APPENDIX A RESEARCH PAPERS PUBLISHED

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International Journal of Poultry Science 9 (5): 482-489, 2010ISSN 1682-8356© Asian Network for Scientific Information, 2010

Corresponding Author: Neeraj Wadhwa, Department of Biotechnology, Jaypee Institute of Information Technology University, A-10 sec62, Noida, Uttar Pradesh, India

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Degradation of Chicken Feather a Poultry Waste Product by Keratinolytic BacteriaIsolated from Dumping Site at Ghazipur Poultry Processing Plant

Sarita Agrahari and Neeraj WadhwaDepartment of Biotechnology, Jaypee Institute of Information Technology University,

A-10 Sec 62, Noida, Uttar Pradesh, India

Abstract: Feathers are byproduct waste of poultry processing plant and produced in large amount. A smallpercentage of feather waste is steamed, chemically treated, ground, to form feather meal a dietary proteinsupplement for animals. Alternatively, keratin can be biodegraded by some Keratinolytic bacteria and in thisstudy Keratinase producing bacteria and their Keratinolytic enzyme production was investigated. Soil samplewas collected from Ghazipur poultry waste site, Ghaziabad, India, a feather dumping site. Soil sample wereinoculated in three enrichment media and colonies producing clear zone in feather meal agar were selectedand identified as B. megaterium SN1, B. thuringenesis SN2, B. Pumilis SN3 were able to degrade chickenand pigeon feathers. They produced extracellularly Keratinolytic enzymes in enrichment media with 10%Feather meal powder. We report that Keratinase and Protease activity were detected in the culturesupernatant and optimal medium for extracellular production of Keratinase and Protease is feather mealmedia 2 at pH (7.5) and temperature (30 C). There was complete degradation of feathers in 120 h ofo

incubation and 0-80% Ammonium sulphate fraction showed 1.5 fold purification for Keratinase and 1.3 foldpurification for Protease over the crude enzyme preparation. The keratinous waste can be biologicallydegraded by enzymes or the microbe itself to form useful products.

Key words: Bacillus, feather, keratinolytic enzymes

INTRODUCTIONWorldwide 24 billion chickens are killed annually andaround 8.5 billion tonnes of poultry feather are produced.According to a recent report in leading news paperIndia's contribution alone is 350 million tonnes. Thepoultry feathers are dumped, used for land filling,incinerated or buried, which involves problems instorage, handling, emissions control and ash disposal.Discarded feather also causes various human ailmentsincluding chlorosis, mycoplasmosis and fowl cholera(Williams et al., 1991).Feather is pure keratin protein and is insoluble and hardto degrade due to highly rigid structure rendered byextensive disulphide bond and cross-linkages. Thekeratin chain is insoluble, high stable structure tightlypacked in the "-helix ("-Keratin) and $-sheets ($-keratin)into super coiled polypeptide chain (Parry and North,1998). 90% of the feather contain $-keratin by mass(Onifade et al., 1998) and $-keratin are extensively crosslinked. Cross-linking of protein chains by cysteinebridges confers high mechanical stability and resistanceto proteolytic degradation by pepsin, trypsin and papain.The disulphide bonds of $-keratin can be reduced by theenzyme disulphide reductase (Yamamura et al., 2002)followed by proteolyitc keratinases (Gupta and Ramnani,2006). Feather can be utilized so that it can be used asanimal feed, this can prevent accumulation of feather in

the environment and decrease the development ofpathogenic strains. Biotechnological processing offeathers for the production of feather meal, instead ofchemical processing is preferred as it preserves theessential amino acids (Methionine, Lysine, Histidine)(Riffel et al., 2003).Innovative solution for waste disposal along withbiotechnological alternative for recycling of such wastesis of utmost importance. Structural keratin can bedegraded by some proteolytic micro-organisms asreported by (Onifade et al., 1998). Keratinase arespecific protease that degrade keratin specifically. It isproduced by Saprophytic and Dermatophytic Fungi andsome Bacillus species. Feather degrading bacteria arephysiologically diverse and approximately 99% ofBacterial species are unculturable because of theirability to enter non culturable state or because no culturemethods have been established (Amann et al., 1995). Anumber of keratinolytic microorganisms have beenreported, including some species of fungi such asMicrosporum (Essien et al., 2009), Trichophyton (Anbuet al., 2008) and from the bacteria Bacillus (Cai andZheng, 2009; Macedo et al., 2005; Pillai and Archana,2008) and Streptomyces (Syed et al., 2009; Szabo et al.,2000; Tatineni et al., 2008) and actinomycetes (Bockle etal., 1995; Young and Smith, 1975). Increase inkeratinolytic activity is also found to be associated with

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thermophilic organisms, which require high energy The culture broth in which feather degradation wasinputs to achieve maximum growth and thedecomposition of keratin wastes (Friedrich andAntranikian, 2002).Till date most of purified keratinizes known cannotcompletely solubilize native keratin (Ignatova et al., 1999;Ramnani et al., 2005), their exact nature anduniqueness for keratinolysis is still not clear There isalways a requirement of isolation of enzymes from newsources to meet the industrial and environmentaldemand.Keratinolytic enzymes have found important utilities inbiotechnological processes involving keratin-containingwastes from poultry and leather industries, through thedevelopment of non-polluting processes. Afterhydrolysis, the feathers can be converted to feedstuffs,fertilizers, glues, films and as the source of rare aminoacids, such as serine, cysteine and proline (Gupta andRamnani, 2006; Cai and Zheng, 2008; Cao et al., 2009).In this study we report the isolation of three mesophilicbacteria that produce Keratinolytic enzymes. which canefficiently degrade chicken and pigeon feather within 120hrs of incubation. Earlier studies from our lab involvingscreening of micro-organism from same soil sample ofdumping site of Gazipur poultry processing plant, wehave reported isolation of Pseudomonas thermaerumGW1, GenBank accession GU95151, this bacteriashowed proteolytic activity but not keratinolytic activity(Gaur et al., 2010).

MATERIALS AND METHODSIsolation and screening of keratinase producingbacteria: Soil was collected from a regular featherdumping site of Ghazipur poultry processing plant,Ghaziabad, India in sterilized sampling bags. Thesamples were brought in winter to the laboratory andprocessed for analysis on the same day. Soil sampleswere suspended in Peptone broth and kept for growth at30 C for 3 days. This suspension was reinoculated ino

three media, Horikoshi media, Feather meal media 1and Feather meal media 2 at 30 C at 160 rpm for 7 days.o

They were used for keratinase production. Theycontained the following constituent:I Horikoshi media (g/l): Soluble starch, 5; Peptone, 5;

Glucose, 5; K2HPO4, 1; MgSO4 7H2O, 0.2; Na2CO3, 1;Yeast extract, 5 and Feathers, 10; pH 7.5.

II The Feather meal media 1 (g/l): NaCl, 0.5; K2HPO4,0.3; KH2PO4, 0.4 and Feather, 10; pH 7.5.

III The Feather meal media 2 (g/l): NH4Cl, 0.5; NaCl,0.5; K2HPO4, 0.3; KH2PO4, 0.4; MgCl2.6H2O, 0.1;Yeast extract, 0.1 and Feather, 10; pH 7.5.

The flask was incubated at temperature of 30 C on ao

rotary shaker at 160 rpm for 7 days. Feather degradationin culture broth was confirmed visually.

confirmed was screened for keratinolytic activity. Feathermeal agar that composed of (g/l): NH4Cl, 0.5; NaCl, 0.5;K2HPO4, 0.3; KH2PO4, 0.4; MgCl2.6H2O, 0.1; Yeast extract,0.1 and Feather meal powder, 10, agar powder, 20 andpH was maintained at 7.5 at 30 C for 72 h, Strain whicho

exhibited the largest clearing zones were selected,identified and grown in cultivation media for enzymeproduction.

Feather meal powder preparation: Poultry feathers waswashed extensively, boiled at 30-40 psi for 2-3h. Driedin hot air oven for 4 h at 50 C. The dried feathers wereo

pulverized and the powder was used as feather meal.

Morphological studies of isolated bacterial strains:Bacterial identification was conducted on morphological,physiological and biochemical tests. Results werecompared with Bergey’s Manual of DeterminativeBacteriology, 8 edition (Buchanan and Gibbons, 1974).th

Genus Bacillus: Agriculture Handbook No. 427 (Gordonet al., 1973). The strain were also identified bychromogenic method on the bacillus differential agarfrom Himedia, India, M1651 recommended for rapididentification of Bacillus species from a mixed culture.The medium contains peptic digest of animal tissuesand meat extract, which provide nitrogenouscompounds. Mannitol serves as the fermentablecarbohydrate, fermentation of which can be detected bythe pH indicator phenol red. Mannitol fermentingorganisms like B. megateruim yield yellow coloredcolonies, B. thuringiensis will grow as blue colonies andB. pumilis will also grow as green colonies on thismedium. Results are summarized in Table 1. Growthdetermination of bacteria was done taking absorbanceat 600 nm of bacterial growth media (Fig. 4) at regularintervals.

Growth condition for Protease and Keratinaseproduction: Seed culture of the three isolated strainswere prepared in 500 ml Erlenmeyer conical flaskcontaining 100 ml of the culture media that wasmaintained at 30 C at 160 rpm and washed feather 10%o

in cultivation media. After five days of incubation, thecrude culture broth was centrifuged (10,000g, 4 C, 30o

min) and cell free supernatant was subjected to 0-80%ammonium sulphate precipitation. After chilling at 4 Co

for 1 h, the resulting precipitate was collected bycentrifugation (10,000 g, 4 C, 30 min) and dissolved ino

a minimal volume of Tris-Cl buffer 10 mM (pH 8.0) anddialyzed overnight against 4 liters Tris-Cl buffer 10 mm(pH 8.0). The dialysed protein fraction was checked forprotease and keratinase activity by the modified methodof Tsuchida et al. (1986) and Cheng et al. (1995)respectively. Standard strain of Bacillus. licheniformis(MTCC 1483) was also studied for comparativepurposes.

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Table 1a: Results of morphological, physiological, cultural, biochemical characteristic of three isolated bacterial strain SN1, SN2, SN3were coducted. Collectively these characteristics indicated that the isolates were of genus Bacillus

Observations--------------------------------------------------------------------------------------------------------------------------

Details of experiment B. megaterium SN1 B. thuringenesis SN2 B. Pumilis SN3Shape of Bacteria Rod Short rod RodEndospore formation + + +Motility Motile Highly motile MotileGram character + + +Anaerobic growth _ _ _Colony characteristicsGrowth Rapid Rapid RapidShape Circular Irregular CircularSurface Smooth shiny Smooth Smooth shinyMargin Entire Entire EntireColor Cream White CreamElevation Convex Flat ConvexConsistency Buttery Viscous ButteryOpacity Opaque Opaque OpaqueBiochemical characteristicsGlucose -/- A/- -/-Lactose -/- A/- -/-Mannitol -/- A/- -/-Indole production _ _ _Methyl red reaction _ + +Voges-proskaure reaction _ _ _Citrate utilization _ _ _Catalase + + +Gelatinase _ + +Caesinase + + +Amylase _ _ _Cellulase _ _ _Deaminase _ _ _Symbol: +: Positive; -: Negative; A/- :Acid/No gas; -/- :No acid/No gas

Table 1b: Table depicts that growth on Triple sugar iron (TSI) agar. B. megaterium SN1, B. Pumilis SN3 showed alkaline reaction onslant and acidic reaction on butt. B. thuringenesis SN2 showed acidic reaction both on slant and butt

Hydrogen sulphideBacterial Isolates Color and reaction of slant Color and reaction of butt Gas production productionB. megaterium SN1 Red, Alkaline Yellow, Acidic _ _B. thuringenesis SN2 Yellow, Acidic Yellow, Acidic _ _B. Pumilis SN3 Red, Alkaline Yellow, Acidic _ _

Protein concentration: Protein concentration was fold diluted Folin-Ciocalteau reagent was measured atdetermined by the method of Bradford (1976) with bovine 660 nm. All assays were done in triplicate. serum albumin as a standard.

Hydrolysis of protein substrates: Protease activity with activity was assayed by the modified method of Chengvarious protein substrates including keratin, casein, et al. (1995) by using keratin as a substrate. Thegelatin and bovine serum albumin (2 mg/ml) was reaction mixture contained 200 µl of enzyme preparationassayed by mixing 100 µl of the enzyme and 900 µl of and 800 µl of 20 µg/ml keratin in 10 mm Tris buffer, pHassay buffer containing the protein substrates. After 8. The reaction mixture was incubated at 45 C for 20 minincubation at 50 C for 20 min, Reaction was terminated and the reaction was terminated by additing 1 ml of 10%o

by the addition of an equal volume of 10% chilled chilled trichloroacetic acid. The mixture was centrifugedtrichloroacetic acid then the reaction mixture was at 10,000 g for 5 min and the absorbance of theallowed to stand in ice for 15 min to precipitate the supernatant fluid was determined at 440 nm. All assaysinsoluble proteins. The undigested proteins were were done in triplicate. One Unit (U) of enzyme activityremoved by filtration or centrifugation at 10,000 rpm for was the amount of enzyme that caused a change of5 min and amino acid released was assayed. The absorbance of 0.01 at 440 nm in 20 min at 45 C.supernatant was separated by centrifugation at 10,000rpm for 10 min at 4 C; the acid soluble product in theo

supernatant was neutralized with 5 ml of 0.5 M Na2CO3

solution. The color developed after adding 0.5 ml of 3

Determination of keratinase activity: The keratinase

o

o

Determination of protease activity: Protease activitywas assayed by a modified method of Tsuchida et al.(1986) by using casein as substrate. 100 µl of enzyme

A B

SN1

SN2

SN3

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solution was added to 900 µl of substrate solution (2mg/ml casein in 10 mM Tris-Cl buffer, pH 8.0). Themixture was incubated at 50 C for 20 min. Reaction waso

terminated by the addition of an equal volume of 10%chilled trichloroacetic acid then the reaction mixture wasallowed to stand in ice for 15 min to precipitate theinsoluble proteins. The supernatant was separated bycentrifugation at 10,000 rpm for 10 min at 4 C, the acido

soluble product in the supernatant was neutralized with5 ml of 0.5 M Na2CO3 solution. The color developed afteradding 0.5 ml of 3 fold diluted Folin-Ciocalteau reagentwas measured at 660 nm. All assays were done intriplicate. One protease unit is defined as the amount ofenzyme that releases 1 µmol of tyrosine per ml perminute under the above assay conditions. The specificactivity is expressed in the units of enzyme activity permilligram of protein. Fig. 1: Degradation of chicken feathers by the bacterial

RESULTSIsolation, characterization of keratinolytic strains: Itwas found that the enriched feather degrading culturecontained micro-organism exhibited keratinolytic activityThe feathers were fully solubilized within 120 h ofincubation with the microbes from selected soil (Fig. 1).Three bacterial strains that visually degraded featherwere isolated allowed to grow on medium containingfeather meal powder as sole carbon and nitrogensource. The strain SN1, SN2, SN3 were selected as theyproduced clear zones on incubation at 30 C for 72 ho

suggesting the presence of keratinolytic activity (Fig. 2).The identification of the keratinolytic bacteria was basedon cell morphology, colony morphology, and severalbiochemical tests (Table 1a). Isolates SN1, SN2, SN3were determined to be Gram-positive, sporulating,motile bacilli. The isolate SN1, SN2 formed yellowcolored colonies and SN3 showed white colored colonyon feather meal agar plate. These results suggestedthat these three strains belong to genus Bacillus. On thebasis of morphological characteristic and culturalcharacteristic on Hicrome Bacillus agar and wasidentified (Table 1b). They (SN1, SN2 and SN3) werefurther identified to be as sample B. megaterium, B.thuringenesis, B. Pumilis respectively (Fig. 3). Thesestrains degraded the chicken feathers and pigeonfeathers (figure not shown) completely. Their culturalcharacteristic in the differential media is given in Table2 with B. megaterium SN1 showing yellowish green,irregular colonies, B. thuringenesis SN2 showed bluecircular colonies, B. Pumilis SN3 showed green, flat,circular shiny colonies.

Factors affecting growth and enzyme production:Bacterial growth and enzyme production of keratinaseand protease by the microbes of soil was monitoredduring growth in Horikoshi media, Feather meal

strain isolated from soil of Ghazipur poultrydumping site, India, in submerged cultivation at30 C. (A) Feather control without the bacterialo

strain, (B) feather after 120 hrs of incubation withthe bacterial strain showed completedegradation

Fig. 2: Production of clear zones in feather mealpowder (10g/l) agar plates by keratinolyticbacteria. Three strains were identified BacillusSN1, Bacillus SN2 and Bacillus SN3 thatproduced clear zone. Microbial culture wasspreaded on feather meal agar plate andincubated at 30 C for 72 ho

media 1. Growth determination of three bacteria wasdone taking absorbance at 600 nm of bacterial growthmedia at regular intervals from 0-144 h (Fig. 4). Logphase of growth is from 24 h till 48 h for all the strainsand in stationary phase till 120 h. Hydrolysis of proteinsubstrates (Keratin, Casein, Gelatin, Bovine Serum

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Table 2: Cultural characteristics on Hicrome Bacillus agar. Morphological characteristic and cultural characteristic of the three isolateson Hicrome Bacillus agar Himedia M1651. Strains SN1, SN2, SN3 were classified as B. megaterium, B. thuringenesis, B.Pumilis respectively

Colony characteristics B. megaterium SN1 B. thuringenesis SN2 B. Pumilis SN3Growth Luxuriant Luxuriant LuxuriantShape Irregular, large Circular, Small Circular, SmallSurface Smooth Smooth Smooth shinyMargin Irregular Entire EntireColor Yellowish green Blue GreenElevation Convex Flat FlatOpacity Opaque Opaque Opaque

Fig. 3: Growth of bacterial strain SN1, SN2, SN3 onchromogenic differentiation agar Hicromebacillus agar from Himedia M1651. Yellowishgreen colonies represent B. megaterium SN1;Blue colonies represent B. thuringenesis SN2;Green colored colonies represent B. PumilisSN3

Fig. 4: Growth determination of bacterial strains B.megaterium SN1, B. thuringenesis SN2, B.Pumilis SN3, Bacillus licheniformis 1483(Standard strain collected from MTCC was usedfor comparative study) were done takingabsorbance at 600nm of bacterial growth media.It is observed that Log phase is from 24 h till 48h and stationary phase is till 120 h for all the 3strains

Albumin 2 mg/ml) by cell free supernatant collected after120 h of bacterial growth {strains identified were B.megaterium SN1, B. thuringenesis SN2, B. Pumilis SN3,Bacillus licheniformis 1483 (Standard strain collected

Fig. 5: Hydrolysis of protein substrates (Keratin, Casein,Gelatin, Bovine Serum Albumin 2mg/ml) by cellfree supernatant collected after 120 h of bacterialgrowth {strains identified were B. megateriumSN1, B. thuringenesis SN2, B. Pumilis SN3,Bacillus licheniformis 1483 (Standard straincollected from MTCC was used for comparativestudy)}. All strains preferred Keratin and caseinas preferred substrate Gelatin was leastpreferred source as substrate. Maximumutilization of keratin and casein was by strainSN2 as seen by the absorbance

from MTCC was used for comparative study)}. All strainspreferred Keratin and casein as substrate Gelatin wasleast preferred source as substrate. Maximum utilizationof keratin and casein was by strain SN2 as seen by theabsorbance (Fig. 5).The maximum protease activity was seen at 120 h (5th

day) of incubation in feather meal media 2 (Fig. 6) andthe maximum keratinase activity was seen on the 144 h(6 day) of incubation in feather meal media 2 (Fig. 7).th

Relatively the bacterial culture could produce 2.4 timesmore protease in feather meal media 2 and 2.53 timesmore keratinase in feather meal media 2 than inpeptone broth (Table 3 and 4). Comparison was madestandard strain of Bacillus. licheniformis (MTCC 1483).

DISCUSSIONAnimal feed typically includes a carbohydrate source anda protein source. Common protein sources used in

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Table 3: Purification table that compares the yield, Specific Activity of protease from micro-organisms grown in Peptone Broth and theenriched media (Hori koshi, Feather meal media 1 and Feather meal media 2) Enzyme assay was determined as mentionedin Material and Methods. The Standard strain B licheniformis (Crude and 0-80% Ammonium sulphate) was kept as control

Total activity Specific activity Purification Purification step Protein (mg) (U ) (U/mg) Yield (%) (Fold)a

Peptone broth 138.75 1095250 7893.69 100 1Horikoshi media (0-80% A.S.) 217.5 1194075 5503 1.090 0.697Feather meal media 1 (0-80% A.S.) 63 51770 1297 0.0746 0.1644Feather meal media 2 (0-80% A.S.) 279.5 2850750 10199 2.6028 1.292Crude (B.lic) 137.95 782177.5 5670.05 100 1B.lic (0-80% A.S.) 176.25 509362 2890.81 0.6512 0.5098

Table 4: Purification table that compares the yield, Specific Activity of keratinase from micro-organisms grown in Peptone Broth andthe enriched media (( Hori koshi, Feather meal media1 and Feather meal media 2 ) The Standard strain B licheniformis ( Crudeand 0-80% Ammonium sulphate ) was kept as control Enzyme assay was determined as mentioned in Material and Methods

Total activity Specific activity PurificationPurification step Protein (mg) (U ) (U/mg) Yield (%) (Fold)a

Peptone broth 138.75 11,500 82.88 100 1Horikoshi media (0-80% A.S.) 217.5 23,900 110.138 207.82 1.328Feather meal media 1 (0-80% A.S.) 63 4,650 73.80 40.80 0.890Feather meal media 2 (0-80% A.S.) 279.5 33,900 121.28 294.78 1.463Crude (B.lic ) 137.95 9,050 65.61 100 1B.lic (0-80% A.S.) 176.25 18,500 104.96 204.41 1.596

Fig. 6: Protease activity produced by feather degradingbacterial strains of feather dumping site, activitywas measured by a modified method of Tsuchidaet al. (1986) by using casein as substrate. Oneprotease unit is defined as the amount of enzymethat releases 1 µmol of tyrosine per ml perminute under the above assay conditions. Thespecific activity is expressed in the units ofenzyme activity per milligram of protein. Indifferent culture media Horikoshi media, Feathermeal media 1, Feather meal media 2. Maximumprotease units 54 U/ml were seen in feather mealmedia 2 (Media III in graph)

animal feed include soy meal; fish meal; blood meal;meat or poultry by-products and meat and poultry meal.These protein sources are generally expensive Featherwaste too is high in protein and very inexpensive, butcannot be used directly in animal feed, as it is difficult foranimals to digest. Typical treatments to f orm feather

Fig. 7: Keratinase activity produced by feather degradingbacterial strains dumping site, activity wasmeasured by a modified method of Cheng et al.(1995) by using keratin as a substrate. One unit(U) of enzyme activity was the amount of enzymethat caused a change of absorbance of 0.01 at440 nm in 20 min at 45 C. In different cultureo

media Horikoshi media, Feather meal media 1,Feather meal media 2. Maximum activity 1.2 U/mlwas produced in feather meal media 2 (Media IIIin graph)

meal are expensive. These treatments also tend todestroy some amino acids, which are heat-sensitiveamino acids. This lowers the quality of the protein in thefeed. Due to these problems, feather meal is notextensively used in feed, despite the expense of othersources of dietary protein. It is reported that keratinolyticbacteria can degrade feathers.

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We attempted to isolate feather degrading bacteria from definitely find biotechnological use in various industrialsoil samples collected from regular dumping site for processes involving keratin hydrolysis. It would alsopoultry waste. From these soil samples, one soil solve the waste disposal problem of poultry waste andsample showed ability to degrade chicken feathers. with limited resources recycling of Keratinacious wasteFeather was completely degraded in Feather meal would be beneficial financially and environmentally. media 2 at 30 C within 5 days of incubation. This mediao

showed the presence of keratinase and protease activityinto the cell culture supernatant. Other samples were notdegraded feather showed no enzyme activity and theywere discarded. Micro-organisms of the selected soilsample was further maintained in enrichment mediasHorikoshi media, Feather meal media 1, Feather mealmedia 2. Growth of bacteria, keratinase and proteaseproduction was monitored at regular intervals. Ourresults depict that optimal medium for protease andkeratinase enzyme production is feather meal media 2.The Keratinolytic activity of crude enzyme was confirmedbecause the activity of enzyme on he feather was visiblydegraded with the formation of soluble feather productand reduction in weight (Moreira et al., 2007). It is alsoknown that most microbial Keratinase are inducible andit is substrate specific (Cheng et al., 1995) and variouskeratinous material chicken feather, feather meal, wool,bovine hair, humans foot skin are used as inducer forKeratinase. (Kumar et al., 2008; DeToni et al., 2002;Ignatova et al., 1999) keratinase has been purified usingvarious strategies and molecular weight determined(Fuhong et al., 2010). Lower level of degradation ofbuffalo horn, goat hair, duck feathers is reported bySingh (1997). It is also reported that Keratinolytic activityof micro-organism is associated with the production ofserine protease, metalloprotease (Gupta and Ramnani,2006) with the exception of yeast which produce asparticproteases (Monod et al., 2002).Ammonium sulphate fraction of the crude cell freesupernatant which was seeded with the soil inoculumshowed 1.5 fold purification for Keratinase and 1.3 foldpurification. for Proteases Bacteria were isolated from apoultry processing plant, that showed keratinolytic activityand ability to degrade chicken and pigeon feathers.Preliminary identification tests indicate that bacterialstrains B. megaterium SN1, B.thuringenesis SN2, B.Pumilis SN3.

Conclusion: Through the strategy of isolation ofKeratinolytic microorganisms utilized in this work,bacteria presenting high Keratinolytic activity wereselected. Considering that feather protein has beenshowed to be an excellent source of metabolizableprotein (Klemersrud et al., 1998) and that microbialKeratinases enhance the digestibility of feather keratin(Lee et al., 1991), these Keratinolytic strains could beused to produce animal feed protein. In addition, theselected isolates were able to grow and displayKeratinolytic activity in keratin wastes (feathers).Utilization of these potential keratin degraders will

ACKNOWLEDGEMENTWe are thankful to Jaypee Institute of InformationTechnology University, Noida, India for providing theinfrastructure facilities for this study.

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Monod, M., S. Capoccia, B. Lechenne, C. Zaugg, M.Holdom and O. Jousson, 2002. Secreted proteasefrom pathogenic fungi. Int. J. Med. Microbiol., 292:405-419.

Moreira, F.G., C.G.M. Souza, M.A.F. Costa, S. Reis andR.M. Peralta, 2007. Degradation of keratinousmaterials by the plant pathogenic fungusMyrothecium verrucaria. Mycopathologia, 163: 153-160.

Choe, J.K. Hwang, M.T. Suhartono and Y.R. Pyun,2002. Native feather degradation by Fervido-bacterium islandicum AW-1 a new isolatedkeratinaseproducing thermophilic anaerobe. Arch.Microbiol., 178: 538-547.

Onifade, A.A., N.A. Al-Sane, A.A. Al-Musallan and S. Al-Zarban, 1998. Potentials for biotechnologicalapplications of keratin-degrading microorganismsand their enzymes for nutritional improvement offeathers and other keratins as livestock feedresources. Bioresour. Technol., 66: 1-11.

Parry, D.A.T. and A.C.T. North, 1998. Hard-keratinintermediate filament chains: substructure of theNand C-terminal domains and the predictedstructure and function of the C-terminal domains oftype and type II chains. J. Struct. Biol., 122: 67-75.

Pillai, P. and G. Archana, 2008. Hide depilation andfeather disintegration studies with keratinolyticserine protease from a novel Bacillus subtilisisolate. Appl. Microbiol. Biotechnol., 78: 643-650.

Ramnani, P., R. Singh and R. Gupta, 2005. Keratinolyticpotential of Bacillus licheniformis RG1: Structuraland biochemical mechenism of featherdegradation. Can. J. Microbiol., 51: 191-196.

Riffel, A., A. Brandelli, P. Heeb and F. Lucas, 2003.Characterization of a new keratinolytic bacteriumthat completely degrades native feather keratin.Arch. Microbial., 179: 258-265.

Singh, C.J., 1997. Characterization of an extracellualarkeratinase of Trichophyton simii and its role inkeratin degradation. Mycopathologia, 137: 13-16.

Syed, D.G., J.C. Lee, W.J. Li, C.J. Kim and D. Agasar,2009. Production, characterization and applicationof keratinase from Streptomyces gulbargensis,Bioresour. Technol., 100: 1868-1871.

Szabo, I., A. Benedek, I.M. Szabo and G. Barabas, 2000.Feather degradation with a thermotolerantStreptomyces graminofaciens strain. World J.Microbiol. Biotechnol., 16: 253-255.

Tatineni, R., K.K. Doddapanem, R.C. Potumarthi, R.N.Vellanki, M.T. Kandathil, N. Kolli and L.N.Mangamoori, 2008. Purification and characterizationof an alkaline keratinase from Streptomyces sp.,Bioresour. Technol., 99: 1596-1602.

Tsuchida, O., Y. Yamagota, J. Ishizuka, J. Arai, J.Yamada, M. Ta-keuchi and E. Ichishima, 1986. Analkaline proteinase of an alkalophilic Bacillus sp.Curr. Microbiol., 14: 7-12.

Williams, C.M., C.G. Lee, J.D. Garlich and J.C.H. Shih,1991. Evolution of bacterial feather fermentationproduct, feather lyaste, as a feed protein. Poult. Sci.,70: 85-94.

Yamamura, S., Y. Morita, Q. Hasan, K. Yokoyama, E.Tamiya, 2002. Keratin degradation: a cooperativeaction of two enzymes from Stenotrophomonas sp.Biochem. Biophys. Res. Commun., 294:1138-1143.

Young, R.A. and R.E. Smith, 1975. Degradation offeather keratin by culture filtrates of Streptomycesfradiae. Can. J. Microbiol., 21: 583-586.

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Research ArticleISSN: 0974-6943 Available online through

www.jpronline.info

*Corresponding author.Neeraj WadhwaDepartment of Biotechnology,Jaypee Institute of Information Technology (Deemed University)A-10 sec 62, Noida, Uttar Pradesh, India.Tel.:+ (91)-120-2594207;Telefax:+ (91)-120-2400986.E-mail:neeraj.wadhwa@jiit.ac.in

INTRODUCTION

Production of extra cellular milk clotting enzyme from isolated Bacillus sp.Sarita Agrahari , Neeraj Wadhwa*Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University), A-10 sec 62, Noida, Uttar Pradesh, India.

Received on: 20-08-2010; Revised on: 16-10-2010; Accepted on:15-11-2010

ABSTRACTBacillus megaterium SN1, Bacillus thuringiensis SN2, Bacillus pumilus SN3 were isolated from soil samples of Ghazipur poultry waste site, Ghaziabad, India. Theisolates showing positive test on skim milk agar (10%) produced caesinolytic and keratinolytic enzyme in their culture media Optimum medium for keratinase andprotease production was feather meal media containing feather (10 g/L) at pH 7.5, 30ºC with shaking at 160rpm for 7 day. The protease isolated also showed milkclotting properties. Highest keratinolytic and caesinolytic activity was noted in Bacillus sp. SN2, followed by Bacillus sp. SN1 and then Bacillus sp. SN3. Remarkablemilk-clotting activity (MCA), 520.84 SU/ml, was obtained in Bacillus sp. SN1 (30-60% AS fraction) followed by Bacillus sp. SN3, Bacillus sp. SN2. The MCA (SU/ml) and MCA/CA ratio of the enzyme obtained are comparable with these three bacterial strains. Comparative accounts on enzymes viz., keratinolytic activity,caseinolytic activity, gelatinolytic activity, milk clotting activity were studied. The result showed that Bacillus sp . Under study are good producers of extra cellularprotease.

Keywords: Bacillus sp., Caesinolytic activity (CA), Keratinolytic activity, Milk clotting activity (MCA)

Proteases represent one of the three largest groups of industrial enzymes. Proteases(serine protease (EC. 3.4.21), cysteine (thiol) protease (EC.3.4.22), asparticproteases (EC. 3.4.23) and metallo-protease (EC. 3.4.24) constitute one of themost important groups of industrial enzymes accounting for about 60% of thetotal worldwide enzyme sales[1, 2, 3]. Proteases have failed applications inphysiological and commercial fields. They catalyze the cleavage of peptide bonds.Proteases are degradative enzymes which catalyze the total hydrolysis of proteins.The current estimated value of the worldwide sales of industrial enzymes is $1.8billion. Of the industrial enzymes, 75% are hydrolytic. Bacillus species are specificproducers of extra cellular proteases. These proteases have wide applications inpharmaceutical, leather, laundry, food and waste processing industries [4, 5, 6].

Protease has been routinely used for food industry such as cheese making, baking,preparation of soya hydrolysates, and meat tenderization. The major applicationof proteases in the dairy industry is in the manufacture of cheese. The milk-coagulating enzymes fall into three main categories, animal rennets, microbialmilk coagulants, and genetically engineered chymosin. Rennet extracted from thefourth stomach of unweaned calves is used. It contains the highest ratio of chymosin(EC 3.4.23.4) to pepsin activity. Alternatively microbial milk coagulants can beused. It is reported that instead of calf rennet the microbial enzymes but exhibitedtwo major drawbacks, i.e., (i) the presence of high levels of nonspecific and heat-stable proteases, which leads to the development of bitterness in cheese afterstorage; and (ii) a poor yield. In cheesemaking, proteases are used to hydrolyze thespecific peptide bond (the Phe105-Met106 bond) to generate para- -casein andmacropeptides. Chymosin is preferred due to its high specificity for casein, whichis responsible for its excellent performance in cheesemaking. The proteasesproduced by GRAS (genetically regarded as safe)-cleared microbes such as Mucormichei, Bacillus subtilis, and Endothia parasitica are gradually replacing chymosinin cheesemaking to cheesemakers for evaluation. Genencor International increasedthe production of chymosin by recombinant DNA technology in Aspergillus nigervar. awamori to commercial levels [7]. Although there are many microorganismsthat produce MCEs [8], only the MCEs produced by strains of Rhizomucor miehei,Rhizomucor pusillus var. Lindt, Aspergillus oryzae and Enthothia parasitica arewidely used [9, 10].

Milk-clotting enzymes has been isolated also for plant materials such as Withaniacoagulans[11], Ficus carica[12,13,14,15], Carica papaya[16], Cynara species[17] and pump-

kin [18]. Various fungi, particularly Entomophthorales[19,20], are potent sources sug-gested for cheese making. The bacteria investigated for this purpose are Bacillusbrevis, B. cereus, B. mesentericus, B. subtilis and Streptococcus liquefaciens [21, 22,

23, 24]. Microorganisms are known to be highly versatile in producing a wide range ofenzymes, with varied patterns of activity.

The present report deals with the screening of various bacteria for the productionof milk-clotting enzymes and proteolytic enzymes. The effect of calcium andmagnesium metal ion on the production of milk-clotting enzymes was also stud-ied. The capacity of selected Bacillus strains to produce and secrete large quanti-ties of extracellular enzymes has led them to be among the most importantindustrial enzyme producers.

MATERIALS AND METHOD

Isolation and Screening microorganism

Bacterial strainsBacillus sp. SN1,Bacillus sp. SN2, Bacillus sp. SN3. were isolated from soil andmaintained in the lab. Standard strain of Bacillus licheniformis (MTCC 1483) wasalso studied for comparative purposes [25].

Growth condition for caesinolytic and keratinolytic enzymeproductionSeed culture of the bacterial strains were prepared in 500 ml Erlenmeyer conicalflask containing 100 ml of the culture media containing washed feather (10%)that was maintained at 30°C at 160 rpm for 7 days and casinolytic and keratinolyticactivity was checked by regular interval. After incubation, the crude culture brothwas centrifuged (10,000g, 4ºC, 30 min) and cell free supernatant was subjected toammonium sulphate precipitation. After chilling at 4ºC for 1 hr, the resultingprecipitate was collected by centrifugation (10,000g, 4ºC, 30 min) and dissolved ina minimal volume of Tris–Cl buffer 10mM (pH 8.0) and dialyzed overnightagainst 4 liters Tris–Cl buffer 10mM (pH 8.0). The dialysed protein fraction waschecked for caesinolytic activity and keratinolytic activity by the modified methodof Tsuchida et. al., 1986 [26] and Cheng et. al.,1995 [27] respectively.

Soil microbes from Ghazipur poultry processing plant site were identified andmaintained in the laboratory. They were inoculated in Feather meal media thatcomposed of (g/L):NH

4Cl,0.5;NaCl,0.5;K

2HPO

4,0.3;KH

2PO

4 ,0.4; MgCl

2.6H

2O,

0.1; Yeast extract, 0.1and Feather meal powder, 10 and pH was maintained at 7.5at 30°C at 160rpm for 7 days. They were checked for keratinolytic activity andcaesinolytic activity production. Feather degradation in culture broth was con-firmed visually [25]. Further the extracellular supernatant was also tested for milkclotting activity.

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Protein concentrationProtein concentration was determined by the method of Bradford with bovineserum albumin as a standard [28].

Determination of caesinolytic activityCaesinolytic activity was assayed by a modified method of Tsuchida et al., 1986 byusing casein as substrate [26]. 100µl of enzyme solution was added to 900µl ofsubstrate solution (2 mg/ml casein in 10 mM Tris–Cl buffer, pH 8.0).The mixturewas incubated at 50°C for 20 min. Reaction was terminated by the addition of anequal volume of 10% chilled trichloroacetic acid then the reaction mixture wasallowed to stand in ice for 15 min to precipitate the insoluble proteins. Thesupernatant was separated by centrifugation at 10,000 rpm for 10 min at 4°C, theacid soluble product in the supernatant was neutralized with 5 ml of 0.5 M Na

2CO

3solution. The color developed after adding 0.5 ml of 3 fold diluted Folin–Ciocalteaureagent was measured at 660 nm. All assays were done in triplicate. One caesinolyticactivity unit is defined as the amount of enzyme that releases 1 µmol of tyrosineper ml per minute under the above assay conditions.

Determination of keratinolytic activityThe keratinolytic activity was assayed by the modified method of Cheng et.al.,1995 by using keratin as a substrate [27]. The reaction mixture contained 200µlof enzyme preparation and 800µl of 20µg/ml keratin in 10mM Tris buffer, pH 8.The reaction mixture was incubated at 45ºC for 20min and the reaction wasterminated by additing 1ml of 10% chilled trichloroacetic acid. The mixture wascentrifuged at 10,000g for 5min and the absorbance of the supernatant fluid wasdetermined at 440 nm. All assays were done in triplicate. One unit (U) of enzymeactivity was the amount of enzyme that caused a change of absorbance of 0.01 at440 nm in 20 min at 45ºC.

Hydrolysis of protein substrate

Determination of activity against gelatin and BSAEnzyme activity was assayed using gelatin and BSA as substrate. 100 µl of theenzyme and 900 µl of assay buffer containing the protein substrates (2 mg/mlgelatin and BSA in 10 mM Tris–Cl buffer, pH 8.0). After incubation at 50°C for 20min, Reaction was terminated by the addition of an equal volume of 10% chilledtrichloroacetic acid then the reaction mixture was allowed to stand in ice for 15min to precipitate the insoluble proteins. The undigested proteins were removedby filtration or centrifugation at 10,000 rpm for 5 min and amino acid releasedwas assayed. The supernatant was separated by centrifugation at 10,000 rpm for10 min at 4°C; the acid soluble product in the supernatant was neutralized with 5ml of 0.5 M Na

2CO

3 solution. The color developed after adding 0.5 ml of 3 fold

diluted Folin–Ciocalteau reagent was measured at 660 nm. All assays were done intriplicate.

Determination of milk clotting activity

I. Preparation of milk clotting activity substrate10 g of skimmed milk powder was dissolved by stirring on magnetic stirrer in100ml of 10mM CaCl

2 and MnSO

4. The pH of milk substrate was adjusted to 6.5

with 0.1 N NaOH or HCl.

II. Assay protocolMilk-clotting activity was determined according to the method of Arima [8], whichis based on the visual evaluation of the appearance of the first clotting flakes, andexpressed in terms of Soxhlet units (SU). One SU is defined as the amount ofenzyme which clots 1ml of a solution containing 0.1 g skim milk powder in 40min at 35ºC. In brief, 0.5 ml of tested materials was added to a test-tube containing5ml of reconstituted skim milk solution (10 g dry skim milk/100 ml, 10mMCaCl2 and 10mM MnSO

4) preincubated at 35ºC for 5min. The mixture was mixed

well and the clotting time T (s), the time period starting from the addition of testmaterial to the first appearance of clots of milk solution,was recorded and theclotting activity was calculated using the following formula:

SU = 2400×5×D/T×0.5; T = clotting time (s); D= dilution of test material [29]

Determination of antimicrobial activityAntimicrobial tests were then carried out by disk diffusion [30]. The sensitivity ofvarious microorganism like Micrococcus luteus, Bacillus subtilis, Bacillusamyloliqifaceance, Pseudomonas fluroscence, E. coli. 100 µl of suspension con-taining 108 colony forming units (CFU)/ml of bacteria spread on nutrient agar(NA) medium. The sterilized filter paper discs (Whatman filter paper no.1, 6mmin diameter) impregnated with 10 µl of dialyzed ammonium sulphate fractionpurified from all three bacterial samples and Bacillus licheniformis (50 µg/disk)

were placed on the bacteria inoculated agar. Negative controls were prepared withthe same medium. .Tetracyclin (30 mcg) was used as standard antibiotics (HIMEDIALabratories, Mumbai, India). The positive controls were used to determine thesensitivity of one strain/isolated in each microbial species tested. The inoculatedplates were incubated aerobically at 30°C (Gram negative) and 37°C (Gram posi-tive) according to strain for 24 h.

Antimicrobial activity was evaluated by measuring the zone of inhibition that hadbeen caused by the enzyme preparation against the test organisms [31, 32]. Eachassay was repeated twice.

RESULTS AND DISCUSSION:

Isolation and Screening microorganismBased on morphological and biochemical characteristics as reported in Bergey’sManual of Determinative Bacteriology and further selective growth studies onHicrome bacillus agar, isolate SN1, SN2, SN3 grew as yellowish green, blue, greencolonies respectively. Hicrome bacillus agar studies confirmed that these strainswere Bacillus megaterium, Bacillus thuringiensis, Bacillus pumilus respectively.They are gram-positive, rod shaped, highly motile and sporulating bacteria de-tected clearly under light microscope [28].These colonies were separately grown inoptimal media for growth and assayed for presence of enzyme.

Caesinolytic and Keratinolytic enzyme activityCaesinolytic and keratinolytic enzyme activity in the growth culture supernantfor all three isolated bacterial strains and standard Bacillus licheniformis strain waschecked separately at regular time interval interval. Maximum caesinolytic activ-ity of Bacillus sp . SN1, SN3 was found at 96hr and Bacillus sp . SN2 at 72 hr thenBacillus licheniformis at 144 hr (Fig.1). Whereas maximum keratinolytic activityof Bacillus sp . SN3, Bacillus licheniformis was found at 72 hr, Bacillus sp . SN2 at96 hr, Bacillus sp . SN1 at 120 hr (Fig.2).

0

10

20

30

40

50

60

70

24 48 7 2 9 6 1 2 0 1 4 4 1 6 8T i m e ( H r )

Pro

teas

e ac

tivity

(U/m

l)

I s o l a t e S N 1

I s o l a t e S N 2

I s o l a t e S N 3

B . l i c .

Fig 1. Caesinolytic activity activity

Figure depicts that the incubation period is important for production of caseinolyticenzyme Maximum caesinolytic activity of Bacillus sp . SN1, SN3 (60 Units /ml)was found at 96hr of growth in the cell free culture supernatant. Bacillus sp . SN2showed presence of caseinolytic activity at 72 hr of growth . Whereas Bacilluslicheniformis (standard strain) showed maximum activity at 144 hr of incubation

0

0.1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

2 4 4 8 72 9 6 1 2 0 1 4 4T i m e ( H r )

Ker

atin

ase

activ

ity (U

/ml)

I s o l a t e S N 1I s o l a t e S N 2

I s o l a t e S N 3

B . l i c .

Fig.2 Keratinolytic activity activityFigure depicts that the incubation period is also important for production ofkeratinolytic enzyme. Maximum keratinolytic activity is seen for Bacillus sp .SN3 (0.99 Units /ml) at 72 hrs of incubation, Further for Bacillus sp . SN2 it is 96

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hr, Bacillus sp . SN1 is 120 hr and standard Bacillus licheniformis 72 hr wheremaximum keratinolytic activity is reported.

Hydrolysis of protein substrateHighest enzyme activity against substrate casein, keratin and gelatin was demon-strated in Bacillus sp . SN2 whereas highest enzyme activity against substrate BSAwas found in Bacillus licheniformis (Table 1).

Table 1: Table depicts relative enzyme activity (%) with keratin, casein,gelatin and BSA of bacterial isolated strain by taking Bacillus licheniformisas 100%

Bacterial strains Relative Relative Caesinolytic Relative Enzyme Relative EnzymeKeratinolytic activity activity activity against activity againstactivity activity (%) gelatin substrate BSA substrate(%) (%) (%)

Bacillus 100 100 100 100licheniformisBacillus sp. SN1 133.75 106.87 90 27Bacillus sp. SN2 152.22 182.5 145 85.58Bacillus sp. SN3 96.17 92 26.76 45

Milk clotting activityThe result in Table 2 summarizes all bacterial isolate and their ammonium sul-phate fractions show presence of caesinolytic activity. Milk clotting activity wasseen in crude and ammonium sulphate fraction for Bacillus sp. SN1, SN2, SN3.Crude and ammonium sulphate fraction of standard strain Bacillus licheniformisdid not show the presence of milk clotting activity. Interestingly highest ratio ofmilk clotting activity (with CaCl

2 and MnSO

4) to caesinolytic activity in Bacillus

sp. SN1 (30-60% ammonium sulphate cut) followed by Bacillus sp. SN1 (0-30%ammonium sulphate cut), Bacillus sp. SN1 (60-90% ammonium sulphate cut),Bacillus sp. SN3 (0-80% ammonium sulphate cut), Bacillus sp. SN2 (0-80%ammonium sulphate cut). 30-60% ammonium sulphate fraction of Bacillus sp.SN1 showed highest milk clotting activity while 0-80% ammonium sulphatefraction of Bacillus sp. SN3 showed highest caesinolytic activity (Table 2). It isnot clear whether the coagulation of skimmed milk by bacterial source of enzymeis due to single action of enzyme or the synergistic action of many enzymes. Butthis bacterial strain showed promising proteolytic activity.

Table 2: Milk clotting activity, Caesinolytic activity activity, Ratio ofmilk clotting units to caesinolytic activity of bacterial strain

Bacillus licheniformis ND ND 0.474 0 0(Crude)Bacillus licheniformis ND ND 0.426 0 0(0-80%)Bacillus sp. SN1 1.429 2.5 0.250 5.716 10(Crude)Bacillus sp. SN1 10 12.5 0.115 86.96 108.69(0-30%)Bacillus sp. SN1 50 100 0.192 260.4 520.84(30-60%)Bacillus sp. SN1 9.09 11.1 0.132 68.87 84.09(60-90%)Bacillus sp. SN2 0.83 1.11 0.458 1.81 2.42(Crude)Bacillus sp. SN2 1.11 8.33 0.816 1.36 10.20(0-80%)Bacillus sp. SN3 1.25 1.25 0.435 2.87 2.87(Crude)Bacillus sp. SN3 1.66 14.28 0.644 2.58 22.17(0-80%)

Milk-clotting activity (MCA),520.84 SU/ml, was obtained in Bacillus sp. SN1 (30-60% dialysed ammonium sulphate fraction)

Fig 1. Milk coagulation: B. megaterium SN1, B. thuringenesis SN2, B. Pumilis SN3 strainshowed milk clotting activity. C- Control; 1- Crude enzyme of isolate SN1; 2- 0-30% A. S.sample of isolate SN1; 3- 30-60% A. S. sample of isolate SN1; 4- 60-90% A. S. sample ofisolate SN1; 5- Crude enzyme of isolate SN2; 6- 0-80% A. S. sample of isolate SN3; 7- Crudeenzyme of isolate SN3; 8- 0-80% A. S. sample of isolate SN3.

Fig 2. Milk coagulation: 30-60%, 60-90% A. S. fraction of B. megaterium SN1, crude and 0-80% A. S. fraction of B. thuringenesis SN2 showing floating pellet. C- Control; 1- Crude enzymeof isolate SN1; 2- 0-30% A. S. sample of isolate SN1; 3- 30-60% A. S. sample of isolate SN1; 4-60-90% A. S. sample of isolate SN1; 5- Crude enzyme of isolate SN2; 6- 0-80% A. S. sample ofisolate SN3; 7- Crude enzyme of isolate SN3; 8- 0-80% A. S. sample of isolate SN3.

Antimicrobial activityA visually clear zone around enzyme disk indicates the presence of antibacterialcomponents against bacterial strain in the enzyme preparation (Table 1). Bacillussp. SN2 showed antimicrobial activity against M. luteus and Bacillus subtilis,Bacillus amyloliquifaceance, Escherichia coli.

Table 3. Antimicrobial activity is represented as Inhibition (mm) ofbacterial growthReference Strain Bacillus Bacillus sp. SN1 Bacillus sp. SN2 Bacillus sp. SN3

licheniformis

Te TS Te TS Te TS Te TSMicrococcus luteus 18 ND 17 ND 20 12 ND ND(MTCC 106)Bacillus subtilis 20 ND 20 17 25 10 20 16(MTCC 1789)Bacillus 15 10 30 16 20 10 23 NDamyloliquifacience(MTCC 1270)Pseudomonas 25 ND ND ND 15 ND 25 22fluroscence(MTCC 2421)Escherichia coli 25 13 20 12 20 15 10 ND

(MTCC 1695)

Te: Standard antibiotic Tetracycline; TS: Test samples; Values given in mm; ND:Not determinedCONCLUSIONWe adopted the strategy of isolating the keratinolytic and caesinolytic producingmicroorganism by selective growing them in enriched media and found that featherprotein can be metabolized which is used as animal feed protein [38, 39] Remarkablemilk-clotting activity (MCA), 520.84 SU/ml, was obtained in Bacillus sp. SN1(30-60% AS fraction).The caesinolytic activity was 0.816 ODU and the milkclotting activity was 10.20 SU/ml obtained for Bacillus sp. SN2 can be furtheroptimized. The thermostability and wide pH range shown for the caesinolyticactivity suggests its application in dairy industry. Since the caesinolytic activity isinhibited by barium chloride (result not shown) it is suggest that the addition of adietary important salt for example barium selenate can be used to inhibit theenzyme activity in the manufacturing process of cheese thus increasing itsnutraceutical value.

ACKNOWLEDGEMENTWe are thankful to Jaypee Institute of Information Technology University, Noida,India for providing the infrastructure facilities for this study.

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26. Tsuchida O, Yamagota Y, Ishizuka J, Arai J, Yamada J, Ta-keuchi M, Ichishima E. An alkaline proteinaseof an alkalophilic Bacillussp. Current Microbiol. 1986; 14: 7-12.

27. Cheng SW, Hu HM, Shen SW, Takagi H, Asano M, Tsai YC. Production and characterization of keratinaseof a feather-degrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem. 1995; 59: 2239–2243.

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The Open Microbiology Journal, 2010, 4, 67-74 67

1874-2858/10 2010 Bentham Open

Open Access

Purification of Protease from Pseudomonas thermaerum GW1 Isolated from Poultry Waste Site

Smriti Gaur, Sarita Agrahari and Neeraj Wadhwa*

Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) A-10, sec 62, Noida, Uttar Pradesh, India

Abstract: An extracellular protease was purified from Pseudomonas thermaerum GW1 a new strain identified by morphological, biochemical and 16S rDNA sequencing. It was isolated from soil of Poultry waste site at Ghazipur near Ghaziabad, Delhi. The strain produces extra cellular protease in the culture media that was maintained at 37°C, 140 rpm. The media was harvested for protease after 48 hrs of incubation at 37°C in basal media supplemented with 1% casein. We report 6.08 fold purification of enzyme following ammonium sulphate precipitation and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated to be approximately 43,000 daltons as shown by casein zymography studies. The optimum pH for the proteolytic activity was pH 8.0 and enzyme remained stable between pH 5 -11 at 60°C. Interestingly Mn2+ (5mM) activated enzyme activity by 5 fold, while Cu2+, Mg2+and Ca2+ moderately activated enzyme activity, where as Zn2+, Fe2+ and Hg2+ inhibited enzyme activity. The protease produced was stable in presence of 50 % (v/v) ethylacetate and acetone. Isopropanol, methanol and benzene increased protease activity by 2.7, 1.3 and 1.1 fold respectively but was inhibited in presence of glycerol and DMSO. This organic solvent-stable protease could be used as a biocatalyst for enzymatic peptide synthesis

Keywords: Protease, Pseudomonas, Casein zymography

INTRODUCTION

Proteases covers up to 60% of total enzyme market and are valuable commercial enzyme that have biotechnological as well as industrial applications however the present known proteases are not sufficient to meet most of the industrial demands It is desirable to have new proteases with novel properties from different sources. Alkaline proteases hold a great potential for application in the detergent and leather industries [1-3] and are also reported to have been isolated from microbes, plants and animals. Proteases from plant sources have application in food industry [4]. Previous studies in our lab have shown that proteases from senesced leaves of Lantana camara can have application in detergent industry as the enzyme is thermosta-ble [5]. Microbes are the preferred source of proteases because of their rapid growth, and the ease with which they can be genetically manipulated to generate new enzymes with altered properties [6, 7]. Proteases have been purified and characterized from several bacteria [8-12]. However there are only few reports on Pseudomonas thermaerum, Yang et al., [13] have identified two strains of Psudomonas thermaerum isolated from activated sludge that could use lignin as sole carbon source and excrete peroxi-dases. A novel antimicrobial peptide (30 kDa) produced by a this bacterium isolated from the effluent pond of a bovine abattoir showed inhibition to a broad range of indicator *Address correspondence to this author at the Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) A-10, sec 62, Noida, Uttar Pradesh, India; Tel: + (91)-120-2400973-976; Fax: + (91)-120-2400986; E-mail: neeraj.wadhwa@jiit.ac.in

strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococ-cus aureus has been reported by Fontoura et al., [14]. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases. S28 strain isolated from Nanyang oil field was identified to be as P. thermaerum based on homology studies and could degrade 82.02% of phenenthrene within 10 days [15]. In our attempt to screen for microbial isolate that can provide stable enzyme, soil sample of the poultry waste site which is rich in organic waste was selected. In this investiga-tion, we report the production and effect of pH, temperature, metal ions and substrate concentration on protease secretion whereby the culture condition were manipulated for maxi-mum protease production. Pseudomonas thermareum GW1 was isolated from soil of poultry waste site and it extracellu-larly secreted this protease which was purified to homogene-ity, characterized and found to be stable in the presence of several organic solvents. Ours is the first report that shows extracellular production of proteases from Pseudomonas thermaerum GW1 strain.

MATERIALS AND METHODS

Isolation and Screening of Protease-Producing Strains

Soil was collected in sterilized sampling bags of Ghazipur poultry waste site, India. The microbe responsible for the production of protease was identified. The classifica-tion was based on gram strain (-), catalase (+) and oxidase (+) reactions, using morphological and biochemical

68 The Open Microbiology Journal, 2010, Volume 4 Gaur et al.

characteristics based on Bergey’s Manual of Determinative

Bacteriology [16]. Soil was selected from areas where

naturally degraded feather were seen. The samples were

brought to the laboratory and processed for the analysis the

same day. Soil samples were suspended in basal media and

kept for growth at 37°C for 6 days. At regular intervals of 6

hrs the activity of protease was measured and sample

showing maximum activity was screened for protease

producing strains. Samples of repeated batch cultures were

plated on skim milk agar. After 24–48 hrs at 37°C, colonies

which exhibited the largest cleared zones were selected and

was further incubated in cultivation media for further 48 hrs

and checked for protease production.

Identification of Protease Producing Bacteria

The isolate GW1 was identified originally as a strain of

Pseudomonas by our laboratory based on Morphological,

Physical, Biochemical characteristics and the single colony

was subcultured on bacterial culture plates supplemented

with casein. The culture was sent for identification till

species level to Bangalore GeneI India by partial 16S r DNA

sequence analysis.

Production of Enzyme in Cultivation Media

The Basal media for protease production composed of

(g L-1

): Peptone, 5; Glucose, 10; NaCl, 0.5; CaCl2.2H2O, 0.1;

K2HPO4, 0.3; KH2PO4, 0.4; MgSO4 · 7H2O, 0.1; and yeast

extract, 5. The pH was maintained at 7.5. Microbes were

allowed to grow in 500 ml conical flask containing 50 ml of

the culture media that was maintained at 37°C at 140 rpm.

5% (v/v) of the 20 hrs old culture was inoculated in cultiva-

tion media [17].

i Tryptic soy broth (TSB)

ii Gelatin (1%) + basal medium for protease production

iii Casein (1%)+ basal medium for protease production

iv Skim milk powder (1%)+ basal medium for protease

production

v Pigeon feathers (1%)+ basal medium for protease pro-

duction

The above cultivation media were checked for enzyme

activity at regular intervals of 6 hrs by the modified method

of Tsuchida et al. [18].

Protease Purification

The bacterial strain was grown for 48 hrs at 37°C in the

selected cultivation media. The culture medium was

centrifuged at 10,000 rpm for 10 min at 4°C and the cell-free

supernatant was precipitated with 0-60% ammonium sulfate.

The precipitate was collected by centrifugation and dissolved

in a small volume (1/50) of 10 mM Tris-HCl buffer (pH 8.0),

and dialyzed against 4 liters of same buffer for 12 hrs at 4°C.

This step was repeated twice. The dialyzed enzyme prepara-

tion was applied on a DEAE-cellulose column (2 X 24 cm)

pre-equilibrated with 10 mM Tris-HCl (pH 8.0). The unad-

sorbed protein fraction was eluted with the same buffer (150

ml). The enzyme was eluted with a gradient of 2mM and 4

mM NaCl in the same buffer at a flow rate of 1ml/min.

Active fractions that contained (80%) of the enzyme activity

were pooled, and subsequently used for characterization. All

steps were conducted at 4°C.

Determination of Protease Activity

Protease activity was assayed by a modified method of

Tsuchida et al. [18] by using casein as substrate. 100 μl of

enzyme solution was added to 900 μl of substrate solution (2

mg/ml (w/v) casein in 10 mM Tris–HCl buffer, pH 8.0).The

mixture was incubated at 45°C for 30 min. Reaction was

terminated by the addition of an equal volume of 10% (w/v)

chilled trichloroacetic acid then the reaction mixture was

allowed to stand in ice for 15 min to precipitate the insoluble

proteins. The supernatant was separated by centrifugation at

10,000 rpm for 10 min at 4°C; the acid soluble product in the

supernatant was neutralized with 5 ml of 0.5 M Na2CO3

solution. The colour developed after adding 0.5 ml of 3-fold-

diluted Folin–Ciocalteau reagent was measured at 660 nm.

All assays were done in triplicate. One protease unit is

defined as the amount of enzyme that releases 1 g of tyro-

sine per ml per minute under the above assay conditions. The

specific activity is expressed in the units of enzyme activity per milligram of protein.

Protein Concentration

Protein concentration was determined by the method of Bradford [19] with bovine serum albumin as standard.

Polyacrylamide Gel Electrophoresis and Zymogram

SDS-PAGE was performed on a slab gel containing 10%

(w/v) polyacrylamide by the method of Laemmli [20].

Casein zymography was performed in polyacrylamide slab

gels containing SDS and casein (0.12% w/v) as co-

polymerized substrate, as described by Choi et al., [21]. Af-

ter electrophoresis, the gel was incubated for 30 minutes at

room temperature on a gel rocker in 50 mM Tris-Cl (pH

7.4), which contained 2.5% Triton X-100 to remove SDS.

The gel was then incubated in a zymogram reaction buffer

(30 mM Tris-HCl, pH 7.4, 200 mM NaCl and 10 mM CaCl2)

left at 37°C for 12 hrs on rocker shaker.The gel was stained

with Coomassie brilliant blue (0.5% w/v) for 30 min. The

activity band was observed as a clear colourless area

depleted of casein in the gel against the blue background

when destained in 10% methanol and 5% acetic acid for a

limited period of time.

Effect of pH on Enzyme Activity

Effect of pH on the purified enzyme activity was meas-

ured at various pH ranges (3.0 – 12). Reaction mixtures were

incubated at 45°C for 30 min and the activity of the enzyme was measured as described previously.

Effect Of Temperature On Enzyme Activity And Stability

The activity of the enzyme was determined by incubating

the reaction mixture at different temperatures ranging from

20, 30, 40, 50, 60, 70 and 80°C were studied. The activity of the enzyme was measured as described previously.

Purification of Protease from Pseudomonas Thermaerum GW1 Isolated The Open Microbiology Journal, 2010, Volume 4 69

Effect of Various Metal Ions on Protease Activity

The effects of metal ions on enzyme activity (e.g., Ca2+, Mg2+, Fe2+, Mn2+, Zn2+, Hg2+, and Cu2+ [5 mM]) were investigated by adding them to the reaction mixture and pre-incubated for 30 min at 45°C pH 10.0. The activity of the enzyme was measured as described previously.

Effect of Organic Solvents on the Protease Stability

The organic solvents used were Methanol, Ethyl acetate, Benzene, Glycerol, Sucrose, Toluene, Acetone, Hexane, DMSO, Isopropanol and Ethanol. In the stability test, 1.0 ml of organic solvent (100% v/v) was added to 1 ml of the reaction mixture and pre incubated at 37°C for 30 min. The remaining proteolytic activity was measured as de-scribed previously. Stability was expressed as the remaining proteolytic activity relative to the solvent-free controls (0%, v/v).

RESULTS AND DISCUSSION

Isolation and Identification of Protease-Producing Bacterial Strains

Soil samples were analyzed for isolation of proteolytic bacterial cultures. Screening of microorganisms that pro-duced protease was done on cultures isolated from soil of Ghazipur poultry waste site. Organic waste such as feathers and other poultry waste is essentially composed of proteins. Protease producing strains were selected by growth on skim milk agar, as described in Methods. Among the cultures tested, the laboratory isolate GW1 showed highest zone of clearance. The purity of the isolated bacteria was ascertained through repeated streaking (Fig not shown).

Microscopic observation of the isolate showed a non sporulating gram negative rods, the bacterium grew aerobically and formed typical blue green, flat, large, grape like odour colonies. The strain showed positive reaction for catalase, oxidase, citrate, nitrate, motility, and production of pyoveridin and pyocyanin. Negative reactions were observed for indole, urea and starch hydrolysis (Table 1). These phenotypic characteristics based on Bergey’s Manual of Determinative Bacteriology [16] suggest the Pseudomonad-aceae family genus Pseudomonas.

Strain Identification by 16S rDNA Sequencing

The GW1 strain was identified to be as Pseudomonas thermaerum as predicted by 16S rDNA studies. Studying the Alignment view of the sequence of the isolated microbe using combination of NCBI GenBank and RDP database using 10 examples and nucleotide similarity. Nearest ho-molog was found to be Pseudomonas aeruginosa strain EKi (Accession No. FJ685995). The details are given in Table 2, 3 respectively.

The sequence of the isolate GW1 was submitted to the GenBank (Accession Number GU951516)” and based on nucleotide homology and phylogenetic analysis it is found to have close similarity to Pseudomonas thermaerum strain EKi (GenBank Accession Number: F3816019) (Fig. 1).

Protease Production and Effects of Different Parameters

Protease production was tested at various time interval (1–7 days) and influence of addition of various nutrient sources (TSB, Gelatin, Casein, Skim milk, Pigeon feathers) were evaluated in relation to enzyme yield. Pseudomonas thermaerum strain GW1 grew in five nutrient sources and produced protease. The highest protease production 32 units/mg occurred in Basal medium supplemented with casein whereas lowest in basal medium supplemented with Pigeon feathers 9.7units/mg protein after 48 hrs of cultiva-tion (Fig. 2).

Table 1. Morphological and Biochemical Characteristics of Isolate

Morphological and Biochemical Characteristics

Results

Colony morphology Irregular,Undulated,slimy and flat

Pigment Light green to blue green

Texture Shiny, smooth

Odour Sweet grapey

Gram staining Gram Negative rods

Spores –

Aerobic growth +

Motility +

Catalase +

Oxidase +

Glucose –

Lactose –

Sucrose –

Methyl Red –

V-P test –

Indole –

Citrate +

Nitrate reduction +

Urea Test –

Starch Hydrolysis –

Pseudomonas agar P + blue green pigment

Cetrimide Agar +, Pyocyanin (blue green pigment) production

Identification of organism Genus Pseudomonas

Purification of Protease

The extracellular protease produced by Pseudomonas thermaerum strain GW1 was purified in two steps by 0-60%

70 The Open Microbiology Journal, 2010, Volume 4 Gaur et al.

ammonium sulphate precipitation followed by anion exchange chromatography on DEAE- cellulose resin (Fig. 3). The recovered active fraction from 0-60% ammonium sulphate of culture broth was adsorbed on the DEAE- cellu-lose matrix. The bound protease was eluted with 0.2 and 0.4 M NaCl (in 10 mM Tris–HCl buffer, pH 8.0). The prote-ase was purified 6.08 fold and about 9.3% of the total activ-ity units was recovered. The specific activity of the purified enzyme was 137.54 units/mg. The purified enzyme could be

stored in 10 mM Tris-HCl buffer, pH 8.0, at -80ºC for 3 months without any apparent loss of activity. The results of purification of protease from Pseudomonas thermaerum strain GW1 are summarized in Table 4.

SDS-PAGE and Zymogram Analysis

The DEAE fraction was analysed on SDS PAGE (10%), showed presence of single band indicating a homogeneous

Table 2. Table gives the Alignment view of the Sequence of the Isolated Microbe Using Combination of NCBI GenBank and RDP Database Using 10 Examples. Nearest Homolog was Found to be Pseudomonas Aeruginosa Strain EK1 (Accession No. FJ685995)

Alignment View ID Alignment Results Sequence Description

PGW1B 0.81 Studied sample

FJ816019 0.83 Pseudomonas thermaerum strain EKi

FJ685995 0.83 Pseudomonas aeruginosa strain EK1

FJ948174 1.00 Pseudomonas aeruginosa strain WJ-1

FJ864676 0.99 Pseudomonas aeruginosa strain pp1a

EU352760 1.00 Pseudomonas aeruginosa strain NK 2.1B-1

EU331416 1.00 Pseudomonas aeruginosa strain pY11T-3-1

EU099381 0.99 Pseudomonas sp. J16

AB305018 1.00 Pseudomonas aeruginosa strain PA1

EU603683 0.99 Pseudomonas aeruginosa strain XJTUMS3

EF551040 0.93 Pseudomonas sp. GZ1

Table 3. Indicates Nucleotide Similarity (Above Diagonal) and Distance (Below Diagonal) Identities Between the Studied Sample ‘PGW1B’ and Ten other Closest Homologs Microbe

Distance Matrix

1 2 3 4 5 6 7 8 9 10 11

FJ948174 1 --- 0.996 0.949 0.999 0.999 1 0.949 0.999 0.960 0.996 0.940

EU331416 2 0.004 --- 0.944 0.995 0.995 0.996 0.944 0.996 0.955 1 0.936

FJ685995 3 0.051 0.056 --- 0.948 0.949 0.949 1 0.948 0.906 0.944 0.982

EU099381 4 0.001 0.005 0.052 --- 0.999 0.999 0.948 0.999 0.959 0.995 0.939

FJ864676 5 0.001 0.005 0.051 0.002 --- 0.999 0.949 0.999 0.959 0.995 0.940

AB305018 6 0.000 0.004 0.051 0.001 0.001 --- 0.949 0.999 0.960 0.996 0.940

FJ816019 7 0.051 0.056 0.000 0.052 0.051 0.051 --- 0.948 0.906 0.944 0.982

EU603683 8 0.001 0.004 0.052 0.002 0.002 0.001 0.052 --- 0.959 0.996 0.940

EF551040 9 0.041 0.045 0.094 0.041 0.041 0.041 0.094 0.041 --- 0.955 0.894

EU352760 10 0.004 0.000 0.056 0.005 0.005 0.004 0.056 0.004 0.045 --- 0.936

PGW1B 11 0.060 0.064 0.018 0.061 0.060 0.060 0.018 0.060 0.106 0.064 ---

Purification of Protease from Pseudomonas Thermaerum GW1 Isolated The Open Microbiology Journal, 2010, Volume 4 71

preparation. The enzyme has a low molecular weight of approximately 43Kda (Fig. 4 Lane 2). Zymogram activity staining also revealed one clear zone of proteolytic activity against the blue background for purified sample at corre-sponding positions in SDS-PAGE (Fig. 4 Lane 3).

Fig. (2). Study of effect of various nutrient sources on protease production by Pseudomonas thermaerum GW1. The culture was grown for production of protease as described in “Methods”. 1% of the above mentioned nutrient sources were added to basal media. pH of the media was adjusted to 7.5.

pH Optimum and pH Stability

Activity of the enzyme was determined at different pH ranging from 3.0-12.0. The optimum pH recorded was 8.0 for protease activity. Protease activity was found to be stable in the alkaline range starting from the pH 5-11 at 45°C (Fig. 5).

Temperature Optimum and Thermal Stability

The thermal stability of the enzyme was also tested at different temperatures 20°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C on incubation for 60 minutes (Fig. 6). The opti-

mum temperature recorded was at 60°C for protease activity and Protease activity was found to be stable in the tempera-ture range from 40°C - 70°C. The enzyme activity gradually declined at temperatures beyond 70°.

Fig. (3). The bound protease on DEAE column was eluted with 0.2 and 0.4 M NaCl (in 10 mM Tris–HCl buffer, pH 8.0) Fraction of 0.4M NaCl showed a single peak of caseinase activity.

Effect of Metal Ions

Fe2+ has a strong inhibitory effect, whereas Zn2+

and Hg2+

have mild effects on protease activity. Interestingly Mn2+

strongly activated enzyme activity by 5 fold (Table 5).

Effect of Organic Solvents on the Protease Stability

Ten organic solvents were used to study the effect on protease activity. As shown in (Fig. 7) the protease has ability to act in the presence of solvents in reaction system. The enzyme retained 78% and 75 % of activity in the pres-ence of ethylacetate and acetone respectively. The presence of isopropanol, methanol and benzene increased the activity of isolates GW1 by 2.7, 1.3 and 1.1 fold, respectively.

Fig. (1). Phylogenetic tree made in MEGA 3.1 software using neighbor joining method.

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72 The Open Microbiology Journal, 2010, Volume 4 Gaur et al.

Enzyme lost 60% of total activity in presence of DMSO and hexane and was not stable in the presence of glycerol, tolu-ene and sucrose. Ogino et al., [22] have reported importance of disulfide bonds for stability of the protein in presence of solvents. Jorden et al., [23] have reported that 61% of the activity of HIV1 protease was lost in presence of 12% Me2SO; similar results were also reported from protease thrombin [24]. This loss of hydrolytic activity over time as reported by the authors is not due to slow dissociation of enzyme dimer into inactive monomer. The activity of HIV1 is also sensitive to glycerol and the hydrolytic efficiency of this enzyme decreases with the increasing concentration of glycerol.

Fig. (4). Acitvity gel electrophoresis of the purified protease, M: molecular weight markers, Lane 1: purified protease on 10% SDS-PAGE and Lane 2: Zymography of purified protease from Pseudo-monas thermaerumGW1. Zymography was done by the method of Choi et al.

Table 6 reveals that if the concentration of glycerol is as low as10%v/v. then there is 38% decrease in Protease activity whereas 45 %decrease in enzyme activity is reported in presence of 20%v/v glycerol. Ours is the first report that shows extracellular produc-tion of proteases from Pseudomonas thermaerum. Protease from GW1 strain lost its activity in the presence of glycerol, sucrose and metal ion iron.

CONCLUSIONS

Enzyme activity from Pseudomonas thermaerum is lost in the presence of glycerol and sucrose. The buffer best suited for Pseudomonas thermaerum protease should minimize the use of glycerol and sucrose during dialysis. So far, several well-known proteases such as thermo-lysin, papain, and chymotrypsin have been used as biocata-

lysts of peptide synthesis in the presence of organic solvents. Protease from Pseudomonas thermaerum retained its activity in organic solvents; these results are in line with many stud-ies that report protease stability in the presence of organic solvents [25-29] promising potential industrial application of protease from Pseudomonas thermaerum.

Fig. (5). Effect of pH on the activity of the purified Protease from Pseudomonas thermaerum GW1 pH optima was measured by incu-bating the enzyme with the substrate at different pH values at 45°C. The maximum activity obtained at pH 8.0 was considered as 100% activity. The treated enzyme solution was cooled rapidly in ice and the relative activity was measured under standard condition.

Fig. (6). Effect of temperature on protease activity from Pseu-domonas thermaerum GW1. The relative activity was defined as the percentage of activity detected with respect to the maximum protease activity. The maximum activity obtained at temperature 60°C was considered as 100% activity. The treated enzyme solu-tion was cooled rapidly in ice and the relative activity was meas-ured under standard condition.

Table 4. Summary of Purification Steps of Alkaline Protease from Pseudomonas thermaerum

Purification Step Total Protein(mg) Total Enzyme Activity(U) Specific Activity(U/mg) % Recovery Purification Fold

Culture supernatant 117.4 2645 22.6 100 1

(NH4)2SO4 Precipitation, dialyzed (0-60%)

36.70 1940.40 52.87 73.3 2.33

DEAE –cellulose 1.79 246.15 137.54 9.3 6.08

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Purification of Protease from Pseudomonas Thermaerum GW1 Isolated The Open Microbiology Journal, 2010, Volume 4 73

Table 5. Enzyme was Incubated with Various Metal Ions at 45°C for 30 Min

Metal Ion (5 mM) Relative protease Activity (%)

Control

CuSO4

MnSO4

HgCl2

FeCl3

CaCl2

MgCl2

ZnSO4

100

109.57

546.80

69.14

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174.46

80.85

Fig. (7). Figure denotes Influence of different solvents on activity of the purified protease from Pseudomonas thermaerum GW1: puri-fied enzyme (10 g) was preincubated with 100% v/v of various solvents vis Isopropanol (IP), Methanol (M), Benzene (B), Ethy-lacetate (EA), Acetone (A), DMSO (D), Hexane (H), Glycerol (G), Toluene (T), Sucrose (S) at 37°C for 30 minutes. The control (without organic solvent) and treated enzyme solution was cooled rapidly in ice and the relative activity was measured under standard condition as described in methods. Note: the relative enzyme activ-ity of control fraction without organic solvent (0% v/v) was denoted as 100% The relative activity of treated fractions was defined as the percentage of activity detected with respect to the control fraction.

Table 6. Stability of Enzyme in the Presence of Glycerol

Glycerol (%) Relative Protease Activity (%)

Control (without glycerol) 100

10 62.6

20 55.2

40 25.2

60 23.9

80 17.3

100 0

ACKNOWLEDGMENT

We are thankful to Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) Noida, India for providing infrastructure facilities for this study.

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74 The Open Microbiology Journal, 2010, Volume 4 Gaur et al.

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Received: June 07, 2010 Revised: June 22, 2010 Accepted: June 25, 2010 © Gaur et al.; Licensee Bentham Open.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

Paper Inpress

Applied Biochemistry and Microbiology

[Indexed in Scopus, Impact factor 0.67]

Isolation and characterization of Feather degrading enzymes from

Bacillus megaterium SN1 isolated from Ghazipur poultry waste site Sarita Agrahari and Neeraj Wadhwa*

Department of Biotechnology, Jaypee Institute of Information Technology University, A-

10 sec 62, Noida, Uttar Pradesh, India.

Abstract

The strain Bacillus megateruim SN1, isolated from soil of Poultry waste site produced

extra cellular caseinolytic enzyme and keratinolytic enzymes in basal media at 30°C, 160

rpm in presence of 10% feather. Feathers were completely degraded after 72 hrs of

incubation. The caesinolytic enzyme was separated from the basal media following

ammonium sulphate precipitation and ion exchange chromatography. We report 29.28

fold purification of protease after 25 Q sepharose ion exchange chromatography. Protease

activity increased by two fold in presence of Mn2+ (10mM) whereas Ba2+, Hg2+ inhibited

protease activity. Ratio of milk clotting activity to caseinolytic activity of the 30-60%

ammonium sulphate fraction was found to be 520.84 in presence of Mn2+ ion suggesting

potential application in dairy industry. Keratinase was purified to 655.64 fold with

specific activity of 544.69U/mg protein and 12.4% recovery. The molecular weight of

this enzyme was estimated to be 30 kDa as shown by SDS PAGE and zymography

studies.

Paper InPress

Journal of Pharmacy Research

[Indexed in SCOPUS, DOAJ, Impact factor: 1.09]

Response Surface Methodology and Resilient Back Propagation Based

Yield Prediction of Protease from Bacillus megaterium SN1 Wadhwa N., Asawa K., Agrahari S.

Jaypee Institute of Information Technology University, A-10, Sector 62, Noida, Uttar

Pradesh, India.

Abstract

A Bacillus strain was isolated from soil of a regular feather dumping site of

Ghazipur poultry processing plant, Ghaziabad, India. Strain was identified as Bacillus

megaterium SN1 after morphological, biochemical and cultural characteristic and was

found to produce protease and keratinase extracellularly in the media. Cell free media of

twelve experimental setups that varied in their medium components viz. NaCl, Yeast

extract and Feather were checked for specific activity of protease and keratinase. To

obtain the best prediction by the neural network several architectures were trained and

evaluated using these obtained 12 experimental data set. Resilient Backpropagation

(RPROP) was concluded as best architecture with the order of error 1.88e-18. Codes were

developed by MATLAB 2007. Optimum production of protease forecasted according to

ANN studies could be generated when factors Yeast extract is 3.75 gms, NaCl is 0.1875

gms and Feather is 1.25 gms. These combinations yielded a specific activity of 4.6

units/mg protein.

APPENDIX B INTERNATIONAL CONFERENCES

223

Production of industrially important food and feed enzymes from

Bacillus thuringiensis SN2 isolated from Ghazipur poultry waste site.

Sarita Agrahari , Neeraj Wadhwa*

Department of Biotechnology, Jaypee Institute of Information Technology University, A-

10 sec 62, Noida, Uttar Pradesh, India.

*Corresponding author. Tel.: + (91)-120-2594207 fax: + (91)-120-2400986.

E-mail address: neeraj.wadhwa@jiit.ac.in; hisarita@gmail.com

Abstract:

Four bacterial isolates were screened from soil sample of Ghazipur poultry waste site,

Ghaziabad, India. One isolate Pseudomonas thermaerum GW1 was not able to degrade

feather and rest three isolates designated as SN1, SN2, SN3 were able to degrade feather.

The isolate SN2 was identified as B. thuringenesis. The strain SN2 was maintained at

30°C for 96 hrs in feather meal media 2 and then centrifuged and supernatant was

checked for caesinolytic activity and keratinolytic activity. Partial purification of enzyme

using 0-80% ammonium sulphate precipitation resulted in 1.8 fold increase in enzyme

activity. We report maximum caesinolytic activity on 3rd

of incubation at pH 5 and 40ºC

where as keratinolytic activity on 4th

day of incubation at pH 3 and 50ºC. Interestingly

Mn2+

and Ba2+

strongly activated caesinolytic activity and keratinolytic activity

respectively. Further investigation on milk clotting showed the presence of 8.33 SU/ml

suggesting potential application in dairy industry.

Keywords: Bacillus, Feather, Milk clotting activity, Keratinase

World Academy of Science, Engineering and Technology 69 2010

1355

includes: Antennas, Radar, and Wave Propagation Robotics and Applications Nanotechnology and Applications and

Computational Bioscience

IASTED TechnologyConferences 2010

November 1 – 3, 2010 | Cambridge, Massachusetts, USA

The International Association of Science and Technology for Development

Conference Program

SPONSORSThe International Association of Science and Technology for Development (IASTED) • Technical Committee on RoboticsInternational Journal of Robotics and Automation World Modelling and Simulation Forum (WMSF)

LOCATIONLe Méridien Cambridge-MIT, 20 Sidney Street, Cambridge, MA 02139 USA Phone: (617) 577-0200 Fax: (617) 494-8366

705-7, 728

Cambridge, Massachusetts, USANovember 1 – 3, 2010

http://sim.confex.com/sim/2009/techprogram/P10906.HTM

PRODUCTION OF ENZYMES AND DEGRADATION OF

FEATHERS BY SOIL MICROBES

Synopsis submitted in fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY By

SARITA AGRAHARI

Department of Biotechnology

JAYPEE INSTITUE OF INFORMATION TECHNOLOGY (DECLARED DEEMED TO BE UNIVERSIY U/S 3 OF THE UGC ACT 1956)

A-10, SECTOR-62, NOIDA,U.P., INDIA

February 2011

SYNOPSIS - 1 -

Enzymes are a very well established product in biotechnology [1], sales from US have

been from $1.3 billion in 2002 to US $5.1 billion in 2009 and is anticipated to reach $7 billion by

2013 [2, 3, 4, 5, 6]. A recent survey on world sales of enzymes ascribes 31% for food enzymes,

6% for feed enzymes and the remaining for technical enzymes [7]. This pattern corresponds to a

rise in global demand of about 6% yearly [4, 6]. This includes enzymes required in large scale

for application in food and feed [8, 9], where the amino acids segment will have the share of the

market at $7.8 billion in 2013 [10]. Other technical enzymes are used in the detergent, personal

care, leather, textile and pulp, and paper industries [11]. Major enzyme producers are located in

Europe, USA and Japan. Denmark is dominating, with major players like Novozymes (45%),

Danisco (17%), Genencor (USA), DSM (The Netherlands) and BASF (Germany) [7, 8, 10]. The

pace of development in emerging markets suggested that companies from India and China can

join this restricted party in a very near future [12, 13, 14, 15].

Feathers are produced in large amount as a waste by poultry product processing

plants; it reaches millions of tons per year worldwide [16]. They can be degraded by

keratinolytic bacteria. A number of keratinolytic microorganisms have been reported, including

some species of fungi such as Microsporum [17], Trichophyton [18], Bacillus [19, 20, 21],

Streptomyces [22, 23, 24] and Actinomycetes [25, 26].Till date most of the purified keratinase

cannot completely degrade keratin, their exact nature and uniqueness for keratinolysis is still not

clear, so there is a requirement to isolate new sources of microbial keratinases to meet the

industrial demand.

The innovative aspect of the present work is to identify new sources of keratinases producing

microbes from soil of feather dumping sites and this can have positive effect in solid waste

management.

The objective of the present work was

i. To identify the new sources of keratinolytic bacteria from soil sample of feather dumping

site at Ghazipur poultry waste site near our institute.

ii. Isolation, characterization, purification and optimization of enzymes produced by isolated

bacteria and check for its application

SYNOPSIS - 2 -

We describe the protocol for production of caesinolytic enzymes and keratinolytic

enzymes from microbes of soil found at dumping site of Ghazipur poultry processing plant,

Ghaziabad. The purified enzyme has application in food and feed industry. Optimization studies

for production of enzyme were also performed.

The strategy followed is as below

SAMPLE:

Soil and feather waste sample from poultry waste

site

A

Screening, Isolation & Identification of

new feather degrading bacteria

B

Isolation, Purification and

Characterization of extracellular enzyme

C

Industrial application and

Optimization of enzyme

We describe in our work that

On Screening for keratinolytic enzyme and caesinolytic enzyme producing microbes.

Three microbial sources after initial screening the enzyme were selected and identified

as B. megaterium SN1, B. thuringenesis SN2, B. Pumilis SN3, these produced acidic

enzyme extracellulary as reported by Agrahari et. al. 2010 [28].

Our Studies involving application of isolated enzymes: We report that feather was

degraded in cultivation media with the isolated microbes separately and enzymes

SYNOPSIS - 3 -

isolated from this media was found to have milk clotting activity Agrahari et. al. 2010

[29]. These enzymes have been purified and characterized. Agrahari et. al. 2010 [32].

Our studies involving optimization of enzyme production from B. megaterium SN1. We

have varied the various components in the media and the specific activity of both the

enzymes was determined. Resilient back propagation- RPROP, neural network was used

to predict the best combination. Asawa et. al. 2010 and Wadhwa et. al. 2010 [31, 35].

Soil sample was collected from Ghazipur poultry waste site, Ghaziabad, India, a feather

dumping site. Soil sample was inoculated in three enrichment media, our results depict that

optimal medium for caesinolytic enzyme and keratinolytic enzyme production is feather meal

media 2 and colonies producing clear zone in feather meal agar were selected and identified as

B. megaterium SN1, B. thuringenesis SN2, B. Pumilis SN3 were able to degrade chicken and

pigeon feathers. They produced extracellularly keratinolytic enzymes in enrichment media with

10% Feather meal powder [28]. Earlier studies from our lab involving screening of micro-

organism from same soil sample of dumping site of Ghazipur poultry processing plant, we have

reported isolation of Pseudomonas thermaerum GW1, GenBank accession GU95151, this

bacteria showed proteolytic activity but not keratinolytic activity [30].

All bacterial isolates of Bacillus sp. SN1, SN2, SN3 crude should presence of

caesinolytic activity and milk clotting activity in crude and ammonium sulphate fraction. Highest

ratio (520.84) of milk clotting activity to caesinolytic activity was seen in presence of CaCl2 and

MnSO4. 30-60% ammonium sulphate of Bacillus sp. SN1, 0-30% ammonium sulphate fraction

of Bacillus sp. SN1, 0-80% ammonium sulphate fraction of Bacillus sp. SN3 and Bacillus sp.

SN2 too showed milk clotting activity. Antibacterial activity against Bacillus subtilis (MTCC

1789), Bacillus amyloliquifaceance (MTCC 1270) and Escherichia coli (MTCC 1695) and

Bacillus sp. SN2 could inhibit M. luteus and Bacillus subtilis, Bacillus amyloliquifaceance,

Escherichia coli whereas Bacillus sp. SN3 showed against Bacillus subtilis (MTCC 1789),

Pseudomonas fluroscence (MTCC 2421) as reported by Agrahari et. al. 2010 [29].

SYNOPSIS - 4 -

The strain B. megaterium SN1, B. thuringenesis SN2 produces extracellular caesinolytic

enzyme and keratinolytic enzyme in feather meal media 2 that was maintained at 30°C, 160 rpm

for 72 hrs and 96hrs respectively. Enzyme of B. megaterium SN1 was purified by ammonium

sulphate precipitation and 25Q sephrose chromatography and casein zymography studies showed

that enzyme is having molecular weight 30 kDa. The optimum pH for the proteolytic and

keratinolytic activity was pH 3 and at 60°C and 70°C temp respectively. Interestingly Mn2+

(10mM) strongly activated caesinolytic enzyme and keratinolytic enzyme activity by 2.1, 1.17

fold respectively. While Hg2+ strongly inhibited caesinolytic enzyme and keratinolytic enzyme

activity [32, 33].

Caseinolytic activity from B. thuringenesis SN2 is reported in 0-80% ammonium

sulphate precipitation. Casein zymography studies showed that enzyme has molecular weight of

80 kDa, 60 kDa and 40 kDa. We report optimum pH for caesinolytic enzyme activity was at pH

5 and 40ºC where as keratinolytic enzyme activity at pH 3 and 50ºC. Interestingly Mn2+ strongly

activated caesinolytic enzyme activity by 3.74 fold. Ba2+ strongly activated keratinolytic enzyme

activity by 1.9 fold. Whereas Ba2+ and Fe2+ strongly inhibited caesinolytic enzyme activity and

keratinolytic enzyme activity respectively [34].

To develop a process for the optimum production of caseinolytic enzyme from poultry

feather, standardization of media components is crucial. We selected Bacillus megaterium SN1

that is competent of rapidly degrading native feather for our optimization studies. The various

components in the media was varied and the specific activity of the enzyme was determined.

Resilient back propagation- RPROP, neural network was used to predict the best combination

and validated. To optimize these three significant medium constituents viz., NaCl, Yeast extract

and Feather were chosen in our experimental design. The optimization studies suggest NaCl,

Yeast extract are insignificant variables, however Feather had a profound effect on yield of

keratinolytic enzymes NaCl 0.5gm, Yeast extract 0.13 gm and Feather 15g (-1, 1, 1) yielded the

maximum amount of caesinolytic enzyme (24.292 U/mg) and for keratinolytic enzyme

production, it is evident that presence of feather is significant. The optimum combination being

NaCl 0.5gm, Yeast extract 0.1 gm and Feather 10g (-1,-1, 0) yielded the maximum amount of

keratinolytic enzyme (17.2314 U/mg protein).

SYNOPSIS - 5 -

The prediction method used is found that the trained network is a better option to predict new

data points thus providing a mathematical alternative to quadratic polynomial required for data

derived from statistically designed experiment [31, 35].

In future optimization with other factors can be studied and predicted. Amino acid

sequence determination of purified enzyme from B. megaterium SN1, B. thuringenesis SN2

would be performed and checked for innovative application in other biotechnology industries.

Future studies regarding upgrading the caesinolytic and keratinolytic enzyme production

technology from laboratory to a large-scale process is to be performed.

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28. Agrahari S., Wadhwa N., “Degradation of chicken feather a poultry waste product by

keratinolytic bacteria isolated from dumping site at Ghazipur poultry processing plant”,

International Journal of Poultry Science, vol. 9(5), pp. 482-489, 2010.

29. Agrahari S., Wadhwa N., “Production of extra cellular milk clotting enzyme from

isolated Bacillus sp.”, Journal of Pharmacy Research, vol.3 (12), pp. 2924-2927, 2010.

30. Gaur S., Agrahari S., Wadhwa N., “Purification of protease from Pseudomonas

thermaerum GW1 isolated from poultry waste site”, The Open Microbiology, vol. 4, pp.

67-74, 2010.

31. Asawa K., Wadhwa N., Agrahari S., “Resilient Back Propagation Based Yield Prediction

of Keratinase from Bacillus megaterium SN1” (Oral presentation) at IASTED

Technology Conferences 2010 November 1–3, 2010 Cambridge, Massachusetts, USA

32. Agrahari S., Wadhwa N., “Isolation and characterization of Feather degrading enzymes

from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site”, Submitted in

Applied Biochemistry and Microbiology. (Inpress)

33. Gupta S., Gaur S., Wadhwa N., “Production of extracellularly secreted keratinase and

protease from bacteria of poultry waste site” (Oral presentation) at International

Conference on Emerging trends in Environmental Research (St Albert's College,

Ernakulam) Kerala, 14th -16th August 2009.

34. Agrahari S., Wadhwa N., “Production of industrially important food and feed enzymes

from Bacillus thuringiensis SN2 isolated from Ghazipur poultry waste site” WASET

proceeding year 6 issue 69 August 2010. (Oral presentation) at ICBFE 2010:

"International Conference on Biotechnology and Food Engineering" Singapore, 25th -27th

August 2010.

SYNOPSIS - 9 -

35. Wadhwa N., Asawa K., Agrahari S., “Response Surface Methodology and Resilient Back

Propagation Based Yield Prediction of Protease from Bacillus megaterium SN1”, Journal

of Pharmacy Research (Inpress).

SYNOPSIS - 10 -

AUTHOR’S PUBLICATION

Journals (International)

[1] Agrahari S., Wadhwa N., “Isolation and characterization of Feather degrading

enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site”,

Applied Biochemistry and Microbiology (InPress) [Indexed SCOPUS, Impact factor

0.67] (Accepted on 7th December 2010)

[2] Agrahari S., Wadhwa N., “Production of extra cellular milk clotting enzyme from

isolated Bacillus sp.”, Journal of Pharmacy Research, vol.3 (12), pp. 2924-2927, 2010.

[Indexed in SCOPUS, DOAJ Impact factor: 1.09].

[3] Agrahari S., Wadhwa N., “Degradation of Chicken Feather a Poultry Waste Product

by keratinolytic Bacteria Isolated from Dumping Site at Ghazipur Poultry Processing

plan”, International Journal of Poultry Science vol. 9 (5), pp. 482-489, 2010. [Indexed

in DOAJ and SCOPUS]

[4] Wadhwa N., Asawa K., Agrahari S., “Response Surface Methodology and Resilient

Back Propagation Based Yield Prediction of Protease from Bacillus megaterium SN1”,

Journal of Pharmacy Research. (InPress) [Indexed SCOPUS and DOAJ, Impact factor

1.09] (Accepted on 27th December 2010)

[5] Kaushik P., Batra E., Juneja N., Tushar, A., Kohli S., Suchit, A., Agrahari S., Rani V.

and Wadhwa N., “Phytochemical screening of developing garlic and effect of its

aqueous extracts on viability of cardiac cell line: A comparative study”, Journal of

Pharmacy Research. (InPress) [Indexed SCOPUS and DOAJ, Impact factor 1.09]

(Accepted on 26th January 2011)

[6] Gaur S., Agrahari S., Wadhwa N., “Purification of protease from Pseudomonas

thermaerum GW1 isolated from poultry waste site,” The Open Microbiology vol. 4, pp.

67-74, 2010. [Indexed in Pubmed Central, Cab Abstracts and DOAJ]

SYNOPSIS - 11 -

International Conference (Abroad)

[7] Agrahari S., Wadhwa N., “Production of industrially important food and feed enzymes

from Bacillus thuringiensis SN2 isolated from Ghazipur poultry waste site” (Oral

presentation) at ICBFE 2010: “International Conference on Biotechnology and Food

Engineering” Singapore, 25th -27th August 2010. (Travel grant was awarded by

Department of Biotechnology) [Indexed in DOAJ]

[8] Wadhwa N., Asawa K., Agrahari S., “Optimization studies of medium components for

protease production from Pseudomonas thermaerum GW1”, Submitted to (Oral

Presentation) Enzyme Engineering XXI , An ECI Conference Series, September 18-23,

2011, Vail, Colorado. USA

[9] Asawa K., Wadhwa N., Agrahari S., “Resilient Back Propagation Based yield

prediction of keratinase from Bacillus megaterium SN1” (Oral presentation) at

IASTED Technology Conferences 2010 November 1–3, 2010 Cambridge,

Massachusetts, USA [Indexed in Web of Science, Scopus]

[10] Gaur S., Gupta S. and Wadhwa N., “Isolation of Protease and Keratinase from

Microbes Isolated From Ghazipur Poultry Waste Site, Ghaziabad, India”, (Oral

Presentation), at SIM Annual Meeting and Exhibition Industrial Microbiology and

Biotechnology, Toronto, Canada. July. 26–30, 2009.

International Conference (India)

[11] Gupta S., Gaur S. and Wadhwa N., “Production of extracellularly secreted keratinase

and protease from bacteria of poultry waste site” (Oral presentation) at International

Conference on Emerging trends in Environmental Research (St Albert's College,

Ernakulam) Kerala, 14th -16th August 2009.

[12] Gupta S., Gupta P., Tyagi S., Gupta S., Gaur S. and Wadhwa N., “Potential

application of protease from senesced leaves of banana (Musa paradisiaca)” (Poster

presentation) at International conference on Emerging trends in Biotechnolgy (ETBT)

and 6th annual convention of the Biotech Reserch Society India (BRSI) at Banaras

Hindu University, Varanasi, 4-6 December 2009.

SYNOPSIS - 12 -