Proteolytic Enzyme Production by Isolated Serratia sp RSPB11

AbstractAn effective proteolytic enzyme

producing microbial strain has been isolated frommarine habitats and evaluated its extracellularprotease production properties with respect todifferent fermentative physiological parameters.The strain has been identified based onbiochemical tests according to Bergey’s Manualof Systematic Bacteriology as Serratia sp anddesignated as RSPB11. This strain has potentialto hydrolyze chitin, gelatin and casein revealingits industrial potential for production of multi-enzyme complex. Since the isolated strainbelongs to Serratia genus, the protease producedby this strain is considered as serralysin andwhich is not inhibited by PMSF suggesting theenzyme belongs to other than serine type ofprotease. Further analysis denoted that thisenzyme belongs to metalloprotease which isconfirmed based on negatively regulation ofcaseinolytic (proteolytic) activity by EDTA. Themaximized enzyme production occurred atmedium initially adjusted to pH 7.0 incubated at33oC under aerated environment. Analysis ofthe pH profile before and after fermentationdepicted that irrespective of initial medium pH,it is shifted to pH 9.0 after fermentationsuggesting the enzyme produced is alkaline innature.

Keywords: Enzyme, Fermentation, Isolation,Protease, Serratia sp.

IntroductionProteases/ proteinases are a group of

hydrolytic enzymes (catalyse the reaction ofhydrolysis of bonds with the participation of awater molecule) which are specific for digestionof proteins in to peptides and amino acids.Proteases differ in their ability to hydrolyzevarious peptide bonds hence, specificityassociated with each enzyme differs based oncatalytic site. Several classification systemscurrently available, provides rich and vastinformation about each and every identifiedprotease. These schemes can be categorizedunder 4 major categories based on thecharacteristic features like: pH (acidic/neutral/alkaline), peptide bond specificity (endo/exopeptidases), functional group present at activesite (serine/ cysteine/ aspartic/ metalloproteases)of proteolytic activity with respect to functionalgroup present at the active site. The inability ofthe plant and animal proteases to meet currentworld demands has led to an increased interestin microbial proteases.

Most commercially available proteasesbelong to the class serine proteases, produced

Proteolytic Enzyme Production by Isolated Serratia spRSPB11: Role of Environmental Parameters

P. Lakshmi Bhargavi and R. S. Prakasham*Bioengineering and Environmental Centre, Indian Institute of Chemical Technology

Hyderabad – 500 607, India*For Correspondence - [email protected]

Proteolytic Enzyme Production

Current Trends in Biotechnology and PharmacyVol. 6 (1) 55-65 January 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)

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by organisms belonging to the genus Bacillus,Apergillus and Streptomyces, Alkaline proteasesare more preferable at industrial scale comparedother acidic proteases. Metalloproteases arethose enzymes whose catalytic mechanisminvolves a metal which plays an important rolein pathogenesis hence have advantage in healthcare sector. Most of pharma relatedmetalloproteases are dependent on zinc (1) anda few uses cobalt, iron, manganese, etc (2).

Serratiapeptidase is a type of metallo-protease (EC 3.4.24.40) originally isolated fromdigestive system of silkworm (3). Subsequently,this enzyme also reported from different bacterialstrains including Pseudomonas auri Serratiamarcescens, Proteus mirabilis and Escherichiafreundii (4). Functionally this enzyme breakspeptide bond of non-terminal amino acids underalkaline environment hence also referred asalkaline-endopeptidase. Serrapeptidase differsfrom other metalloend-opeptidases in thecatalytic reaction, where the enzymepreferentially cleaves the peptide bondsassociated with hydrophobic residues (5).

Initially this peptidase production wasnoticed during infection by Serratia sp. as wellas other bacterial strains such as Pseudomonasaeruginosa or Erwinia chrysanthemi. Thephysiological function of serratia peptidase inthese microbial strains is yet to be identified,however the enzyme seems to play a significantrole in nutrient digestion/uptake in these bacterialstrains (6). Maeda and coworkers reported thatthis protease plays a critical role in pathogenesisof Serratia marcescens (4). Interestingly, thisenzyme production is mostly reported fromclinical isolates (4, 7) however, reports frommarine microbial strain YS-80-122,Pseudomonas sp., also noticed in the literature(8, 9).

Much attention has been focused onalkaline metalloprotease production by speciesof Serratia especially Serratia marcescens dueto its potential for higher enzyme yieldscompared to literature reports and has been usedas an anti-inflammatory agent all over the world(10). Alkaline metalloprotease production fromdifferent microbes has been evaluated by variousresearchers with respect to enzyme regulationand excretion mechanisms (11, 12),characterization and purification (10, 13), andgenetic analysis of these enzymes (14, 15).Efforts also have been made to improve theeconomics of the bioprocess by using nonconventional media components such as wheypowder or squid pen powder (16-18). At present,Serratiapeptidase, a major alkalinemetalloprotease is commercially produced witha SMP-overproducing mutant of S. marcescensATCC-21074 (19).

Analysis of biochemical and biocatalyticproperties of alkaline metalloprotease producedby different microbial strains revealed variationin specificity of action, metal component, optimalpH, temperature, etc (10) which influencesbiotechnological application specificity. This,in addition to the high price for this enzyme inthe market are some of the powerful appeals thatlead to search for new protease producingsources and subsequent bioprocess development.Microbial enzyme production is highlyinfluenced by media components like carbon andnitrogen sources besides several other factorssuch as aeration/agitation, pH and temperature,salinity and incubation time (20, 21, 22). Hence,the present study focused on isolation of abacterial strain with high protease productionproperty, growth characterization, and enzymeproduction properties in addition toidentification. The data indicated that the isolatebelongs to Serratia sp and has higher enzymetiters compared to literature reports therefore this

Lakshmi Bhargavi and Prakasham


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isolate could be effective strain for industrialproduction. This strain’s protease production ishighly regulated by chelating agents, incubationtemperature, hydrogen ion concentration in themedium, and incubation temperature.

Materials and MethodsScreening of chitinase and protease producingmicrobes: Soil samples collected from marineenvironment (contaminated with sewage fromfish processing plants) located near Bapatla,Andhra Pradesh, India were used in this study.A serial dilution method has been followed afterenrichment technique for isolation of microbialstrains. In brief, one gm of soil sample was addedto sterile 100 ml media containing mineral salts((NH

4)

2SO

4 0.1 g, KH

2PO

4 0.02 g, NaCl 0.5g,

MgSO4 0.05g) and 5gm dried shrimp as well as

crab shell powder followed by incubation for 48hat 30oC. After serial dilution, the obtainedsamples were spread over chitin agar plate(Colloidal Chitin 1.5 g, mineral salts, agar 20 g,distilled water 50 ml, sea water 50 ml, pH 7.0)and incubated for 5 days at 30oC. A clear- zoneforming bacteria were selected and inoculatedon 2% casein and 1% gelatin agar plates at pH7.0 for identification of protease production.After incubation for 2 days at room temperature,clear hydrolytic zone forming bacterial strainswere selected for further studies and maintainedon agar slants.

Biochemical and phenotypic characterization:Selective isolate was identified through itsbiochemical and physiological propertiesaccording to Bergey’s Manual of SystematicBacteriology (23).

Scanning Electron Microscope analysis(SEM): SEM was used to investigate themorphology of isolated strain. The sample forSEM was prepared by transferring the microbialstrain to a clean eppendorff tube containingapproximately 1.5 ml of 3.5% glutaraldehyde

solution. Then, culture was incubated for 4 h atroom temperature followed by wash withphosphate buffer (100 mM, pH 7.2). The cultureis then dehydrated using alcohol gradient from10 to 100%. The dehydrated samples were thenair dried and fixed on the stubs using doubleadhesive tape. A thin layer of gold was coatedover the sample using HUS-5GB Hitachi vacuumevaporator for 90 sec. These samples were thenobserved under scanning electron microscopy(Hitachi S- 3000N, Japan) at variousmagnifications at acceleration voltage of 10.0KV.

Production media and culture conditions : Oneof the bacterial isolate designated as RSPB11 wasmaintained regularly on nutrient agar slants andused in this study. For enzyme production, yeastextract – peptone media consisting (%, g/100ml)of yeast extract – 1.0, Peptone – 1.0, Dextrose -0.2, MgSO

4 - 0.02, KH

2PO

4 - 0.02, NaCl -0.02 at

pH 7.0 was used. Inoculum (OD600nm

~1.5) wasdeveloped by growing the isolate in nutrientbroth for 18h. For production of enzyme, 1.0%inoculum was added to 50ml production mediumin 250ml conical flasks and then incubated at30oC for 3-4 days. Samples withdrawn at specifictime intervals were centrifuged at 10,000 rpmfor 10 min and the supernatant has been used asenzyme source for assay. All the cultureconditions were same unless otherwisementioned.

Protease assay: Protease activity was assayedaccording to Anson method and was slightlymodified (24). The reaction mixture contained2.5 ml of 0.65% Hammerstein casein and 0.5 mlof appropriately diluted enzyme in the presenceof 50 mM Glycine NaOH buffer pH 9.0. Thereactants were incubated at 37°C for 10 min andthe reaction was stopped by adding 2.5 ml of110 mM trichloroacetic acid (TCA). A suitableblank was run simultaneously, in which TCA wasadded to the enzyme solution, followed by



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substrate addition. After incubating at roomtemperature for 30min both test and blanksolutions were centrifuged at 10,000g for 10min.To the 0.4ml supernatant, 1.0ml 50mM Na

2CO

3

and 0.2ml Folin-ciocalteau reagent was added,the reaction mixture was incubated at roomtemperature for 30 min and the absorbance wasmeasured at 660nm. One unit (U) of proteolyticenzyme activity was defined as the amount ofenzyme that liberated 1μg tyrosine per ml perminute from casein under specified assayconditions.

Optimization of culture conditions for thebacterial growth and the protease productionby isolated strain: To select the optimum pH,temperature, aeration and agitation, enzymeproduction was investigated at different pHenvironments (pH 5.0-9.0), at differenttemperatures (27oC-40oC), aeration conditionswith respect to volume of media in 250ml conicalflasks (25ml-125ml) and speed of agitation fromstatic to 200 rpm, respectively in separate flasks.The samples were collected every 24 h for 72hto measure the enzyme activity.

Results and DiscussionIsolation and screening of protease producingmicrobes: Various soil samples collected frommarine environment were used for isolation of

effective protease producing microbe. Chitin inthe exoskeleton of shrimp and crab shells isassociated intimately with proteins therefore forthe isolation of chitionolytic and proteolyticbacteria, chitin utilization in the screening mediaoffers a great advantage (18, 25). Therefore,enriched microbial population from the shrimpand crab shell media has been spread over chitinagar plate and incubated for 5 days until thevisibility of hydrolytic zones. Selected microbesfrom the chitin agar plates were again spotinoculated on casein agar plate containing 1mMPMSF (serine protease inhibitor) and gelatin agarplate. After 2 days colonies showing maximumproteolytic zones were picked and purified overnutrient agar plates. Among the 10 potentchitinase producers only 3 microbes showed highcasein hydrolytic zones, revealing that theseisolated strains were not serine proteaseproducers. This is concluded based on the factthat these colonies are showing the proteolyticactivity in presence of PMSF which bindsspecifically to the active site serine residue in aserine protease leading to inactivation of serineproteases (26). As our protease of interest ismetalloprotease due to their pharmaceuticalimportance, addition of PMSF in the casein agarplate during the screening of microbes helped indifferentiating serine protease producers fromother class of protease producers. All three

Fig 1. Petri plates showing the marine isolate with a potential to produce extracellular protease (a) and chitinase (b)


Fig 1(a) Fig 1(b)


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isolates were named and sub cultured in nutrientbroth. Based on the larger hydrolytic zones onchitin (Fig 1a), casein (Fig 1b) agar plates andprotease assay, one isolate designated as RSPB11was selected for further studies.

Biochemical, phenotypic and morphologicalcharacteristics of the RSPB11: Biochemical andphysiological properties of RSPB11 wasidentified according to Bergey’s Manual ofSystematic Bacteriology (2). Table 1 indicatesthe isolated strain physical properties and Table2 denote biochemical characterization tests andtheir results. Colonies of RSPB11 on nutrientagar plates were white, round with smooth andglossy surface as well as slightly opaque innature. The isolate is characterized as aerobic,gram negative and rod shaped bacteria. Fig 2shows the scanning electron microscope pictureof the isolate and cells were visulaized as single,short rod shaped bacteria. Physiological testsshow that the cells could survive and grow inthe medium pH ranging from 5.0 to 11.0 andunder saline conditions of 3.0% NaCl. From thebiochemical tests, it was concluded that thisisolate RSPB11 belong to Enterobacteriacefamily, and it is a member of Serratia genus. Thisgenus is characterized with ten species (strains)distributed in two sub species (27).

Optimization of process parameters for proteaseproduction: Protease production by the isolate,Serratia sp RSPB11, was noticed at 12 hours ofgrowth and enzyme production reached to themaximum level after 48h of cultivation. Growthof the isolate, monitored by taking theabsorbance of media against blank at 600nm atspecific time intervals shows a gradual increasein biomass production. Protease production wasalso noticed in correlation with biomassproduction (Fig 3) but a notable protease activityhas been observed after 24h. This data suggestedthat the protease production by this strain is

growth associated. This is further supported bythe fact that a constant protease activity wasobserved during stationary phase. The resultsfurther suggested that improved level of proteaseproduction could be possible with high activebiomass production.

The impact of incubation time as wellas other physical parameters like initial pH,temperature, and agitation on proteaseproduction by isolated Serratia sp RSPB11 wasinvestigated. Initial pH of the media was adjustedwith either 1M HCl or 1M NaOH for attainingdesired pH conditions. From the results it is clearthat initial pH of 7.0 supported the growth ofbacterium, where a maximum biomass andenzyme activity (5230U/ml) was noted (Fig 4).Analysis of the pH of the fermentation mediumat specific time intervals indicated a gradual risein pH and reached alkaline condition (pH ~ 9.0)after 48h (data not shown) indicating that thisenzyme is an alkaline protease. Media adjustedwith different pH conditions (pH 5.0, 6.0, 8.0,

Fig 2. Scanning electron micrograph of marine isolateSerratia sp. RSPB11



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Fig 4. The effect of pH, temperature, and speed of agitation on production of protease by the marine isolateSerratia sp. RSPB11

Fig 3. Effect of incubation time on growth and protease production by the marine isolate Serratia sp. RSPB11



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9.0) also reached a final pH ~ 9.0 at the end ofthe fermentation, but the enzyme production waslow at other than pH 7.0 conditions. Thisincrease in pH value along with fermentationprocess hence this protease productionbioprocess may be attributed to ammoniaproduced as a consequence of the aminoacidscatabolism released by protein hydrolysis byproduced protease enzyme as reported (28). pHdependent protease production by differentmicrobial strains were also observed by severalinvestigators, where the initial pH 6.0 (29) to8.0 (30) could support the increase in biomassas well as production of protease. The above dataalso depict that higher yields may be effectivelyobtained by constant maintenance of pH of themedium during fermentation process as noticedby Venil, (31) and Panasuriya (32). Efforts arebeing made in this direction.

Among the physical factors, temperatureis one of the most critical parameters that couldaffect the bioprocessing. To evaluate the same,media inoculated with Serratia sp RSPB11 hasbeen incubated at different temperatures rangingfrom 27oC to 40oC. The enzyme production hasbeen noticed in all tested temperature conditionssuggesting at this temperature range the isolatedSerratia sp. RSPB11 survives and producesmetabolism linked protease production. Thoughprotease production noticed all most all studiedenvironments higher production was noticed at33oC (5400U/ml). A very low enzyme activityof 2385U/ml observed at 40oC (Fig 4). Severalstudies revealed that temperature was found toinfluence extracellular enzyme secretion,possibly by changing the physical properties ofthe cell membrane (33). The optimumtemperature of Serratia sp RSPB11 was slightlyhigher than Serratia marcescens NRRL B-23112at 25oC (32), Serratia sp DT3 at 28oC (29), andlower than Serratia marcescens sp7 at 40oC (30),Pseudomonas aeruginosa MTCC 7926

metalloprotease at 40oC (13). A temperaturerange of 30-37oC has been employed in severalworks (17, 34).

In general, it is well known that allorganisms and microbes vary in their aerationrequirement. In particular, during aeratedenvironment, oxygen acts as a terminal electronacceptor for oxidative reactions to provideenergy for cellular activities. The variation in theagitation speed has been found to influence theextent of mixing in the shake flasks and alsoaffect the nutrient availability (35). In view ofthe above, speed of agitation was studied byincubating the flasks on orbital shakers at variousrpm (25-200 rpm) conditions. The data depictedthat fermentation processed at 25 rpm, a verylow production (1460U/ml) of protease wasnoted and a maximum production of 5363U/mlobserved at 150rpm. This data denote that thisisolate needs a particular speed of agitation forachieving the maximum biomass along with theproduction of enzyme.

Partial characterization of protease producedby isolated Serratia sp. RSPB11 : To evaluatethe nature of proteolytic enzyme produced byisolated Serratia sp. RSPB11, the enzymeactivity studied in the presence of 1 mM EDTAto know whether this enzyme belongs tometalloprotease family. Use of 1mM EDTA inthe reaction medium inhibited the proteolyticactivity of the produced enzyme, indictingnegative regulation of protease activity by thischelating agent. The result suggested that theenzyme produced by isolated Serratia sp.RSPB11 belongs to metalloprotease. This wasfurther confirmed from the literature reportswhere protease activity was observed to beinhibited by a metalloprotease inhibitor likeEDTA (36). To evaluate further the nature ofenzyme, the enzyme activity was measured interms of zone of inhibition in the presence of



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PMSF (results not shown). This furtherconfirmed that this enzyme is not belonging toserine type of protease as reported in otherproteases (37).

The protease produced by isolatedmicrobial strain is considered as one of theserralysin type of protein. This is confirmedbased on the following observations- a) Theisolated strain belongs to genus Serratia, b) Theproduced proteolytic enzyme is negativelyregulated by EDTA, c) all serralysin typeproteases are metalloproteases and not belongsto serine type of proteases, d) The proteolyticenzyme production is observed in presence ofserine protease inhibitor, PMSF and e) Theenzyme production is maximum at alkalineenvironment, hence this protease does notbelongs to aspartate type.

ConclusionIn the present investigation, a serralysin

type of protease producing microbial strain hasbeen isolated using marine soil samples. Thestrain has been purified and characterized interms of its biochemical and physiologicalgrowth properties. Based on biochemical tests,the isolate has been identified up to genus leveland observed that this strain belongs to Serratiasp. Extracellular enzyme production propertieswere studied and observed that this strainproduces more than one extracellular enzymes;such as chitinase and gelatinase depending upon the nutritional conditions. This strains abilitytowards protease production and since the strainbelongs to Serratia sp. hence, the producedprotease is considered as serralysin type ofprotease which is known for its commercialimportance. In this context, the proteaseproduction was investigated further in terms ofoptimal requirements for physiological growthfactors. The optimized protease production hasbeen noticed at a physiological pH of 7.0 and at

temperature 33oC as well as at 150 rpm therefore,these parameters are crucial for effectiveproduction yields.

AcknowledgementOne of the authors, P.Lakshmi Bhargavi

is greatly indebted to Council of Scientific andIndustrial Research, New Delhi for financialsupport in the form of Senior ResearchFellowship.

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Proteolytic Enzyme Production by Isolated Serratia sp RSPB11

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