SCREENING, ISOLATION AND CHARACTERISATION OF PROTEASE PRODUCING MODERATELY HALOPHILIC MICROORGANISMS INTRODUCTION

Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (4) : 2012 : 603-612© Global Science PublicationsISSN-0972-3005

SCREENING, ISOLATION AND CHARACTERISATION OFPROTEASE PRODUCING MODERATELY HALOPHILIC

MICROORGANISMS

ANNAPURNA S.A. 1, AMARNATH SINGH 1, SHASHANK GARG 1,ANUPAM KUMAR 1 AND HARSH KUMAR 1

1 Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India

(Received 20 October, 2012; Accepted 10 November, 2012)

Key words : Moderately halophiles, Proteases, Silver recovery, Dehairing, Blood destaining

Abstract - The present study is focused on isolation of moderately halophilic bacteria and fungi that arecapable of producing protease from the samples collected from Sambhar lake of Rajasthan andMumbai seashore. Extremely and moderately halophilic bacteria dominate in saline environments(0.5% to saturated NaCl). Moderately halophilic microorganisms include a broad variety of bacteriaand fungi that are able to grow in media containing a wide range of elevated NaCl concentrations (3-15% NaCl). Considerable attention has been focused on enzymes of moderately halophilic bacteria,since they have substantial biotechnological potential. While several proteases from extremehalophiles, members of the haloarchaea have been characterized, fewer proteases from moderatelyhalophilic bacteria have been purified and studied in depth. These microorganisms use differentstrategies for preserving their cell structure and function in highly saline conditions. They may producecompounds of industrial interest, such as extracellular hydrolytic enzymes with diverse potentialapplications in the industries. Therefore, an attempt is done in the present study for isolation ofmoderately halophilic bacteria and fungi able to produce protease which is of industrial importance.The samples collected from Sambhar lake of Rajasthan and Mumbai seashore were processed for theisolation and characterization of the bacteria and fungi able to produce the protease enzyme whichfurther was shown to have the proteolytic activity demonstrated by using blood stained surgicalinstruments, hair, and casein. The recovery of silver from the X ray photographic film was alsoattempted. The molecular sequencing of the fungus and bacteria with the phylogenetic tree formulatedrevealed the presence of Aspergillus flavus in the sample from Mumbai sea shore and Bacillus subtilisfrom Sambhar lake of Rajasthan. In the subsequent phase effect of temperature, pH, activator andinhibitor was also studied on the activity of protease enzyme by using casein and hair as substrates.The enzyme further was shown to have the ability to remove the blood stain from the surgicalinstruments as well as to recover silver from the X ray film. The isolate Bacillus subtilis optimally grownat pH-8 and temperature 37°C and 10% NaCl with protease activity on casein and human hair. Proteaseenzyme obtained in the present study by using Bacillus subtilis and Aspergillus flavus is found to be stableand active so could be of significant use for detergent and leather processing technology as well as fordehairing and silver recovery.

*Corresponding authors email : [email protected]

INTRODUCTION

Microbial proteases account for approximately60% of the total enzyme sales in the world.According to the market research report availableon world enzymes published in 2007, the worldmarket for enzymes is expected to grow 7.6% peryear to $6 billion in 2011, (Shama and Hameed

Abdul, 2011). The vast variety of proteases, withthe specificity of their action and application hasattracted worldwide attention to exploit theirphysiological as well as biotechnologicalapplications (Poldermans, 1990). The enzymesproduced by halophilic microorganisms haveidentical enzymatic features like their non-halophilic equivalent, but they exhibit different

604 ANNAPURNA ET AL.

properties due to structure difference.Adaptation to such high salt containing

environments has evolved unique properties inthese microorganisms with considerable biotech-nological potential. Halophiles produce a range ofunique and stable biomolecules which havepractical applications including hydrolyticenzymes like lipases, DNAases,, geltinases,amylases and proteases. Therefore, proteaseproducing strains of halophilic archaea can beutilized for commercial salt-based applicationssuch as detergent industry and silver recovery fromX-ray photographic film.

There is numerous numbers of enzymes of thiskind produced by some halophilic microorganismsthat have optimal activity at high salinities and itcan be used in many harsh industrial processeswhere the concentrated salt solutions used wouldotherwise inhibit many enzymatic conversions(Dodia et al.2006; Gupta et al., 2002). Halophilicorganisms have evolved in saline environment andare able to strongly resist the deleterious effects ofsalt. A number of halophilic proteases have beenpurified, characterized, showed to possessmaximum activity at neutral pH and at atemperature ranging from 55 ºC -66ºC, (Kamekuraand Seno, 1990). Halophilic proteases haveextensive application in the processing of food,leather and detergents. Potential applications ofhalophilic proteases and the need to investigate fornew halophilic organisms producing maximumenzyme production is a continuous process.

The majority of the Gram positive or Gram-variable, endospore forming rods with halotolerantproperties have been assigned to the genus Bacillus.Bacillus subtilis grows in a pH range of 7.0-11.0 andproduces extracellular protease. At present, a largesection of commercially available alkalineproteases are derived from different strains ofBacillus subtilis, Aspergillus flavus and Trichodermaspecies.

Halophilic microorganisms grow in unfavorablecondition like high salt concentration, so theyhaving potential to utilize natural carbon sourcevery efficiently to produce enzyme. Proteases havebeen used in the hide-derailing process carried outat pH values between 8 and 10. Proteases are alsouseful and important components in biopharma-ceutical products such as contact-lens enzymecleaners and enzymic debriders (Gupta et al., 2002).Kuberan et al., (2010) reported screening ofhalophylic proteolytic bacteria from the salt pan in

Tuticorin, Tamilnadu and using wheat bran andmung dal husk as substrate for solid statefermentation. In 2007, Sreenivas et al., 2007reported that screening of the brine sample of asolar saltern that yielded one moderately halophilicprotease producing bacteria identified asPseudomonas sp., which produced an extracellularprotease and an amylase. Optimum proteaseproduction was shown to occur in stationaryphase.

Due to natural and man-made global changes,hyper saline environments are increasing day today. Moreover, hyper saline environments caneasily be created by the concentration of sea waterin arid environments.

There is numerous numbers of enzymes of thiskind produced by some halophilic microorganismsthat have optimal activity at high salinities and itcan be used in many harsh industrial processeswhere the concentrated salt solutions used wouldotherwise inhibit many enzymatic conversions.Halophilic microorganisms play an essential role ina variety of fermentation processes that occur inthe presence of salt (Malashetty et al., 2009).Halophilic proteases have extensive application inthe processing of food leather and detergents.Potential applications of halophilic proteases andthe need to investigate for new halophilicorganisms producing maximum enzymeproduction is a continuous process. Proteases fromthese organisms are one interesting group ofhydrolytic enzymes with wide range ofcommercial applications that have not beenextensively studied.

Isolation and characterization of the halophilic-alkalophilic bacteria from saline habitat in westernIndia was reported by Dodia et al., (2006). Differentbacterial strains isolated using enrichmenttechnique at 20% (w/v) NaCl and pH 10. Theactivity and stability of the alkaline protease in abroader range of pH and salt was found to makethis enzyme an important candidate for variousindustrial applications.

Production dynamics of extracellular proteasefrom Bacillus species was done by Olajuyigbe andAjele (2005).They isolated 25 strains of Bacillus andcompared the characteristics of proteases producedby them. Sharmin and Rahman (2007) worked onthe isolation and characterization of proteaseproducing Bacillus strain and it was concluded thatthe enzyme isolated seem to be the alkalinemetalloprotease and was capable of dehairing of

605Screening, Isolation and Characterisation of Protease Producing Moderately

skin and hides. In a study on the partialcharacterization of bacterial proteases done byKaur and Pandey (2009), the crude bacterialproteases produced by Bacillus sp. was partiallycharacterized by optimizing pH and temperaturefor the proteolytic enzyme activity and the studyof thermo stability and pH stability profile of theproteases. In the same study the effect of chelatingand oxidizing agents on enzyme activity was alsoreported. The findings indicated that the enzymecould be used in industrial applications afterfurther characterization studies.

Isolation of halophilic fungi was carried out byEl-Meleigy et al., (2010) from Sharm El-Sheikh andfrom Raas Sader regions at Sinai-Egypt. The fungalisolates tolerated till 30% and 25 % (W/v) NaClrespectively. The isolates were found to beTrichoderma piluliferum fs. halophila AZ and Aspergillusrestrictus and were shown to have manybiotechnological applications. Kamath et al., (2010)reported that paddy soil fungal isolate identified tobe a strain of Aspergillus niger from Manipal. Charleset al., (2010) reported that Aspergillus nidulans is ahighly potent fungus used in the production ofalkaline protease and was isolated from poultryfarm soil. Extracellular alkaline protease waspurified from Aspergillus nidulans in a two-stepprocedure involving ammonium sulphateprecipitation and Sephadex G-100 columnchromatography. The enzyme was more stableover a wide range of pH (6-10) and thetemperatures up to 50 °C. It showed optimumenzyme activity at pH 8.0 and a temperature of 35°C.Oyeleke, et al., (2010) reported the isolation ofAspergillus fumigates and Aspergillus fumigatusfrom soil. Alkaline and acidic proteases producedby these fungi were characterized. Different agro-industrial waste products were evaluated to checkthe possibility of potential utilization of substratesin SSF for protease production by Aspergillus flavususing wheat bran as a substrate (Chinnasamy, etal., 2011). The optimum incubation temperature forthe production of protease was found as 30°Cduring this study.

In this present study an effort is done to isolateand characterise the microorganisms from the soilsample collected from Sambar lake of Rajasthanand Mumbai sea shore and the conditions wereoptimized for the production of protease in thefermentation medium. The enzyme was furthercharacterized to know the influence of pH,

temperature, activator and inhibitor. Thedehairing, caseinolytic, blood removing and silverrecovering properties of the enzyme was studiedindicating the possible application of the enzyme inthe industries.

MATERIALS AND METHODS

The soil samples were collected from Sambhar lake(Rajasthan) and Mumbai Seashore. After collectionthese samples were air dried and then processedfurther. Serial dilution was done for isolation oforganisms. Halophilic medium consisting of Yeastextract 1% (w/v), Peptone 0.2% (w/v),NaCl 10%(w/v), MgSO40.2% (w/v),Tri sodium citrate 0.1% (w/v),Agar 2% (w/v) pH 7.5 (Lakshmi Sreenivas etal.,2007) was used for the isolation of bacteria andthe medium with Yeastextract-1%(w/v),Glucose-6%(w/v),Maltextract-1%(w/v),FeSO4.7H2O-01%(w/v),MgSO4-0.5%(w/v), K2HPO4-0.5%(w/v), Peptone-2%(w/v), NaCl-10%(w/v), Agar 2% (w/v),(Srinubabu et al., 2007) was used for the isolation offungi.

For bacteria the Petriplates were incubated at37°C for 24 hours and for fungus the petriplateswere incubated at 27°C for 4 to 6 days. Theobtained colonies were checked for culturalcharacteristics, morphology. For bacteria variousbiochemical reactions were conducted. Both theisolates were checked for the production ofprotease on skim milk agar plate (Aneja, 2003).

Both bacteria and fungi were outsourced formolecular characterization for the confirmation ofthe isolates by 16S rRNA &18S rRNA gene sequenceanalysis (Courtesy: Bioserve lab, Hyderabad).Protease production was studied at different pH (5-10) and temperature (20°C ,27°C, 37°C) for fungiand at different pH (5-10) and temperature (30°C,37°C, 47°C, 57°C) for bacteria by inoculating intothe medium mentioned above without agar. Theinoculated medium with fungus was incubated for168-192 hours on shaker and for bacteria for 48-72hours on shaker. At the end of fermentation period,the culture medium was centrifuged at 10,000 rpmfor 15 min to obtain the crude extract, which wasused as enzyme source.

Assay for protease activity was done by usinghair as substrate, (Raju et al., 2007) and casein assubstrate, (Kanmani et al.,2011). The enzymeactivity was calculated by using standard graph oftyrosine (Clark et al., 1977; Raju et al., 2007). In thesubsequent phase, various surgical instument


stained with blood were taken and subjected toevaluate the washing performance at 400C bycommercial detergent and the enzyme and checkedfor the cleansing property. (Vishalakshi et al., 2009).Silver recovery from X-ray photographic film byhydrolysis of gelatin layer was done by using thebacterial and fungal proteases, (Shankar et al.,2010). X-ray films were washed with distilledwater and wiped with cotton impregnated withethanol; washed film was dried at 40°C for 30 min.One g of X-ray film cut into 2 x 2 cm pieces wasthen incubated with 10 mL of crude protease (suchthat the film is completely immersed in thesolution) at 40°C, (pH 10) in a water bath withcontinuous shaking. Turbidity of the reactionmixture (hydrolysate) increased with time (as thehydrolysis progressed) and no further increase inturbidity was observed when hydrolysis wascomplete. Hence, progress of hydrolysis i.e.turbidity was monitored by measuring theabsorbance at 660 nm.

Detection of Silver was done by using Nitric Acid -(http://www.howtotestsilver.com/)

A drop of stripping solution along with enzymewas taken on a slide and a drop of nitric acid wasapplied and waited for change in colour.

The interpretation was done as below based onthe colour of the reaction.

Creamy colour - High quality silver, Black colour- Coin quality silver ,Green colour -High amountsof copper ( poor quality silver) ,Gold colour- lot ofbrass (it means this is silver plated item).

Effect of pH (5-10), Temperature ranging from27°C, 37°C, 47°C, 57°C for fungus and 37°C, 47°C,57°C, 67°C for bacteria were also studied as wellas effect of inhibitor (Hg Cl2 ) and effect of activator(CaCl2) on the activity of protease was also studiedin the subsequent phase.

RESULTS

Processing of soil sample collected from Sambharlake (Rajasthan) has resulted in the isolation ofBacillus subtilis and soil sample collected fromMumbai Sea shore, has resulted in the isolation ofAspergillus flavus and both were confirmed bymolecular characterization in addition to the studyof morphology by lacto phenol blue staining forfungus and by Gram’s staining and biochemicalreactions for bacteria. Both the isolates werepositive for casein hydrolysis indicating theproteolytic activity.

Optimum pH and temperature for growth andprotease production was studied in halophilicmedium with pH (5-10) and temperature (300C,370C, 470C, 570C) respectively. The Fig. 1 depicts theinfluence of pH and Fig. 2 depicts the influence oftemperature on the production of protease enzymeby Bacillus subtilis.

The culture supernatant containing protease ispartially purified by 40% and 70% ammoniumsulphate precipitation and was used for checkingthe blood stain removing property.

The influence of pH and temperature on theproduction of protease enzyme by Aspergillus flavusis shown in Fig. 3 and 4.

DISCUSSION

Although proteases are widespread in nature,microbes serve as a preferred source of theseenzymes because of their rapid growth, the limitedspace required for their cultivation and the easewith which they can be genetically manipulated togenerate new enzymes with altered properties thatare desirable for their various applications.Proteolytic bacteria are widespread in nature andare able to grow under various growth conditions,such as different temperatures, pH and ionicstrength. The presence of halotolerant protease inthe halophilic bacteria could be applied inindustrial processes where the concentrated saltsolution used would inhibit ordinary proteases. Inthe present the isolates Bacillus subtilis and Aspergillusflavus have shown optimum pH and temperaturefor protease production with protease activity pH8 and temperature 37°C (8.27 µg/min/mL) and pH 6and temperature 27°C (4.91µg/min/mL). In a similarstudy Vijay anand et al., (2010) reported optimumpH and temperature to be 8 and 40°C respectively.Benazir et al., (2011) reported that optimum pH forprotease production was 8 and temperature was40°C. Similarly Dodia et al., (2006) reportedoptimum pH 10 and temperature was 37°Crespectively.

Temperature also plays an important role inactivation, inactivation and stability of enzymes.Each enzyme has an optimum temperature formaximum enzyme activity. Effect of temperatureon protease activity (caseinolytic and keratinolyticactivity) studied between ranges of 27°C -67°C forbacterial and fungal isolates in presence ofsubstrate casein and hair. The maximum enzymeactivity of Bacillus sp. was obtained at temperature-


Fig. 1 Influence of pH on the production ofprotease by Bacillus subtilis

Fig. 2 Influence of temperature on the productionof protease by Bacillus subtilis

Fig. 3 Influence of pH on the production ofproteases enzyme by Aspergillus flavus

Fig. 4 Influence of temperature on the productionof protease enzyme by Aspergillus flavus

Fig. 5 Effect of pH on protease activity ofBacillus subtilis using casein as substrate

Fig. 6 Effect of temperature on protease activityof Bacillus subtilis using casein as a substrate

Fig. 7 Effect of pH on protease activity ofBacillus subtilis using Hair as substrate

Fig. 8 Effect of temperature on protease activityof Bacillus subtilis using hair as a substrate


Fig. 9 Effect of pH on protease activity ofAspergillus flavus using casein as substrate

Fig. 10 Effect of temperature on protease activity ofAspergillus flavus using casein as a substrate

Fig. 11 Effect of pH on protease activityAspergillus flavus using hair as substrate

Fig. 12 Effect of temperature on protease activity ofAspergillus flavus using hair as a substrate

Fig. 13 Effect of inhibitor (HgCl2) and activator(CaCl2) on protease activity of Bacillus subtilis

using casein as substrate

Fig. 14 Effect of inhibitor (HgCl2) and activator(CaCl2) on protease activity of Bacillus subtilis

using hair as substrate

37°C (i.e.3.60 µg/min/mL & 99.19 µg/min/mL),Aspergillus flavus was at temperature 57°C (i.e. 12.224µg/min/mL and 516.80 µg/min/mL). Similarly,Gehan et al., (2011) reported that the enzyme wasstable at a temperature range from 50°C - 70°C.And maximum enzyme stability was detected at50°C.Small alteration of the macromolecules structuremay have profound effect on protein behavior asthe biological function and native structure ofprotein are closely interconnected. The effect of

activator and inhibitor tested revealed that CaCl2(10mM) only increased the relative enzyme activityup to 12% to 25% whereas HgCl2 (10mM) act asinhibitor and reduced the protease activity ofBacillus sp., and Aspergillus flavus.. Gehan et al.,(2006) reported such activation and inhibition ofhalophilic protease by CaCl2 and HgCl2. SimilarlyJoshi et al., (2007) reported that, enzyme activitywas accelerated by the addition of CaCl2 andunusual inhibition by HgCl2 .The nature of thehalophilic and alkaline protease is assayed in


Fig. 15 Effect of inhibitor (HgCl2) and activator (CaCl2)on protease activity of Aspergillus flavus using

casein as a substrate

Fig. 16 Effect of Inhibitor ( HgCl2 ) and activator(CaCl2) on protease activity of Aspergillus flavus

using hair as a substrate

Fig. 17 Silver recovery from X-ray film byhydrolysis of gelatin layer

Fig.18 Nitric acid test (HB: Halophilic bacteria-Bacillus subtilis)

Fig. 19 Silver recovery from X-ray film byhydrolysis of gelatin layer

Fig. 20 Nitric acid test (HF: Halophilicfungus-Aspergillus flavus)

Fig. 22 Surgical instrument with Blood &detergent Fig. 21 Evaluation of washing performance of enzymewith detergent on surgical instrument


Fig. 23 Surgical instrument with Blood &detergentand partially purified enzyme

presence of inhibitor HgCl2 (10mM).Treatment of X-Ray films with protease resulted

in the silver bound with gelatin being stripped offin to the reaction mixture at pH-10 andtemperature - 400C from 20 to 30 minutes. Presenceof silver in to stripped of reaction mixture wasconfirmed with nitric acid test. Similarly Shankaret al., (2010) reported silver recovered from X-Rayphotographic film by crude protease enzymetreatment at pH-10 and temp- 400C from 6-7minutes.

Evaluation of washing performance of surgicalinstrument with partially purified enzyme alongwith detergent found that after an incubation of 20minutes stain could not be removed completely byindividual detergent treatment but combination ofenzyme with commercial detergent removed theblood stain from the surgical instrument veryeffectively. This is similar to the observation doneby Vishalakshi et al., (2009).

The increased usage of these proteases asdetergent additives is mainly due to the cleaningcapabilities of these enzymes, environmentallyacceptable. As against traditional chemicalmethods, enzymatic processes yield products ofimproved quality and reduce the use of hazardousand polluting chemicals. Properties of thisproteases such as alkaline pH, thermostability,detergent resistance, make the enzyme very usefulfor different applications. Fujiwara and co-workers(Ishikawa et al. 1993). They reported the use of analkaline protease to decompose the gelatinouscoating of X-ray films, from which silver wasrecovered. Different reports are available onindustrial application of proteases (Gupta et al.2002) with respect to properties of enzymes. Also itis useful for cleaning DNA during isolation of theDNA. Designed by nature to digest protein,

carbohydrates, fats and cellulose enzymes makecleaning easier without compromising theenvironment.

Members of the genus Bacillus are used for thesynthesis of a very wide range of importantmedical, agricultural, pharmaceutical and otherindustrial products. These include a variety ofantibiotics, enzymes, amino acids and sugars. Theresults of this work indicate that the moderatelyhalophilic bacterium, Bacillus subtilis could displaya potential role in protease production inbiotechnological processes.

In comparison, haloalkaliphilic bacteria havebeen relatively less attended, as only few alkalineproteases are reported from these organisms.Besides the novel catalytic applications, the wideoccurrence of many of such enzymes among thehaloalkaliphilic bacteria holds ecologicalsignificance. The present isolate Bacillus subtilis in thepresent study is found to have the ability toproduce the alkaline protease and could berecommended of further industrial applications.

The haloalkaliphilic Bacillus subtilis isolated inthis study, produced substantial amounts ofalkaline protease, further investigations on thefactors affecting its synthesis would be of greatsignificance. Moreover, some of the novel features ofthe enzyme such as stability over the wide range ofpH and dehairing and blood destaining propertymake it an attractive candidate for future studiesand process development.

Fungi elaborate a wide variety of proteolyticenzymes than bacteria. The filamentous fungi havea potential to grow under varying environmentalconditions such as time course, pH andtemperature, utilizing a wide variety of substratesas nutrients (Haq et al., 2006). The enzyme wasfound to have thermostability at 570 oC whencasein and hair were used as substrates. The studydone by Chinnasamy et al. (2011) showed Aspergillusflavus strain with a maximal protease productionat pH 4 and temperature 30oC which is differentfrom our study where the maximum proteaseproduction from Aspergillus flavus occurred at pH6and 27oC.

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SCREENING, ISOLATION AND CHARACTERISATION OF PROTEASE PRODUCING MODERATELY HALOPHILIC MICROORGANISMS INTRODUCTION

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