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New Biotechnology �Volume 28, Number 2 � February 2011 RESEARCH PAPER

A novel extracellular protease fromPseudomonas aeruginosa MCM B-327:enzyme production and its partialcharacterizationVasudeo Zambare, Smita Nilegaonkar and Pradnya Kanekar

Microbial Sciences Division, Agharkar Research Institute, Pune 411004, India

The focus of this study was on production, purification and characterization of dehairing protease from

Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity,

395 U mL�1, was observed in the medium containing soybean meal and tryptone, at pH 7 and 308C. The

crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of

dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively.

Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular

weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56 kDa protease (PA02)

was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7–9 and temperature 20–

508C with optimum pH of 8 and temperature 358C. Moreover, the enzyme activity of PA02 protease was

not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine,

cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02

protease was towards various natural and synthetic proteolytic substrates but inactive against collagen

and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application

in dehairing for environment-friendly leather processing.

IntroductionMicrobial proteases are among the most important hydrolytic

enzymes and have been studied extensively. This group of

enzymes represents one of the three largest groups of industrial

enzymes and accounts for approximately 60% of the total enzyme

sales in the world [1]. They have numerous applications in the

industrial production of different items, viz. detergents, foods,

pharmaceutical, leather, diagnostics, including waste manage-

ment and silver recovery [2].

Leather-making is a processing industry with both socio-eco-

nomic and environmental implications. Leather is a by-product of

meat industry. It offers more benefits through employment and

from sales of leather goods. By contrast, it has negative implica-

tions emanating from the wastes associated with industrial pro-

cessing [3]. In a tannery, a raw hide is subjected to a series of

chemical treatments before tanning and finally converted to

Corresponding author: Nilegaonkar, S. (ssnilegaonkar@gmail.com)

1871-6784/$ - see front matter � 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.10.002

finished leather. Proteases are envisaged to have extensive appli-

cations in leather industry. Alkaline proteases may play a vital role

in these treatments by replacing these hazardous chemicals espe-

cially involved in soaking, dehairing and bating [4]. Increased

usage of enzymes for dehairing and bating not only prevents

pollution problems, but also is effective in saving time with better

quality leather. Recently, several research papers were published

on enzymatic leather processing especially on dehairing operation

of leather manufacturing [5–11]. All these protease were obtained

from Bacillus sp. Pseudomonas aeruginosa produced protease which

is recently studied by Yadav et al. [12] and Jain et al. [13] but not for

dehairing operation. At present only one report is available of

dehairing operation of protease by Najafi et al. [14]. Apart from

these some microorganisms such as Streptomyces nogalator [15],

Conidiobolus coronatus [16] and Aspergillus flavus [17] produced

extracellular protease with dehairing activity. However, some

proteases were not suitable for dehairing, since they have collagen

and keratin degrading activities, which destroys the collagen

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RESEARCH PAPER New Biotechnology �Volume 28, Number 2 � February 2011

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structure of hide and avoid keratin recovery. Therefore, it is

essential to explore proteases with highest dehairing activity,

without collagenolytic and keratinolytic activities.

In this investigation, the production and effect of different

parameters on bacterial growth and protease secretion are studied.

The cell-free broth, comprising of two extracellular proteases, pos-

sessed dehairing activity. Here wealso report purificationand partial

characterization of one of the dehairing proteases produced by a

mesophilic bacterium Pseudomonas aeruginosa MCM B-327.

Materials and methodsIsolation and identification of microorganism producingextracellular proteaseA mesophilic bacterium Pseudomonas aeruginosa MCM B-327 was

isolated from vermicompost pit soil sample collected from Pune,

India. The morphological, physiological and biochemical proper-

ties of the isolate were compared with Bergey’s manual of systema-

tic bacteriology [18]. Analytical Profiling Index (API) system and

16S rRNA sequencing were also carried out for the identification of

the strain at species level. The sequence was submitted to GenBank

database to get the accession number. The stock culture was

maintained on Nutrient agar (Himedia, India) at 48C and as a

glycerol stock at �208C. Isolate was deposited in MACS Collection

of Microorganisms (MCM) at Agharkar Research Institute, Pune

and Microbial Type Culture Collection (MTCC) at Institute of

Microbial Technology (IMTECH), Chandigarh.

Optimization of protease productionVarious media were used for the production of protease in 250 mL

Erlenmeyer flasks containing 100 mL of the liquid medium: syn-

thetic medium-casein (SMC, 0.7% K2HPO4, 0.3% KH2PO4, 0.01%

MgSO4, and 1% casein), Nutrient broth supplemented with 1%

casein (NBC), starch–soybean meal (SS, 2% starch, 1% soybean

meal), soybean meal–tryptone (ST, 1% soybean meal, 1% tryp-

tone) and nutrient broth (NB, 1.3%). Protease production was

optimized with respect to various environmental and nutritional

parameters such as shaking speed (100–200 rpm), cultivation

volume (25–200 mL in 250 mL capacity flask), inoculum size

(0.5–10%, v/v), initial pH (6–12) and temperatures (25–408C),

nitrogen and carbon sources. The inorganic nitrogen sources

(1%, w/v) such as ammonium chloride, ammonium sulphate,

ammonium phosphate, ammonium nitrate and sodium nitrite

were used. Tryptone, beef extract, yeast extract, casein, soybean

meal and peptone served as organic nitrogen sources (1%, w/v).

Starch, glucose, sucrose, fructose, lactose and maltose were used at

1% (w/v) as supplementary carbon sources. CaCO3, CaCl2,

K2HPO4, KH2PO4, FeSO4, ZnSO4, MgSO4, NaCl and MnSO4 were

used as metal ions sources (0.3%). All incubation experiments

(except the temperature experiment) were carried out at 308Cfor 72 h. All the analyses were conducted independently in tripli-

cates and the data presented here are the mean value � standard

deviations (SD). Analysis of variance (ANOVA) with repeated mea-

sures was carried out using GLM command of software SPSS (SPSS

version 10, Windows 98 version).

Dehairing of buffalo hide and pollution load analysisCrude enzyme was concentrated by ammonium sulfate precipita-

tion at 60% saturation. The precipitate of enzyme (1%, w/w in 20%

174 www.elsevier.com/locate/nbt

water) was applied on flesh side of tap water soaked buffalo hide

(3 cm � 3 cm) and kept at ambient temperature (28 � 28C) in a dry

place. Control with conventional chemical dehairing was carried

out by 10% lime, 2.5% sodium sulphide and 20% water [19].

Loosening of hair and epidermis were observed by mechanical

means at an hourly interval. The effluent obtained after chemical

and enzymatic dehairing was collected and analyzed for total

dissolved solids (TDS), total suspended solids (TSS) and chemical

oxygen demand (COD) using standard analytical procedures [20].

Measurement of enzyme activity and proteinProtease activity was measured using caseinolytic assay [21]. The

culture supernatant (1 mL) was incubated in 4 mL of 0.625%

casein at 378C for 30 min. The reaction was stopped by the addi-

tion of 5 mL of trichloroacetic acid (5%) and the casein hydrolysis

product was measured by modified Folin–Ciocalteu method,

against inactive enzyme. A standard graph was generated using

standard tyrosine of 10–50 mg mL�1. One unit (U) of protease

activity was defined as the amount of enzyme, which liberated

1 mg tyrosine per min at 378C. Protein concentration was mea-

sured by the method of Biuret [22] using BSA as the standard.

Purification of dehairing proteaseThe enzyme from cell-free broth was partially purified by ammo-

nium sulphate precipitation (60% saturation) at 48C and dialyzed

against 50 mM Tris–HCl buffer (pH 8) for 24 h. The enzyme pre-

paration was loaded on a Sephadex G 50 column (2.5 � 15.0 cm),

previously equilibrated with the same buffer. The active fractions

were pooled and subjected to preparative native-polyacrylamide

gel electrophoresis (PAGE). Zymogram of a small PAGE strip was

carried out to detect protease activity bands [25] and Rf values were

recorded. Corresponding to the Rf values, active protein band from

gel was cut and enzyme was eluted in 50 mM Tris–HCl buffer (pH

8). All the purification steps were performed at 48C. All bands with

protease activity were screened for their dehairing activity on

buffalo hide by Zambare et al. [23]. Protease band exhibiting

potential dehairing activity was used for further characterization.

Characterization of purified proteaseThe molecular weight of purified protease was determined by

comparing with mobility of standard molecular weight marker

proteins of Sigma (St. Louis, MO, USA). The purified enzyme of P.

aeruginosa MCM B-327 was characterized with respect to its activity

(100 U and 8.5 mg protein) under different pH values in the range

of 6–12 (pH 6, 50 mM acetate buffer; pH 7–8, 50 mM phosphate

buffer; pH 9–10, 50 mM Tris–HCl buffer; pH 11–12, glycine–NaOH

buffer at 308C) and temperature range of 20–508C with 50 mM

phosphate buffer (pH 7) conditions. The effect of Na+, Ca2+, Zn2+,

Mg2+, Fe2+, Cr2+, Hg2+, dithiothreitol (DTT), 2-mercaptoethanol,

sodium dodecyl sulphate (SDS), ethylenediaminetetraacetic acid

(EDTA), iodoacetamide, phenylmethylsulphonyl fluroride (PMSF)

and pepstatin A on the enzyme activity was also studied. The

substrate specificity of the purified protease was examined against

casein, bovine serum albumin, gelatin, elastin-orcein, keratin

azure, and collagen. N-benzoyl-L-tyrosine ethyl ester (BTEE), N-

benzoyl-L-arginine ethyl ester (BAEE), N-succinyl-Ala-Ala-Ala-p-

nitroanilide (SAAAPNA), N-succinyl-Ala-Ala-Pro-Phe-p-nitroani-

lide (SAAPPPNA), N-benzoyl-DL-arginine-p-nitroanilide (BAPNA),

New Biotechnology �Volume 28, Number 2 � February 2011 RESEARCH PAPER

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FIGURE 1

Effect of pH (at 308C) and temperature (at pH 7) on protease production fromP. aeruginosa MCM B-327 (soybean–trytone medium, 1% inoculum, 36 h,

150 rpm, CV of 100 mL in 250 mL capacity flask). Error bar represents the

mean of triplicate analysis � standard deviation.

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N-benzoyl-Pro-Phe-Arg-p-nitroanilide (BPPAPNA) and N-[3-(2-fur-

yl)acryloy]-Leu-Gly-Pro-Ala (FALGPA) were purchased from Sigma

Chemicals (St. Louis, MO, USA). The kinetic parameters used in

this study were 100 U enzymes, substrate concentration in the

range of 0–20 mg/mL for casein and 0–10 mM for SAAAPNA,

SAAPPPNA, 100 mM phosphate buffer (pH7) and at 358C and

the enzyme kinetic were performed according to Bharathiraja

and Jayamuthunagai [24].

Results and discussionIsolation and identification of bacteriaAn organism was isolated from vermicompost pit soil. It was

identified as Pseudomonas aeruginosa on the basis of its morpho-

logical and physiological characteristics, biochemical tests, API

system and 16S rRNA sequencing. A comparison of the DNA

sequence with the sequences in the National Center for Biotech-

nology Information (NCBI) database with BLAST software showed

100% sequence identity with the published 16S rRNA sequences of

P. aeruginosa PA01. The 16S rRNA sequence of the isolate is

deposited in GenBank database with accession number

DQ473435. Culture deposition numbers for P. aeruginosa were

MCM B-327 and MTCC-5270.

Optimization of growth conditions for protease productionThe protease production by P. aeruginosa MCM B-327 was carried

out with various media (SMC, NBC, SS, ST and NB). The use of

soybean meal in combination with tryptone produced signifi-

cantly maximum protease activity of 299.85 � 6.2 U mL�1

(p < 0.001) at 72 h compared to NB (120.51 � 2.2 U mL�1), NBC

(44.84 � 5.9 U mL�1), SSM (22.99 � 6.3 U mL�1) and SS

(14.46 � 2.7 U mL�1). Likewise different production media were

used for protease production from various microbes by various

researchers [14,36] Based on the results, ST medium was selected

for further optimization studies.

A significantly higher protease activity, that is 2.79-fold was

observed under shaking culture condition (399.05 � 11.8 U mL�1)

than static culture condition (142.68 � 3.9 U mL�1). In the litera-

ture, shake culture condition has been reported to favour protease

production [25,26]. The cultivation volume (CV) of 100 mL med-

ium in 250 mL capacity Erlenmeyer flask, that is 1.0:1.5 was found

to be a suitable cultivation condition. This was probably due to the

compromise between the mass transfer and/or shear stress at this

volume. In another report on the effect of oxygen supply on

protease production from P. aeruginosa K-187, the best results were

observed when the ratio was 1:5 [27].

The effect of different inoculum sizes (0.5–10%) with initial cell

count 27.57 � 109 cells mL�1 on the protease production by P.

aeruginosa MCM B-327 was studied up to 72 h. Interestingly,

maximum protease activity was observed at inoculum size of

1% and a further increase in the inoculum size up to 10% did

not influence the enzyme yield. Rahman et al. [26,28] reported

that the increase of inoculum size up to 4% and 10% was beneficial

for protease production from P. aeruginosa. In our study, we report

different behaviors of P. aeruginosa where an increase in protease

activity was only observed at inoculum size up to 1% and were

similar to our published work for Bacillus cereus [25].

Any fermentation process is known to be influenced by the pH

and temperature of environment. Our results showed that max-

imum protease secretion by P. aeruginosa was observed after 72 h

incubation at pH 7 and 308C (Fig. 1). Similar optimum pH and

temperature were observed and reported for P. aeruginosa strain K

by Rahman et al. [26]. Also, P. aeruginosa MCM B-327 showed two

peaks at pH 7 and 11 which are similar to study on protease from P.

aeruginosa MN1 by Bayoudh et al. [29].

Effect of nitrogen sources on enzyme production by P. aeruginosa

MCM B-327 was studied (Fig. 2a) Maximum protease production

was obtained with soybean meal–tryptone (371.86 � 5.4 U mL�1)

followed by beef extract (196.33 � 5.3 U mL�1) and peptone

(210.71� 4.4 U mL�1). P. aeruginosa produced markedly less pro-

tease activity in the presence of sole soybean meal

(9.17 � 4.4 U mL�1) or sole tryptone (100.31 � 5.4 U mL�1), but

the combination of both (1:1 ratio) showed synergistic effect on

activity. Deshpande et al. [30] reported the use of soybean meal for

induced protease production by Conidiobolus coronatus. Thus, our

findings on the positive impact of soybean meal during protease

production are supporting literature reports. Protease production

from P. aeruginosa was studied with various easily available and cost-

effective nitrogen sources such as crab shell waste, deoiled Jatropha

seed cake, fish protein hydrolysate and animal fleshing waste by

several researchers [31–33]. Protease production from P. aeruginosa

MCM B-327 at 72 h incubation was very low in the presence of

inorganic nitrogen sources and an organic source such as casein.

Similar observations for inorganic nitrogen sources and casein were

observed by Fortelius and Markkanen [34] for protease from Tritir-

achium album.

As poor growth in any fermentation system is normally asso-

ciated with the low nutritional capacity of solid substrates, addi-

tional carbon-containing compounds are used in media

formulation to enhance microbial growth and subsequently

improved enzymes. In our study, carbohydrate supplementation

in ST medium had no enhancing effect on protease production

(Fig. 2b). In fact, supplementation with monosaccharide such as

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RESEARCH PAPER New Biotechnology �Volume 28, Number 2 � February 2011

[()TD$FIG]

Nitrogen sources (1%)

ST

Bee

f ex

trac

t

Yea

st e

xtra

ct

Pep

tone

Try

pton

e

Cas

ein

Soyb

ean

mea

l

Am

mon

ium

chl

orid

e

Am

mon

ium

sul

phat

e

Sodi

um n

itri

te

Pot

asiu

m n

itri

te

Am

mon

ium

pho

spha

te

Pro

teas

e ac

tivi

ty (

Um

L-1

)

0

100

200

300

400

Carbon sources (1%)

ST

Glu

cose

Star

ch

Sucr

ose

Fru

ctos

e

Lac

tose

Mal

tose

Pro

teas

e ac

tivi

ty (

U m

L-1

)

0

100

200

300

400

500

Metal ions (0.3%)

a

c

ST

CaC

O3

CaC

l2

FeS

O4

ZnS

O4

MnS

O4

MgS

O4

K2H

PO

4

KH

2PO

4

NaC

l

Pro

teas

e ac

tivi

ty (

U m

L-1

)

0

100

200

300

400

FIGURE 2

Effect of nutritional parameters (a: nitrogen sources, b: carbon sources, c: metal ions) on protease production from P. aeruginosa MCM B-327 (pH 7, 308C, 1%inoculum, 36 h, 150 rpm, CV of 100 mL in 250 mL capacity flask). Error bar represents the mean of triplicate analysis � SD.

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glucose and fructose suppressed the protease production by 95%

and 60%, respectively. It is reported that the enzyme production is

regulated by physiological mechanisms and the production of

hydrolytic enzyme is often repressed by the catabolites of glucose

(catabolite repression) in liquid culture [35].

Sometimes, metal ions are important for enzyme actions and

their structural modifications. Effect of supplementation of metal

ions in ST medium was studied for P. aeruginosa and had no

enhanced effect on protease production (Fig. 2c). In fact, addition

of FeSO4, MnSO4 and ZnSO4 in the medium resulted in low

protease activities. Calcium ion neither enhanced nor suppressed

protease activity. This may suggest that the enzyme production is

calcium independent. Nicodeme et al. [36] have also reported that

the Pseudomonas sp. LBSA1 did not show a significant increase in

the specific proteolytic activity when CaCl2 was added in the

medium.

A maximum protease production (395 U mL�1) occurred at

308C after 72 h of incubation under following liquid fermentation

176 www.elsevier.com/locate/nbt

conditions: soybean meal (1%), tryptone (1%), pH 7, 1% inoculum

(27.57 � 109 cells mL�1). Initially the protease production by P.

aeruginosa was dependent on cell growth up to 48 h but, after

wards the cell growth decreased and protease production increased

(Fig. 3). Similar pattern of growth and protease production were

observed for novel psychro-tolerant Curtobacterium luteum (MTCC

7529) by Kuddus and Ramteke [37]. Maximum protease produc-

tion was observed in decline growth phase of P. aeruginosa. Simi-

larly Bacillus sp. strain GX6638 showed maximum protease

production in decline phase of the organism [38]. However, there

is no hard and fast rule for relationship between time of growth

and extracellular enzyme production; it varies with the organism,

the enzyme and the conditions of growth.

Dehairing of buffalo hide and pollution loadThe enzyme precipitate (1%) dehaired the buffalo hide

(3 cm � 3 cm) piece within 16–21 h at neutral pH (tap water)

and ambient temperature 28 � 28C. Enzyme treated buffalo hide

New Biotechnology �Volume 28, Number 2 � February 2011 RESEARCH PAPER

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FIGURE 3

Time course for protease production (–*–) and growth (. . .&. . .) of P.

aeruginosa MCMB-327 in optimized liquid medium containing, 1% soybeanmeal, 1% tryptone (pH 7, 308C, 1% inoculum, 150 rpm, CV of 100 mL in

250 mL capacity flask).

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piece was white, smooth and silky (Fig. 4b) as compared to

chemically treated (Fig. 4a) and untreated control (Fig. 4c). Enzy-

matic process loosens hair and epidermis because of degradation of

specific proteins, glycoproteins and proteoglycans in the basal

membrane [39]. The present results are similar to those of our

earlier studies on dehairing of buffalo hide by protease from B.

cereus MCM B-326 [25]. In dehairing process, pH tolerance for the

enzyme is an important factor. In earlier report, an alkaline

protease from Aspergillus tamarri dehaired the goat skin at pH 9–

11, temperature 30–378C with 1% enzyme concentration and

incubation period of 18–24 h [40].

[()TD$FIG]

FIGURE 4

Dehairing of buffalo hide (a) chemical treated, (b) crude enzyme treated, (c)control-water treated buffalo hide pieces, (d) purified PA02 protease of P.

aeruginosa MCM B-327 (enzyme untreated- with water as control; PA02

proteases used 100 ml per 3 cm � 3 cm buffalo hide).

The spent liquor from lime–sulphide and enzymatic dehairing

process was collected separately and analyzed for pollution para-

meters such as COD, TDS, and TSS. As compared to chemical

method, enzymatic dehairing showed reduction in COD from

8575 to 5635 mg L�1, that is 34.28%; TDS 26,160 to

16,398 mg L�1, that is 37.32% and TSS from 22,110 to

10,708 mg L�1, that is 51.58%. This indicates that enzymatic

dehairing under study has reduced the pollution load of the

dehairing process effluent. In previous report, substantial reduc-

tion of COD, TDS and TSS was observed in enzymatic dehairing

process [40]. Saravanabhavan et al. [41] reported a lime and sulfide-

free dehairing process for cow hides to reduce the pollution load as

well as to achieve better quality of leather primarily through the

use of enzyme and sodium metasilicate.

Purification and characterization of proteaseThe protease was purified 3.05-fold and about 2.71% of the total

activity units were recovered (Table 1). The specific activity of the

purified enzyme was 11264.66 U mg�1. Proteases purification from

other P. aeruginosa has been carried out using various chromato-

graphic techniques. Bayoudh et al. [29] purified 25-fold an alkaline

metalloproteases from P. aeruginosa MN1 by gel filtration and ion

exchange chromatography to a specific activity 82,350 U mg�1.

Lama et al. [42] obtained 2.97% yield and 86.6-fold purification of

a protease produced by Salinivibrio sp. strain 18AG by ammonium

sulfate precipitation, Q-Sepharose, and Superdex G 200 gel filtra-

tion chromatography.

Molecular weight and zymogram of the enzymeThe crude enzyme of P. aeruginosa MCM B-327 migrated on native-

PAGE as several bands with different molecular weights (Fig. 5).

Zymogram analysis of crude enzyme revealed two bands staining

for protease activity (PA01, PA02) where clear hydrolytic activity

zone was formed against a dark background. PA01 and PA02

migrated with molecular masses of 67 and 56 kDa, respectively.

Molecular weights were calculated by plotting a graph of mole-

cular weights of standard protein against Rf values as shown in

Fig. 5. Likewise high molecular weight proteases from P. aeruginosa

were reported by various researchers [43,44]. The protease PA02 of

molecular weight 56 kDa showed potent dehairing ability on

buffalo hide when compared to water as control (Fig. 4d). The

hair was removed along with intact hair root. There was only one

report available on dehairing protease (36 kDa) from P. aeruginosa

PD100 [45]. However, we report on a 56 kDa protease from P.

aeruginosa for dehairing activity.

pH optimaThe PA02 protease was active in pH range of 6–12 and showed

optimum activity at pH 8 and stability in pH range of 7–9

(Fig. 6). This indicates that PA02 protease has slightly alkaline

nature. Likewise Oh et al. [27] published a report on a protease

from P. aeruginosa K-187 which was active in the pH range (7–9)

and had an optimum pH of 8. Gupta et al. [46] reported an

alkaline protease from P. aeruginosa PseA having enzyme stabi-

lity in pH range of 6–9 with an optimum pH of 8. In earlier

reference of protease from P. aeruginosa San-ai has optimum pH

of 9 in 50 mM Tris–HCl buffer and a broader stability range from

pH 5 to 12 [47].

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RESEARCH PAPER New Biotechnology �Volume 28, Number 2 � February 2011

TABLE 1

Purification steps of dehairing protease from P. aeruginosa MCM B-327

Purification steps Total activitya

(U min�1)Total proteina

(mg)Specific activitya

(U mg�1)Yielda (%) Purificationa

(fold)

Culture supernatant 141,008 � 1408 38.25 � 2.9 3685.70 � 327 100 1

Ammonium sulphate precipitation 80,034 � 1000 21.03 � 6 3805.31 � 6.3 56.75 � 0.7 1.03 � 0.001

Sephadex G50 29,669 � 934 6.0 � 0.6 4111.53 � 296.7 17.49 � 0.6 1.11 � 0.07

Preparative PAGEb PA02 Protease 3823 � 171 0.33 � 0.3 11,264.66 � 0.5 2.71 � 0.1 3.05 � 0.0001

a Values expressed mean of triplicate � standard deviation.b PAGE, polyacryalamide gel elctrophoresis.R

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Temperature optima and stabilityPA02 protease was active in temperature range of 20–508C with

optimum activity at 358C. The enzyme PA02 was stable in tem-

perature 20–508C range and retained 80% of protease activity at

508C for 30 min (Fig. 6). In comparison, Najafi et al. [14] reported

optimum temperature for a protease from P. aeruginosa PD 100 of

608C and completely stable at 558C for 60 min. Also, Ogino et al.

[48] reported a protease from P. aeruginosa PST01 which was active

at 558C and stable below 508C. Thus the enzyme has activity and

stability at 20–508C which is suitable for tanneries.

Substrate specificity and enzyme kineticsPA02 protease of P. aeruginosa MCM B-327 exhibited the max-

imum affinity for casein followed with gelatin, BSA and elastin-

orcein but were not able to hydrolyze the collagen and keratin

(Table 2). These properties suggested that the PA02 protease has an[()TD$FIG]

FIGURE 5

Native-PAGE of crude and purified protease from P. aeruginosaMCMB-327, A, silver s

weights versus Rf values (1, molecular weight markers; 2, crude protease; 3, partial

zymogram of crude protease with casein; 6, zymogram of purified PA02 with case

ovalbumin, 45 kDa; bovine serum album, 66 kDa; phosphorylase B, 97.4 kDa).

178 www.elsevier.com/locate/nbt

endopeptidase activity. PA02 protease activities reported were

non-collagenase and non-keratinase type with potent dehairing

activity among the best described in the literature for P. aeruginosa.

By contrast, protease from P. aeruginosa PD100 has strong collage-

nase and gelatinase activities [14].

PA02 protease was also able to hydrolyze synthetic substrates

such as BTEE, BAEE, SAAAPNA, SAAPPPNA but not BAPNA,

BPPPANA and FALGPA (Table 2). According to these results, the

PA02 protease preferentially catalyzed non-polar amino acid resi-

dues of L-Ala at P1 site. The positional substrate specificity has

confirmed that the enzyme cleaved at Ala-Ala showing elastase

type of protease. Kocabiyik and Ozdemir [49] reported that the

purified enzyme from Thermoplasma volcanium, was preferentially

catalyzed the hydrolysis of non-polar amino acid residues such as

L-Ala, L-Leu, and L-Phe at P1 site, suggesting the chymotrypsin as

well as elastase type of protease.

tained gel; B, zymogram of protease with casein; C, plot of standardmolecular

ly purified protease-fraction 2 from G50 column; 4, purified PA02 protease; 5,

in; molecular weight proteins: cytochrome C, 12.4 kDa; trypsinogen, 24 kDa;

New Biotechnology �Volume 28, Number 2 � February 2011 RESEARCH PAPER

[()TD$FIG]

FIGURE 6

Effect of pH (at 378C) and temperature (at pH 8) on activity and stability of

PA02 proteasewith casein as substrate (relative protease activity expressed aspercentage of maximum activity).

TABLE 3

Km and Vmax of purified PA02 protease from P. aeruginosaMCM B-327 with different substrates

No. Substrates Kma Vmax

a (U min�1)

1 Casein 2.788 � 0.20 mg mL�1 830 � 2.35

2 N-Succinyl-Ala-Ala-Ala-p-nitroanilide

18.08 � 0.68 mM 68.96 � 2.12

3 N-Succinyl-Ala-Ala-

Pro-Phe-p-nitroanilide

8.81 � 0.54 mM 36.90 � 1.63

a Values expressed mean of triplicate � standard deviation.

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The results of kinetic study of PA02 protease using casein, N-

Succinyl-Ala-Ala-Ala-p-nitroanilide and N-Succinyl-Ala-Ala-Pro-

Phe-p-nitroanilide substrates, in terms of Michaelis–Menten’s con-

stant (Km) and maximum velocity (Vmax) are calculated and sum-

marized in Table 3. On casein, PA02 protease had Km and Vmax

values of 2.788 mg mL�1 and 830 U min�1, respectively. Dehair-

ing protease reported from Alcaligen faecalis had Km

(1.66 mg mL�1) and Vmax (526 U min�1 mg�1) on casein substrate

[50]. The Km and Vmax for protease from P. aeruginosa PseA were

2.7 mg mL�1 casein and 3 mmol min�1, respectively [46]. Also, the

Km (18.08 mM and 8.81 mM) and Vmax (68.96 and 36.90 U min�1)

of PA02 protease were also calculated for synthetic substrates

TABLE 2

Substrate specificity profile of purified PA02 protease by P.aeruginosa MCM B-327

Substrates Enzyme activitya

Natural substrates U mL�1 min�1

Casein 753.96 � 28.05

Bovine serum albumin 625 � 14.02

Gelatin 677.91 � 32.73

Elastin-orcein 1.0 � 0.02

Keratin azure 0

Collagen 0

Synthetic substrates U mg�1 min�1

N-Benzoyl-L-tyrosine ethyl ester (BTEE) 8.80 � 0.64

N-Benzoyl-L-arginine ethyl ester (BAEE) 120 � 8.12

N-Succinyl-Ala-Ala-Ala-p-nitroanilide 42.58 � 4.96

N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide 39.75 � 3.47

Na-Benzoyl-DL-arginine-p-nitroanilide (BAPNA) 0

N-Benzoyl-Pro-Phe-Arg-p-nitroanilide 0

N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) 0

a Values expressed mean of triplicate � standard deviation.

SAAAPNA and SAAPPPNA respectively. The enzyme hydrolyzed

PhepNP and AlapNP, indicating the specificity for hydrophilic

residues as an N terminal residue. Also, the Km value of an alkaline

protease from Bacillus pumilus MK6-5 was observed 1.1 mM for

Glu-Gly-Ala-Phe-p-nitroanilide, 8.0 mM for Glu-Gly-Ala-Phe-p-

nitroanilide and 3.7 mM for Glu-Ala-Ala-Ala-p-nitroanilide at

378C [51].

Effect of metal ions and inhibitors on enzyme activityThe influence of various metal ions and inhibitors on the protease

activity of PA02 protease from P. aeruginosa MCM B-326 was

investigated (Table 4). A significant inhibitory effect on the pro-

tease activity was observed with Hg2+ (0% relative activity) and

Fe3+, Zn2+, Cr2+ (20–30% relative activity). Other metal ions which

had a negative impact included Na+, Ca2+ and Mg2+. So this

indicated that PA02 protease does not require any metal ions

for its activity. Likewise, Najafi et al. [14] reported a metal-inde-

pendent protease from P. aeruginosa PD100. But, Gupta et al. [46]

reported that in case of protease from P. aeruginosa PseA, 46, 44 and

36% inhibition was observed by Ca2+, Mg2+ and Zn2+, respectively.

Proteolytic enzyme types were classified on the basis of their

inhibitory actions. About 25% inhibition of PA02 protease was

observed by EDTA, 15% by PMSF, 45% by SDS and 4% by pepstatin

A (Table 4). Thus the protease could not be completely inhibited by

serine, aspartyl, cysteine or metalloprotease inhibitors and this is

the novelty of PA02 protease. It can be suggested that the protease

configuration is in such a way that it does not allow the inhibitors

to reach the active site residues and affect its activity or the

protease is not inhibited by such type of inhibitors [45]. According

to previous report of Gupta et al. [46] inhibitors, PMSF and iodoa-

cetate had no effect on protease from P. aeruginosa PseA, but was

inhibited by DTT and EDTA. Ogino et al. [52] included a report on

four different types of endopeptidases produced from P. aeruginosa.

The first one is a 33 kDa Zn-metallopeptidases, which is termed

pseudolysin and generally called as elastase. The second one is a

20 kDa Zn-metallopeptidase which is a staphylolysin and gener-

ally called as LasA protease. The third one is a 50 kDa Zn-metallo-

peptidase which is an alkaline protease. The last one is a lysine-

specific endopeptidase. Comparatively PA02 protease from P.

aeruginosa under study showed pseudolysin type elastase but it

was neither a metalloprotease. Other property of molecular weight

of the said protease is that it is close to 50 kDa of alkaline protease

but again it was not a metalloprotease. Thus, PA02 protease from P.

aeruginosa MCM B-327 showed novel nature which differentiates it

from other types of proteases such as pseudolysin, staphylolysin,

alkaline protease and lysine-specific endopeptidase.

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RESEARCH PAPER New Biotechnology �Volume 28, Number 2 � February 2011

TABLE 4

Effect of various metal ions and protease inhibitors on purified PA02 protease activity

Metal ion/inhibitor Specific action Concentration Relative activity (%)a

Control (without additives) – – 100

Na+ (NaCl) – 5 mM 88 � 1.6

Ca2+ (CaCl2) – 5 mM 89 � 4.4

Fe2+ (FeSO4) – 5 mM 20 � 1.1

Zn2+ (ZnSO4) – 5 mM 31 � 3.3

Mg2+ (MgSO4) – 5 mM 96 � 0.5

Hg2+ (HgCl2) – 5 mM 2 � 1.1

Cr2+ (CrSO4) – 5 mM 23 � 3.8

2-Mercaptoethanol – 5 mM 3 � 0.4

DTTb – 5 mM 2 � 1.6

SDSc – 1% 57 � 2.7

EDTAd Metalloprotease inhibitor 5 mM 76 � 3.3

PMSFe Serine protease inhibitor 5 mM 87 � 3.3

Iodoacetamide Cysteine protease inhibitor 5 mM 86 � 3.3

Pepstatin A Aspartic protease inhibitor 1 mM 98 � 0.4

2 mM 97 � 1.79

a Relative activity expressed as percentage of maximum activity � standard deviation.b DTT, dithothreitol.c SDS, sodium dodecyl sulphate.d EDTA, ethylenediaminotetra acetic acid.e PMSF, phenymethylsulphonyl fluoride.

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ConclusionsThe exploitation of biodiversity may result in the discovery of

microorganisms that produce enzymes with novel properties. In

this study we describe the novel protease produced by Pseudomonas

aeruginosa MCM B-327 for dehairing of buffalo hide. Purification of

protease of P. aeruginosa revealed the presence of a 56 kDa enzyme

with deharing potential. Based on the inhibitory actions, protease

was found to be of novel nature with non-collagenase, non-ker-

atinase but strong dehairing activities. This protease is removing

hair from root because of hydrolysis of cementing substance of

180 www.elsevier.com/locate/nbt

root sheath. The dehairing activity at ambient temperature with-

out the addition of buffer or chemicals could have important

implications of the P. aeruginosa MCM B-327 protease in the

enzymatic dehairing operation for the establishment of robust

and cost efficient process for leather industry.

AcknowledgementThe work was financially supported by the NMITLI project on,

‘Biotechnology for leather towards cleaner processing.’ sponsored

by CSIR, Govt. of India.

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