Biochemical Engineering Journal Statistical optimization of production ...

Biochemical Engineering Journal 48 (2010) 173–180

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Statistical optimization of production, purification and industrial application of a laundry detergent and organic solvent-stable subtilisin-like serine protease (Alzwiprase) from Bacillus subtilis DM-04 Sudhir K. Rai, Ashis K. Mukherjee ∗ Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Assam, India

a r t i c l e

i n f o

Article history: Received 1 July 2009 Received in revised form 3 September 2009 Accepted 9 September 2009

Keywords: Enzyme Submerged Microbial Bacillus subtilis Chromatography Protease Kinetics Solid-state fermentation Optimization

a b s t r a c t Optimum protease production of 518 U by Bacillus subtilis DM-04 in submerged fermentation was attained by response surface method. An alkaline protease, exists as zwitterionic form at pH 7.0 was purified to 23.5-fold by a combination of cation and anion exchange chromatography, ethanol precipitation followed by reverse-phase HPLC. The purified protease (Alzwiprase) contributes 29.0% of overall extracellular proteases of B. subtilis DM-04, has a subunit molecular mass of 16.9 kDa and exists as a monomer. It shows optimum activity at 45 ◦ C and pH 10.0, respectively. The Km and Vmax values of Alzwiprase towards casein were determined as 59 ␮M and 336 ␮g min−1 , respectively. Irreversible inhibition of enzyme activity of Alzwiprase with serine protease inhibitors demonstrates that it belongs to serine protease family, more particularly endopeptidase K and/or subtilisin-like protease. The significant stability and compatibility towards organic solvents, urea, surfactants, commercial laundry detergents as well as excellent stain removal and dehairing properties of Alzwiprase hold a tremendous promise for its industrial application. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The enzymes are considered as “green chemicals” owing to their eco-friendly in nature and possess wide range of applications from industrial sector to house-hold products. Microbial proteases represent one of the three largest groups of industrial enzymes and account for approximately 60% of the total enzyme sale in the world and they are the leaders of the industrial enzyme market worldwide [1]. Microbial proteases are classified as acidic, neutral and alkaline depending on the pH at which they show maximum activity. Amongst these, alkaline proteases find a wide range of applications in laundry detergent, textile, food processing, pharmaceuticals, and leather, paper and pulp industries [1–3]. Since the enzyme based detergents can function better at room temperature (∼23 ◦ C) and possess pollution-alleviating capacity over conventional synthetic detergents; therefore, alkaline proteases have made their way as key-ingredients in detergent formulations [2,3]. It is worthwhile to mention that there are many parameters involved in the selection of an ideal detergent protease,

∗ Corresponding author. Tel.: +91 3712 267007x5405; fax: +91 3712 267005/267006. E-mail address: [email protected] (A.K. Mukherjee). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.09.007

such as compatibility with the detergent components, good activity at relevant washing pH and temperature, compatibility with the ionic strength of the detergent solution, stain degradation and removal potential, stability and half-life. This has stimulated the scientists to search for newer microbial proteases with novel properties that can further improve the wash performance of enzyme based laundry detergents. In our previous study, we report the purification, characterization, physiological significance and industrial application of a 33.1 kDa anionic, alkaline serine protease (Bsubap-I) from Bacillus subtilis DM-04 [3]. It is to be noted that a bacterium produces arrays of protease isoenzymes for its survival and growth and many of these proteases may be explored for the industrial purpose [3]. Our subsequent study has shown that apart from secreting the anionic and cationic proteases, the same bacterium produces another major group of proteases, which is zwitterionic in nature at pH 7.0 (protease does not bind to any cation or anion exchanger at pH 7.0) and holds a tremendous promise for industrial application. This has prompted us to purify, characterize and explore the possible biotechnological potential of an organic solvent-stable protease which exists as zwitterionic form at pH 7.0 (Alzwiprase) but shows optimum activity at pH 8.0 from B. subtilis DM-04. Further, we also improved the protease yield in SmF by statistical optimization of process parameters using response surface method. Characteriza-

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S.K. Rai, A.K. Mukherjee / Biochemical Engineering Journal 48 (2010) 173–180

Table 1 Observed responses and predicted values. Run no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Independent variables

Y response (protease yield in U)

C1

C2

1.5 (−1) 2.5 (+1) 1.5 (−1) 2.5 (+1) 1.5 (−1) 2.5 (+1) 1.5 (−1) 2.5 (+1) 2.0 (0) 2.0 (0) 1.0 (−2) 3.0 (+2) 2.0 (0) 2.0 (0) 2.0 (0) 2.0 (0) 2.0 (0) 2.0 (0) 2.0 (0) 2.0 (0)

0.75 (−1) 0.75 (−1) 0.75 (−1) 0.75 (−1) 0.75 (−1) 0.75 (−1) 1.25 (+1) 1.25 (+1) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 0.5 (−2) 1.5 (+2) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0)

C3 48 (−1) 48 (−1) 48 (−1) 48 (−1) 72 (+1) 72 (+1) 72 (+1) 72 (+1) 48 (−1) 72 (+1) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0) 60 (0)

Residual value

Observed value

Predicted value

428.129 452.629 458.629 484.129 453.129 472.629 478.629 508.290 456.486 477.486 443.371 487.370 440.370 493.371 466.086 460.270 463.492 467.211 466.871 470.910

431.193 452.008 457.258 483.653 453.645 474.040 479.290 505.266 455.888 477.920 441.954 488.744 438.204 495.494 465.819 465.819 465.819 465.819 465.819 465.819

−3.065 0.621 1.371 0.476 −0.516 −1.411 −0.661 3.024 0.598 −0.435 1.416 −1.375 2.166 −2.125 −2.125 −5.550 −2.330 1.390 1.050 5.090

The observed values are average of triplicate determinations. Boundaries of the experimental domain and spacing of levels are expressed in coded (within parenthesis) and natural units. C1 , IC:PP level (%, w/v); C2 , beef-extract level (%, w/w); C3 , incubation time (h).

tion of biochemical properties and evaluation of biotechnological potential advocated the application of Alzwiprase in laundry detergent formulations and leather industry. 2. Materials and methods 2.1. Microorganism Isolation of a protease producing thermophilic B. subtilis DM04 from the soil sample of Assam was reported previously [2,4]. The procedure for the preparation of low cost fermentable substrates, viz., IC and PP used in the present study has been described elsewhere [2]. 2.2. Screening of process parameters influencing protease yield in SmF To check the effect of pH and temperature on growth and alkaline protease production, the bacteria were propagated at different temperatures (30–60 ◦ C) and pH ranges (8–12) for different time intervals (24–120 h) in 0.1 l M9 media supplementing with a combination of IC:PP (1:1, %, w/w) at a concentration of (2.5%, w/v) as a carbon source, placed in a 0.5 l flask with constant shaking (200 rev min−1 ) on a rotary shaker. For the initial screening purpose, any one of the following at a concentration of 1% (w/v) served as the nitrogen source-beef extract, yeast extract, peptone, tryptone, casein, ammonium chloride (NH4 Cl), potassium nitrate (KNO3 ), ammonium nitrate (NH4 NO3 ), sodium nitrate (NaNO3 ), and ammonium sulphate (NH4 )2 SO4 . At a regular time interval sample collection was carried out followed by assay of protease activity (see below) and protein estimation [5]. 2.3. Statistical optimization of protease production in SmF using response surface methodology As shown in Table 1, response surface methodology (RSM) was used to estimate main effects on response, i.e. protease yield. Central composite design consisting of three main critical independent variables, (i) the concentration (%, w/v) of (1:1, w/w) IC:PP (C1 ) and (ii) concentration (%, w/v) of beef extract (C2 ) and incubation time (C3 ) were chosen based on the initial screening since these inde-

pendent variables were capable of influencing the alkaline protease production (Y) by B. subtilis DM-04 in SmF at pH 8.0 and 50 ◦ C temperature (data not shown). For each factor, a conventional level was set to zero as a coded level. These three factors, each with five coded levels consisting of 20 experimental runs, were used to analyze the experimental data to allow better estimate of the experimental error and to provide extra information about yields in the interior of the experimental region [6]. The experimental data were fitted according to Eq. (1) as a second-order polynomial regression equation including individual and cross effect of each variable. Y = a0 +

3

ai Ci +

i=1

3

aii Ci2 +

i=1

3 2

aij Ci Cj

(1)

i=1 j=i+1

where Y is the predicted response (total protease production in U), a0 is the intercept term, ai is the linear effect, aii is the square effect, aij is the interaction effect, and Ci and Cj are the variables. The above equation was used to optimize the values of independent parameters for the response. Multiple regression analysis, response surface plots and statistical analyses were performed using Minitab 15 Statistical Software® (Minitab Inc., PA, USA). 2.4. Batch fermentation Batch fermentation was carried out in a 5 l Bioflow 110 Fermenter (New Brunswick Scientific, USA) with a working volume of 3 l, operating with foam/anti-foam probe system and the M9 medium composed of 2.5% (w/v) of PP and IC mixed in a ratio of 1:1 [2] and 1.25% (w/v) beef extract, adjusted to pH 8.0 with 0.1N NaOH. The agitation speed was 200 rpm, provided by a centrifuge propeller. O2 and pH electrodes were used for the control of the conditions at 50 ◦ C. The cells were harvested at different time periods (24 h, 48 h, 60 h, 72 h and 96 h post inoculation) and the cell-free clear supernatant was used to determine the protease yield from B. subtilis DM-04. 2.5. Protease assay Unless otherwise stated, cell-free culture supernatant was used for the assay of protease activity as described previously [2] using 1.0% (w/v) casein as a substrate at pH 10.0 (100 mM Glycine–NaOH)


and at 45 ◦ C temperature for 30 min. The unit (U) of protease activity is defined as ␮g of tyrosine liberated per min per ml of enzyme. The keratinase activity assay was done as described previously [7]. For the assay of substrate specificity of protease enzyme, the above procedure was followed except casein was replaced with the desired protein substrate. 2.6. Isolation and purification of a zwitterionic protease at pH 7.0 Unless otherwise stated, all the fractionation was carried out at room temperature (∼23 ◦ C). One hundred milliliter (equivalent to 240.0 mg protein) of cell-free culture supernatant (post 60 h incubation) was applied to a CM-cellulose column (1 cm × 20 cm) pre-equilibrated with 20 mM K-phosphate buffer, pH 7.0. The flow through (unbound proteins) was collected in a single tube and then the column was washed with three column volume of equilibration buffer to elute the non-specifically bound proteins. The combined wash fraction and flow-through fraction was applied to a DEAE-Sephadex A-50 column (1 cm × 20 cm) pre-equilibrated with 20 mM K-phosphate buffer, pH 7.0. The unbound proteins as well as proteins eluted post washing the column with equilibration buffer were collected in a single tube, desalted on a pre-packed desalting column (16 cm × 2.5 cm, Bangalore Genei, India) and then pre-chilled ethanol (final concentration 66%) was added and the mixture was kept at 4 ◦ C for 12 h. The mixture was centrifuged at 10,000 × g for 10 min and the pellet obtained was dried in vacuum, then re-suspended in 20 mM K-phosphate buffer, pH 7.0 followed by re-fractionation on Waters Nova-Pak reverse-phase C18 column (3.0 mm × 300 mm). Proteins were eluted with a linear gradient from 5% to 70% (v/v) acetonitrile containing 0.1% (v/v) TFA at a flow rate of 1.0 ml h−1 and elution was monitored at 280 nm. Protein peaks were collected manually, dried in a vacuum centrifuge at −20 ◦ C, dissolved in minimal amount of 20 mM K-phosphate buffer, pH 7.0, and the protease activity as well as protein content was determined [5]. The fraction showing the highest protease activity was checked for homogeneity by 12.5% SDS-PAGE of protein(s) under reducing as well as and non-reducing conditions [8]. 2.7. Biochemical characterization The optimum pH and temperature for protease activity were determined by incubating enzyme with casein at different pH (from 6.0 to 13.0) at 45 ◦ C, and different temperatures (from 25 ◦ C to 60 ◦ C) at optimum pH (10.0) for 30 min followed by and assaying the protease activity against the control (substrate without enzyme). For heat inactivation study, purified enzyme (2 mg ml−1 ) was incubated at 60 ◦ C, and after a specified time period the required volume was withdrawn for enzyme assay against control (unheated enzyme which was considered as 100% activity). The Km and Vmax values of the protease were calculated by using a Line weaverBurk plot using different concentrations of casein (0.1–2%, w/v) as substrate and by plotting the values of 1/v as a function of 1/S. 2.8. Effect of organic solvents, chemical inhibitors, metal-ion chelator and surfactants on protease activity To test the effect of different organic solvents on protease activity, the procedure described by Rahman et al. [9] was followed. Irreversible chemical modification of histidine, cysteine and serine residues was performed by pre-incubating the protease (at a concentration of 2.0 mg ml−1 dissolved in 100 mM Glycine–NaOH, pH 10.0) for 30 min with 4-bromophenacyl bromide, iodoacetamide (IAA), phenyl methylsulfonyl fluoride (PMSF), Tosyl-l-lysine chloromethyl ketone (TLCK), Tosyl-l-phenylalanine chloromethyl ketone (TPCK), respectively, at a final inhibitor concentrations of 2 mM and 4 mM at room temperature (∼25 ◦ C). To

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investigate the effect of various metal ions and chemicals (EDTA, surfactants, urea, DTT) on protease activity, casein hydrolysis was performed with 5 ␮g of purified enzyme in the presence of 2 mM concentration of different metal ions (Ni2+ , Co2+ , Mg2+ , Mo2+ , Fe2+ , Hg2+ , Cu2+ , Zn2+ , Mn2+ , Ca2+ and Cd2+ ) or different concentrations of chemicals as described in Table 5. The enzymatic activity without metal ions/surfactants/inhibitors/urea/EDTA/DTT served as control and was considered as 100% activity. 2.9. Detergent compatibility and removal of blood stain from cotton fabrics The compatibility and stability of various fractions of protease enzymes with some commercial laundry detergents available in the local market such as Surf excel® , Rin advanced® , (Hindustan Lever Ltd, India), Ghadi® (Calcutta detergent Pvt. Ltd, India), Safed® (Safechem Industry, India), Fena Ultra® (Jericho Detergent Pvt. Ltd, India), Wheel® (Hindustan Lever Ltd, India) and Tide® (Procter and Gamble, India) were assessed by our previously elucidated procedure [2,3]. Before protease stability assay, the detergents solutions (7 mg ml−1 ) were pre-heated at 100 ◦ C for 60 min to destroy the endogenous protease activity, if any (which was reconfirmed by protease assay of heated detergent solution). The relative enzyme activity in the presence of detergent was expressed in percentage activity considering the activity of control (enzyme in the presence of 100 mM Glycine–NaOH, pH 10.0) as 100% [2]. Wash performance of purified protease was evaluated by subjecting the blood stain removal test from cotton fabrics as described previously [10]. Each experiment was repeated in triplicate to assure the reproducibility and the standard error in all the experimental results was within 5%. Percent increase of stain removal by purified protease solution (0.1 mg ml−1 prepared in tap water) compared to stain removal by control (tap water) was calculated as described previously [10]. 2.10. Dehairing activity Goat skin was cut to 5 cm2 pieces and incubated with 10.0 ml of purified protease of (50 U ml−1 in 100 mM Glycine–NaOH buffer, pH 10.0) for 6 h at 37 ◦ C or with sterilized bacterial media or 100 mM Glycine–NaOH buffer, pH 10.0 as control. The skin pieces were then virtually analyzed for dehairing activity as described by Rai and Mukherjee [3]. 2.11. Statistical analysis Results are represented as mean ± S.D. of at least three experiments. Statistical analysis was done by ANOVA for analysis of the results. A probability level of p < 0.01 was considered statistically significant. 3. Results and discussion 3.1. Optimization of protease production by response surface method Designing an appropriate fermentation medium is of critical importance in optimizing the product yield; however, the conventional experimental approaches for the optimization of media are time consuming and require a large number of experiments to study the influence of each factor of the media on enzyme production. On the contrary, statistical optimization of media components can take into account the interactions of variables in generating process responses. Response surface methodology (RSM), which is the most accepted statistical technique for bioprocess optimization, can be used to examine the relationship between a set of controllable experimental factors and observed results. Initial screening

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result showed beef extract was the most effective nitrogen source for enhancing the protease yield post 72 h of incubation at pH 8.0 of M9 medium and 50 ◦ C incubation temperature. The results of the observed and predicted values of alkaline protease yield (response) in submerged fermentation (SmF) as a function of the chosen variables with reference to the experiments performed according to the CCD are shown in Table 1. Maximum protease yield (508 U) obtained with the data set of 2.5% (w/v) of IC:PP (1:1), 1.25% (w/v) of beef extract post 72 h incubation was significantly higher compared to protease production by using the same substrates in solid-state fermentation (SSF) system (2382.0 ± 40.0 U g−1 of dry substrate which was equivalent to 9.5 U in submerged fermentation) as reported in our earlier study [2]. It is interesting to note that in case of biosurfactant production by the same strain, no significant difference in yield was observed irrespective of the fermentation systems used [11]. However, Sandhya et al. [12] reported 3.5-fold more protease production by Aspergillus oryzae in SSF compared to SmF. The parameters of Eq. (1) were determined by multiple regression analysis by the application of RSM. The overall second-order polynomial regression equation showing the empirical relationship between protease activity (Y) and three test variables in coded units is represented by Eq. (2). Y = 465.820 + 11.698C1 + 14.323C2 + 11.016C3 − 0.117C1 2 + 0.258C2 2 + 1.085C3 2 + 1.395C1 C2 − 0.105C1 C3 − 0.105C2 C3 (2) Multiple regression model assumes a linear relationship between some variable Y (dependent variable) and n independent variables C1 , C2 , C3 , . . ., Cn [13]. Based on the result obtained with the multiple regression analysis, it was observed that interaction of incubation time with the fermentation substrates had a negative impact on protease production. The analysis of variance (ANOVA) by Fisher’s statistical test was conducted for the second-order response surface model and the result showed that the computed F value for linear regression was much greater than the tabulated (P) > F value; however, the F values for the square

Table 2 Model coefficients estimated by multiple linear regressions (significance of regression coefficients) for Alzwiprase from Bacillus subtilis DM-04 in SmF under shake-flask study. Factor

Coefficient

SE coefficient

Computed t-value

p-Value

Constant C1 C2 C3 C1 2 C2 2 C3 2 C1 C2 C1 C3 C2 C3

465.820 11.698 14.323 11.016 −0.117 0.258 1.085 1.395 −0.105 −0.105

1.2698 0.8008 0.8008 1.0130 0.6240 0.6240 1.4326 1.1325 1.1325 1.1325

366.837 14.607 17.885 10.875 −0.188 0.413 0.757 1.232 −0.093 −0.093

0.000 0.000 0.000 0.000 0.854 0.689 0.466 0.246 0.928 0.928

R2 = 98.49%, R2 (pred) = 92.06%, R2 (adj) = 97.14%.

and interaction effects were less than the tabulated (P) > F value (Table 2). Therefore, the model terms C1 , C2 and C3 were found to be the significant and not their interaction effects (Fig. 1). The goodness-of-fit of the model was checked by determining the coefficient of determination (R2 ) and adjusted R2 . When R2 is large, then, the regression has accounted for a large proportion of the total variability in the observed value of Y which favors the regression equation model [6,14]. The observed values of R2 explain that the fitted model could explain 98.49% of the total variation and hence vouches for adequacy of the model (Table 2). The adjusted R2 corrects the R2 value for the sample size and for the number of terms in the model. The adjusted R2 value (97.14%) in the present study advocated for a high significance of the model. These results reinforced that the response equation provided a suitable model for the CCD experiment. 3.2. Batch fermentation The validation of the statistically optimized condition for the production of alkaline protease by B. subtilis DM-04 was verified by carrying out batch fermentation in a 5-l fermenter. It was interesting to observe that maximum protease yield of 518 U obtained by batch culture post 60 h of incubation was slightly higher than the

Fig. 1. Response surface plots for alkaline protease production by Bacillus subtilis DM-04. The interaction between (a) concentration (%, w/v) of (1:1, w/w) IC:PP and concentration (%, w/v) of beef-extract level, (b) concentration (%, w/v) of (1:1, w/w) IC:PP and incubation time, and (c) concentration (%, w/v) of beef extract and incubation time.


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Fig. 2. Reverse-phase HPLC of ethanol precipitated protease fraction. The detail procedure is described in the text.

observed highest experimental value in shake-flask study as well as the predicted value of the protease yield by response surface method. Furthermore, the optimal time requirement for maximum protease production in a process-controlled fermenter was less than that observed under shake-flask study (60 h vs 72 h). This discrepancy might be due to slight variation in experimental conditions in a fermenter and EM flasks, because the concentration of dissolved oxygen as well as the pH of the medium could be maintained at the desired level in a bioreactor that favors the higher protease production in less time [15]. Although the protease yield by B. subtilis DM-04 in SmF was found to be lower than the reported yield of alkaline protease from Bacillus sp. RKY3 [16] and Bacillus sp. [17]; however, use of cheaper substrates (IC and PP) for protease production could lead to a considerable reduction in production cost which may favor the commercialization of alkaline protease production by B. subtilis DM-04. 3.3. Isolation and purification of a zwitterionic protease The CM-Cellulose and DEAE-Sephadex A-50 unbound fraction (DEAE-Sephadex A-50 flow through) containing the neutral/zwitterionic proteases at pH 7.0 with a specific activity of 397.0 U mg−1 of protein represented 57.0% of total extracellular proteases of B. subtilis DM-04. It is noteworthy to mention that when the extracellular proteases of B. subtilis DM-04 were separated on the basis of their overall net charge at pH 7.0, the zwitterionic proteases (which did not bind to cation and anion exchangers) were found to be the major protease group compared to anionic and cationic proteases (data not shown). By RP-HPLC, the ethanol precipitated proteins were separated into five major peaks (HP-I to HP-V). The HP-I fraction with a retention time of 5.371 min (Fig. 2) demonstrated maximum protease specific activity (5 × 103 U mg−1 ) and was found to be homogenous by 12.5% SDS-PAGE (Fig. 3). A summary of purification of this protein is presented in Table 3. SDS-PAGE of about 15 ␮g protein under both reduced and non-reduced conditions displayed a single band of

Fig. 3. 12.5% SDS-polyacrylamide gel electrophoresis of purified protease. Molecular weight markers are phosphorylase b (97,400 Da), BSA (66,000 Da), ovalbumin (43,000 Da), carbonic anhydrase (29,000 Da), soybean trypsin inhibitor (20,100 Da) and lysozyme (14,300 Da); lane 1, crude protease (40 ␮g); lane 2, CM-Cellulose flow through (CMFT) (30 ␮g); lane 3, DEAE-Sephadex A-50 Flow through (DEFT) (30 ␮g); lane 4, ethanol precipitated fraction (8 ␮g); lane 5, HP-I fraction under reduced condition (15.0 ␮g); lane 6, HP-I fraction under non-reduced condition (15.0 ␮g).

16.9 kDa (Fig. 3) suggesting a monomer protease. This protein was subsequently named as Alzwiprase. The molecular weight of Alzwiprase differs from the reported molecular weight of alkaline proteases isolated from other Bacillus species such as B. subtilis DM04 anionic protease [3], B. subtilis RM-01 [6], B. clausii GMBAE 42 [18], and B. mojavensis A21 [19]. 3.4. Biochemical characterization Before assessing the biotechnological potential of any enzyme, characterization of biochemical properties pertinent to industrial application is utmost important and advantageous. Alzwiprase showed maximum activity at alkaline range of pH (8.0–12.0) and in a broad range of temperature (30–55 ◦ C); however, optimum activity was observed at 45 ◦ C and pH 10.0 (Table 4). The optimum pH for protease activity of Alzwiprase is comparable with the pH optima of other microbial proteases [19], including the anionic protease Bsubap-I isolated from the same strain [3], but higher than the pH optima of alkaline ␤-keratinase isolated from B. subtilis RM-01 strain [7]. Some of the biochemical properties of Alzwiprase are shown in Table 4. Casein was found to be the most preferred substrate for this enzyme followed by hemoglobin, gelatin, chicken-feather keratin (␤-keratin), and bovine serum albumin; however human hair (␣-keratin) and collagen were not hydrolyzed by this enzyme (Table 4). The substrate (casein) specificity of Alzwiprase is significantly higher than that exhibited by anionic protease (Bsubap-I) isolated from the same bacterium [3]. Heating the Alzwiprase at 60 ◦ C for 10 min did not affect the pro-

Table 3 Summary of purification of Alzwiprase from Bacillus subtilis DM-04. Purification step

Total protein (mg)

Enzyme yield (%)

Total enzyme activity (U)

Specific activity (U mg−1 )

Purification fold

Cell-free supernatant CM-Cellulose flow throw DEAE-Sephadex A-50 flow throw Ethanol precipitate HP-I (Alzwiprase)

240.0 153.0 73.0 18.0 3.0

100 66.7 57.0 45.0 29.0

5.1 × 104 3.4 × 104 2.9 × 104 2.3 × 104 1.5 × 104

213 222.0 397.0 1278.0 5000.0

1 1.04 1.9 6.0 23.5

Values are from a typical experiment.

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Table 4 Biochemical properties and substrate specificity of Alzwiprase from Bacillus subtilis DM-04.

Table 5 Effect of inhibitors, EDTA and surfactants on residual activity of Alzwiprase from Bacillus subtilis DM-04 at pH 10.0 and 45 ◦ C temperature.

Biochemical property

Value

Reagents/effectors

Residual activity (%)

Optimum pH Optimum temperature Km (␮M) for casein Vmax (␮g min−1 ) for casein

10.0 45 ◦ C 59.0 336.0

Control

100

Substrate specificity (U mg−1 protein) (mean ± S.D., n = 3) (a) Casein 5000.00 ± 250 (b) Hemoglobin 2560.00 ± 126 (c) Gelatin 1300.00 ± 12 (d) Chicken-feather keratin 433.00 ± 31 (e) Bovine serum albumin 149.00 ± 11 (f) Human-hair keratin 0.0 (g) Collagen 0.0

tease activity; however heating it for 90 min and 120 min at the same temperature resulted in reduction of 75% and 92% of original protease activity, respectively. The above mentioned properties of the purified protease such as thermostability even in the absence of Ca2+ , high specificity activity as well as the property to function at alkaline pH have the added advantages that advocated the use of this purified protease in detergent formulations and other biotechnological processes. A significant reduction in enzyme activity in the presence of DTT suggested the presence of intramolecular disulfide bonds in the enzyme molecule. 3.5. Effect of chemical reagents and organic solvent on protease activity The effect of different chemicals, surfactant and urea on enzyme catalyzed reaction is shown in Table 5. The purified protease retained 92.0 ± 1.8% of their original activity in the presence of 2 mM EDTA, indicating the metallic ions were not necessary for enzyme activity. It was interesting to note that SDS (40 mM), urea (6 M), Tween-20 (1%, v/v) and Triton-X 100 (1%, v/v) did not affect enzymatic activity of Alzwiprase. The serine protease inhibitor PMSF at a concentration of 2 mM significantly reduced the activity of Alzwiprase whereas TPCK and TLCK the inhibitors of chymotrypsin and trypsin- like serine protease at 2 mM concentration could not modulate the activity of this enzyme. The Alzwiprase retained 90.0 ± 3.0% of initial activity in the presence of histidine inhibitor pBPB (2 mM). These results indicated that the Alzwiprase resembled endopeptidase K and/or subtilisin-like proteases because they were inhibited by PMSF but not inactivated by metal chelator (EDTA), TLCK and TPCK [1,20]. It has been reported

PMSF 2 mM 4 mM

83.0 ± 4.2 33.0 ± 1.2

4-Bromophenacyl bromide 2 mM 4 mM

90.0 ± 3.0 79.0± 2.5

IAA 2 mM 4 mM

75 ± 4.0 51.0 ± 3.0

EDTA 2 mM 4 mM

92.0 ± 5.0 89 ± 3.4

TLCK 2 mM 4 mM

100 ± 5.0 97 ± 1.0

TPCK 2 mM 4 mM

100 ± 5.0 98 ± 1.2

SDS (40 mM) Triton-X 100 (1%, v/v) Tween-20 (1%, v/v) Tween-80 (1%, v/v) Urea (6 M)

97.6 ± 2.0 100 ± 5.0 95.1 ± 5.0 95.1 ± 5.0 126.0 ± 6.0

DTT 2 mM 4 mM

90.0 ± 5.0 10.0 ± 1.0

Values are mean ± S.D. of three determinations.

that this class of serine proteases is most active around pH 10, with a molecular weight range of 15–30 kDa. The partial inhibition by DTT (2 mM) might be due to the reduction of intramolecular disulfide bonds required to maintain the activity and stability of Alzwiprase. Study on the influence of metal ions on activity of Alzwiprase showed that all the tested cations, viz., Ni2+ , Cd2+ , Co2+ , Mg2+ , Fe2+ , Hg2+ , Cu2+ , Mn2+ , Ca2+ and Zn2+ inhibited the enzyme activity to a various extent (data not shown). Maximum inhibition of protease activity was observed in the presence of Mg2+ and Cu2+ (100% inhibition). There is a dearth of report on organic solvent tolerant microbial proteases and those enzymes that function in non-aqueous solvents offer new avenues of their industrial applications [3,19,21].

Fig. 4. Organic solvent stability of Alzwiprase. Enzyme activity in the absence of solvents was considered as 100% activity and other values were compared with that. Each value represents mean ± S.D. of three experiments.


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Fig. 5. Detergent stability and compatibility of Alzwiprase at 37 ◦ C. Enzyme activity in the absence of detergent was considered as 100% activity and other values were compared with that. The values represent mean ± S.D. of three determinations.

Fig. 6. Dehairing activity of Alzwiprase. (a) Goat skin incubated in 100 mM Glycine–NaOH buffer, pH 10.0 for 6 h at 37 ◦ C (control) and (b) enzymatically dehaired goat skin incubated with Alzwiprase (50 U ml−1 ) for 6 h at 37 ◦ C.

The organic solvent (at a final concentration of 20% (v/v)) stability of Alzwiprase was observed in the following order: n-hexane (log P 3.5) > methanol (log P −0.764) > xylene (log P 3.1) > ethanol (log P −0.235) ∼ acetonitrile (log P −0.394) > benzene (log P 2.0) (Fig. 4). It is interesting to note that Alzwiprase demonstrated higher organic solvents stability compared to anionic protease Bsubap-I purified from the same strain [3] which vouches for the industrial application of Alzwiprase, for example, synthesis of peptides in the presence of organic solvents. It may be assumed that like organic solvent-stable PST-01 protease from Pseudomonas aeruginosa, presence of disulfide bonds in Alzwiprase plays an important role in the organic solvent stability of the enzyme [22,23]. Inhibition of protease activity in the presence of disulfide bond reducing agent DTT supports the above argument. In general, higher is the log P of solvent, which is defined as the logarithm of the partition coefficient P of the solvent between octanol and water [24], greater is the stability of protease in that particular solvent [9,22]. Interestingly, Alzwiprase demonstrated higher stability in methanol compared to xylene although log P-value of former was higher than the latter solvent. Noteworthy to mention that we could not detect the presence of biosurfactant in this protease preparation (unpublished observation); therefore, the possibility of interference of microbial surfactant on the solvent stability of Alzwiprase may be excluded.

(Fig. 5). The stability of any enzyme is influenced by the ingredients of the detergents, such as surfactants particularly the anionic surfactants, sequestrates, bleaching agents and stabilizers [14,25]. Therefore, partial loss of protease activity of Alzwiprase in some of the detergents may be attributed to inhibitory effect(s) of component(s) of these detergents. In contrast, some of the components of the detergent(s), for example, ethoxylated surfactants and sucrose may have a stimulatory effect [25,26] resulting in observed increase in enzyme activity in the presence of some of the detergents as compared to control [3,14]. This excellent laundry detergent stability of Alzwiprase prompted us to evaluate its stain removal potency for application in commercial laundry detergent formulations. It was observed that Alzwiprase at a concentration of 0.1 mg ml−1 could remove 28 ± 2.1% (mean ± S.D., n = 3) of blood stain from cotton fabrics. It has been recommended that proteases or other hydrolytic enzymes to be used in detergent formulations should be effective at low levels ranging from 0.4% to 0.8% [27,28] and therefore, it is reasonable to assume that Alzwiprase is an ideal candidate for use in laundry detergent. The efficient stain removal capacity as well as the ability to retain the protease activity even in the presence of metal chelator (EDTA), surfactants and urea reinforced the inclusion of Alzwiprase in commercial laundry detergent formulations. 3.7. Dehairing activity

3.6. Detergent stability and stain removal from cotton fabrics Enzyme based laundry detergents find a wide range of applications in laundry, dishwashing, textile and other related industries. The Alzwiprase demonstrated significant stability and compatibility with all the tested commercial laundry detergents at 37 ◦ C

Enzymatic dehairing process has been gaining importance as an alternative chemical methodology in present day scenario as this process is significant in the reduction of toxicity in addition to improvement of leather quality [29]. However, most of the proteases reported so far were unsuitable for dehairing pur-

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pose because of the associated collagen-degrading activity [30]. Proteases such as Alzwiprase possessing keratinolytic activity but lacking collagenolytic activity are having the great demand in the leather industry for the depilating process [31]. They selectively degrade the soft keratin tissue in the follicle, thereby pulling out intact hairs without affecting tensile strength of the leather [32]. Dehairing result showed that hairs from goat skin could be removed very easily 6 h post incubation of goat skin with Alzwiprase as compared to skin treated with buffer only (control) confirming the dehairing potency of this protease (Fig. 6). This dehairing potency of Alzwiprase is comparable to the same property exhibited by Bsubap-I purified from the same strain [3]. 4. Conclusion In conclusion, Alzwiprase which is zwitterionic protease at pH 7.0 possesses several interesting properties which may lead us to conclude that apart from use in laundry detergent and in leather industries, the future industrial application of Alzwiprase in ultrafiltration membrane cleaning as well as in peptide synthesis is highly promising. Acknowledgements Mr. S.K. Rai was a recipient of Senior Research Fellowship from the Department of Biotechnology, New Delhi. This work was supported by financial assistance to A.K.M from the Department of Biotechnology, Ministry of Science and Technology, New Delhi. References [1] R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline protease: molecular approaches and industrial applications, Appl. Microbiol. Biotechnol. 59 (2002) 15–32. [2] A.K. Mukherjee, H. Adhikari, S.K. Rai, Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: characterization and application of enzyme in detergent formulation, Biochem. Eng. J. 39 (2008) 353–361. [3] S.K. Rai, A.K. Mukherjee, Ecological significance and some biotechnological application of an organic -solvent stable alkaline serine protease from Bacillus subtilis strain DM-04, Bioresour. Technol. 100 (2009) 2642–2645. [4] A.K. Mukherjee, K. Das, Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strain in a particular habitat, FEMS Microbiol. Ecol. 54 (2005) 479–489. [5] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with folin-phenol reagent, J. Biol. Chem. 193 (1951) 275–276. [6] P.D. Haaland, Experimental Design in Biotechnology, Elsevier Science Publishing Co., New York, USA, 1990. [7] S.K. Rai, R. Konwarh, A.K. Mukherjee, Purification, characterization and biotechnological application of an alkaline ␤-keratinase produced by Bacillus subtilis RM-01 in solid-state fermentation using chicken-feather as substrate, Biochem. Eng. J. 45 (2009) 218–225. [8] U.K. Laemmli, Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [9] R.N.Z.R.A. Rahman, S. Mahamad, B.A. Salleh, M. Basri, A new organic solvent tolerant protease from Bacillus pumilus 115b, J. Ind. Microbiol. Biotechnol. 34 (2007) 509–517. [10] A.K. Mukherjee, Potential application of cyclic lipopetide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations, Lett. Appl. Microbiol. 45 (2007) 330–335.

[11] K. Das, A.K. Mukherjee, Comparison of lipopetide biosurfactant production by Bacillus subtilis strain in submerged and solid state fermentation systems using a cheap carbon source: some industrial applications of biosurfactants, Process Biochem. 42 (2007) 1191–1199. [12] C. Sandhya, A. Sumantha, G. Szakaes, A. Pandey, Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation, Process Biochem. 40 (2005) 2689–2694. [13] W.W. Daniel, A foundation for analysis in the health sciences. Hypothesis testing, in: W.W. Daniel (Ed.), Biostatistics, 7th edition, John Wiley and Sons, Inc., New York, 2000, pp. 166–167. [14] A.K. Mukherjee, M. Borah, S.K. Rai, To study the influence of different components of fermentable substrates on induction of extra cellular ␣-amylase synthesis by Bacillus subtilis DM-03 in solid state fermentation and exploration of feasibility for inclusion of ␣-amylase in laundry detergent formulations, Biochem. Eng. J. 43 (2009) 149–156. [15] S. Saran, J. Isar, R.K. Saxena, Statistical optimization of conditions for protease production from Bacillus sp. and its scale-up in a bioreactor, Appl. Biochem. Biotechnol. 141 (2007) 229–239. [16] L.V.A. Reddy, Y.J. Wee, J.S. Yun, H.W. Ryu, Optimization of alkaline protease production by batch culture of Bacillus sp. RKY 3 through Plackett-Burman and response surface methodological approaches, Bioresour. Technol. 99 (2008) 2242–2249. [17] S. Puri, Q.K. Beg, R. Gupta, Optimization of alkaline protease production from Bacillus sp. By response surface methodology, Curr. Microbiol. 44 (2002) 286–290. [18] D. Kazan, A.A. Denizei, M.N. Kerimak Oner, A. Erarslan, Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42, J. Ind. Microbiol. Biotechnol. 32 (2005) 335–344. [19] A. Haddar, R. Agrebi, A. Bougatef, N. Hmidet, A. Sellami-kamoun, M. Nasri, Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive, Bioresour. Technol. 100 (2009) 3366–3373. [20] J.M. Bankus, J.S. Bond, in: R. Beynon, J.S. Bond (Eds.), Proteolytic Enzymes: A Practical Approach, 2nd edition, Oxford University Press, Oxford, 2001, pp. 293–316. [21] A. Gupta, S.K. Khare, Enhanced production and characterization of a solvent stable protease from solvent tolerant Pseudomonas aeruginosa Pse A, Enzyme Microb. Technol. 42 (2007) 11–16. [22] H. Ogino, K. Yasui, T. Shiotani, T. Ishihara, H. Ishikawa, Organic solvent-tolerant bacterium which secretes an organic solvent-stable proteolytic enzyme, Appl. Environ. Microbiol. 61 (1995) 4258–4262. [23] H. Ogino, T. Uchiho, J. Yokoo, R. Kobayashi, R. Ichise, H. Ishikawa, Role of Intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability, Appl. Environ. Microbiol. 67 (2001) 942–947. [24] C. Laane, S. Boeren, K. Vos, C. Veeger, Rules for optimization of biocatalysis in organic solvents, Biotechnol. Bioeng. 30 (1987) 81–87. [25] M.R. Stoner, D.A. Douglas, P.J. Gualfetti, T. Becker, M.C. Manning, J.F. Carpenter, T.W. Randolph, Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors, Enzyme Microb. Technol. 34 (2004) 114–125. [26] G.L. Russell, L.N. Britton, Use of certain alcohol ethoxylates to maintain protease stability in the presence of anionic surfactants, J. Surfact. Deter. 5 (2002) 5–10. [27] C.G. Kumar, H. Takagi, Microbial alkaline proteases: from a bioindustrial viewpoint, Biotechnol. Adv. 17 (1999) 561–594. [28] S.K. Rai, J.K. Roy, A.K. Mukherjee, Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. ASS24-II. Appl. Microbiol. Biotechnol., doi:10.1007/s00253-009-2145-y (in press). [29] S. Sivasubramanian, B. Murali Manohar, A. Rajaram, R. Puvanakrishna, Ecofriendly lime and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease, Chemosphere 70 (2008) 1015–1024. [30] Q. Huang, Y. Peng, X. Li, Purification and characterization of extracellular alkaline serine protease with dehairing function from Bacillus pumilis, Curr. Microbiol. 43 (2003) 169–173. [31] V.P. Zambare, S.S. Nilegaonkar, P.P. Kanekar, Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agents, World J. Microbiol. Biotechnol. 23 (2007) 1569–1574. [32] P. Thanikaivelan, J.R. Rao, B.U. Nair, T. Ramasami, Progress and recent trends in biotechnological methods for leather processing, Trends Biotechnol. 22 (2004) 181–188.