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Process Biochemistry 46 (2011) 1430–1435

Contents lists available at ScienceDirect

Process Biochemistry

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novel psychrotrophic, solvent tolerant Pseudomonas putida SKG-1 and solventtability of its psychro-thermoalkalistable protease

antosh Kumar Singha, Sanjay Kumar Singha, Vinayak Ram Tripathia, Sunil Kumar Khareb,atyendra Kumar Garga,∗

Center of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Allahabad Road, Faizabad 224001, UP, IndiaDepartment of Chemistry, Indian Institute of Technology, Hauz-Khas, New Delhi 110016, India

r t i c l e i n f o

rticle history:eceived 12 January 2011eceived in revised form 17 March 2011ccepted 21 March 2011

eywords:sychro-thermoalkaline protease6S rDNAAME analysisseudomonas putida

a b s t r a c t

It is the first time when psychrotrophic, solvent tolerant, psychro-thermoalkaline protease produc-ing strain of Pseudomonas putida isolated from dairy sludge was capable of growing in the presenceof 30% (v/v) organic solvents. The strain exhibited resistance against heavy metals (Cr6+, As3+, Pb2+,Cs1+) and various antibiotics. The isolate was able to grow at wide range of temperature (10–40 ◦C)with maximum growth at 25 ◦C. In glucose gelatin yeast extract (GGY) broth (pH 9.0 and 25 ◦C),the strain produced 514 U Protease ml−1. The presence of organic solvents n-dodecane, n-decane,isooctane and n-octane, enhanced the protease production. The protease was not only stable butalso its activity enhanced in the presence of 25% (v/v) solvents n-dodecane, n-decane, isooctane,n-octane, n-hexane, n-butanol, n-heptane, cyclohexane and xylene, after prolonged incubation of four-

olvent stability teen days. The molecular weight of purified protease was ∼53 kDa as revealed by SDS-PAGE andactivity gel analysis. The protease was active in broad pH and temperature range of 8.0–12.0 and10–70 ◦C, respectively, with maxima at pH 9.5 and 40 ◦C. The unique property of solvent tolerance,antibiotic and heavy metal resistance proves the potential candidature of this isolate not only for pep-tide synthesis, but also for industrial use and bioremediation strategies involved in environmentalclean up.

. Introduction

In recent years, there has been a phenomenal increase in the usef enzymes as industrial catalysts. These enzymes offer advantagesver the use of conventional chemical catalysts for various reasons,.g. they exhibit high catalytic activity, a high degree of substratepecificity, can be produced in large amounts, etc. [1]. Proteasesepresent one of the three largest groups of most important indus-rial enzymes, and account for ∼60% of the total worldwide sale ofhe enzymes [2].

Extreme environmental conditions prevail on the earth, andow temperature is of most common occurrence. Cold envi-onment on the earth represents about 80% of the biospherend 90% of the marine environment in which cold adapted

icrobes (psychrophiles and psychrotrophs) are present which

roduce psychro-tolerant enzymes [3]. Psychrotrophs are alsoredited to produce heat-stable extracellular proteases [4], but

∗ Corresponding author. Tel.: +91 9454755166/5278 245330;ax: +91 5278 246330.

E-mail address: sk garg001@yahoo.com (S.K. Garg).

359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.03.012

© 2011 Elsevier Ltd. All rights reserved.

are less studied compared to thermo-stable proteases of ther-mophilic/thermotolerant microbes. When compared to theirmesophilic counterpart, these enzymes display high catalytic effi-ciencies in order to maintain appropriate metabolic fluxes atlow temperatures [5,6]. Proteases have application in proteinhydrolysis, meat tenderization, cheese making, detergents, tan-ning, photography and pharmaceuticals, etc. [2]. They are also usedfor peptide synthesis in non-aqueous environments. Generally, thepeptide bonds of proteins are hydrolyzed by proteases in aqueousmedium and synthesized in non-aqueous environment. Therefore,the formation of peptide bonds in peptide synthesis necessitatesthe activity and stability of proteases in non-aqueous solvents. It isgenerally observed that enzymes are unstable in organic solvents,which leads to the slow rate of reaction catalyzed by them [7]. So,to overcome this limitation, search for solvent stable proteases ismuch needed [5].

Microbes, especially bacteria and fungi are well known sourcesfor protease production at industrial scale [2]. Several bacterial

isolates have been studied by many researchers for their solvent tol-erance as well as production of solvent stable proteases. Gupta andKhare [8] have reported a solvent tolerant Pseudomonas aeruginosaPseA, capable of alkaline protease production in the presence of

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everal solvents with its remarkable stability. A solvent tolerant P.eruginosa PST-01 produced a thermoalkaline protease, which wasound significantly stable in the presence of many organic solvents9]. A protease of P. aeruginosa strain K exhibited enhanced pro-ease activity in the presence of organic solvents [10]. An elevatedctivity of thermostable solvent tolerant protease of Bacillus subtilistrain Rand has been reported in the presence of various solventsy Abusham et al. [11].

Keeping the above in view, the present study was aimed at isola-ion and identification of a psychrotrophic solvent tolerant bacterialsolate from a chilling unit of a dairy plant. Its solvent tolerance,ntibiotic and heavy metal resistance pattern was studied. Therowth response at different temperatures was studied to validatets psychrotrophic nature. The effect of various organic solvents onrowth and thermoalkaline protease production was investigated.he solvent stability of this protease was also studied to reveal itsossible application in several non-aqueous systems. The proteaseas purified using Sephadex G-75 and effect of pH and temperature

n protease activity was studied.

. Materials and methods

.1. Isolation, screening and identification of organic solvent tolerant proteaseroducer

Samples were collected locally from various cold stores, stored food in deepreezer(s) and chilling unit of dairy plant. Samples were taken in sterile plas-ic vials and carried to the laboratory in cool packs. Samples (soil and liquid)ere diluted ten times with sterile distilled water and 0.1 ml of this dilution was

pread over skimmed milk agar plates (pH 9.0) containing (g l−1 distilled water):kimmed milk, 20.0; agar, 15.0. The inoculated plates were overlaid with 7.0 mlf cyclohexane, and incubated at 7 ◦C, till sufficient growth appeared. The colonieshowing proteolysis, as indicated by clearing of casein around the colonies wereelected as psychrotrophic solvent tolerant alkaline protease producer [8,12]. Theolony having maximum zone of clearance was designated as strain 3a, purifiedy repeated streaking [13] and maintained on nutrient agar slants at 4 ◦C till fur-her use. The isolate 3a was characterized first by morphological and biochemicaltudies as per the Bergey’s Manual of Systematic Bacteriology [14]. Molecular char-cterization was done by 16S rDNA sequence analysis using universal primers7F and 1492R. The strain identification was further authenticated by fatty acidethyl ester (FAME) analysis at Institute of Microbial Technology, Chandigarh,

ndia.

.2. Solvent tolerance, antibiotic and heavy metal resistance studies on efficientacterial strain

In order to determine organic solvent tolerance, the strain 3a was grown inodified GYE broth (pH 9.0, adjusted using sterile 1 M Na2CO3 solution) containing

g l−1): glucose, 10.0; peptone, 10.0; yeast extract, 5.0 and NaCl, 5.0 [13]. The broth50 ml) was inoculated with a loopful of bacterial culture and incubated at 25 ◦Cn incubator shaker (150 rpm). From this cultured broth, 5.0 ml mother culture of.8 OD (A660; 1 cm cuvette) containing 2.8 × 108 cfu ml−1 was transferred to 250 mlterile modified GYE broth containing 30% (v/v) cyclohexane. To prevent the solventvaporation, the mouth of Erlenmeyer flask was plugged with butyl rubber stoppers.rowth in the absence of solvent under similar conditions served as control. Theulture broth (5.0 ml) was drawn aseptically from each flask periodically after every2 h and growth was assessed spectrophotometrically by turbidity measurementA660; 1 cm cuvette).

The antibiotic sensitivity pattern was studied by disc diffusion method [15].he antibiotics (�g/disc) used were: amikacin (30), cefaclore (30), cefadroxil (30),efazoline (30), cefotaxime (30), ceftaxidime (30), ceftriaxone (30), cefuroxime (30),iprofloxacin (5), norfloxacin (10), ofloxacin (5) and pefloxacin (5). On Mueller Hin-on agar plates, fresh bacterial lawn was prepared and antibiotic impregnated discsere placed over it and incubated at 25 ± 1 ◦C for 24–48 h. The bacterial isolateas classified as sensitive/resistant by the presence/absence of inhibition zone of

acterial growth around the discs.For heavy metal resistance pattern, 100 �l of bacterial culture of 0.8 OD

A660; 1 cm cuvette) was spread on Mueller Hinton agar plates having different−1

oncentrations (�g ml ) of different heavy metals [Cs (0.0–2500), Co (0.0–200),

b (0.0–1000), Hg (0.0–50), Ni (0.0–300), Cr (0.0–5000), Se (0.0–300) and As0.0–3000)]. The metal salts used were cesium chloride, cobaltous chloride, leadcetate, mercuric chloride, nickel chloride, potassium dichromate, selenium sul-hide and sodium arsenate. The growth was observed during 48–72 h at 25 ± 1 ◦C.

istry 46 (2011) 1430–1435 1431

2.3. Growth pattern of strain SKG-1 at different temperatures

The isolate was grown at temperatures 10, 15, 20, 25, 30, 35 and 40 ◦C to studytheir effect on its growth pattern. Two ml bacterial mother culture (0.8 OD at A660

containing 2.8 × 108 cfu ml−1) prepared in modified GYE broth was transferred to100 ml sterile glucose gelatin yeast extract (GGY) broth containing (g l−1): glucose,10.0; gelatin, 20.0; yeast extract, 5.0 and MgSO4·7H2O, 0.1 (pH 9.0). The culture brothwas incubated at above temperatures on incubator shaker (200 rpm) for 96 h. At 12 hinterval, 5 ml culture broth was withdrawn and growth was assessed by turbiditymeasurement at 660 nm.

2.4. Bacterial growth, protease production and effect of organic solvents

As this strain was isolated in the presence of cyclohexane, it was imperativeto study the effect of other organic solvents on bacterial growth. Hence, to studythe effect of various organic solvents on growth and protease production, bacterialgrowth was carried out for 72 h at 25 ◦C in 100 ml GGY broth (as above) in the pres-ence of 50 ml of various individual organic solvent. Each flask was inoculated andincubated as described earlier. Mouth of each flask was tightly plugged using butylrubber stoppers to avoid solvent evaporation. A flask with no solvent served as con-trol. At 12 h interval, 5 ml culture broth was withdrawn, centrifuged at 12,000 rpm(4 ◦C) for 10 min and cell free supernatant was used as enzyme for protease estima-tion. The cell pellet obtained was dried and bacterial growth was assessed in termsof cell dry weight (mg ml−1).

2.5. Enzyme assay

The protease activity was assayed by casein digestion method of Shimogaki [16].Crude enzyme (0.5 ml) was added to 3.0 ml casein solution of 0.6% (w/v), prepared in100 mM sodium carbonate–bicarbonate buffer of pH 9.5. After incubation for 10 minat 40 ◦C, the reaction was stopped by addition of 3.2 ml stopper solution (containing0.11 M trichloro acetic acid, 0.22 M sodium acetate and 0.33 M acetic acid) and keptat room temperature for 30 min. The reaction mixture was then filtered using What-man No. 1 filter paper and absorbance of the filtrate was measured at 280 nm. Oneunit of protease activity was defined as the amount of enzyme required to liberate1 �g of tyrosine min−1 ml−1.

2.6. Effect of organic solvents on protease stability

Cell-free supernatant having maximum protease activity was filtered with nitro-cellulose membrane (pore size 0.22 �m) and incubated with 25% (v/v) of differentorganic solvents viz., n-dodecane, n-decane, isooctane, n-octane, xylene, n-hexane,n-butanol, cyclohexane, n-heptane, benzene, toluene and ethanol for 2 weeks inscrew capped tubes at 30 ◦C and 120 rpm. The residual protease activity was esti-mated against the control, in which solvent was not present.

2.7. Protease purification

The protease produced by Pseudomonas putida SKG-1 was fractionated byammonium sulphate (40–60%) and the precipitate was dissolved in Tris–HCl buffer(50 mM, pH 8.0). This solution was dialyzed against the same buffer for 24 h at 4 ◦Cby changing the buffer at every 6 h. The dialyzate was concentrated by lyophiliza-tion and loaded on sephadex G-75 column (30 cm × 2.0 cm). Prior to protein loading,the column was equilibrated with Tris–HCl buffer (50 mM, pH 8.0) and protein waseluted with the same buffer. The fractions having protease activity were pooled,lyophilized and stored at −20 ◦C. The purified protease was resolved by sodiumdodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) according to themethod of Laemmli [17] using 4% stacking gel and 12% resolving gel. Electrophore-sis was carried out at 100 V and the protein bands were visualized by staining thegel with Coomassie Brilliant Blue R-250. The molecular weight of the protease wasdetermined by comparison with standard molecular weight markers (97.4, 66, 43,29, 20.1 and 14.3 kDa). All the purification steps were performed at 4 ◦C. To detectprotease activity in gel, SDS-PAGE was performed using 12% polyacrylamide gelcontaining 0.1% gelatin in separating gel [18]. The native protease sample was elec-trophoresed, and the gel was washed for 30 min twice in 2.0% (v/v) Triton X-100 atambient temperature to remove SDS. The gel was then incubated at 40 ◦C for 2 h inassay buffer (pH 9.5) followed by staining as above to visualize the zone of gelatinhydrolysis.

2.8. Protease characterization

The above protease was characterized to ascertain its psychro-thermoalkalinenature.

2.8.1. Effect of pHThe protease activity at different pH was measured using casein (0.6%, w/v) as

substrate at 40 ◦C. Casein solution was prepared using the following buffers: citratebuffer (pH 6.0, 100 mM), phosphate buffer (pH 6.5, 100 mM), Tris–HCl buffer (pH7.0–9.0, 100 mM), sodium carbonate–bicarbonate buffer (pH 9.5–11.0, 100 mM) and

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orax–NaOH buffer (pH 11.5–12.0, 20 mM). The protease activity was determineds described earlier [16].

.8.2. Effect of temperatureThe effect of temperature on protease activity at optimized pH was determined

sing casein (0.6%, w/v, pH 9.5) as substrate at different incubation temperatures (10,5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ◦C). Prior to treatment, the temperaturef each substrate solution was stabilized at required value and then protease activityas measured.

.9. Statistical analysis

Each experiment was performed twice, each in triplicate and standard deviationor every experimental result was calculated using the Microsoft Excel available inhe Microsoft Office.

. Results and discussion

.1. Isolation, screening and identification of organic solventolerant protease producer

Eight bacterial cultures were isolated from various samples,mong which two were found solvent tolerant psychrotrophic goodlkaline protease producers as they exhibited clear zone diameter3.5 mm on milk agar plates (pH 9.0) in the presence of cyclohex-ne, after one week of incubation at 7 ◦C [13]. Of the two efficientsolates, 3a strain was found the most potent protease producers evident by maximum (4.8 mm) caseinolytic zone of clearance.he capability to grow at 7 ◦C reveals psychrotrophic nature ofhe isolate. Poffe and Mertens [12] also isolated psychrotrophicseudomonas fluorescens and Achromobacter xylosoxidans fromooled raw milk on milk agar plates after 10 days of incubationt 7 ◦C.

The efficient strain 3a was Gram-negative, aerobic, motile rodith positive catalase and oxidase activity. The isolate exhibitedegative response for IMViC test, except for citrate utilization.itrate reductase, urease and H2S production were not detected

n the growing culture. It grew over a wide pH range (6.0–9.5),emperature (5–40 ◦C), NaCl concentration (0.0–3.0%, w/v), andas able to hydrolyze starch, casein, gelatin and produced acid

rom glucose. Its psychro-thermoalkaline protease was activen temperature range of 10–70 ◦C with optimum at 40 ◦C. Onccount of morphological and biochemical characteristics, it wasdentified as Pseudomonas sp. The partial 16S rDNA sequence ofhis isolate (1488 bp) was compared using BLAST program, andas confirmed as Pseudomonas sp. as it revealed 99% homol-

gy with different Pseudomonas strains. The 1488 bp 16S rDNAequence was submitted to Genbank [HQ: 259593]. The 16S rDNAequence analysis is the most broad-spectrum method employedor bacterial classification and identification. Gupta et al. [6] haveescribed 16S rDNA sequence analysis as species-specific iden-ification tool which can provide a very fine sensitivity downo single-cell detection. On the basis of FAME analysis results,he isolate was identified as P. putida and designated as strainKG-1. The isolate was deposited to MTCC (accession numberTCC 10510) at the Institute of Microbial Technology, Chandigarh

India).Several solvent tolerant strains of P. aeruginosa have been

eported for the production of organic solvent stable protease8–10]. Other organic solvent tolerant microbes are also stud-ed, but only by few workers [11]. Several other researchers haveeported P. putida strains capable of growing in the presence of dif-erent organic solvents. A solvent tolerant P. putida IH-2000 strain

as able to grow in the presence of toluene [19]. However, it is

or the first time when a thermoalkaline protease is being reportedrom a psychrotrophic solvent tolerant P. putida SKG-1, having sol-ent stability.

istry 46 (2011) 1430–1435

3.2. Solvent tolerance, antibiotic and heavy metal resistancestudies on efficient bacterial strain

The bacterial growth was slightly reduced (10–20%) in thepresence of cyclohexane (30%, v/v) as compared to the con-trol. Although, the initial growth was slow up to 24 h in thepresence of cyclohexane, it was fairly high compared to theextent of growth reported by other researchers [8,9]. Similargrowth behavior by P. aeruginosa PST-01 was reported by Oginoet al. [9], where 23% (v/v) cyclohexane was supplemented forgrowth and tolerance studies. Gupta and Khare [8] also reported adecreased growth of P. aeruginosa PseA strain in the presence of 33%(v/v) cyclohexane who observed ∼50% reduction in dry cell mass.Therefore, the tolerance and growth efficiency of our strain SKG-1was certainly better than that reported by other researchers. Thereduced availability of air and hence oxygen may not be the reasonof decreased growth response in the presence of cyclohexane, asboth the control as well as organic solvent containing media flaskswere plugged with butyl rubber stoppers. It was, therefore, cyclo-hexane responsible for the reduced growth in the experimentalflask compared to the control.

Among all the antibiotics tested, the isolate was resistant tocefaclore (30), cefadroxil (30), cefazoline (30) and cefuroxime(30). Other antibiotics created variable inhibition zones of bac-terial growth around the discs. The antibiotics may be arrangedin the following order of growth inhibition zones (mm dia.):amikacin (18) > ciprofloxacin (17) > ceftaxidime (13) > pefloxacin(12) > cefotaxime (10) > norfloxacin (9) > ofloxacin (9) > ceftriaxone(8). Since the strain was isolated from sludge of dairy chilling plant,the indiscriminate use of antibiotics in dairy products for preser-vation creates a selective pressure for survival on the microbes. Itultimately results in creation of antibiotic resistant strains either bymutation or by transfer of resistance genes among bacteria. Lopeset al. [20] screened enterococci isolated from Portuguese dairyproducts (milk and cheese) for gentamicin resistance. Althoughenterococci are generally regarded as being intrinsically resistantto low levels of gentamicin, a high-level gentamicin resistance wasdetected in many dairy isolates. D’Aimmo et al. [21] have also iso-lated 34 bacterial strains belonging to the genera Bifidobacteriumand Lactobacillus from commercial dairy and pharmaceutical prod-ucts. All tested strains were susceptible to ampicillin, bacitracin,clindamycin, dicloxacillin, erythromycin, novobiocin, penicillin G,rifampicin (MIC90 ranging from 0.01 to 4 �g ml−1); resistant toaztreonam, cycloserin, kanamycin, nalidixic acid, polymyxin B andspectinomycin (MIC90 ranging from 64 to >1000 �g ml−1). Knee-bone et al. [22] have detected the presence of beta-lactam andtetracycline antibiotics in five powdered milk products named asCarnation (Nestlé USA Inc., Solon, OH), Nido youth and Nido adult(Nestlé Mexico Inc., Mexico City, Mexico), ELK (Campina, Eind-hoven, The Netherlands), and Regilait (Saint-Martin-Belle-Roche,France). Antibiotics, for which the isolate is resistant, may beemployed in fermentation medium to check the contamination byother sensitive bacterial strains.

A variable level of tolerance was noted among various heavymetals under study. The strain SKG-1 tolerated very high concen-trations (�g ml−1) of certain heavy metals which may be arrangedin the following order of tolerance: chromium (4300) > arsenic(2500) > cesium (1750) > lead (800). It also exhibited toleranceagainst fairly high concentrations of cobalt (100), nickel (275) andselenium (275), but was sensitive to even very low concentra-tion of mercury (20). Protease producing bacteria having antibioticresistance and heavy metal tolerance was reported by other work-

ers also [13]. The very high level of tolerance against chromium,arsenic, lead and cesium reveals the possible application of this iso-late in bioremediation of such metals from natural contaminatedsites.

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Fig. 1. Effect of organic solvents on growth of strain SKG-1. Bacterial growth in theabsence ( ) and presence of solvent n-dodecane (�), n-decane ( ), isooctane (�),n-octane ( ), n-heptane ( ), n-hexane ( ), cyclohexane ( ), xylene ( ), toluene(@(

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) and benzene ( ). Individual solvent was added to sterile GGY broth (pH 9.0)33% (v/v), inoculated with SKG-1 strain @ 2% (v/v) and incubated in rotary shaker

150 rpm) at 25 ◦C.

.3. Growth pattern of strain SKG-1 at different temperatures

The bacterial strain was grown at different temperatures10–40 ◦C) to observe the growth pattern. The isolate was able torow at all the temperatures employed with maximum growtht 25 ◦C. At lower temperatures (10 and 15 ◦C), the growth waselatively slow, and attained the maxima at 72 h, while at otheremperatures (>15 ◦C), the growth rate was faster with maximumrowth at 48 h (not shown). So, it is deduced that the isolate is a psy-hrotroph; growth pattern at wide temperature range confirmedhe assumption from isolation studies at 7 ◦C. Olson and Notting-am [23] have described that psychrotrophs and psychrophilesave the same minimum temperature range of (−) 5–(+) 5 ◦C, butnly psychrotrophs grow at above 20 ◦C with optimum growth at5–30 ◦C, while psychrophiles have 12–15 ◦C as an optimum tem-erature range for maximum growth.

.4. Bacterial growth, protease production and effect of organic

olvents

Bacterial growth (Fig. 1) and protease production (Fig. 2) in GGYroth was observed after 12 h onwards with a maximum 514 U of

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ig. 2. Effect of organic solvents on protease production. Different organic solvents,iz., control (–�–), n-dodecane (–�–), n-decane (–�–), isooctane (· · ·�· · ·), n-octane· · ·�· · ·), n-heptane (–�–), n-hexane (–�–), cyclohexane (–♦–), xylene (· · ·�· · ·),

oluene (· · ·�· · ·) and benzene (· · ·♦· · ·), were added to sterile GGY broth (pH 9.0)33% (v/v), inoculated with SKG-1 strain @ 2% (v/v) and incubated in rotary shaker

150 rpm) at 25 ◦C for 72 h.

istry 46 (2011) 1430–1435 1433

protease ml−1 at 60 h incubation. The effect of each organic solventon bacterial growth was studied by measurement of cell dry weight(Fig. 1). As the isolate was cyclohexane tolerant, the effect of variousother organic solvents on growth as well as protease productionwas expected. Among various solvents studied, n-dodecane, n-decane, isooctane and n-octane enhanced the bacterial growth(Fig. 1), as well as protease production up to approximately 123,119, 118 and 117%, respectively (Fig. 2). Other solvents employedwere found slightly inhibitory for bacterial growth as well as pro-tease production at varied extent. In the presence of n-hexane,cyclohexane, n-heptane, xylene and toluene, protease productionwas 94, 91, 48, 19 and 15%, respectively (Fig. 2). Although in thepresence of benzene, the isolate was able to grow up to some extent(Fig. 1), no protease production was evident. Ethanol and n-butanolcompletely inhibited the bacterial growth, and hence enzyme pro-duction. This revealed that in the presence of hydrophilic solvents(log P value < 1.0), the strain neither produced protease nor exhib-ited growth. But, in the presence of hydrophobic solvents (log Pvalue > 2.0), the isolate exhibited better growth pattern and pro-tease production as well. log P value is the logarithmic value of thepartition coefficient (P) of solvent between n-octanol and water.It is a quantitative measure of the polarity of solvent [24], andincreasing log P corresponds with greater hydrophobic nature ofthe solvent, which in turn results in reduced toxicity. Bacterialgrowth in terms of cell dry weight (Fig. 1) directly correlated withhigher protease production (Fig. 2) i.e. higher the bacterial growthmore the protease production. Gupta and Khare [8] have isolated asolvent tolerant P. aeruginosa PseA strain, able to produce proteasein the presence of various organic solvents. The extent of cell dryweight as well as protease production reported by them was less inthe presence of all organic solvents employed as compared to thecontrol without organic solvent. Similar pattern of protease andcell dry weight reduction in the presence of solvents was reportedfrom P. aeruginosa PST-01 strain by Ogino et al. [9]. Usually, bacte-ria are less capable of growth in the presence of organic solvents.The enzymes are also generally inactivated, and exhibit reducedrate of catalytic reactions under such conditions. Proteases to beemployed as biocatalyst in peptide synthesis are required to bestable in the presence of organic solvents in non-aqueous environ-ments [7]. Therefore, the alkaline protease produced by our strainSKG-1 in the presence of organic solvent is highly suitable for thepurpose of peptide synthesis. Additionally, the property of solventtolerance makes the possible application of our strain for biore-mediation of different solvents from contaminated and oil spillagesites.

3.5. Effect of organic solvents on protease stability

In another approach, the effect of various organic solvents (25%,v/v) on protease stability was also investigated for two weeks, andthe results are depicted in Table 1. The protease of P. putida SKG-1 is extraordinarily stable in the presence of all organic solventsunder study. It was observed that except benzene, toluene andethanol, presence of other solvents enhanced the protease activity.After incubation with n-dodecane, isooctane, n-octane, n-decane,xylene, n-hexane, n-butanol, cyclohexane and n-heptane, the pro-tease activity increased to 128, 121, 121, 118, 113, 109, 105, 104 and102%, respectively. The presence of benzene, toluene and ethanolmarginally reduced the protease with residual activities of 98, 94and 91%, respectively (Table 1). An organic solvent stable alkalineprotease has been reported from P. aeruginosa PseA by Gupta and

Khare [8]. After 10 days of incubation with organic solvents (25%,v/v), the residual protease activities were 112, 75, 98, 92, 97, 94, 75,90, 96, 102 and 104 in the presence of ethanol, 1-butanol, benzene,toluene, xylene, cyclohexane, hexane, heptane, isooctane, n-decane

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1434 S.K. Singh et al. / Process Biochemistry 46 (2011) 1430–1435

Table 1Stability pattern of thermoalkaline protease in the presence of organic solvents. Indi-vidual organic solvent was added @ 25% (v/v) to cell free supernatant and incubatedin rotary shaker (120 rpm) at 30 ◦C for 14 days.

Organic solvent log P value Stability (%)

None – 100Ethanol −0.24 91n-Butanol 0.8 105Benzene 2.0 98Toluene 2.5 94Xylene 3.1 113Cyclohexane 3.2 104n-Hexane 3.5 109n-Heptane 4.0 102n-Octane 4.5 121

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Fig. 3. SDS-PAGE of partially purified protease from strain SKG-1. Lane 1: molec-ular weight markers (kDa): phosphorylase b—97.4; bovine serum albumin—66.0;ovalbumin—43.0; carbonic anhydrase—29.0; soya-bean trypsin inhibitor—20.1 andlysozyme—14.3; Lane 2: crude extract; Lane 3: ammonium sulphate precipitate ofprotease; Lane 4: protease after passing through Sephadex G-75; Lane 5: zymogra-phy of purified protease; Lane 6: native PAGE markers (kDa): phosphorylase b—98.0;bovine serum albumin—67.0; ovalbumin—44.0; glutathione S-transferase—29.0;soya-bean trypsin inhibitor—20.1 and lysozyme—16.0.

TP

Isooctane 4.5 121n-Decane 5.6 118n-Dodecane 6.6 128

nd n-dodecane, respectively. Abusham et al. [11] also reported arotease of B. subtilis strain Rand with enhanced activity in the pres-nce of organic solvents (25%, v/v), but only after 30 min incubation.reduced protease activity of 2.0–49% was reported by Ogino et al.

9] after 2 weeks of incubation with various organic solvents (25%/v). Geok et al. [10] have reported a protease of P. aeruginosa strain, which exhibited enhanced activity when incubated for 2 weeksith solvents (25%, v/v) of log P value greater than 4.0. Solvents

elow this log P value reduced the protease activity by 37–65%. Its, therefore, evident from our study that alkaline protease of P.utida SKG-1 is remarkably stable in the presence of broad rangeydrophilic as well as hydrophobic organic solvents employed inhis study.

.6. Protease purification

The purification steps of extracellular protease of strain SKG-are summarized in Table 2. The enzyme was purified 9.7 foldith total recovery and specific activity of 39% and 10,603 U mg−1,

espectively. On SDS-PAGE analysis, the protease appeared asmajor protein band of ∼53 kDa (Fig. 3). Activity gel analysis

onfirmed that the purified protease was consisting of a singleonomeric protein. The molecular weight of our strain SKG-1

rotease (∼53 kDa) was almost similar to that of 50 kDa alkalinerotease family [25].

.7. Protease characterization

Fig. 4 presents the effect of pH and temperature on the activityf Sephadex G-75 purified protease.

.7.1. Effect of pHIn general, bacteria belonging to genus Pseudomonas are known

o produce extracellular alkaline proteases having pH optimum atearly 10. The enzyme produced by strain SKG-1 exhibited its opti-um activity at pH 9.5 indicating its alkaline nature. Any deviation

n pH from optimum reduced the protease activity (Fig. 4). The

ecrease in protease activity was drastic towards acidic pH leadingo no activity at pH 6.0. Contrary to that, the extent of decreasen protease activity was less in alkaline pH range indicating itsobust nature in the pH range of 9.0–11 (Fig. 4). Interestingly, the

able 2urification steps of protease produced by Pseudomonas putida SKG-1.

Purification steps Total protein (mg) Total activ

Crude extract 47 51,400Ammonium sulphate precipitation (40–60%) 4.8 27,658Sephadex G-75 chromatography 1.9 20,146

Fig. 4. Effect of temperature (–�–) and pH (–�–) on protease activity of strain SKG-1.

protease was also active at pH 11.5 and 12.0 with relative activ-ities of 74% and 52%, respectively. Alkaline proteases from otherPseudomonas sp. are also reported by several researchers havingmaximum activity in this optimum pH range [8,25].

3.7.2. Effect of temperatureThe alkaline protease of strain SKG-1 was active at all the tem-

peratures employed with maximum activity at 40 ◦C. It exhibited35, 46, 58, 65, 73, 87, 96, 94, 82 and 71% activity at 10, 15, 20, 25,30, 35, 45, 50, 55 and 60 ◦C, respectively. Even at 65 and 70 ◦C, itexhibited 52 and 26% protease activity, respectively (Fig. 4). Theremarkable activities (≥35%) in the temperature range of 10–65 ◦Creveals psychro-thermoalkaline nature of this protease. Alkalineproteases of psychrotrophs active at high temperatures have been

reported by other researchers also [4]. Rao et al. [18] have puri-fied an alkaline protease from Bacillus circulans having optimumcatalytic activity at pH 9.0–11.5 and 45–70 ◦C.

ity (U) Specific activity (U mg−1) Recovery (%) Fold purification

1094 100 15762 53.8 5.27

10,603 39 9.7

Journal Identification = PRBI Article Identification = 9206 Date: May 21, 2011 Time: 10:36 am

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. Conclusions

It is the first instance when a psychro-thermoalkaline proteaseeing reported from a psychrotrophic solvent tolerant P. putidaKG-1 isolate. The strain is unique with respect to increased pro-ease production in the presence of various solvents of greaterydrophobicity (log P ≥ 4.5). The tolerance to several solvents,eavy metals and antibiotics makes the isolate applicable undertressed conditions. Outstanding solvent stability of the proteaseroves its possible application under anhydrous conditions andor peptide synthesis. The protease activity in broad pH andemperature range of 8.0–12.0 and 10–70 ◦C clearly indicate thesychro-thermoalkaline nature of this enzyme.

cknowledgements

The senior author is thankful to the University Grants Com-ission, Government of India, New Delhi, for providing research

ellowship under the scheme of major research project. The sup-ort of Mr. Surendra Vikram (Institute of Microbial Technology,handigarh, India) in bacterial identification and preparation ofhylogenetic tree is duly acknowledged. The assistance of Uttarradesh Government to the Department of Microbiology under thecheme of Center of Excellence is also acknowledged.

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