Research Article Streptomyces flavogriseus HS1: Isolation ...

Research ArticleStreptomyces flavogriseus HS1Isolation and Characterization of Extracellular Proteasesand Their Compatibility with Laundry Detergents

Sofiane Ghorbel Maher Kammoun Hala Soltana Moncef Nasri and Noomen Hmidet

Laboratoire de Genie Enzymatique et de Microbiologie Ecole Nationale drsquoIngenieurs de Sfax BP 1173 3038 Sfax Tunisia

Correspondence should be addressed to Sofiane Ghorbel so fianhotmailcom

Received 13 January 2014 Revised 22 February 2014 Accepted 5 March 2014 Published 6 April 2014

Academic Editor Neelu Nawani

Copyright copy 2014 Sofiane Ghorbel et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterizationof the secreted proteases By sequencing of its noted 16S rDNA HS1 strain was found to have a 100 identity with Streptomycesflavogriseus The highest protease production was found using FermII media In these conditions maximum protease production(99UmL) was obtained after 96 h incubation at 30∘C and 150 rpm HS1 strain produced at least five proteases as revealed byzymogram technique The enzyme preparation exhibited activity over a broad range of pH (5ndash11) and temperature (25ndash70∘C)Optimum activity was observed at a pH of 70 and a temperature of 50∘C Proteolytic activity was significantly unaffected by Ca2+and Mg2+ EDTA and PMSF highly decreased the original activity The crude extracellular proteases showed high stability whenused as a detergent additive These properties offer an interesting potential for enzymatic hydrolysis at the industrial level

1 Introduction

Actinomycetes a Gram-positive filamentous bacteria candegrade various macromolecules in soil [1] Among actino-mycetes Streptomyces species are the most industrially usefulbecause of their capacity of producing numerous secondarymetabolites particularly antibiotics Similarly these bacteriaoffer a second industrial interesting use by producing largeamounts of proteolytic enzymes with different substratespecificities [2] The investigation of proteases does not takeplace only in scientific fields such as protein chemistry andprotein engineering but also in other industrial uses includ-ing cleaning detergents leather and food additives Sucha wide use of proteases in the industrial field shows theirimportance especially that they represent around 60 of thetotal enzymemarket [3] Unlike proteases fromother bacteriawhich have been extensively characterized proteases fromactinomycetes did not receive a similar attention [4] Stilltheir ability to produce a variety of enzymes may be anattractive phenomenon of these prokaryotes

The composition of the protease complexes secreted byStreptomyces is determined by the taxonomic position ofthe producers [5ndash8] The potential use of Streptomyces for

producing proteases is justified by their ability to releasethe proteins into extracellular media Such a capability isgenerally regarded as safe (GRAS)with food and drug admin-istration Streptomyces spp that produce proteases includeS clavuligerus S griseus S rimouses S thermoviolaceusand S thermovulgaris [7] Some of these proteases like theserine proteases of Streptomyces griseus [8 9] and Strepto-myces fradiae [10] have been characterized structurally andenzymatically There have also been many descriptions ofisolation and partial characterization of alkaline proteaseactivities from other members of the genus Streptomyces likeStreptomyces clavuligerus Streptomyces gulbargensis Strepto-myces viridifaceins and Streptomyces sp [11ndash13]

Present study describes the isolation of a multiple pro-tease producing Streptomyces flavogriseusHS1 strain isolatedfrom a Tunisian soilWe report the biochemical characteriza-tion of the crude enzyme for evaluation of its biotechnologicalpotential as detergent additive

2 Material and Methods

21 Isolation of the Actinomycete Strain The isolation of theActinomycete strains from a soil sample was done by serial

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 345980 8 pageshttpdxdoiorg1011552014345980

2 BioMed Research International

dilution plate technique on ISP4 agar media containing(gL) starch 10 casein 03 KNO

32 NaCl 2 K

2HPO42

MgSO4sdot7H2O 005 CaCO

3002 FeSO

4sdot7H2O 001 and 15

agar [14]

22 Identification of HS1 Strain The 16S rRNA gene ofthe HS1 strain was amplified by PCR using the follow-ing primers F (forward) 51015840-CCGAATTCGTCGACAACA-GAGTTTGATCCTGGCTCAG-31015840 and R (reverse) 51015840-CCC-GGGATCCAAGCTTAAGGAGGTGATCCAGCC-31015840 ThePCR mixture contained 30 pmol of primers 20 pmol of eachdeoxynucleoside triphosphate polymerisation buffer and5U Taq polymerase The PCR program involved 35 cycles ofdenaturing at 94∘C for 1min primer annealing at 55∘C for1min and extension at 72∘C for 90 s The sequencing wasperformed three times using the DNA sequencer ABI PRISM31003100-Avant Genetic Analyser (CA USA) 16S rDNAsequence was searched for similarities to known sequencesin the GenBank database (National Center for BiotechnologyInformation National Library ofMedicine) using the BLASTsearch program The sequence was aligned with those of thereference strains usingClustalW [15] A phylogenetic treewasconstructed by the neighbour-joining method [16]

23 Determination of Protease Activity Measuring of theprotease activity was done as described by Kembhavi et al[17] using casein as substrate 1 (wv) in 100mM Tris-HClbuffer pH 70 The mixture was incubated for 15min at 50∘Cand the reactionwas stopped by addition of 05mL 20 (wv)TCA (trichloroacetic acid) The mixture was left at roomtemperature for 10 minutes and then centrifuged at 10000 gfor 15 minutes to remove the precipitate The absorbance ofthe soluble TCA peptides was recorded at 280 nm One unitof protease activity was defined as the amount of enzymerequired to liberate 1120583g of tyrosine per minute under theexperimental conditions used All measurements were car-ried out in triplicate

24 Protease Production The proteolytic isolates were cul-tured in three different liquid medium FermII media(gL) dextrin 20 tryptone 10 KH

2PO410 K

2HPO434

MgSO4sdot7H2O03 FeSO

4sdot7H2O001 ZnCl

201 CuSO

4sdot7H2O

001 MgCl2sdot4H2O 0003 CaCl

2001 NaCl 003 pH 70 [18]

gelatin containing media (gL) gelatin 10 peptone 5 yeastextract 5 NaCl 50 and pH 9 and liquid ISP4 media [14]

A 1mL spore suspension (104 to 106 sporesmL) wasadded to a 250mL Erlenmeyer flask containing 100mL ofliquid media [14] and the flasks were incubated at 30∘C and150 rpm for 96 h The culture medium was centrifuged at5000 rpm to remove mycelia and medium debris and thesupernatant was used as a crude enzyme preparation

25 Characterization of Proteases Zymography was per-formed on NativePAGE according to the method of Garcia-Carreno et al [19] After electrophoresis the gel was sub-merged in 1 (wv) casein in 100mM glycine-NaOH bufferpH 70 and incubated at 50∘C for 20min After washingthe gel was stained with Coomassie Brilliant Blue R-250 anddestained with 5 ethanol-75 acetic acid A clear zone

appeared on the blue background of the gel which indicatedthe presence of protease activity

26 Effect of Temperature and pH on Protease Activity andStability To investigate the temperature effect the proteaseassay was performed at different temperatures between 20and 80∘C using casein as substrate for 10min at pH 70For thermal stability the enzyme was incubated at differenttemperatures for 60min Aliquots were withdrawn at thedesigned time intervals to test the remaining activity Theresidual activity was assayed at pH 70 and 50∘C for 10minThe nonheated enzyme was considered as control (100activity)

Protease activity was assayed over the pH range 50ndash120at 50∘C for 10min using casein as a substrateThe effect of pHon enzyme stability was evaluated by measuring the residualenzyme activity after incubation at various pH for 60minat 25∘C The following buffer systems were used 100mMglycine-HCl pH 40 and 50 100mM sodium acetate pH60 100mM phosphate-buffer pH 70 100mM Tris-HCl pH80 100mM glycine-NaOH pH 90 and 10 and 100mMNa2HPO4sdotNaOH pH 120

27 Effect of Metal Ions and Other Chemicals on ProteaseActivity and Stability The effects of different monovalent(Na+ or K+) or divalent (Fe2+ Mn2+ Zn2+ Cu2+ Ba2+ Mg2+or Hg2+) metal ions at a concentration of 5mM on proteaseactivity were investigated by adding them to the reactionmixtureThe activity in the absence of any additiveswas takenas 100

The effects of enzyme inhibitors on protease activity werestudied using PMSF and EDTA The crude enzyme waspreincubated with each inhibitor for 30min at 25∘C and thenthe remaining enzyme activity was estimated using caseinas a substrate The activity in the absence of inhibitors wastaken as 100 The effects of some surfactants (Triton X-100Tween 80 and SDS) and oxidizing agents (sodium perborate)on enzyme stability were studied by preincubating the crudeenzyme for 1 h at 25∘C The residual activity was measuredat pH 70 and 50∘C The activity of the enzyme without anyadditive was taken as 100

28 Detergent Compatibility The compatibility of the HS1extracellular proteases with commercial laundry detergentswas studied using Ariel (Procter and Gamble Switzerland)Newdet (Sodet Tunisia) and Dixan (Henkel Spain) as soliddetergents The endogenous proteases contained in thesedetergents were inactivated by heating the diluted detergentsfor 1 h at 65∘Cprior to the addition of the enzymepreparationThe extracellular proteases of Streptomyces flavogriseus HS1were incubated with different diluted detergents (1100) for1 h at 30∘C 40∘C and 50∘C and then the remaining activitieswere determined under the standard assay conditions Theenzyme activity of a control without detergent incubatedunder similar conditions was taken as 100

29 Statistical Analysis Statistical analyses were performedwith Statgraphics ver 51 professional edition (Manugistics

BioMed Research International 3

Table 1 Effect of various culture media on the production of extra-cellular proteases from Streptomyces flavogriseusHS1

Medium Proteolytic activity (UmL)FermII 98Gelatin based medium 47ISP4 518The activity of the proteases was determined at 50∘C and pH 70 after 96 h ofculture

Corp USA) using ANOVA analysis Differences were con-sidered significant at 119875 lt 005 Results represent the meansof at least two determination carried out in duplicate Thedifference between values did not exceed 5

3 Results and Discussion

31 Isolation of the Actinomycete Strain Samples were takenfrom an organic rich soil in Sfax city (Tunisia) Isolation ofthe actinomycete strainswas obtained after 96 h of incubationat 30∘C One isolate was selected for further studies becauseof its important extracellular proteases secretion and namedHS1 strain (Figure 1) HS1 strain was confirmed as belongingto the genus Streptomyces since it possessed nonfragmentedsubstrate mycelia aerial hyphae and smooth spores orga-nized in straight chains Analysis of the 16S rRNA genesequence of this strain showed a high similarity (100) withStreptomyces flavogriseus (Figure 2)

Streptomyces flavogriseus was well known to produceseveral enzymes such as cellulose xylanase and glucose iso-merase [19ndash21] but no data was found describing extracellu-lar proteases

32 Protease Production Three liquid culture media opti-mized for the production of extracellular proteases in Strep-tomyces were tested among them are FermII [18] ISP4 [14]and gelatin based media (this study) In the light of thisexperiment FermII was found to be the best medium forthe production of Streptomyces flavogriseus HS1 extracellularproteases (Table 1)The fermentation time course for proteaseproduction by Streptomyces flavogriseusHS1 (data not shown)indicates that the maximum protease activity (99UmL)was obtained after 96 h of cultivation when cells were inthe stationary phase and its production was not growthassociated Similar results were obtained by Gibb and Strohl[22] who observed that the maximum protease productionby Streptomyces peucetius occurred after 100 h of cultivationat the stationary phase growth This period was shorter thanthat of the well-studied Streptomyces (eg Streptomyces mod-eratus required 120 h of cultivation for maximum proteaseproduction [6]) However Dastager et al [13] showed thatthe protease activity measured in the cell-free supernatantfluid of Streptomyces gulbargensis sp Nov was maximum(1218UmL) after 48 h of growth

33 Zymogram To givemore information about the diversityof extracellular proteases secreted by HS1 strain zymogramanalysis was done as described in the ldquoMaterial andMethodsrdquo

Figure 1 Plate assay showing the zone of proteolytic activity byprotease-producing Streptomyces flavogriseusHS1 strain

section As shown in Figure 3 proteolytic activity profiles ofcell-free enzymatic preparation of Streptomyces flavogriseusHS1 showed at least five major proteases This is a commonfeature for the streptomycetes [6 23] However the natureand characteristics of the enzymes of protease complexderived from streptomycetes have not been widely studied[6] All the thermophilic bacterial extracellular proteases sofar reported are interestingly serine or neutral metallopro-teases [24]

34 Effect of the pH on Activity and Stability of Streptomycesflavogriseus HS1 Extracellular Proteases The pH profile ofprotease activity from Streptomyces flavogriseusHS1 is shownin Figure 4(a) The crude enzyme was highly active betweenpH 60 and 80 having an optimum around pH 70 The rela-tive activities at pH 60 and 80 were about 65 to 75 Similarresults were described for several Streptomyces strains in theliterature with optimumpH range being between 60 and 120[25] The optimum pH activity of Streptomyces flavogriseusproteases was similar to that from other Streptomyces speciessuch as Streptomyces griseus pronase [26] Therefore thisactivity was lower than that of other Streptomyces describedproteases showing maximum activity at pH 80 like Strep-tomyces sp DP2 [27] and Streptomyces sp CN902 [28] ThepH stability test showed that the crude proteases were highlystable over a broad pH range maintaining more than 70 ofits original activity between pH 50 and 90 (Figure 4(b))

35 Effect of Temperature on the Activity and Stability ofStreptomyces flavogriseus HS1 Extracellular Proteases Thetemperature profile of protease activity from Streptomycesflavogriseus HS1 is presented in Figure 5(a) The HS1 crudeextract was active at temperatures from 30 to 70∘C and hadan optimum at 50∘C while activity decreased rapidly above70∘C The relative activities at 40 and 60∘C were about 63


Streptomyces flavoriensStreptomyces flavogriseusHS1

Streptomyces nitroporeusStreptomyces griseolus

Streptomyces halstediiStreptomyces cinereorectusStreptomyces griseus

Streptomyces fulvissimusStreptomyces microflavus

Streptomyces luridiscabieiStreptomyces fulvorobeus

Streptomyces pulveraceusStreptomyces globisporusStreptomyces badiusStreptomyces rubiginosohelvolus

Streptomyces albovinaceusStreptomyces sindensensisStreptomyces pluricolorescens

Streptomyces parvusStreptomyces anulatusStreptomyces fimicarius

Streptomyces mutomycini

0001

Figure 2 Dendrogram showing the relationships between Streptomyces flavogriseus HS1 and other Streptomyces species Topology wasinferred using the neighbour-joining method

Figure 3 Zymography analysis of Streptomyces flavogriseus HS1crude extracellular proteases

and 60 respectively Several actinomycete thermophilicproteases with high activity at 70∘C have been reported forThermoactinomyces vulgaris and Nocardiopsis dassonvillei Scorchorusii [23] S megasporus [29] S thermovulgaris [4]and S thermoviolaceus [7] Above 60∘C protease activityfrom Streptomyces flavogriseus HS1 rapidly fell as shown forproteases of Streptomyces spp and other bacteria

The thermal stability profile of the crude enzyme showeda high stability at temperatures below 40∘C but was inac-tivated at higher temperatures (Figure 5(b)) After 45minof incubation at 60∘C 78 of the initial activity was lostStreptomyces flavogriseusHS1 proteases were stable at 40 and50∘C after 1 h incubation At low temperatures (minus20 and 4∘C)the crude enzymepreparation retained 75of its activity after2 months El-Raheem et al [23] observed that for alkalineproteases from a strain of S corchorusii activity did notdecrease after storage at minus20∘C for one year at pH valuesbetween 40 and 120 and repeated freezing and thawing

36 Effects of Metal Ions The effects of some metal ionsat a concentration of 5mM on the activity of Streptomycesflavogriseus HS1 crude enzyme were studied at pH 70 and50∘C by the addition of the respective cations to the reactionmixture (Table 2) The Ca2+ Mg2+ and Na+ were shownto have no effect on the protease activity The latter wasslightly affected by Ba2+ andK+ and it retains about 791 and666 of its activity respectively The Hg2+ Cu2+ and Zn2+greatly affected the enzymatic activity till the total inhibitionReduction in protease activity of Streptomyces spp has beenpreviously observed [30 31] especially for Cu2+ probablyas a result of the denaturing action of copper [31] and thechelating effect of EDTA Proteases from Streptomyces sppand N dassonvillei were also stimulated by Mg2+ Mn2+Ca2+ and Zn2+ [30] whereas cation-requiring proteases


120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

5 6 7 8 9 10 11

pH

(a)

100

80

60

40

20

0

Rem

aini

ng ac

tivity

()

6 7 8 9 10 11

pH

(b)

Figure 4 Effect of pH on activity (a) and stability (b) of the crude protease from Streptomyces flavogriseus HS1 The protease activity wasassayed in the pH range of 50ndash120 using buffers of different pH values at 50∘CThe maximum activity obtained from pH 70 was consideredas 100 activity The pH stability of the Streptomyces flavogriseus HS1 crude enzyme was assayed in the rage of 50ndash120 and determined byincubating the crude protease in different buffers for 1 h at 25∘C and the residual activity was determined at pH 70 and 50∘CThe proteolyticactivity before incubation was taken as 100

120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

30 40 50 60 70 80 90

Temperature (∘C)

(a)

0

20

40

60

80

100

0 15 30 45 60

Rem

aini

ng ac

tivity

()

Time (min)

40∘C

50∘C

60∘C

(b)

Figure 5 Effect of temperature on activity (a) and stability (b) of the crude protease from Streptomyces flavogriseus HS1 The temperatureprofile was determined by assaying protease activity at temperatures between 30 and 80∘C The activity of the crude enzyme at 50∘C wastaken as 100 The temperature stability was determined by incubating Streptomyces flavogriseus HS1 crude enzyme at temperatures from40 to 60∘C for 1 h The residual proteolytic activity was determined at regular intervals under standard assay conditions The original activitybefore preincubation was taken as 100

from streptomycetes have also been reported [32] HoweverStreptomyces sp 594 protease stability was enhanced onlyby Ca2+ and Ba2+ [33] It was shown that the Ca2+ ionsare important for catalysis James et al [7] suggested thatmost probably they stabilize the protein through specific ornonspecific binding sites and may also allow for additionalbonding within the enzyme molecule preventing unfoldingat higher temperatures as has been demonstrated for proteasefrom thermophilic bacteria mainly thermolysin

37 Effects of Enzyme Inhibitors on Protease Activity Pro-teases can be classified by their sensitivity to variousinhibitors [34] In order to confirm the nature of the extra-cellular proteases the effects of different enzyme inhibitors

such as chelating agent and a specific group of reagents onthe protease activity were investigated (Table 3) The crudeproteolytic preparation was strongly inhibited by the serineprotease inhibitor (PMSF) indicating that the HS1 crudeextract contained serine proteases In addition the enzymaticextract was also inhibited by the chelating agent EDTA(5mM) with 77 of its original activity being lost indicatingthe importance of ions in enzyme stabilizationThese findingsare in line with several earlier reports showing that activestructure of serine proteases containsCa2+ binding site(s) andthe removal of Ca2+ from the strong binding site is associatedwith a significant reduction in thermal stability [35] Fromthis result HS1 can contain serine metalloproteases since thecrude enzyme is strongly inhibited by both PMSF and EDTA


Table 2 Effect of various metal ions (5mM) on the activity of crude enzyme from Streptomyces flavogriseusHS1

Ions (5mM) None Ca2+ Mg2+ Fe2+ Mn2+ Cu2+ Ba2+ Hg2+ Na+ K+

Relative activity () 100 100 100 71 100 0 79 0 100 67The activity of the proteases was determined by incubating the enzyme in the presence of various metal ions for 10min at 50∘C and pH 70

Table 3 Effect of various enzyme inhibitors (5mM) on the activityof extracellular proteases from Streptomyces flavogriseusHS1

Inhibitors Remaining activity ()None 100PMSF 5EDTA 23The secreted proteases from Streptomyces flavogriseus HS1 were preincu-bated with various enzyme inhibitors for 30min at room temperature andthe remaining activity was determined at pH 70 and 50∘C Crude proteaseactivity measured in the absence of any inhibitor was taken as 100

Table 4 Stability of the Streptomyces flavogriseus HS1 extracellularproteases in the presence of various surfactants and oxidizing agents

Detergents Concentrations () Remaining activity ()

Triton X-100 1 6145 405

Tween 80 1 7145 405

Tween 20 1 7025 378

SDS 01 18905 0

Sodium perborate 01 6411 398

H2O201 61405 587

The crude extracellular proteases were incubated with different surfactantsand oxidizing agents for 1 h at 25∘C and the remaining activity was measuredunder standard conditions The activity is expressed as a percentage of theactivity level in the absence of additives

38 Effects of Oxidizing Agents and Surfactants on ProteaseStability In order to be effective during washing a gooddetergent protease must be compatible and stable with allcommonly used detergent compounds such as surfactantsoxidizing agents and other additives whichmight be presentin the detergent formulation [29 30] HS1 protease extractwas preincubated 60min at 25∘C in the presence of SDSTween 20 and 80 and Triton X-100 and the residual activitieswere assayed at pH 70 and 50∘C (Table 4) Interestinglythe Streptomyces flavogriseus HS1 proteases were less stableagainst the strong anionic surfactant (SDS) and retained only19 of its activity in the presence of 01 (wv) SDSHoweverHS1 protease activity was little influenced by oxidizing agentsand retained 641 and 398 of its activity after incubationfor 1 h at 25∘C in the presence of 01 and 1 sodiumperborate respectively (Table 4) The stability of the enzymein the presence of oxidizing agents is a very importantcharacteristic for its eventual use in detergent formulations

0

20

40

60

80

100

120

Ariel Newdet Dixan

Rem

aini

ng ac

tivity

()

30∘C

40∘C

50∘C

Figure 6 Stability of crude protease from Streptomyces flavogriseusHS1 in the presence of various commercial solid detergents Theenzyme at 100UmL was incubated 1 h at 30∘C 40∘C and 50∘Cin the presence of solid detergents The remaining activities weredetermined at pH 70 and 50∘C using casein as a substrate Enzymeactivity of control sample without any detergent incubated underthe similar conditions was taken as 100

Important commercial detergent proteases like SubtilisinCarlsberg Subtilisin BPN1015840 Alcalase Esparase and Saviraseare stable in the presence of various detergent componentsHowever most are unstable in the presence of oxidant agentssuch as hydrogen peroxide [36]

39 Stability of the Streptomyces flavogriseus HS1 Proteaseswith Commercial Solid Detergents The high activity andstability of the Streptomyces flavogriseus HS1 proteases in thepH range 50ndash100 and their relative stability towards sur-factants and oxidizing agents are very useful for its eventualapplication as a detergent additive To check the compatibilityof the proteases with solid detergents the crude enzyme waspreincubated in the presence of various commercial laundrysolid detergents for 1 h at different temperatures (30 40and 50∘C) (Figure 6) The data showed that the proteaseswere stable in Ariel and Dixan retaining 70 of theiractivity at 50∘CThe obtained results clearly indicated that theperformance of enzymes in detergents depends on numberof factors including the detergents compounds since theproteolytic stability ofHS1 proteases variedwith each laundrytested detergent

Singh et al [37] reported that the alkaline protease fromBacillus sp SSR1 retained 37 of its initial activity after 1 hincubation at 40∘C in the presence of Ariel at a concentrationof 5mgmL Alkaline protease from Conidiobolus coronatusretained only 16 activity in Revel 114 activity in Ariel


and 66 activity inWheel at 50∘C in the presence of 25mMCaCl2[38]

4 Conclusion

This work describes the isolation of an actinomycete strainidentified as Streptomyces flavogriseus HS1 which producesat least five proteases as described by zymogram techniqueCrude protease was shown to have optimum activity at pH 7and 50∘C The crude enzyme has a good stability toward theoxidizing agents and was found to be useful as a detergentadditive

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was funded by the Ministry of Higher Educationand Scientific Research Tunisia

References

[1] H Tsujibo K Miyamoto T Hasegawa and Y Inamori ldquoPurifi-cation and characterization of two types of alkaline serineproteases produced by an alkalophilic actinomyceterdquo Journal ofApplied Bacteriology vol 69 no 4 pp 520ndash529 1990

[2] W Peczynska-Czoch ldquoActinomycete enzymesrdquo in Actinomy-cetes in Biotechnology pp 219ndash283 Academic Press London1988

[3] M B Rao A M Tanksale M S Ghatge and V V Desh-pande ldquoMolecular and biotechnological aspects of microbialproteasesrdquoMicrobiology andMolecular Biology Reviews vol 62no 3 pp 597ndash635 1998

[4] KH Yeoman andC Edwards ldquoProtease production by Strepto-myces thermovulgaris grown on rapemeal-derived mediardquo Jour-nal of Applied Bacteriology vol 77 no 3 pp 264ndash270 1994

[5] M Pokorny L Vitale V TurkM Renko and J Zuvanic ldquoStrep-tomyces rimosus extracellular proteases-1 Characterization andevaluation of various crude preparationsrdquo European Journal ofApplied Microbiology and Biotechnology vol 8 no 1-2 pp 81ndash90 1979

[6] S Chandrasekaran and S C Dhar ldquoMultiple proteases fromStreptomyces moderatus II Physicochemical and enzymaticproperties of the extracellular proteasesrdquo Archives of Biochem-istry and Biophysics vol 257 no 2 pp 402ndash408 1987

[7] P D A James M Iqbal C Edwards and P G GMiller ldquoExtra-cellular protease activity in antibiotic-producing Streptomycesthermoviolaceusrdquo Current Microbiology vol 22 no 6 pp 377ndash382 1991

[8] T Muro T Murakami Y Tominaga T Tokuyama and SOkada ldquoPurification and some properties of protease I havingtransfer action from Streptomyces griseus var alcalophilusrdquoAgricultural and Biological Chemistry vol 55 no 2 pp 307ndash3141991

[9] V K AntonovChemistry of Proteolysis Springer NewYork NYUSA 1993

[10] K Kitadokoro H Tsuzuki E Nakamura T Sato and HTeraoka ldquoPurification characterization primary structurecrystallization and preliminary crystallographic study of aserine proteinase from Streptomyces fradiae ATCC 14544rdquoEuropean Journal of Biochemistry vol 220 no 1 pp 55ndash61 1994

[11] J Thumar and S P Singh ldquoTwo-step purification of a highlythermostable alkaline protease from salt-tolerant alkaliphilicStreptomyces clavuligerus strain Mit-1rdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 854 no 1-2 pp 198ndash203 2007

[12] A S Rana I S Rana and L Yadav ldquoAlkaline proteases fromStreptomyces viridifaceins and its application with detergentsrdquoAdvanced Biotechnology vol 10 no 4 pp 10ndash14 2010

[13] S G Dastager A Dayanand W-J Li et al ldquoProteolytic activityfrom an alkali-thermotolerant Streptomyces gulbargensis spnovrdquo Current Microbiology vol 57 no 6 pp 638ndash642 2008

[14] M K Kharel M D Shepherd S E Nybo M L Smith M ABosserman and J Rohr ldquoIsolation of Streptomyces species fromsoilrdquo Current Protocols in Microbiology no 19 Article ID 10E42010

[15] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[16] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[17] A A Kembhavi A Kulkarni and A Pant ldquoSalt-tolerant andthermostable alkaline protease fromBacillus subtilis NCIMNo64rdquo Applied Biochemistry and Biotechnology vol 38 no 1-2 pp83ndash92 1993

[18] W-J Chi J-H Song E A Oh et al ldquoMedium optimizationand application of affinity column chromatography for trypsinproduction from recombinant Streptomyces griseusrdquo Journal ofMicrobiology and Biotechnology vol 19 no 10 pp 1191ndash11962009

[19] 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

[20] W P Chen A W Anderson and Y W Han ldquoExtraction ofglucose isomerase from Streptomyces flavogriseusrdquoApplied andEnvironmental Microbiology vol 37 no 4 pp 785ndash787 1979

[21] R Srivastava S S Ali and B S Srivastava ldquoCloning ofxylanase gene of Streptomyces flavogriseus in Escherichia coliand bacteriophage 120582-induced lysis for the release of clonedenzymerdquo FEMS Microbiology Letters vol 78 no 2-3 pp 201ndash205 1991

[22] G D Gibb and W R Strohl ldquoPhysiological regulation ofprotease activity in Streptomyces peucetiusrdquo Canadian Journalof Microbiology vol 34 no 2 pp 187ndash190 1988

[23] A El-Raheem R El-Shanshoury M A El-Sayed R H Sam-mour andW A El-Shouny ldquoPurification and partial character-ization of two extracellular alkaline proteases from Streptomycescorchorusii ST36rdquo Canadian Journal of Microbiology vol 41 no1 pp 99ndash104 1995

[24] D Cowan R Daniel and HMorgan ldquoThermophilic proteasesproperties and potential applicationsrdquo Trends in Biotechnologyvol 3 no 3 pp 68ndash72 1985

[25] P Sampath C Subramanian and G Chandrakasan ldquoExtracel-lular proteases from streptomyces spp G157 purification and


characterizationrdquo Biotechnology and Applied Biochemistry vol26 no 2 pp 85ndash90 1997

[26] M Trop and Y Birk ldquoThe specificity of proteinases from Strep-tomyces griseus (pronase)rdquo Biochemical Journal vol 116 no 1pp 19ndash25 1970

[27] B K Bajaj and P Sharma ldquoAn alkali-thermotolerant extracellu-lar protease from a newly isolated Streptomyces sp DP2rdquo NewBiotechnology vol 28 no 6 pp 725ndash732 2011

[28] H Lazim H Mankai N Slama I Barkallah and F LimamldquoProduction and optimization of thermophilic alkaline proteasein solid-state fermentation by Streptomyces sp CN902rdquo Journalof Industrial Microbiology and Biotechnology vol 36 no 4 pp531ndash537 2009

[29] D Patke and S Dey ldquoProteolytic activity from a thermophilicStreptomyces megasporus strain SDP4rdquo Letters in AppliedMicrobiology vol 26 no 3 pp 171ndash174 1998

[30] W Aretz K P Koller and G Riess ldquoProteolytic enzymes fromrecombinant Streptomyces lividans TK24rdquo FEMS MicrobiologyLetters vol 65 no 1-2 pp 31ndash35 1989

[31] N S Demina and S V Lysenko ldquoExoproteases of StreptomyceslavendulaerdquoMicrobiology vol 64 no 4 pp 385ndash387 1995

[32] V Bascaran C Hardisson and A F Brana ldquoRegulation ofextracellular protease production in Streptomyces clavuligerusrdquoApplied Microbiology and Biotechnology vol 34 no 2 pp 208ndash213 1990

[33] L A I de Azeredo M B de Lima R R R Coelho and D MG Freire ldquoA low-cost fermentation medium for thermophilicprotease production by Streptomyces sp 594 using feather mealand corn steep liquorrdquo Current Microbiology vol 53 no 4 pp335ndash339 2006

[34] M J North ldquoComparative biochemistry of the proteinases ofeucaryotic microorganismsrdquo Microbiological Reviews vol 46no 3 pp 308ndash340 1982

[35] 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

[36] R Gupta K Gupta R K Saxena and S Khan ldquoBleach-stablealkaline protease from Bacillus sprdquo Biotechnology Letters vol 21no 2 pp 135ndash138 1999

[37] J SinghN Batra andR C Sobti ldquoSerine alkaline protease froma newly isolated Bacillus sp SSR1rdquo Process Biochemistry vol 36no 8-9 pp 781ndash785 2001

[38] S H Bhosale M B Rao V V Deshpande and M C Srini-vasan ldquoThermostability of high-activity alkaline protease fromConidiobolus coronatus (NCL 86820)rdquo Enzyme andMicrobialTechnology vol 17 no 2 pp 136ndash139 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

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Stem CellsInternational



Enzyme Research



Microbiology


dilution plate technique on ISP4 agar media containing(gL) starch 10 casein 03 KNO

32 NaCl 2 K

2HPO42

MgSO4sdot7H2O 005 CaCO

3002 FeSO

4sdot7H2O 001 and 15

agar [14]

22 Identification of HS1 Strain The 16S rRNA gene ofthe HS1 strain was amplified by PCR using the follow-ing primers F (forward) 51015840-CCGAATTCGTCGACAACA-GAGTTTGATCCTGGCTCAG-31015840 and R (reverse) 51015840-CCC-GGGATCCAAGCTTAAGGAGGTGATCCAGCC-31015840 ThePCR mixture contained 30 pmol of primers 20 pmol of eachdeoxynucleoside triphosphate polymerisation buffer and5U Taq polymerase The PCR program involved 35 cycles ofdenaturing at 94∘C for 1min primer annealing at 55∘C for1min and extension at 72∘C for 90 s The sequencing wasperformed three times using the DNA sequencer ABI PRISM31003100-Avant Genetic Analyser (CA USA) 16S rDNAsequence was searched for similarities to known sequencesin the GenBank database (National Center for BiotechnologyInformation National Library ofMedicine) using the BLASTsearch program The sequence was aligned with those of thereference strains usingClustalW [15] A phylogenetic treewasconstructed by the neighbour-joining method [16]

23 Determination of Protease Activity Measuring of theprotease activity was done as described by Kembhavi et al[17] using casein as substrate 1 (wv) in 100mM Tris-HClbuffer pH 70 The mixture was incubated for 15min at 50∘Cand the reactionwas stopped by addition of 05mL 20 (wv)TCA (trichloroacetic acid) The mixture was left at roomtemperature for 10 minutes and then centrifuged at 10000 gfor 15 minutes to remove the precipitate The absorbance ofthe soluble TCA peptides was recorded at 280 nm One unitof protease activity was defined as the amount of enzymerequired to liberate 1120583g of tyrosine per minute under theexperimental conditions used All measurements were car-ried out in triplicate

24 Protease Production The proteolytic isolates were cul-tured in three different liquid medium FermII media(gL) dextrin 20 tryptone 10 KH

2PO410 K

2HPO434

MgSO4sdot7H2O03 FeSO

4sdot7H2O001 ZnCl

201 CuSO

4sdot7H2O

001 MgCl2sdot4H2O 0003 CaCl

2001 NaCl 003 pH 70 [18]

gelatin containing media (gL) gelatin 10 peptone 5 yeastextract 5 NaCl 50 and pH 9 and liquid ISP4 media [14]

A 1mL spore suspension (104 to 106 sporesmL) wasadded to a 250mL Erlenmeyer flask containing 100mL ofliquid media [14] and the flasks were incubated at 30∘C and150 rpm for 96 h The culture medium was centrifuged at5000 rpm to remove mycelia and medium debris and thesupernatant was used as a crude enzyme preparation

25 Characterization of Proteases Zymography was per-formed on NativePAGE according to the method of Garcia-Carreno et al [19] After electrophoresis the gel was sub-merged in 1 (wv) casein in 100mM glycine-NaOH bufferpH 70 and incubated at 50∘C for 20min After washingthe gel was stained with Coomassie Brilliant Blue R-250 anddestained with 5 ethanol-75 acetic acid A clear zone

appeared on the blue background of the gel which indicatedthe presence of protease activity

26 Effect of Temperature and pH on Protease Activity andStability To investigate the temperature effect the proteaseassay was performed at different temperatures between 20and 80∘C using casein as substrate for 10min at pH 70For thermal stability the enzyme was incubated at differenttemperatures for 60min Aliquots were withdrawn at thedesigned time intervals to test the remaining activity Theresidual activity was assayed at pH 70 and 50∘C for 10minThe nonheated enzyme was considered as control (100activity)

Protease activity was assayed over the pH range 50ndash120at 50∘C for 10min using casein as a substrateThe effect of pHon enzyme stability was evaluated by measuring the residualenzyme activity after incubation at various pH for 60minat 25∘C The following buffer systems were used 100mMglycine-HCl pH 40 and 50 100mM sodium acetate pH60 100mM phosphate-buffer pH 70 100mM Tris-HCl pH80 100mM glycine-NaOH pH 90 and 10 and 100mMNa2HPO4sdotNaOH pH 120

27 Effect of Metal Ions and Other Chemicals on ProteaseActivity and Stability The effects of different monovalent(Na+ or K+) or divalent (Fe2+ Mn2+ Zn2+ Cu2+ Ba2+ Mg2+or Hg2+) metal ions at a concentration of 5mM on proteaseactivity were investigated by adding them to the reactionmixtureThe activity in the absence of any additiveswas takenas 100

The effects of enzyme inhibitors on protease activity werestudied using PMSF and EDTA The crude enzyme waspreincubated with each inhibitor for 30min at 25∘C and thenthe remaining enzyme activity was estimated using caseinas a substrate The activity in the absence of inhibitors wastaken as 100 The effects of some surfactants (Triton X-100Tween 80 and SDS) and oxidizing agents (sodium perborate)on enzyme stability were studied by preincubating the crudeenzyme for 1 h at 25∘C The residual activity was measuredat pH 70 and 50∘C The activity of the enzyme without anyadditive was taken as 100

28 Detergent Compatibility The compatibility of the HS1extracellular proteases with commercial laundry detergentswas studied using Ariel (Procter and Gamble Switzerland)Newdet (Sodet Tunisia) and Dixan (Henkel Spain) as soliddetergents The endogenous proteases contained in thesedetergents were inactivated by heating the diluted detergentsfor 1 h at 65∘Cprior to the addition of the enzymepreparationThe extracellular proteases of Streptomyces flavogriseus HS1were incubated with different diluted detergents (1100) for1 h at 30∘C 40∘C and 50∘C and then the remaining activitieswere determined under the standard assay conditions Theenzyme activity of a control without detergent incubatedunder similar conditions was taken as 100

29 Statistical Analysis Statistical analyses were performedwith Statgraphics ver 51 professional edition (Manugistics
























0001







120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

5 6 7 8 9 10 11

pH

(a)

100

80

60

40

20

0

Rem

aini

ng ac

tivity

()

6 7 8 9 10 11

pH

(b)


120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

30 40 50 60 70 80 90

Temperature (∘C)

(a)

0

20

40

60

80

100

0 15 30 45 60

Rem

aini

ng ac

tivity

()

Time (min)

40∘C

50∘C

60∘C

(b)













Triton X-100 1 6145 405

Tween 80 1 7145 405

Tween 20 1 7025 378

SDS 01 18905 0


H2O201 61405 587



0

20

40

60

80

100

120

Ariel Newdet Dixan

Rem

aini

ng ac

tivity

()

30∘C

40∘C

50∘C







4 Conclusion




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























0001







120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

5 6 7 8 9 10 11

pH

(a)

100

80

60

40

20

0

Rem

aini

ng ac

tivity

()

6 7 8 9 10 11

pH

(b)


120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

30 40 50 60 70 80 90

Temperature (∘C)

(a)

0

20

40

60

80

100

0 15 30 45 60

Rem

aini

ng ac

tivity

()

Time (min)

40∘C

50∘C

60∘C

(b)













Triton X-100 1 6145 405

Tween 80 1 7145 405

Tween 20 1 7025 378

SDS 01 18905 0


H2O201 61405 587



0

20

40

60

80

100

120

Ariel Newdet Dixan

Rem

aini

ng ac

tivity

()

30∘C

40∘C

50∘C







4 Conclusion




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

5 6 7 8 9 10 11

pH

(a)

100

80

60

40

20

0

Rem

aini

ng ac

tivity

()

6 7 8 9 10 11

pH

(b)


120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

30 40 50 60 70 80 90

Temperature (∘C)

(a)

0

20

40

60

80

100

0 15 30 45 60

Rem

aini

ng ac

tivity

()

Time (min)

40∘C

50∘C

60∘C

(b)













Triton X-100 1 6145 405

Tween 80 1 7145 405

Tween 20 1 7025 378

SDS 01 18905 0


H2O201 61405 587



0

20

40

60

80

100

120

Ariel Newdet Dixan

Rem

aini

ng ac

tivity

()

30∘C

40∘C

50∘C







4 Conclusion




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Triton X-100 1 6145 405

Tween 80 1 7145 405

Tween 20 1 7025 378

SDS 01 18905 0


H2O201 61405 587



0

20

40

60

80

100

120

Ariel Newdet Dixan

Rem

aini

ng ac

tivity

()

30∘C

40∘C

50∘C







4 Conclusion




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion




Acknowledgment


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 Streptomyces flavogriseus HS1: Isolation ...

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Transcript of Research Article Streptomyces flavogriseus HS1: Isolation ...