ORIGINAL ARTICLE

Production and characterization of a solvent-tolerantprotease from a novel marine isolate Bacillus tequilensis P15

Anjali Bose & Vishal Chawdhary & Haresh Keharia &

Ramalingam Bagavathi Subramanian

Received: 18 January 2013 /Accepted: 2 May 2013 /Published online: 19 May 2013# Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract Thirty-six proteolytic bacteria were isolated fromthe Jakhau coast, Kutch, India, amongst which isolate P15identified as Bacillus tequilensis (JQ904626) was found toproduce an extracellular solvent– and detergent-tolerant pro-tease (116.69±0.48 U/ml) and was selected for further in-vestigation. Deoiled Jatropha seedcake (JSC) was found tobe a suitable substrate for protease production under sub-merged condition. Upon optimization of process parametersfollowing one-factor-at-a-time approach, an overall 6.4-fold(860.27±18.48 U/ml) increase in protease production wasachieved. The maximum protease yield was obtained usinga medium containing 2 % (w/v) deoiled JSC as substrate(pH of 8.0) upon 36 h of fermentation at 30 °C. Theoptimum temperature and pH for activity of B. tequilensisP15 protease was found to be 50 °C and 8.0, respectively.The enzyme exhibited a half-life of 190 min at 50 °C, whichwas enhanced to 270 min in presence of 5 mM Ca2+. Theenzyme exhibited significant stability in almost all the sol-vents tested in the range of log Pow varying from 8.8 to−0.76. The enzyme activity was strongly inhibited by PMSFat 5 mM concentration, whereas the presence of EDTA(5 mM) and pCMB (5 mM) enhanced enzyme activity by20.9 and 13.7 %, respectively. The enzyme was also foundto be stable in the presence of surfactants, commercial de-tergents and bleach-oxidant (H2O2). This protease was dem-onstrated to be effective in removal of blood stains fromfabrics, dehairing of hide, and stripping off the gelatin fromused photographic films.

Keywords Bacillus tequilensis . Organic solvent-tolerantprotease . Deoiled Jatropha seedcake . Blood stain removal .

Dehairing of hide . Gelatin strip-off

Introduction

Proteases (EC 3.4.21; peptidyl-peptide hydrolases) are a classof proteins ubiquitously found in all organisms; they act as anindispensible biocatalyst with applications in detergent, leath-er, pharmaceuticals, food, textile, bakery, soy-processing,meat-tendering, brewery, protein-processing, peptide synthe-sis, ultra filtration membrane cleaning, and extraction of silverfrom used X-ray film industries, as well as in basic research(Gupta et al. 2002). They represent one of the three largestgroups of industrial enzymes that account for approximately60 % of the total enzyme sales in the world and is projected toincrease further in coming years with anticipated applicationsin both physiological and commercial fields (http://www.prlog.org/10039621-enzymes-industryforecasts-to-2010-2015). Normally, proteases catalyze hydrolysis of pep-tide bonds in aqueous environments, but under water-restrictedorganic conditions, reaction equilibria shift in favor of synthe-sis, creating pathways for synthesizing novel compounds likepeptides and esters (Kumar and Bhalla 2005; Maruyama et al.2002; Xu et al. 2010). The use of organic solvents as reactionmedia for enzymatic reactions provide numerous industriallyattractive advantages such as high regio- and stereo-selectivityand minimal side-chain protection requirements (Kumar andBhalla 2005) in comparision to traditional aqueous chemicalreaction systems. However, the major limitation for enzymaticreactions in water-restricted environments is the tendency oforganic solvents to strip water molecules from enzyme sur-faces affecting their activity and stability (Yang et al. 2004). Toovercome these limitations, several strategies like chemicalmodification of amino acids on enzyme surfaces (Davis2003), protein engineering (Wolff et al. 1996), use of ionic

Electronic supplementary material The online version of this article(doi:10.1007/s13213-013-0669-y) contains supplementary material,which is available to authorized users.

A. Bose :V. Chawdhary :H. Keharia (*) :R. B. SubramanianBRD School of Biosciences, Sardar Patel Maidan, SatelliteCampus, Sardar Patel University, Vadtal Road, P.O.Box 39,Vallabh Vidyanagar 388 120 Gujarat, Indiae-mail: haresh970@gmail.com

Ann Microbiol (2014) 64:343–354DOI 10.1007/s13213-013-0669-y

liquids (Noritomi et al. 2009), supercritical fluids (Habulin etal. 2005) and co-lyophilization with inorganic salts (Ru et al.2000) for enhancing enzyme activity and stability in organicsolvents have been employed. Alternatively, it has been pro-posed that, instead of modifying enzymes for increasing sol-vent stability, it would be more desirable to screen naturallyevolved solvent-tolerant enzymes for application in non-aqueous synthetic reactions. Ogino et al. (1994, 1995) reportedfor the first time on the production of organic solvent-tolerantlipolytic and proteolytic enzymes from two organic solvent-tolerant bacteria, viz., Pseudomonas aeruginosa LST-03 and P.aeruginosa PST-01, respectively. Since then, various organicsolvent-tolerant enzymes have been described (Doukyua andOgino 2010). A major work explored solvent stable proteasefrom Pseudomonas sp. (Ogino et al. 1995; Gupta and Khare2006; Tang et al. 2010). Recently, several Bacillus sp., viz. B.pumilus (Rahman et al. 2007),B. sphaericus (Fang et al. 2009),B. subtilis (Rai andMukherjee 2009), B. licheniformis (Jellouliet al. 2011; Li et al. 2009), and B. cereus (Xu et al. 2010), havebeen reported for the production of organic solvent stableproteases with potential industrial applications. Most solventtolerant proteases exhibit stability only in non-polar solvents(Rahman et al. 2007), while few proteases have been reportedto be stable even in polar solvents such as alcohol (Thumar andSingh 2009; Karan et al. 2011) and glycerol (Ruiz and DeCastro 2007).

Halophilic microorganisms generally produce halophilicenzymes as a result of adaptation to high salt conditions.Since salt reduces water activity, a feature common withorganic solvent systems, halophilic enzymes are consideredto be valuable tools as biocatalysts in aqueous organic media(Sellek and Chaudhuri 1999). A protease fromOceanobacillussp. isolated from the coastal region of Gujarat was found to bestable in the presence of various solvents (Pandey et al. 2012).The protease from the haloalkaliphilic archaeon Natrialbamagadii required a high salt concentration (1.5 M NaCl) toexhibit stability in the presence of organic solvents (Ruiz andDe Castro 2007). Gujarat has a coastline of about 1,600 kmand it is the second most industrialized state in India. Jakhaucoast in Kutch is one of the unexplored niches for its microbialdiversity, situated on the Gujarat coast with lot of fishingactivity and was thus considered as a prospective source formicrobes with novel properties.

Large numbers of agricultural/food residues have beenexplored as suitable raw materials for protease production(Raimbault 1998) in order to make the process economicallyviable. Gessesse (1997) reported on the suitability of nug meal(a byproduct of oil extraction fromGuizotia abyssinica seeds)as the sole nitrogen source for the production of extracellularalkaline protease by Bacillus sp. AR-009. Joo et al. (2002)reported maximum enzyme activity by Bacillus horikoshiiwhen grown in soybean cake (1.5 %, w/v) supplemented with1 % casein as the sole energy source. The suitability of several

oil cakes such as coconut oilcake, palm kernel cake, sesameoilcake, and olive oilcake in combination with several agro-industrial residues such as wheat bran, rice husk, rice bran,and spent brewing grain have been evaluated for proteaseproduction (Sandhya et al. 2005). Jatropha seedcake, abyproduct of biodiesel production is composed of 22 % pro-tein, 49 % carbohydrate, 0.8 % fat, 10 % ash, and 14 % fiber(Rakshit et al. 2008). However, the presence of anti-nutritionalcomponents such as phorbol ester, curcin, trypsin inhibitors,saponins, and phytate (Makkar et al. 2009) makes it unsuitableas a feed supplement. Thus, the cake is considered as a wastematerial (Rakshit et al. 2008; Martínez-Herrera et al. 2006)and therefore requires the development of methods for its safedisposal and/or potential utilization. In this context, owing toits nutrient rich profile, Jatropha seedcake has recently beenevaluated as a substrate for microbial production of industri-ally important enzymes such as lipase (Mahanta et al. 2008),xylanase (Joshi and Khare 2011), and cellulase (Dave et al.2012).

In view of the demand for proteolytic enzymes and theneed for added value to toxic biodiesel waste, we investi-gated the potential of Jatropha seedcake as a substrate forthe production of protease by a novel marine isolate Bacillustequilensis P15. This article also describes the characteriza-tion and application of the organic solvent-tolerant proteaseas a detergent additive, for dehairing of animal hides, andstripping off the gelatinfrom photographic films.

Materials and methods

Materials

Bushnell Haas Medium (BHM; 0.2 g/l MgSO4, 0.2 g/l CaCl2,1 g/l KH2PO4, 1 g/l K2HPO4, 1 g/l NH4NO3, 0.05 g/l FeCl3),nutrient agar (5 g/l peptic digest of animal tissue, 3 g/l meatextract, 20 g/l agar), gelatin, soybean meal and bovine serumalbumin (BSA, protein standard) were obtained fromHiMedia,India. Hammerstein casein was procured from Sisco ResearchLaboratories, India. Deoiled Jatropha seedcake (JSC) wasobtained gratis from the Food Processing and Bioenergy divi-sion, Anand Agriculture University, Anand, Gujarat, India.The seedcake was sun dried and then ground to get seedcakemeal which was added to basal medium as a substrate forenzyme production. All other solvents and chemicals usedduring the experiment were of analytical grade.

Isolation of proteolytic bacteria

Proteolytic bacteria were isolated upon enrichment from themarine soil collected from the Jakhau coast (23°13′0″N,68°43′0″E) in Kutch, Gujarat, India. Next, 100-μl aliquotsof serially diluted enrichment culture were spread on NaCl-

344 Ann Microbiol (2014) 64:343–354

containing casein agar plates (NCA; 30 g/l NaCl, 10 g/l ca-sein, 20 g/l agar in BHM). The plates were incubated at 30 °Cfor 48 to 72 h. Individual colonies with clear zones resultingfrom casein hydrolysis were picked up and purified by repeat-ed sub-culturing. The cultures were maintained by on nutrientagar slants amended with 3 % (w/v) NaCl and preserved in arefrigerator at 4 °C.

The proteolytic cultures were further screened for theirability to hydrolyze casein and gelatin in the presence andabsence of 3 % (w/v) NaCl. The gelatin agar plates (10 g/lgelatin and 20 g/l agar in BHM) were flooded with Frazier’sreagent (15 % HgCl2 in 2 N HCl) in order to determinegelatinase activity. The diameters of the zones of proteolyticactivity of all the cultures were measured along with thegrowth diameters, and the ratio of proteolytic zone to growthwas used for selecting potential protease producers. In orderto monitor protease production in liquid medium, cultureswere inoculated in 10 ml of NCBHM (NCA without agarpowder) and incubated on an orbital shaker (150 rpm) at30 °C for 48 h. After incubation, the cells were pelletedby centrifugation at 5,867 g for 10 min and the super-natant was collected in correspondingly labeled cleanglass test tubes and assayed for protease activity.Eighteen bacterial isolates showing significant proteaseactivity on solid as well as liquid media were selectedas potential protease producers for the subsequent exper-iments and subjected to further screening.

Screening based on organic solvent tolerance and salttolerance

The cultures selected by primary screening on the basis oftheir ability to produce protease on casein and gelatin assubstrates were further subjected to screening for their sol-vent tolerance. The cultures were spot inoculated on NCAplates using sterile tooth picks, after which each plate wasflooded with 7 ml of different solvents [n-hexadecane(log Pow=8.8) , 1-dodecanol (log Pow=5.0), iso-octane(log Pow=4.5), n-heptane (log Pow=4.39), n-hexane (logPow=3.86), cyclohexane (log Pow=3.2), xylene (log Pow=3.1), toluene (log Pow=2.64), benzene (log Pow=2.13),chloroform (log Pow=2.0), dichloromethane (log Pow=1.25) and methanol (log Pow=−0.76)] and incubated at30 °C in an air-tight canister in order to prevent the evapo-ration of the solvent from the plates. After incubation, theplates were observed for growth as well as clear zonesaround the colony in the presence of solvents.

Salt tolerance of the 18 selected cultures was investigatedby growing the culture in NCBHM with varying NaClconcentrations of 0, 3, 6, 9, and 12 % (w/v). After incuba-tion at 30 °C for 48 h, the cells were removed by centrifu-gation at 5,867 g and the supernatant thus obtained wasassayed for protease activity.

Identification

The identity of the selected bacterial isolate P15 was re-vealed on the basis of BIOLOG carbon source utilization(using GEN III plate and Microlog 3 identification software,v.5.1.1; Biolog, USA) and further confirmed by 16S rRNAgene sequence analysis.

Profile of protease production by P15

The 500-ml Erlenmeyer flasks containing 200 ml NCBHMwere inoculated with an overnight-grown culture of P15 toobtain an initial culture density (OD600nm) of 0.05 andincubated on an orbital shaker (150 rpm) at 30 °C. Then,2-ml aliquots were withdrawn after 3, 6, 9, 12, 24, 36, 48,60, and 72 h of incubation, and monitored for growth andprotease activity.

Medium component optimization for protease productionby P15

Protease production was carried out in 250-ml Erlenmeyerflasks to study the suitability of substrate (casein, gelatin,soybean meal, corn waste, castor seedcake and JSC; 1 %w/v) for enzyme production. Studies were also performed toevaluate the influence of varying substrate concentration(0.25–5 %, (w/v) JSC), various co-carbon sources(glucose/lactose/sucrose/starch; 0.1 % w/v) and co-nitrogen sources (replacing inorganic nitrogen source inBHM with 25 mM of either NH4NO3/NH4Cl/KNO3) bysupplementing them individually to 3 % (w/v) NaClcontaining BHM broth (NBHM). In all cases, the mediawere inoculated with an overnight grown culture of P15 asdescribed above and incubated on an orbital shaker(150 rpm) at 30 °C for 36 h. After incubation, the flaskswere harvested and monitored for protease activity.

Characterization of crude P15 protease

The overnight-grown culture of P15 was inoculated in opti-mized deoiled Jatropha seedcake medium, incubated for36 h and harvested by centrifugation at 5,867 g for 15 minat 4 °C; the culture supernatant so obtained was used ascrude enzyme for further studies.

Effect of temperature and pH on enzyme activity

The protease activity was monitored at different temperatures(30 –70 °C) in 50 mM Tris-HCl buffer, pH 8. This wasfollowed by studies on thermostability (at 50 and 60 °C),where the enzyme sample was incubated in presence andabsence of 5 mM Ca2+ (CaCl2) in a water bath. Samples weretaken at regular intervals followed by the determination of

Ann Microbiol (2014) 64:343–354 345

enzyme activity, which was expressed as the percentage ofinitial activity.

The effect of pH on protease activity was monitored byassaying P15 protease over a pH range of 5.0–11.0 at 30 °C.Sodium acetate buffer (50 mM) for pH 5.0 and 5.5, sodiumphosphate buffer (50 mM) for pH in the range of 6.0–7.5,Tris-Cl buffer (50 mM) for pH 8.0 and Glycine-NaOH(50 mM) for pH in range of 9.0–11.0 were used to adjustthe pH of the reaction mixtures. The pH stability of theenzyme was estimated by pre-incubating the enzyme atdifferent pH for 1 h at 50 °C and then assayed for proteaseactivity at optimum pH.

Organic solvent stability

The organic solvent stability of protease was investigated byincubating enzyme in buffer containing 25, 50, and 75 %(v/v) of different solvents in screwcap tubes in a shakingwater bath (150 rpm) at 30 °C. After 24 h of incubation,residual protease activity was assayed in the aqueous phase.

Influence of metal ions, inhibitors, detergents and oxidizing-bleaching agent on P15 protease

The effect of various divalent cations (final concentration of5 mM), viz., Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Zn2+, and Hg2+;different enzyme inhibitors (final concentration of 5 mM),namely, ethylene-di-amine-tetra-aceticacid (EDTA), β-mercaptoethanol, phenylmethylsulphonyl fluoride (PMSF),p-chloromercuribenzoic acid (pCMB); detergents, viz., non-ionic detergent (Tween 20, Tween 80, Triton X-100; 1 % v/v),anionic detergent (sodium dodecyl sulphate SDS; 5 mM),cationic detergent (N-Cetyl-N,N,N-trimethylammonium bro-mide (CTAB); 5 mM), and commercial available laundrydetergent formulations (Ariel®, Surf Excel®, Tide®; 1 %w/v); bleach-oxidant H2O2 (1 % v/v) were investigated forprotease activity. This was done by pre-incubating the enzymefor 1 h at 30 °C with these agents and then it was assayedusing casein as a substrate. The enzyme activity wasexpressed as % activity relative to control activity (withoutany of the aforementioned additives), which was consideredas 100 %.

Removal of blood stain, dehairing of sheep skin and X-raydecomposition by P15 protease

A clean piece of cloth was spotted with blood and allowedto dry, followed by 2 % formaldehyde treatment for 30 minand then washed with water to remove excess formaldehydeand blood cells (Subba Rao et al. 2009). The cloth with theblood stain was incubated with: tap water without detergentand protease as experimental control (Treatment 1); only de-tergent (Treatment 2); or crude protease (50 U) (Treatment 3) at

room temperature. After incubation for 10 min, each piecewas rinsed with water for 2 min, dried, and comparedwith the untreated control for evaluating blood stainremoval efficiency.

Dehairing activity of the enzyme was investigatedaccording to Subba Rao et al. (2009). Goat skin was cutinto ∼4×4 cm2 pieces and incubated with the crude P15protease (50 U) in 50 mM Tris-HCl (pH 8) at room temper-ature (∼30–35 °C) under shaking condition for 3 h.Afterwards, dehairing was checked mechanically by gentlerubbing with hands.

A piece of X-ray film (5×5 cm2) was incubated with thecrude protease (10 U) at 50 °C for 20 min. The film waschecked for decomposition of the gelatinous coating byestimating the soluble protein content in the eluate at theend of incubation (Shankar et al. 2010).

Analytical procedures

The protease activity was measured using casein as a sub-strate according to the method of Anson (1939) with a fewmodifications. In brief, 2 ml of reaction mixture containing1 ml of 1 % (w/v) casein (according to Hammerstein) and1 ml of appropriately diluted enzyme in 50 mM Tris-Clbuffer (pH 8.0) were incubated for 20 min at 50 °C. Thereaction was stopped by the addition of 2 ml 10 % (w/v)trichloroacetic acid. A control was run in parallel in whichthe enzyme was added after the addition of trichloroaceticacid. The reaction mixture was then centrifuged at 13,201 gfor 10 min in order to remove precipitates, and the clearsupernatant was then assayed for tyrosine by the method ofLowry et al. (1951). One unit of protease activity wasdefined as the amount of enzyme required to liberate 1 μgof tyrosine/ml/min under specified assay conditions. Theprotein was estimated according to the method of Lowryet al. (1951) using BSA (fraction V) as standard.

Results

A total of 36 proteolytic bacterial strains were isolated fromsoil collected from the Jakhau coast in Kutch, India. Amongstall these organisms, the isolate designated as P15 exhibitedmaximum halotolerant proteolytic activity on solid as well asin liquid media (Supplementary Fig. 1, SupplementaryTables 1, 2). The isolate P15 also exhibited good growth andcomparably higher proteolytic activity in the presence ofalmost all the tested solvents with log Pow values varying from8.8 to 1.25 (Supplementary Table 3) and thus was selected forfurther studies. This isolate P15 was identified as Bacillus sp.using BIOLOG (Biolog) and as Bacillus tequilensis on thebasis of its 16S rRNA gene sequence (NCBI GenBank acces-sion no. JQ904626).

346 Ann Microbiol (2014) 64:343–354

Profile of protease production by B. tequilensis P15

The isolate P15 exhibited maximum cell density (3.07OD600nm) at 24 h incubation while maximum protease pro-duction (116.69±0.48 U/ml) was at 36 h of incubationwhich declined thereafter (Fig. 1). Henceforth, 36 h wasconsidered as the optimized incubation period for proteaseproduction in the subsequent experiments.

Production of protease by B. tequilensis P15 on agrowastes

Recent research efforts have been directed towards the use ofcost-effective agro-industrial wastes, such as crab-shell wastes,deoiled seedcake, fish protein hydrolysate, bird feathers, etc.,for protease production in order to make the process econom-ically viable. In the same context, protease production by B.tequilensis P15 was studied with supplementation of variouseasily available agrowastes to the NBHM broth. Maximumproduction of protease (715.89±3.53 U/ml) was obtained inJSC supplemented broth followed by corn waste (690.37±3.38 U/ml), soybean meal (630.29±2.65 U/ml), and castorseedcake (608.9±4.08 U/ml) (Fig. 2a). In the subsequent ex-periment, it was observed that the B. tequilensis P15 producedincreasing amounts of protease with increasing concentrationsof JSC in medium from 0.25 to 2.0 % (w/v) and further in-creases in seedcake concentration resulted in decreased enzymeproduction (Fig. 2b). Further supplementation of additionalcarbon or nitrogen sources in addition to 2 % (w/v) JSC inthe medium had no influence on the protease production by B.tequilensis P15 (Fig. 2c and d).

Characterization of crude protease from B. tequilensis P15

Influence of temperature on activity of B. tequilensis P15protease

The enzyme exhibited activity over a wide temperature rangeof 30–70 °C, the optimum being at 50 °C (Fig. 3a). The relative

enzyme activity at 60 and 70 °C were about 75 and 60 %,respectively, of that at 50 °C. The half-lives of the protease wasfound to be 190 and 10 min at 50 and 60 °C, respectively(Fig. 3b). The thermal stability of protease activity was signif-icantly improved in the presence of 5 mM Ca2+ with half-livesof 270 and 20 min at 50 and 60 °C, respectively.

Influence of pH on activity of B. tequilensis P15 protease

The pH profile of the enzyme was determined at differentpH values ranging from 5.0 to 11.0. The P15 protease wasfound to be active over a broad range of pH, i.e. 6.0–11.0with maximum activity at pH 8.0 (Fig. 3c). Studies on pHstability revealed that the enzyme was stable in the pH rangeof 7.0–10.0 retaining more than 95 % activity (Fig. 3d).

Organic solvent stability

The effect of various organic solvents (at a final concentra-tion of 25, 50, and 75 %, v/v, in the reaction mixture) onprotease stability was investigated for 24 h (Table 1).Interestingly, the protease activity was slightly improved inthe presence of various hydrophobic, water-immiscible sol-vents (high log Pow), such as n-hexadecane, 1-dodecanol,iso-octane, n-heptane, xylene, and benzene. The P15 pro-tease retained about 90 % or more of its activity in thepresence of polar (low log Pow) solvents such as tolu-ene, chloroform, and dichloromethane in comparison tocontrol (without solvent). However, the presence ofhexane, cyclohexane, and toluene marginally reducedthe enzyme activity at all the solvent concentrationstested (Table 1). Furthermore, 25 % methanol causedan approximately 10 % reduction in activity after 24 hof incubation followed by complete inactivation in thepresence of 50 and 75 % methanol.

Influence of metal ions, divalent metal ion chelators,inhibitors, detergents, and oxidizing agents on P15 proteaseactivity

Amongst different metal ions, Ca2+ and Zn2+ had stimula-tory effects on protease activity, while transition metal ionssuch as Fe2+ and Cu2+ strongly suppressed P15 proteaseactivity, followed by Hg2+, Mg2+, and Mn2+ (Table 2). Theprotease activity was inhibited in the presence of 5 mMPMSF, suggesting it to be a serine hydrolase. Further, theprotease activity remained unaffected on exposure toβ-mercaptoethanol (S-S-reducing agent) and pCMB (acysteine-modifying agent). The presence of EDTA (divalentmetal ion chelators) slightly increased the protease activity.The activity of P15 protease remained unaffected in thepresence of non-ionic detergents like Tween 20, Tween 80,and Triton X-100 at 1 % (w/v), whereas it was inhibited by

Fig. 1 Time course of protease production by Bacillus tequilensis P15.(Data are means of three replicates, error bars SE)

Ann Microbiol (2014) 64:343–354 347

50 and 80 %, respectively, in the presence of SDS andCTAB (both at 1 %, w/v concentration). In the presence ofcommercial laundry detergent formulations Surf Excel®,Tide® and Ariel® at 1 % w/v, the enzyme retained 90.82,54.49, and 25.97 % activity upon 1 h of incubation, respec-tively. Furthermore, the enzyme could retain 91.02 % activ-ity upon 1 h of incubation in the presence of the bleach-oxidant, hydrogen peroxide.

Destaining of blood stains, dehairing of sheep hides,and decomposition of the gelatinous layer of usedX-ray films

The P15 protease revealed robustness towards organic sol-vents, detergents, and bleach-oxidant. Therefore, the use ofenzymes for blood stain removal from cloth was investigat-ed. The incubation of blood-stained cloth pieces with pro-tease showed the removal of the blood stain without usageof any detergents within 10 min of treatment (Fig. 4a),exhibiting the positive effect on protein stain removal.

In addition, the enzyme was examined for dehairinggoat skin without application of sodium sulphide. The

experimentally dehaired goat skin showed removal ofhair (Fig. 4b) within 3 h of treatment with the enzymeat room temperature.

Furthermore, the gelatin strip-off experiment revealedthat the P15 protease can efficiently degrade the gelatinouslayer from the developed X-ray film within 20 min at 50 °C(Fig. 4c), leaving behind the stripped photographic film basefor reuse. Gelatin degradation was monitored by estimatingthe protein content of the hydrolysate which increased withthe course of the experiment (data not shown).

Discussion

Most bacteria and their enzymes are destroyed or inactivatedunder harsh application conditions such as organic solvents,high salt concentration, high or low temperature, alkaline pH,etc. Hence, several attempts have been made to enhance en-zyme activity and stability under conditions of application byprotein engineering approaches (Chen and Arnold 1993;Pantoliano et al. 1989;Wolff et al. 1996). Alternatively, insteadof modifying the enzymes, it would be desirable to screen

Fig. 2 Media optimization: a effect of different proteinaceous substrates, b effect of varying concentration of deoiled Jatropha seedcake, c effect ofco-carbon sources, and d co-nitrogen sources. (Data are means of three replicates, error bars SE)

348 Ann Microbiol (2014) 64:343–354

naturally evolved robust enzymes for industrial applications. Inthe same context, the organic solvent-tolerant bacteria are anovel and unique group of extremophilic microorganisms that

thrive in the presence of very high concentrations of organicsolvents (Adams et al. 1995; Sardessai and Bhosle 2004).There are reports of Bacillus sp. that grow in the presence of

Fig. 3 a Effect of assay temperature, b thermostability of enzyme in absence and presence of 5 mM Ca2+, c effect of assay pH, and d pH stability ofenzyme. (Data are means of three replicates, error bars SE)

Table 1 Organic solvent stabil-ity of P15 protease Organic solvents Log Pow Relative enzyme activity (%)

Solvent (25 %, v/v) Solvent (50 %, v/v) Solvent (75 %, v/v)

None (control) 100 100 100

n-Hexadecane 8.8 104 101 100

1-dodecanol 5.0 108 107 105

iso-Octane 4.5 102 101 103

n-Heptane 4.39 100 101 99

n-Hexane 3.86 88 85 88

Cyclohexane 3.2 87 83 86

Xylene 3.1 100 101 100

Toluene 2.64 95 95 99

Benzene 2.13 102 100 101

Chloroform 2 98 91 95

DCM 1.25 90 91 90

Methanol −0.76 89 2 2

Ann Microbiol (2014) 64:343–354 349

high concentrations of toxic organic solvents such as DMSO,methanol, ethanol, toluene, xylene, cyclohexane, n-hexane,and iso-octane (Li et al. 2009; Rahman et al. 2007; Sana etal. 2006; Xu et al. 2010). The solvent tolerance in such micro-organisms have been attributed to the alteration in cell mor-phology, the composition of the membrane fatty acids, and/orthe presence of active efflux pumps which maintain minimalsub-toxic concentration of organic solvents within cells (Torreset al. 2011).

Similarly, considerable diversity exists in the mecha-nisms of tolerance against salt stress. At high salt concen-trations, physiologically important proteins are in generaldestabilized due to enhanced hydrophobic interactions (VonHippel and Schleich 1969) leading to cell death. However,enzymes in halotolerant/halophilic organisms have evolvedto remain stable and active under high saline conditions(Ruiz and De Castro 2007).

Therefore, the present study was aimed at screening of 36proteolytic bacteria isolated from marine coastal soil for or-ganic solvent- and sodium chloride-tolerant protease producerswith prospective industrial applications. After extensive

screening, isolate P15 identified as B. tequilensis was selectedfor protease production and characterization. The maximumactivity of protease in batch culture of B. tequilensis P15 wasfound at the end of the exponential growth phase. A similarproduction profile of an alkaline protease from alkaliphilicactinomycete just after the exponential growth phase has beenreported by Mehta et al. (2006). Nilegaonkar et al. (2007) alsodemonstrated growth-associated protease production(126.87 U/ml) in optimized medium by B. cereus MCM B-326. Maximum protease production by B. tequilensis P15(∼860 U/ml) was higher in comparison to protease productionreported for B. licheniformis RG1 (161 U/ml) (Ramnani andGupta 2004) andHalobacterium spp. SP1 (69 U/ml) (Akolkaret al. 2009).

The type of substrate and its optimum concentration forgrowth and protease production has been shown to varydepending upon production culture. The JSC was found tobe preferred substrate in comparison to other agrowastes forprotease production by B. tequilensis P15. It should be notedhere that JSC contains approximately 22 % protein alongwith other nutrients (Rakshit et al. 2008), which justifies itto be a good substrate for microbial growth as well asprotease production. Several other deoiled seedcakes suchas coconut oil cake, cotton oil cake, groundnut oil cake,mustard oil cake, olive oil cake, palm kernel cake, sesameoil cake, and soybean cake have been reported for theirsuitability as substrates for protease production using differ-ent bacteria and fungi (Joo et al. 2002; Laxman et al. 2005;Sandhya et al. 2005). In the present study, 2 % (w/v) JSC inliquid medium was found to be optimum for protease pro-duction by B. tequilensis P15. The decrease in enzymeproduction at higher JSC concentrations may be attributedto increased medium viscosity resulting in poor oxygentransfer into the medium, which might have altered bacterialmetabolism and consequently decreased enzyme production(Schügerl 1981).

The enzyme exhibited activity over a temperature rangeof 30–70 °C, with maximum activity at 50 °C. This is inagreement with the report of Xu et al. (2010), who reportedan extracellular organic solvent-tolerant protease from B.cereus having the same temperature optima. In the presentstudy, the presence of Ca2+ ions improved the half-life of theenzyme at its optimum and higher temperatures. The Ca2+

ions have been reported to stimulate enzyme activity bymaintaining the rigid conformation of the enzyme moleculethereby conferring protection against thermal denaturation(Smith et al. 1999).

The P15 protease exhibited activity over a broad range ofpH, 6.0–11.0, with optimum activity at pH 8.0. Organicsolvent-stable proteases from Pseudomonas aeruginosaPST-01 (Ogino et al. 1999) and B. cereus (Xu et al. 2010)were reported to have optimal pH between 8.0 and 9.0, withno activity at pH values over 10.0. Since detergent solutions

Table 2 Effects of various metal ions, inhibitors, detergents, andbleach-oxidants on P15 protease activity

Relative enzyme activity (%)

Control 100

Metal ions (5 mM)

Ca2+ 126.17

Cu2+ 15.80

Fe2+ 9.79

Hg2+ 49.61

Mg2+ 69.0

Mn2+ 61.3

Zn2+ 108.39

Divalent chelator (5 mM)

EDTA 120.92

Inhibitors (5 mM)

PMSF 56.12

pCMB 113.74

β-mercaptoethanol 92.23

Surfactants (1 %; w/v)

Tween 20 Non-ionic 100

Tween 80 91.02

Triton X-100 87.30

SDS (5 mM) Anionic 52.6

CTAB (5 mM) Cationic 21.2

Ariel® 25.97

Surf® 90.82

Tide® 54.49

Bleach-oxidant (1 % w/v)

H2O2 91.02

350 Ann Microbiol (2014) 64:343–354

generally have pH between 8.0 and 10.5, high activity atalkaline pH may prove as an important factor required inalmost all detergent enzymes such as those described by Liet al. (2009) and Cheng et al. (2010).

In the present study, it was observed that the P15 proteaseactivity was enhanced in the presence of non-polar (high logPow value) solvents such as n-hexadecane, 1-dodecanol, iso-octane, n-heptane, xylene, and benzene. A similar effect onprotease produced by an organic solvent-tolerant B. cereusWQ9-2 in the presence of hydrophobic solvents has recentlybeen reported by Xu et al. (2010). This enhancement ofenzyme activity in the presence of solvents might be due tothe replacement of some water molecules of the enzyme withsolvent molecules, thereby causing favorable changes in theenzyme structure (Ogino et al. 1999). The stabilization of

PST-01 protease by the addition of various organic solventshas also been reported by Ogino et al. (1999). Thus, enzymesare sometimes much more stable in solutions containing or-ganic solvents than in organic solvent-free aqueous media.The P15 protease was found to be significantly inhibited in thepresence of methanol, which is in agreement with studies onB. cereus WQ9-2 protease reported by Xu et al. (2010).However, Ogino et al. (1995) reported high methanol andethanol stable protease secreted by the organic solvent-tolerant bacterium P. aeruginosa PST-01.

The increase in enzyme activity in the presence of Ca2+

may be due to stabilization of the enzyme in its activeconformation (Glusker et al. 1999). It probably acts as a saltor ion bridge via a cluster of carboxylic groups and therebymaintains the rigid conformation of the enzyme molecule

Fig. 4 a Blood destainingperformance of P15 protease, benzymatic dehairing of goathide, c enzymatic stripping ofthe gelatinous layer of usedphotographic film

Ann Microbiol (2014) 64:343–354 351

(Smith et al. 1999). A similar trend was observed in thepresent study where an increase in the P15 protease activityin the presence of Ca2+ and Zn2+, but a reduction in thepresence of Fe2+, Cu2+, Hg2+, and Mn2+, were observed.Such a drop in enzyme activity in the presence of transitionmetals has been ascribed to the interaction with chargedside-chain groups of accessible amino acids, thus influenc-ing the conformation and stability of the enzyme (Rahmanet al. 2006). Furthermore, Hg2+ is recognized as an oxidiz-ing agent of the thiol group, and the enzyme inhibition bythis ion could suggest the presence of important –SH groups(such as free cysteine) at or near the active site (Thys andBrandelli 2006).

P15 protease was inhibited by serine inhibitor PMSF,and, therefore, the enzyme was classified under the serineprotease family.

The protease activity in the present study was marginallyaffected by the presence of 1 % non-ionic surfactants likeTween 20, Tween 80, and Triton X-100, whereas anionic(SDS) and cationic (CTAB) surfactants exerted significantinhibitory effects. These results are well in agreement withreports on proteases from other Bacillus sp. (Rai andMukherjee 2009; Sareen and Mishra 2008). The effect ofdetergents on the enzyme can be correlated with theirhydrophilic/lipophilic balance (HLB), which is defined asthe way a detergent is distributed between polar and non-polar phases (Furth 1980). Thus, non-ionic surfactants withlow HLB value (Triton X-100: HLB 13.5; Tween 20: HLB16.7; and Tween 80: HLB 15) were less detrimental on theactivity of P15 protease in comparison to SDS with a higherHLB of 40. Although CTAB accounts for a lowHLB of 10, itsunfavorable electrostatic interaction with the enzyme maycause unfolding and/or disruption of substrate binding thusresulting in diminished enzyme activity (Otzen 2002).Furthermore, the P15 protease was stable in the presence ofa bleach-oxidant like hydrogen peroxide, which is in completeagreement with the report of Sana et al. (2006). Oxidant- andbleach-stability are the pre-requisite for the application ofenzymes in detergent formulation. Several researchers haveengineered proteases in order to make them bleach-stable(Tuschiya et al. 1992; Wolff et al. 1996), whereas the P15protease reported in the present study exhibited inherent sta-bility towards high concentrations of hydrogen peroxide.

Blood stain removal studies demonstrated that the P15protease was more efficient than the earlier reports of SubbaRao et al. (2009) and Jellouli et al. (2011), wherein the enzymerequired 30 min of detergent supplementation for completeremoval of blood stains from cloth. Thus, the detergent stabil-ity and stain-removing ability of P15 protease makes it apotential candidate for use as a detergent additive.

The enzymatic dehairing process has gained impor-tance as an alternative to chemical processes, owing toits eco-friendly nature in addition to improvements in

leather quality (Sivasubramanian et al. 2008). Severalmicrobial proteases have been evaluated for their dehairingcharacter, and it was noticed that only those enzymes with pHstability under alkaline conditions with non-collagenolyticproperties were having an edge over others (Subba Rao et al.2009). The enzymatic treatment loosens the hair and epider-mis because of degradation of specific proteins, glycoproteins,and proteoglycans in the basal membrane (Sivasubramanianet al. 2008). Similar experimental observations were reportedwith protease produced by B. cereus MCM B-326(Nilegaonkar et al. 2007). P15 protease was found to be moreeffective in dehairing of goat skin in comparision to alkalineproteases from B. circulans (Subba Rao et al. 2009) and B.cereusMCM B-326 (Nilegaonkar et al. 2007), which took 12and 21 h, respectively.

In the gelatin strip-off experiment, the P15 protease effi-ciently decomposed the gelatin layer within 20 min at 50 °C.Nakiboğlu et al. (2001) reported B. subtilis ATCC-6633 toproduce neutral and alkaline protease which collectivelydecomposed the gelatin layer within 15 min, while the alkalineprotease took more than 20 min to act and the enzyme rapidlybecome inactivated at temperatures of more than 60 °C.

Conclusion

In the present study, production, optimization, and charac-terization of an organic solvent-tolerant, detergent- andbleach-oxidant-stable protease from Bacillus tequilensisP15 has been described. The maximum production of P15protease was achieved using 2 % (w/v) JSC medium. Itsapplication as a detergent additive, in the dehairing of hide,and in gelatin hydrolysis from used photographic film forthe recovery of silver was demonstrated. The P15 proteaseexhibited good tolerance toward a wide range of organicsolvents (log Pow values varying from 8.8 to −1. 249) whichfurther adds versatility to its industrial applications.

Acknowledgment A. Bose gratefully acknowledges the UniversityGrants Commission, New Delhi, for financial support in the form of ameritorious fellowship.

References

Adams MWW, Perler FB, Kelly RM (1995) Extremozymes: expandingthe limits of biocatalysis. Nature Biotechnol 13:662–668

Akolkar A, Bharambe N, Trivedi S, Desai A (2009) Statistical optimi-zation of medium components for protease production by anextreme haloarchaeon, Halobacterium sp. SP1(1). Lett ApplMicrobiol 48:77–83

Anson ML (1939) The estimation of pepsin, trypsin, papain andcathepsin with hemoglobin. J Gen Physiol 22:79–89

352 Ann Microbiol (2014) 64:343–354

Chen K, Arnold FH (1993) Tuning the activity of an enzyme for unusualenvironments: sequential random mutagenesis of subtilisin E forcatalysis in dimethylformamide. Proc Natl Acad Sci USA90:5618–5622

Cheng K, Lu F-P, Li M, Liu L-L, Liang X-M (2010) Purification andbiochemical characterization of a serine alkaline protease TC4from a new isolated Bacillus alcalophilus TCCC11004 in deter-gent formulations. Afr J Biotechnol 9:4942–4953

Dave BR, Sudhir AP, Pansuriya M, Raykundaliya DP, SubramanianRB (2012) Utilization of Jatropha deoiled seedcake for productionof cellulases under solid-state fermentation. Bioprocess BiosystEng 35:1343–1353

Davis BG (2003) Chemical modification of biocatalysts. Curr OpinBiotechnol 14:379–386

Doukyua N, Ogino H (2010) Organic solvent-tolerant enzymes.Biochem Eng J 48:270–282

Fang Y, Liu S, Wang S, Lv M (2009) Isolation and screening of a novelextracellular organic solvent-stable protease producer. BiochemEng J 43:212–215

Furth AJ (1980) Removing unbound detergent from hydrophobic pro-teins. Anal Biochem 109:207–215

Gessesse A (1997) The use of nug meal as a low-cost substrate for theproduction of alkaline protease by the alkaliphilic Bacillus sp. AR-009 and some properties of the enzyme. Biores Technol 62:59–61

Glusker JP, Katz AK, Bock CW (1999) Metal ions in biologicalsystems. Rigaku J 16:8–16

Gupta A, Khare SK (2006) A protease stable in organic solvents fromsolvent tolerant strain of Pseudomonas aeruginosa. BioresTechnol 97:1788–1793

Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molec-ular approaches and industrial applications. Appl MicrobiolBiotechnol 59:15–32

Habulin M, Primožič M, Knez Ž (2005) Stability of proteinase formCarica papaya latex in dense gases. J Supercrit Fluids 33:27–34

Jellouli K, Ghorbel-Bellaaj O, Ayed HB, Manni L, Agrebi R, Nasri M(2011) Alkaline-protease from Bacillus licheniformis MP1: puri-fication, characterization and potential application as detergentadditive and for shrimp waste deproteinization. ProcessBiochem 46:1248–1256

Joo HS, Kumar CG, Park GC, Kim KT, Paik SR, Chang CS (2002)Optimization of the production of an extracellular alkaline prote-ase from Bacillus horikoshi. Process Biochem 38:155–159

Joshi C, Khare SK (2011) Utilization of deoiled Jatropha curcas seedcake for production of xylanase from thermophilic Scytalidiumthermophilum. Bioresour Technol 102:1722–1726

Karan R, Singh SP, Kapoor S, Khare SK (2011) A novel organic solventtolerant alkaline protease from a newly isolated Geobacteriumsp.EMB2 (MTTC 10310): production optimization by responsesurface methodology. New Biotechnol 28:136–145

Kumar D, Bhalla TC (2005) Microbial proteases in peptide synthesis:approaches and applications. Appl Microbiol Biotechnol 68:726–736

Laxman RS, Sonawane AP, More SV, Seetarama Rao B, Rele MV,Jogdand VV, Deshpande VV, Rao MB (2005) Optimization andscale up of production of alkaline protease from Conidioboluscoronatus. Process Biochem 40:3152–3158

Li S, HeB, Bai Z, Ouyang P (2009) A novel organic solvent-stable alkalineprotease from organic solvent-tolerant Bacillus licheniformisYP1A. JMol Catal B 56:85–88

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Proteinmeasurement with the Folin phenol reagent. J Biol Chem193:265–275

Mahanta N, Gupta A, Khare SK (2008) Production of protease andlipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seedcake as substrate.Biores Technol 99:1729–1735

Makkar H, Maes J, Greyt WD, Becker K (2009) Removal and degrada-tion of phorbol esters during pre-treatment and transesterification ofJatropha curcas oil. J Am Oil Chem Soc 86:173–181

Martínez-Herrera J, Siddhuraju P, Francis G, D vila-Ortíz G, Becker K(2006) Chemical composition, toxic/antimetabolic constituents,and effects of different treatments on their levels, in four prove-nances of Jatropha curcas L. from Mexico. Food Chem 96:80–89

Maruyama T, Nagasawa S-I, Goto M (2002) Enzymatic synthesis ofsugar amino acid esters in organic solvents. J Biosci Bioeng94:357–361

Mehta VJ, Thumar JT, Singh SP (2006) Production of alkaline proteasefrom an alkaliphilic actinomycetes. Biores Technol 97:1650–1654

Nakiboğlu N, Toscali D, Yaşa I (2001) Silver recovery from wastephotographic films by an enzymatic method. Turk J Chem25:349–353

Nilegaonkar SS, Zambare VP, Kanekar PP, Dhakephalkar PK, SarnaikSS (2007) Production and partial characterization of dehairingprotease from Bacillus cereus MCM B-326. Biores Technol98:1238–1245

Noritomi H, Suzuki K, Kikuta M, Kato S (2009) Catalytic activity ofα-chymotrypsin in enzymatic peptide synthesis in ionic liquids.Biochem Eng J 47:27–30

Ogino H, Miyamoto K, Ishikawa H (1994) Organic-solvent-tolerantbacterium, which secretes organic-solvent-stable lipolytic en-zyme. Appl Environ Microbiol 60:3884–3886

Ogino H, Watanabe F, Yamada M, Nakagawa S, Hirose T, Noguchi A,Yasuda M, Ishikawa H (1999) Purification and characterization oforganic solvent-stable protease from organic solvent-tolerantPseudomonas aeruginosa PST-01. J Biosci Bioeng 87:61–68

Ogino H, Yasui K, Shiotani T, Ishihara T, Ishikawa H (1995) Organicsolvent tolerant bacterium, which secretes an organic solvent-stable proteolytic enzyme. Appl Environ Microbiol 61:4258–4262

Otzen DE (2002) Protein unfolding in detergents: Effect of micellestructure, ionic strength, pH, and temperature. Biophys J83:2219–2230

Pandey S, Rakholiya K, Raval VH, Singh SP (2012) Catalysis andstability of an alkaline protease from a haloalkaliphilic bacteriumunder non-aqueous conditions as a function of pH, salt andtemperature. J Biosci Bioeng 114:251–256

Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD,Rollence ML, Bryan PN (1989) Large increases in general stabil-ity for subtilisin BPN’ through incremental change in the freeenergy of unfolding. Biochemistry 28:7205–7213

Rahman RNZRA, Bahrum SN, Salleh AB, Basri M (2006) S5 lipase:an organic solvent tolerant enzyme. J Microbiol 44:583–590

Rahman RNZRA, Mahamad S, Salleh AB, Basri M (2007) A neworganic solvent tolerant protease from Bacillus pumilus 115b. JInd Microbiol Biotechnol 34:509–517

Rai SK, Mukherjee AK (2009) Ecological significance and somebiotechnological application of an organic -solvent stable alkalineserine protease from Bacillus subtilis strain DM-04. BioresTechnol 100:2642–2645

Raimbault M (1998) General and microbiological aspects of solidsubstrate fermentation. Electron J Biotechnol 1:3

Rakshit KD, Darukeshwara J, Raj KR, Narasimhamurthy K, Saibaba P,Bhagya S (2008) Toxicity studies of detoxified Jatropha meal(Jatropha curcas) in rats. Food Chem Toxicol 46:3621–3625

Ramnani P, Gupta R (2004) Optimization of medium composition forkeranitase production on feather by Bacillus licheniformis RG1using statistical methods involving response surface methodology.Biotechnol Appl Biochem 40:191–196

Ru MT, Hirokane SY, Lo AS, Dordick JS, Reimer JA, Clark DS (2000)On the salt-induced activation of lyophilized enzymes in organicsolvents: effect of salt kosmotropicity on enzyme activity. J AmChem Soc 122:1565–1571

Ann Microbiol (2014) 64:343–354 353

Ruiz DM, De Castro RE (2007) Effect of organic solvents on theactivity and stability of an extracellular protease secreted by thehaloalkaliphilic archaeon Natrialba magadii. J Ind MicrobiolBiotechnol 34:111–115

Sana B, Ghosh D, Saha M, Mukherjee J (2006) Purification and charac-terization of a salt, solvent, detergent and bleach tolerant proteasefrom a new gamma-proteobacterium isolated from the marine envi-ronment of the Sundarbans. Process Biochem 41:208–215

Sandhya C, Sumantha A, Szakaes G, Pandey A (2005) Comparativeevaluation of neutral protease production by Aspergillus oryzae insubmerged and solid-state fermentation. Process Biochem 40:2689–2694

Sardessai YN, Bhosle S (2004) Industrial potential of organic solventtolerant bacteria. Biotechnol Prog 20:655–660

Sareen R, Mishra P (2008) Purification and characterization of organicsolvent stable protease from Bacillus licheniformis RSP-09-37.Appl Microbiol Biotechnol 79:399–405

Schügerl K (1981) Oxygen transfer into highly viscous media. AdvBiochem Eng Biotechnol 19:71–174

Sellek GA, Chaudhuri JB (1999) Biocatalysis in organic media usingenzymes from extremophiles. Enzym Microb Technol 25:471–482

Shankar S, More SV, Seeta Laxman R (2010) Recovery of silver fromwaste X-ray film by alkaline protease from Conidioboluscoronatus. Kathmandu Univ J Sci Eng Technol 6:60–69

Sivasubramanian S, Murali Manohar B, Rajaram A, Puvanakrishna R(2008) Ecofriendly lime and sulfide free enzymatic dehairing ofskins and hides using a bacterial alkaline protease. Chemosphere70:1015–1024

Smith CA, Toogood HS, Baker HM, Daniel RM, Baker EN (1999)Calcium-mediated thermostability in the Subtilisin superfamily:the crystal structure of Bacillus Ak.1 protease at 1.8 Å resolution.J Mol Biol 294:1027–1040

Subba Rao C, Sathish T, Ravichandra P, Prakasham RS (2009)Characterization of thermo- and detergent stable serine protease

from isolated Bacillus circulans and evaluation of eco-friendlyapplications. Process Biochem 44:262–268

Tang XY, Wu B, Ying HJ, He BF (2010) Biochemical properties andpotential applications of a solvent-stable protease from the high-yield protease producer Pseudomonas aeruginosa PT121. ApplBiochem Biotechnol 160:1017–1031

Thumar JT, Singh SP (2009) Organic solvent tolerance of an alkalineprotease from salt-tolerant alkaliphilic Streptomyces clavuligerusstrain Mit-1. J Ind Microbiol Biotechnol 36:211–218

Thys RCS, Brandelli A (2006) Purification and properties of akeratinolytic metalloprotease from Microbacterium sp. J AppMicrobiol 101:1259–1268

Torres S, Pandey A, Castro GR (2011) Organic solvent adaptation ofGram positive bacteria: applications and biotechnological poten-tials. Biotechnol Adv 29:442–452

Tuschiya K, Nakamura Y, Sakashita H, Kimura T (1992) Purificationand characterization of a thermostable alkaline protease fromalkalophilic Thermoactinomyces sp. HS682. Biosci BiotechnolBiochem 56:246–250

Von Hippel PH, Schleich T (1969) The effects of neutral salts on thestructure and conformational stability of macromolecules in solu-tion. In: Timasheff SN, Fasman GD (eds) Structure and stabilityof biological macromolecules, vol 2. Marcel Dekker, New York,pp 417–574

Wolff AM, Showell MS, Venegas MG, Barnett BL, Wertz WC (1996)Laundry performance of subtilisin proteases. Adv Exp Med Biol379:113–120

Xu J, Jiang M, Sun H, He B (2010) An organic solvent-stable proteasefrom organic solvent-tolerant Bacillus cereus WQ9-2: purifica-tion, biochemical properties, and potential application in peptidesynthesis. Biores Technol 101:7991–7994

Yang L, Dordick JS, Garde S (2004) Hydration of enzyme in non-aqueous media is consistent with solvent dependence of its activ-ity. J Biophys 87:812–821

354 Ann Microbiol (2014) 64:343–354