered to be 100 [25] ZnCl

2

MgCl2 CuSO

4 Urea HgCl

2 CaCl

2 BaCl

2 Guanidin HCl

and MnCl2were used

2812 Effect of Solvents The effect of the different solvents(Methanol Ethanol DMSO Isopropanol Tween 20 andTriton X100) on protease activity was investigated using aconcentration of 1 and 05 (final concentration) [25]

29 First-Dimension Protein Electrophoresis

291 SodiumDodecyl Sulfate Polyacrylamid Gel Electrophore-sis (SDS-PAGE) Characterization of proteins and evaluationof the protein enrichment process SDS-PAGEwas performedin a discontinuous SDS-PAGEvertical slab gel electrophoresisapparatus as described by Laemmli [26] Discontinuous SDS-PAGE consisted of a stacking gel (5 wv pH 68) and aseparating gel (12 wv pH 88) The separating gel wasprepared in a 1mm slab gel (10 times 10 cm)

210 Gel Staining Using Silver Stain The gels were stainedusing silver nitrate staining methods as described by Blumet al [27]

211 Zymogram for the Detection of Protease Activity UsingSDS-PAGE As above in the case of protease zymographyusing SDS PAGE and the separating gel concentration was12 The samples were mixed with 5X sample buffer without120573-mercaptoethanol and without boiling

After running the gel at 80V the SDS-PAGE gel wasstripped off from the gel plate and soaked in 1 triton X100for 2 hrs to change the solution every 1 hour or in 25tritonX100 for 30min for removing SDS and renaturating theenzymes

The gel was washed three times with tape water andsoaked in 1 gelatin powder solubilized in GlycineNaOHbuffer pH9 for 90min at 60∘CThegelwaswashedwith bufferfor 10min before staining

Then the gel was stained with Coomassie blue stain for2 hrs After that the gel was destained till the active bandsappear

212 Zymogram for Detecting Protease Activity Using NativePAGE The gel was carried out using all components of theSDS-PAGE and the same conditions but without SDS as wellas the buffers described aboveThe samples were prepared bymixing with 5X sample buffer for native PAGE and withoutboiling The gel was washed with tap water and soaked into1 gelatin powder as mentioned previously Then the gel wasstained with Coomassie blue stain for 2 hrs After that the gelwas destained till the active bands appear

213 Two-Dimension Polyacrylamide Gel Electrophoresis (2D-PAGE) First-dimension isoelectric focusing (IEF) was per-formed using Ettan IPGphor3 and the second-dimensionSDS-PAGEwas applied using vertical electrophoresis of EttanDALTtwelve System The experiment was carried out usingthe operation instruction of manufacturer (GE healthcarecompany) The gels were stained with Coomassie stain whereCoomassie blue R-250 (002 g) in methanol-glacial aceticacid-distilled water (10mL 5mL and 100mL resp) was used

3 Results and Discussion

31 Strain Isolation Microbial keratinolytic protease hasbeen described for various biotechnological applications infood detergent textiles and leather industries and yet the


growing demand for these enzymes necessitates the screeningfor novel keratinolytic microorganisms with potential appli-cations [28 29] Keratinolytic protease has been describedfor several species of Bacillus [30 31] due to the broaddistribution of keratinase among these genera and this studyfocused on keratinolytic protease production from them

A total of 48 pure cultures of spore-forming bacteriawere isolated and purified which obtained from differentsamples collected from Governorates of Egypt All isolateswere screened using selective method for Bacillus isolationThe proteolysis activities of all the isolates were detectedusing the plate test method containing LB agar mediumwith 1 (wv) skim milk Among the isolates analyzed 12isolates exhibited proteolytic activity in which they had ahalo diameter of fivefold longer than the colony diameterAll isolates have the proteolytic activity but do not have theability to degrade wool for that the twelve selected isolateswere grown using a medium which contain (wv) NaCl05 gL K

2HPO4 03 gL KH

2PO4 04 gL wool 10 gL and

the pH was adjusted at 70ndash72 using 2N NaOH and HClThe flasks were incubated for 5 days until the wool has beencompletely degraded by some isolates Three Bacillus isolatesshow the ability to degrade the wool and gained the namesBacillus sp MA20 Bacillus sp MA21 and Bacillus sp MA10In the last screening step the obtained supernatants from thecultivation of three above Bacillus strains culture media wereassayed using agar well diffusion in Petri dishes includinggelatin powder plate which suspended in phosphate bufferpH 7 as in Figure 1 Bacillus sp MA20 and Bacillus sp MA21were selected for studying the keratinolytic protease enzymebased on the diameter of clear zone (Figure 1) By refering totheir isolation source the two selected strains were found tobe isolated from Minia Governorates This method is simplebut proves to be efficient for determining the best enzyme-producing isolates Out of the three strains the two whichwere given the best result have been subjected to furtheridentification

32 Strains Identification

321 Identification by Morphological and Biochemical TestsThe morphological and physiological characteristics of theisolated strains were compared with the data from BergeyrsquosManual of Determinative Bacteriology [17] who revealed thatBacillus sp MA20 and Bacillus sp MA21 are matching withthose of B subtilis group In previous articles on taxonomyspecies included in the B subtilis group are the followingB velezensis B atrophaeus B mojavensis B malacitensis Baxarquiensis B nematocida B vallismortis B subtilis and Bamyloliquefaciens [32ndash34]

The two strains are aerobic motile and Gram-positiverods The smears of Bacillus sp MA20 and Bacillus spMA21 were scanned by scanning electron microscope whichindicates the bacterial size of the two Bacillus strains whichwas measured by slime view program software

The results of biochemical tests of the Bacillus sp MA20and Bacillus sp MA21 indicated that they are related to

Figure 1 Proteolytic activity of the supernatants obtained fromthree Bacillus sp on gelatin suspended in phosphate buffer pH 7Thewell number (1) is supernatant from Bacillus spMA21 number (2)is fromBacillus spMA10 number (3) is fromBacillus spMA20 and(4) is control free of enzyme

B subtilis and B amyloliquefaciens which summarized inTable 1

33 Identification Based on Genetic Materials (DNA)

331 DNA Extraction The isolated DNAwas analyzed by gelelectrophoresis and the quality of the DNA for each samplehas been identified for further investigation

332 Identification by 16S Ribosomal RNA (rRNA) Theamplified 16S rRNA gene from the DNA of Bacillus sp MA20and Bacillus sp MA21 was determined using 2 agarosegel The size of the amplified fragments was determined byusing size standard (Gene ruler 50 bpndash1031 bp DNA ladder)The PCR products were visualized under UV light and pho-tographed using gel documentation system Approximately380 bp of 16S rRNA gene was amplified

The PCR products were purified and sequenced using16S forward primer The sequences of Bacillus sp MA20and Bacillus sp MA21 were deposited in national centerfor biotechnology information (NCBI GenBank) under theAccession numbers (HQ1155991ndashHQ1156001) respectivelyThe basic local alignment search tool (BLAST) algorithmwasused to retrieve for homologous sequences in GenBank

The Bacillus sp MA20 revealed 98 identity to Bacillusamyloliquefaciens andBacillus subtiliswhileBacillus spMA21revealed 97 identity to Bacillus subtilis and Bacillus amy-loliquefaciens Based on the morphological biochemical andmolecular characteristics the Bacillus spMA20 and BacillusspMA21 were designated as B amyloliquefaciensMA20 andB subtilisMA21 respectively

A phylogenetic tree based on the comparison of 16SrRNA sequences of reference strains was constructed Thephylogenetic analysis was performed with 341 bp sequencesusing the softwareMEGA5 [20] using the neighbour-joiningmethod and based on Jukes-Cantor distancesThe branching


Table 1 Morphological and biochemical properties of Bacillus spMA20 and Bacillus sp MA21

Characteristics Bacillus sp MA20 Bacillus sp MA21Morphological

Shape Rods RodsGram stain G+ve G+veMotility Motile Motilespore formation +ve +ve

GrowthGrowth temperature 15∘Cndash50∘C 15∘Cndash60∘CGrowth pH 5ndash8 5ndash8

Biochemical testsOxidase +ve +veCatalase +ve +veVoges-Proskauer +ve +veIndol production minusve minusveNitrate reduced to nitrite +ve +ve

Hydrolysis ofCasein +ve +veGelatin +ve +veWool +ve +veStarch +ve +ve

Acid fromGlucose +ve +veArabinose +ve +veXylose +ve +veMannitol +ve +veGas from glucose minusve minusve

Utilization ofCitrate +ve +vePropionate minusve minusve

pattern was checked by 500 bootstrap replicates (Figures 2and 3)

34 Media Screening for Keratinolytic Protease ProductionKeratinolytic proteases are largely produced in a basalmedium with keratinous substrates and most of the organ-isms could utilize keratin sources such as feather and woolas the sole source of carbon and nitrogen [35 36] BamyloliquefaciensMA20 and B subtilisMA21 were tested oneight nutrientmediaThe selectedmedia ismedium (1) whichis containing (wv) NaCl 05 gL K

2HPO4 03 gL KH

2PO4

04 gL wool 10 gL and the pH was adjusted 70ndash72 using2N of NaOH and HCl

Korniłłowicz-Kowalska (1997) and Amara and Serour(2008) reported that the mass loss of the keratin substrate isthe most reliable indicator of microbial keratinolytic abilitiesB amyloliquefaciens MA20 and B subtilis MA21 have theability to degrade wool completely after incubation for 5 daysand remain it as powders in the bottom of flasks [24 37]

35 Enzyme Production and Characterization The kerati-nolytic protease enzyme was produced using productionmedium mentioned above for each of B amyloliquefaciensMA20 and B subtilis MA21 and the crude enzymes werecharacterized

36 Detection of the Proteolytic Activity on Plates The crudeenzymes of B amyloliquefaciens MA20 and B subtilis MA21were assayed by agar well diffusion methods on platescontaining gelatin soluble in glycineNaOH buffer of pH 9The Coomassie blue and amido black bind to gelatin andwhole plate giving the colour of dye except the hydrolysisareas which appear as transparent without dye (Figure 4)

37 Characterization of Keratinolytic Protease Enzyme

371 Influence of pH and Temperature The effect of tem-perature and pH on enzymes activity and stability wasdetermined The optimum temperature and pH for proteaseactivity of enzymes produced by B amyloliquefaciens MA20and B subtilisMA21 was found to be 60∘C 90 respectively

The enzymes activity was investigated at pH 7 and dif-ferent temperatures (Figure 5)The proteolytic activities weredetermined at optimized temperature 60∘C and different pH(Figure 6) For confirmation from the optimized reactionthe enzymes activity was assayed at pH 9 with differenttemperatures (Figure 7)

Results revealed that the keratinolytic protease from Bamyloliquefaciens MA20 and B subtilis MA21 is similar tothose produced using bacteria actinomycetes and fungi andhas a pH optimum in a neutral-to-alkaline range [38 39]Theoptimal temperature for activity was also found in the usualrange for keratinolytic protease (30ndash80∘C)

The maximum activity of protease enzyme produced byB amyloliquefaciens MA20 was 922UmL at pH 9 and 60∘Cwhile the maximum activity of protease enzyme produced byB subtilis MA21 was determined as 814UmL at the sameconditions The alkaline pH of the keratinolytic proteaseenzyme fromB amyloliquefaciensMA20 andB subtilisMA21suggests a positive biotechnological potential

The enzymes were stable at temperatures between 4 and70∘C The enzyme produced by B amyloliquefaciens MA20is more thermostable than enzyme produced by B subtilisMA21 (Figures 8 and 9) The results of thermal stability indi-cated that the keratinolytic protease fromB amyloliquefaciensMA20 is more thermostable than the enzyme produced by BsubtilisMA21

The thermal stability studies give the enzyme from BamyloliquefaciensMA20 the advantage for using in industrialapplications which are the main objective of this study

The crude enzyme from B amyloliquefaciens MA20 isdescribed as stable over a broad pH range of 40ndash120 but thebest stable pH is 9 and 10 (Figure 10) Figure 11 indicates thatthe enzyme fromB subtilisMA21was stable in pH range (50ndash120) with high stability at pH 9 The stability of keratinolyticproteases produced by B amyloliquefaciens MA20 and BsubtilisMA21 has been suggested to offer great advantages for


Bacillus sp Q-12 (AB1993171)

Bacillus subtilis B-16 (EU5613471)

Bacillus amyloliquefaciens SE-01 (AB2011211)

Bacillus subtilis LB-01 (AB2011201)

Bacillus sp HNR03 (EU3733401)

Bacillus sp TPL08 (EU3733781)


Bacillus sp TPR06 (EU3734021)

Bacillus amyloliquefaciens TUL308 (JF4125461)

Bacillus subtilis ITS1 US116 (AJ5445381)

Bacillus subtilis SSCS9 (AB2110331)

Bacillus subtilis SSCT68 (AB2109681)

Bacillus subtilis DL-3-4-3 (GQ1995931)

Bacillus subtilis subsp Inaquosorum type DSM 22148T (HE5827811)

Bacillus subtilis HFBL124 (HQ1531041)

Bacillus sp AMX-4 (AY6017241)

Bacillus amyloliquefaciens BDT-7 (GU2695721)

Bacillus sp 70A-7 (GU2695711)

Bacillus amyloliquefaciens SB 3232 (GU1919191)

Bacillus subtilis SSCS21-1 (AB2110231)

Bacillus sp K7SC-9A (JF7999621)

Bacillus sp TCCC11045 (EU2316371)

Bacillus subtilis TCCC11008 (EU2316221)

Bacillus sp 477BP (AJ5364251)

Bacillus subtilis SSCT51 (AB2109821) Bacillus amyloliquefaciens MA20 (HQ1155991)

Bacillus subtilis SM98011 (DQ0115221)

Bacillus amyloliquefaciens Chilli-1 (HQ0214201)

Bacillus subtilis AA4 (JN2024421)

Bacillus amyloliquefaciens scs (JF4996551)

Bacillus subtilis ET (HQ2666691)

Bacillus subtilis pC (HQ2666661)

Bacillus subtilis A97 (AB5011131)

Bacillus amyloliquefaciens DM-3 (HM0348171)

Bacillus sp DR13 (FJ4649871)

100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05

Figure 2 Phylogenetic position of Bacillus amyloliquefaciens MA20 within the genus Bacillus The branching pattern was generated byneighbor-joining tree methodThe Genbank accession numbers of the 16S rRNA nucleotide sequences are indicated in bracketsThe numberof each branch indicates the bootstrap values The bar indicates a Jukes-Cantor distance of 05

industrial purposes such as wastewater treatment and leathertanning [40]

38 Influence of Protease Inhibitors Solvents and Metal Ionson Enzymes Activity Mostly keratinolytic proteases belongto the subtilisin family of serine proteases with cysteine

proteases which have higher activity on casein [41] The ker-atinolytic proteases produced by Bacillus sp are often serine-proteases such as the enzymes produced by B licheniformis[42] B pseudofirmus [43] and B subtilis [44 45] Proteaseactivity of enzymes prepared fromB amyloliquefaciensMA20and B subtilis MA21 was completely inhibited by serineprotease inhibitor (PMSF) The result indicated the presence


Bacillus sp 3639ABRRJ (JF3092571)

Bacillus sp FIA02 2 (EU3082821)

Bacillus sp CC15 (AB5760951)

Bacillus amyloliquefaciens H102 (HQ4072771)

Bacillus subtilis (AB3831351)



Bacillus subtilis BL6 (GU8261611)

Bacillus atrophaeus JCM9070 (NR 0246891)


Bacillus subtilis RSNPB27 (HM5881671)



Bacillus subtilis F321122 (EF4235901)

Bacillus subtilis SWB8 (HM2106361)





Bacillus subtilis DM-1 (HM0348161)

Bacillus subtilis EDR4 (EF5020201)

Bacillus subtilis CICCHLJ Q60 (EF5282801)

Bacillus subtilis M2 (HM2243871)




Bacillus sp 3639BBRRJ (JF3092581)

Bacillus amyloliquefaciens LSC04 (EF1767721)

Bacillus subtilis O9 (AF2870111)

Bacillus subtilis MA21 (HQ1156001)


Bacillus subtilis ZJ06 (EU2660711)

Bacillus sp NZT-10-56 (GU2696131)

Bacillus sp NZT-55 (GU269616)

Bacillus sp XJT-7 (GU2696151)

Bacillus sp HMB-6338 (GU2696031)


100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02

Figure 3 Phylogenetic position of Bacillus subtilisMA21 within the genus BacillusThe branching pattern was generated by neighbor-joiningtree method The Genbank accession numbers of the 16S rRNA nucleotide sequences are indicated in brackets The number of each branchindicates the bootstrap values The bar indicates a Jukes-Cantor distance of 02

of the serine group in the enzyme active site The enzymesactivity was partially inhibited by EDTA (Tables 2 and 3)This suggests that the keratinolytic protease from the Bacillusstrain belongs to keratinolytic serine protease family

The stability of keratinolytic proteases in presence of SDSacts as a positive advantage of enzymes feature because itindicated the possibility of using them in different industrialpurposes as detergent industry leather industry and wool


Figure 4 Proteolytic activity of crude enzymes produced by B amyloliquefaciensMA20 and B subtilisMA21 using gelatin as substrate andboth of Coomassie blue and amido black stains Blue plate stained with Coomassie blue while green plate stained with Amido blackThe well(1) refers to enzymes produced by B amyloliquefaciensMA20 (2) refers to enzymes produced by B subtilisMA21 and (3) is inactive enzyme

0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)

B amyloliquefaciens MA20B subtilis MA21

Figure 5 Effect of temperatures at pH 7 on protease activity of crudeenzymes produced by B amyloliquefaciens MA20 and B subtilisMA21

improvement The stability toward SDS is important becausea few authors reported that SDS-stable enzymes are also notgenerally available except for a few strains such as Bacillusclausii I-52 [46] and Bacillus sp RGR-14 [47] The effectsof various solvents and metal ions on enzyme activity wereexamined in order to find which ions are stimulators andwhich are inhibitors of the catalytic process

The metal ions were used with two final concentrations(50mM and 10mM) while the used solvents with finalconcentrations (1 and 05) The effects of metal ions andsolvents on enzyme activities are summarized in Tables 2 and3

39 First-Dimension Protein Electrophoresis The productionof extracellular keratinolytic proteases byB amyloliquefaciensMA20 and B subtilis MA21 was evaluated by SDS-PAGEand zymogram analysis Giongo et al found multiple bandsafter zymogram of keratinolytic protease produced by three

pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Figure 6 Effect of pH at 60∘C on protease activity of crude enzymesproduced by B amyloliquefaciensMA20 and B subtilisMA21

strains of Bacillus sp P6 P7 and P11 using feather degradingmedium [25] Growth of the two strains in presence 10 gLof wool resulted in the production of multiple proteasesas observed by a zymogram on gelatin (Figures 12 and 13)Multiple clear zoneswere observed anddetected using (PAGERuler Prestained Protein Ladder) which indicated that theproteolytic activity was not due to a single protein Thenative-PAGE zymogram of enzymes from the 2 strains wasperformed and compared with protein ladder (Figures 14and 15) The zymogram using native page was carried outfor detecting the bands of protein where the purificationperforms using native protein Multiple bands could bedetected on native-PAGE zymogrm

310 Two-Dimension Protein Electrophoresis The two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)


Table 2 Effect of protease inhibitors metal ions and solvents on proteolytic activity of B amyloliquefaciensMA20

Substance Final concentration Relative activity () Final concentration Relative activity ()Control 0 100 0 100120573-mercaptoethanol 5mM 18 1mM 98PMSF 5mM 0 1mM 0EDTA 5mM 35 1mM 97SDS 05 54 01 122ZnCl2 10mM 153 5mM 156MgCl2 10mM 119 5mM 95CuSO4 10mM 266 5mM 204Urea 10mM 114 5mM 139HgCl2 10mM 1004 5mM 107CaCl2 10mM 104 5mM 107BaCl2 10mM 115 5mM 116Guanidin HCl 10mM 103 5mM 132MnCl2 10mM 71 5mM 162Methanol 1 90 05 100Ethanol 1 103 05 100DMSO 1 90 05 103Isopropanol 1 98 05 108Tween 20 1 86 05 94Triton X100 1 61 05 65

Table 3 Effect of protease inhibitors metal ions and solvents on proteolytic activity of B subtilisMA21


Table 4 Two-dimension protein gel electrophoresis report of crude enzymes produced by B amyloliquefaciensMA20 and B subtilisMA21

Gels Spots Minimum gray Maximum gray Columns Rows Pixel width Pixel heightB amyloliquefaciensMA20 237 23 209 2512 1510 353 353B subtilisMA21 291 0 255 2336 1452 353 353


0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)

Figure 7 Effect of temperatures at pH9onprotease activity of crudeenzymes produced by B amyloliquefaciens MA20 and B subtilisMA21

0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C

Figure 8 Thermal stability of crude enzyme produced by BamyloliquefaciensMA20

is an advanced technique that depends on protein separationfirstly according to the pH and secondly based on themolecular weight This technique was carried out todifferentiate between the crude keratinolytic proteasesobtained from B amyloliquefaciens MA20 and B subtilisMA21 After the separation based on the pH the strips wereapplied on gel for separating according to molecular weightThe gels were stained with Coomassie blue for detecting theprotein spots

In order to classify the keratinolytic protease enzyme asystemic comparison of 2Dmaps of proteases was conducted

Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C

Figure 9Thermal stability of crude enzyme produced by B subtilisMA21

Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

pH 4pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12

Figure 10 pH stability of crude enzyme produced by B amylolique-faciensMA20

with imagemaster 2D Platinum 6 softwareThe two gels werephotographed by high quality scanner image (Figure 16)

The results were analyzed using image master 2D Plat-inum 6 software The spots were detected before matchingbetween the two gels and the report was obtained (Table 4)Every spot refers to the presence of one proteinThe gel whichwas loaded with keratinolytic protease from B amyloliquefa-ciensMA20 had 237 spots while the gel appliedwithB subtilisMA21 had 291 spots


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12

Figure 11 pH stability of crude enzyme produced by B subtilisMA21

1510

Z C M(kDa)

17013010070

55

40

35

25

Figure 12 SDS-PAGE and zymogram analysis of keratinolyticprotease enzyme from B amyloliquefaciens MA20 The lane (M)is prestained protein ladder lane (C) is protein pattern of crudeenzyme and lane (Z) is zymogram of enzyme

The 2 gels were matched and consider gel resulted fromcrude enzymes of B amyloliquefaciens MA20 as referenceand the final report that summarizes the difference between 2gels was obtained (Table 5) The 2 gels were matched and thepercent of matching between them was 1325

4 Conclusion

This paper described in details different methods that lead tothe production of keratinolytic protease from two Bacillus spstrains Different methods and assays ranging from simple toadvanced were carried out to prove that the enzymes have

1510

Z C M

(kDa)

17013010070

55

40

35

25

Figure 13 SDS-PAGE and zymogram analysis of keratinolyticprotease enzyme from B subtilis MA21 The lane (M) is prestainedprotein ladder lane (C) is protein pattern of crude enzyme and lane(Z) is zymogram of enzyme

20 M(kDa)

100

70

55

403525

1510

Figure 14 Native-PAGE zymogram analysis of keratinolytic pro-tease enzyme from B amyloliquefaciens MA20 The lane (M) isprestained protein ladder and lane (20) is zymogram of enzyme

Table 5 Match statistics report of crude enzymes produced by Bamyloliquefaciens MA20 and B subtilis MA21 after two-dimensionprotein gel electrophoresis

Gel name Gel name Number ofmatches

Percentmatches

B amyloliquefaciensMA20 (reference) B subtilisMA21 35 132576

the ability to degrade wool The two used strains have beenselected based on that they belong to Bacillus sp and showthe best keratinolytic activitiesThe study includedmolecularand bioinformatic tools to identify the twoBacillus sp strains16S rRNA phylogenetic tree Blast search for nucleotidesimilarity scanning electron microscope SDS-PAGE and2D-PAGE have been used to differentiate the both strains


21 M

(kDa)

100

70

55403525

1510

Figure 15 Native-PAGE zymogram analysis of keratinolytic pro-tease enzyme from B subtilis MA21 The lane (M) is prestainedprotein ladder and lane (21) is zymogram of enzyme

Bacillus amyloliquefaciens MA20 Bacillus subtilis MA21

Figure 16 Two-dimension gel of enzymes fromB amyloliquefaciensMA20 and B subtilisMA21

and their produced enzymesThe study succeeded in charac-terization of Bacillus sp strains which were named Bacillusamyloliquefaciens MA20 and Bacillus subtilis MA21 Theenzyme activities either on agar diffusion plates or throughthe enzyme activity bioassays under different experimentalconditions proved that the both strains are able to producedifferent keratinolytic protease enzymes B amyloliquefaciensMA20 and B subtilis MA21 could be used in large-scaleproduction of keratinolytic protease enzymes where theseenzymes were stable at temperature range between 4 and70∘C and over a wide range of pH values (4ndash12) as well asstable against organic solvents and detergentsThe charactersof keratinolytic enzymes produced by B amyloliquefaciensMA20 and B subtilis MA21 could play an important roleespecially in industrial applications therefore this researchacts as preliminary studies for applying the keratinolyticproteases in wool quality improvement

References

[1] T Korniłłowicz-Kowalska and J Bohacz ldquoBiodegradation ofkeratinwaste theory andpractical aspectsrdquoWasteManagementvol 31 pp 1689ndash1701 2011

[2] R D B Fraser and D A D Parry ldquoMacrofibril assembly intrichocyte (hard 120572-) keratinsrdquo Journal of Structural Biology vol142 no 2 pp 319ndash325 2003

[3] L N Jones ldquoHair structure anatomy and comparative anatomyrdquoClinics in Dermatology vol 19 no 2 pp 95ndash103 2001

[4] P Pillai and G Archana ldquoHide depilation and feather disinte-gration studies with keratinolytic serine protease from a novelBacillus subtilis isolaterdquo Applied Microbiology and Biotechnol-ogy vol 78 no 4 pp 643ndash650 2008

[5] V FilipelloMarchisio ldquoKaratinophilic fungi their role in natureand degradation of keratinic substratesrdquo in Biology of Dermato-phytes and Other Keratinophilic Fungi R K S Kushawaha andJ Guarro Eds vol 17 pp 86ndash92 Revista Iberoamericana deMicologıa 2000

[6] R Gupta and P Ramnani ldquoMicrobial keratinases and theirprospective applicationsan overviewrdquo Applied Microbiologyand Biotechnology vol 70 pp 21ndash33 2006

[7] S Sangali and A Brandelli ldquoFeather keratin hydrolysis by aVibrio sp strain kr2rdquo Journal of Applied Microbiology vol 89no 5 pp 735ndash743 2000

[8] C H De ToniM F Richter J R Chagas J A P Henriques andC Termignoni ldquoPurification and characterization of an alkalineserine endopeptidase from a feather-degrading Xanthomonasmaltophilia strainrdquo Canadian Journal of Microbiology vol 48no 4 pp 342ndash348 2002

[9] F S Lucas O Broennimann I Febbraro and P Heeb ldquoHighdiversity among feather-degrading bacteria from a drymeadowsoilrdquoMicrobial Ecology vol 45 no 3 pp 282ndash290 2003

[10] S Yamamura Y Morita Q Hasan et al ldquoCharacterization ofa new keratin-degrading bacterium isolated from deer furrdquoJournal of Bioscience and Bioengineering vol 93 no 6 pp 595ndash600 2002

[11] E H Brutt and J M Ichida ldquoKeratinase produced by Bacilluslicheniformisrdquo US Patent 5877000 1999

[12] A Gousterova D Braikova I Goshev et al ldquoDegradationof keratin and collagen containinq wastes by newly isolatedthermoactinomycetes or by alkaline hydrolysisrdquo Letters inApplied Microbiology vol 40 no 5 pp 335ndash340 2005

[13] K L Evans J Crowder and E S Miller ldquoSubtilisins of Bacillusspp hydrolyze keratin and allow growth on feathersrdquo CanadianJournal of Microbiology vol 46 no 11 pp 1004ndash1011 2000

[14] I N S Dozie C N Okeke and N C Unaeze ldquoA thermostablealkaline-active keratinolytic proteinase from ChrysosporiumkeratinophilumrdquoWorld Journal of Microbiology and Biotechnol-ogy vol 10 no 5 pp 563ndash567 1994

[15] A Riffel F Lucas P Heeb and A Brandelli ldquoCharacterizationof a new keratinolytic bacterium that completely degradesnative feather keratinrdquo Archives of Microbiology vol 179 no 4pp 258ndash265 2003

[16] B Zhang D Jiang W Zhou H Hao and T Niu ldquoIsolationand characterization of a new Bacillus sp 50-3 with highlyalkaline keratinase activity from Calotes versicolor faecesrdquoWorld Journal of Microbiology and Biotechnology vol 25 no 4pp 583ndash590 2009

[17] J G Holt N R Krieg P H A Sneath J T Staley and ST Williams Bergeyrsquos Manual of Determinative BacteriologyWilliams ampWilkins Baltimore Md USA 9th edition 1994

[18] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbor Laboratory ColdSpring Harbor New York NY USA 2nd edition 1989


[19] F Sanger S Nicklen and A R Coulson ldquoDNA sequencingwith chain-terminating inhibitorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 74 no12 pp 5463ndash5467 1977

[20] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 pp 2731ndash2739 2011

[21] A A Amara R S Slem and S AM Shabeb ldquoThe possibility touse crude proteases and lipases as biodetergentrdquo Global Journalof Biotechnology amp Biochemistry vol 4 no 2 pp 104ndash114 2009

[22] WMao R Pan andD Freedman ldquoHigh production of alkalineprotease by Bacillus licheniformis in a fed-batch fermentationusing a synthetic mediumrdquo Journal of Industrial Microbiologyvol 11 no 1 pp 1ndash6 1992

[23] A B Vermelho M N L Meirelles A Lopes S D G PetinateA A Chaia and M H Branquinha ldquoDetection of extracellularproteases from microorganisms on agar platesrdquo Memorias doInstituto Oswaldo Cruz vol 91 no 6 pp 755ndash760 1996

[24] AAAmara andA E Serour ldquoWool quality improvement usingthermophilic crude proteolytic microbial enzymesrdquo American-Eurasian Journal of Agricultural amp Environmental Sciences vol3 no 4 pp 554ndash560 2008

[25] J L Giongo F S Lucas F Casarin P Heeb and A BrandellildquoKeratinolytic proteases of Bacillus species isolated from theAmazon basin showing remarkable de-hairing activityrdquo WorldJournal of Microbiology and Biotechnology vol 23 no 3 pp375ndash382 2007

[26] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[27] H Blum H Beier and J H Gross ldquoImproved silver stainingof plant proteins RNA and DNA in polyacrylamide gelsrdquoElectrophoresis vol 8 pp 93ndash99 1987

[28] R Gupta Q Beg and P Lorenz ldquoBacterial alkaline proteasesmolecular approaches and industrial applicationsrdquo AppliedMicrobiology and Biotechnology vol 59 no 1 pp 15ndash32 2002

[29] J Cortez P L R Bonner and M Griffin ldquoTransglutaminasetreatment of wool fabrics leads to resistance to detergentdamagerdquo Journal of Biotechnology vol 117 no 1 pp 379ndash3862005

[30] J M Kim W J Lim and H J Suh ldquoFeather-degrading Bacillusspecies from poultry wasterdquo Process Biochemistry vol 37 no 3pp 287ndash291 2001

[31] M Rozs L Manczinger C Vagvolgyi and F Kevei ldquoSecretionof a trypsin-like thiol protease by a new keratinolytic strain ofBacillus licheniformisrdquo FEMS Microbiology Letters vol 205 no2 pp 221ndash224 2001

[32] M S Roberts L K Nakamura and F M Cohan ldquoBacillusmojavensis sp nov distinguishable from Bacillus subtilis bysexual isolation divergence in DNA sequence and differencesin fatty acid compositionrdquo International Journal of SystematicBacteriology vol 44 no 2 pp 256ndash264 1994

[33] M S Roberts L K Nakamura and F M Cohan ldquoBacillusvallismortis sp nov a close relative of Bacillus subtilis isolatedfrom soil in Death Valley Californiardquo International Journal ofSystematic Bacteriology vol 46 no 2 pp 470ndash475 1996

[34] L T Wang F L Lee C J Tai and H Kasai ldquoComparisonof gyrB gene sequences 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis grouprdquo International

Journal of Systematic and Evolutionary Microbiology vol 57 no8 pp 1846ndash1850 2007

[35] I Szabo A Benedek I Mihaly Szabo and G Barabas ldquoFeatherdegradation with a thermotolerant Streptomyces graminofa-ciens strainrdquo World Journal of Microbiology and Biotechnologyvol 16 no 3 pp 253ndash255 2000

[36] A Gushterova E Vasileva-Tonkova E Dimova P Nedkov andT Haertle ldquoKeratinase production by newly isolated Antarc-tic actinomycete strainsrdquo World Journal of Microbiology andBiotechnology vol 21 no 6-7 pp 831ndash834 2005

[37] T Korniłłowicz-Kowalska ldquoStudies on the decomposition ofkeratin wastes by saprotrophic microfungi PI Criteria forevaluating keratinolytic activityrdquo Acta Mycologica vol 32 pp51ndash79 1997

[38] B Bockle B Galunsky and R Muller ldquoCharacterization of akeratinolytic serine proteinase from Streptomyces pactum DSM40530rdquo Applied and Environmental Microbiology vol 61 no 10pp 3705ndash3710 1995

[39] P Bressollier F LetourneauMUrdaci and B Verneuil ldquoPurifi-cation and characterization of a keratinolytic serine proteinasefrom Streptomyces albidoflavusrdquo Applied and EnvironmentalMicrobiology vol 65 no 6 pp 2570ndash2576 1999

[40] H Takami Y Nogi and K Horikoshi ldquoReidentification of thekeratinase-producing facultatively alkaliphilic Bacillus sp AH-101 as Bacillus haloduransrdquo Extremophiles vol 3 no 4 pp 293ndash296 1999

[41] S Sangali and A Brandelli ldquoIsolation and characterization of anovel feather-degrading bacterial strainrdquo Applied Biochemistryand Biotechnology A vol 87 no 1 pp 17ndash24 2000

[42] X LinDWKelemen E SMiller and J CH Shih ldquoNucleotidesequence and expression of kerA the gene encoding a kerati-nolytic protease of Bacillus licheniformis PWD-1rdquo Applied andEnvironmental Microbiology vol 61 no 4 pp 1469ndash1474 1995

[43] M Kojima M Kanai M Tominaga S Kitazume A Inoueand K Horikoshi ldquoIsolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01rdquoExtremophiles vol 10 no 3 pp 229ndash235 2006

[44] T I Zaghloul ldquoCloned Bacillus subtilis alkaline protease (aprA)gene showing high level of keratinolytic activityrdquo AppliedBiochemistry and Biotechnology A vol 70ndash72 pp 199ndash205 1998

[45] H J Suh and H K Lee ldquoCharacterization of a keratinolyticserine protease from bacillus subtilis KS-1rdquo Protein Journal vol20 no 2 pp 165ndash169 2001

[46] 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

[47] R Oberoi Q K Beg S Puri R K Saxena and R GuptaldquoCharacterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sprdquo World Journal ofMicrobiology and Biotechnology vol 17 no 5 pp 493ndash497 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Kocuria andMicrobacterium as well as a few strains of Gram-negative bacteria such as Xanthomonas Stenotrophomonasand Chryseobacterium are confined as keratin degraders [7ndash10] Most of keratin degrading bacteria belong to the genus ofBacillus [11]

The keratinous substrate such as feather and wool canbe degraded in basal medium by microorganisms which arecapable of utilize keratin as sole carbon and nitrogen source[12] Keratinolytic proteases have broad substrate specificitywhere they have the ability to hydrolyze soluble protein suchas casein gelatin and bovine serum albumin Additionallythey can hydrolyze the insoluble protein including feathersilk and wool [13] Keratinolytic proteases mostly belong toserine or metalloproteases showing sequence similarity withsubtilisin group of proteases [14 15] In recent years moredemands to keratinolytic proteases are increasing due to theirmultitude in industrial applications such as the feed fertilizerdetergent and textile industries The present study describesthe isolation and identification of new Bacillus amylolique-faciens MA20 and B subtilis MA21 strains from Egyptianecosystem that grow well on wool as sole carbon sourceMoreover the two strains are able to degrade wool and theirenzymes can be used to improve the wool quality This paperincludes full characterization of the keratinolytic proteasewhich explains that the best environmental conditions can beused to improve the wool quality in industry

2 Materials and Methods

21 Sample Collection Different types of samples were col-lected from different Governorates of Egyptian ecosystemincluded Alexandria Behera Qaliubia Mania and AsiutThese fresh sampleswere varied such as soil sand soil humuswaste wool and rhizosphere under olive treesThese sampleswere collected in sterile falcon tubes and transported tothe microbiological lab in (City of Scientific Research andTechnological Applications)

22 Strains Isolation The bacterial strains were isolated bysuspending 1 g of soil samples in a 10mL sterile 085 (wv)saline solution and then treated for 20min at 80∘C This willenable the isolation of the spore-forming bacteria Luria-Bertani (LB) agar medium with 1 (wv) skim milk wasused for their cultivation by spreading 01mL of each 10minus5and 10minus6 dilutions The plates then were incubated for 24hours at different temperatures [4 16] The colonies whichgive clear zones formed by hydrolysis of skim milk werepicked Pure bacterial isolates were obtained by reculturingindividual colonies several times on fresh LB agar medium toproduce single colony from each

23 Strains Selection Twelve selected strains isolated accord-ing to the diameter of clear zone were cultured on mediumcontaining 05 g NaCl 03 g K

2HPO4 04 g KH

2PO4 and

10 g wool per liter pH 7 and incubated for 5 days at 37∘CThe wool was used as sole carbon and nitrogen source fordetecting potent strains that have the ability to degrade thewool completely Three strains degraded the wool and the

supernatants of their culture were assayed on plate containing1 gelatin powder which is soluble in phosphate buffer pH 7After determining the existence of the activity (by the clearzone of the supernatants) Bacillus sp MA20 and Bacillus spMA21 were selected and preserved for further investigation

24 Bacterial Identification While the phenotypic charac-teristics and isolation method of the two selected isolatesindicate that they are related to Bacillus group but furtheridentification was conducted

The strains identification are included the spore mor-phology Gram stain and motility The morphological andphysiological characteristics of the bacterial isolates werecompared with the data from Bergeyrsquos Manual of Determi-native Bacteriology [17]

25 Scanning of Bacillus sp MA20 and Bacillus sp MA21by Scanning Electron Microscope The bacterial smear wasprepared by centrifuging the bacterial cultures at 12000 rpmfor 20minThepellets werewashed 2 times by saline solutionThe pellet was suspended in sterilized distilled H

2O The

bacterial film was prepared and fixed on glass slides tillcomplete drying The smear was coated with gold usingsputter coater The golden coated sample was scanned at20KV acceleration voltages at room temperature

26 Genetic Identification and Differentiation

261 DNA Extraction The genomic DNA of Bacillus strainswas isolated usingmodificationmethod from Sambrook et al[18]

262 Identification by 16S Ribosomal RNA (rRNA)

PCR Amplification according to Sambrook et al [18] The PCRamplification reactions were performed in a total volumeof 50120583L Each reaction mixture contained the followingsolutions 2 120583L of DNA (40 ng) 1 120583L of 10 pmol forwarded16S-rRNA primer (51015840-AAATGGAGGAAGGTGGGGAT-31015840) 1 120583L of 10 pmol reverse 16S rRNA primer (51015840-AGGAGGTGATCCAACCGCA-31015840) 08120583L of 125mM(dNTPrsquos) 5 120583L of PCR buffer included MgCl

2 and 02 120583L

Taq polymerase (1 Unit) and water-free DNAse and RNAsewere added up to 50 120583LThePCR apparatus was programmedas follows 3min denaturation at 95∘C followed by 35 cyclesthat consisted of 1min at 95∘C 1min at 58∘C and 1min at72∘C and a final extension was 10min at 72∘C The productsof the PCR amplification were analyzed by agarose gelelectrophoresis (2)

263 PCR Cleanup and 16S rRNA Sequencing The PCRproducts were cleaned up for DNA sequencing following themethod described by Sambrook et al [18] Automated DNAsequencing based on enzymatic chain terminator techniquedeveloped by Sanger et al [19] was carried out using 3130XDNA Sequencer (Genetic Analyzer Applied BiosystemsHitachi Japan)




2HPO4 03 gL KH

2PO4 04 gL wool


28 Enzymes Assay

























2

MgCl2 CuSO

4 Urea HgCl

2 CaCl

2 BaCl

2 Guanidin HCl

and MnCl2were used
















2HPO4 03 gL KH


























2HPO4 03 gL KH

2PO4
















































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2HPO4 03 gL KH

2PO4 04 gL wool


28 Enzymes Assay

























2

MgCl2 CuSO

4 Urea HgCl

2 CaCl

2 BaCl

2 Guanidin HCl

and MnCl2were used
















2HPO4 03 gL KH


























2HPO4 03 gL KH

2PO4
















































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












2

MgCl2 CuSO

4 Urea HgCl

2 CaCl

2 BaCl

2 Guanidin HCl

and MnCl2were used
















2HPO4 03 gL KH


























2HPO4 03 gL KH

2PO4
















































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2HPO4 03 gL KH


























2HPO4 03 gL KH

2PO4
















































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












2HPO4 03 gL KH

2PO4
















































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































100

100

25

98

9

9

30

2

107

0

0

26

0

7

0

2

00

0

98

44

05











































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































100

99

100

99

100

99

24

22

30

10

53

4

16

5

1

0

0

19

0

0

2

0

0

9

95

48

02






0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0100200300400500600700800900

1000

Temperature (∘C)






pH5 6 7 8 9 10 11 12 13

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000













0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









0 10 20 30 40 50 60 70 80 90

Prot

ease

activ

ity (U

mL)

0

100

200

300

400

500

600

700

800

900

1000


Temperature (∘C)


0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

Time (hrs)

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C




Time (hrs)0 5 10 15 20 25 30

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120

70∘C60∘C50∘C40∘C

30∘C20∘C4∘C


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0102030405060708090

100110120


pH 9pH 10pH 11pH 12





Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Time (hrs)0 5 10 15 20 25 30 35 40 45 50 55

Resid

ual a

ctiv

ity (

)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8

pH 9pH 10pH 11pH 12


1510

Z C M(kDa)

17013010070

55

40

35

25



4 Conclusion


1510

Z C M

(kDa)

17013010070

55

40

35

25


20 M(kDa)

100

70

55

403525

1510




Percentmatches




21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


21 M

(kDa)

100

70

55403525

1510





References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article production and characterization of keratinolytic...

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