Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

Transcription

1 Appl Biochem Biotechnol (2012) 168: DOI /s Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications Renganathan Jayakumar & Shanmugam Jayashree & Balumuri Annapurna & Sundaram Seshadri Received: 14 November 2011 / Accepted: 18 September 2012 / Published online: 18 October 2012 # Springer Science+Business Media New York 2012 Abstract This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid 1.5 % (w/w); inducer, soyabean meal 2 % (w/w); ph 11.0; shaking condition 37 C for 48 h. The enzyme had ph and temperature optima of 9.0 and 60 C, respectively. The enzyme displayed the molecular mass of 36 kda in sodium dodecyl sulphate polyacrylamide gel electrophoresis study and exhibited activity at a wide range of ph ( ) and thermostability (20 70 C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H 2 O 2 for 30 min. The protease activity was also enhanced by divalent cations such as Ba 2+, Ca 2+ and Mg 2+ and was strongly inhibited by Fe 2+,Zn 2+,Sr 2+,Hg 2+ andurea.theenzymeretainedmore than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v)organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the ecofriendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications. R. Jayakumar: S. Jayashree : B. Annapurna : S. Seshadri (*) Shri AMM Murugappa Chettiar Research Center, Taramani, Chennai , India energy@mcrc.murugappa.org Sundaram. Seshadri tsvisesh@yahoo.co.in

2 1850 Appl Biochem Biotechnol (2012) 168: Keywords Serine alkaline protease. Dehairing. Pulicat Lake. Soyabean meal. Stain removal Introduction Proteases are one of the important industrial enzymes which occupy significant part of the world s enzyme production [1, 2]. Alkaline proteases produced by bacteria contribute 60 % of total world market share [1, 3] and became inevitable in the production of detergents, food processing, medical formulations, tannery industries, bioremediation etc. [4, 5]. Though a number of microorganisms are known to produce protease with various industrial applications, there is still a need for novel enzymes with specific properties. Since proteases could withstand wide ph range ( ) of laundry detergent solution, it is widely employed in the detergent industries [1, 6, 7]. Further, for an enzyme to be a successful component in the modern bleachbased detergent formulation, it should be stable at elevated temperature, in the presence of nonionic detergents, surfactants and peroxide agents [4, 8]. On the other hand, stability of protease in the presence of organic solvents is necessary because of their wide use in the synthetic reactions [9]. Therefore, it is recommended that, for the isolation of the microorganisms to be employed in the industrial oriented applications, exploitation of the sources like alkaline habitats is much preferred as the strains produce enzymes which are stable in high alkaline conditions and could resist chemical denaturants present in the detergents [10]. Among the bacterial group, interest in Bacilli gained much importance as it accounts for 35 % of total microbial enzyme sale and it was widely used in the detergent formulation since1960s [1, 3]. Besides, protease from Bacillus sp. was also used as cleansing additives in detergents to facilitate the release of proteinaceous materials in stains such as grime, blood, milk etc. Among the Bacillus sp., Bacillus pumilus was well exploited by various researchers, like purification, characterization, role in industrially important protease production and its applications were well documented [6, 10 17]. However, to the best of our knowledge, there is no information regarding the optimization of media components and the cultivation conditions suitable for the B. pumilus strain to obtain maximum yield. Further, the protease production varies significantly with each strain and the kind of media employed. Hence, to obtain maximum yield, it is mandatory to optimize the media components and the cultivation conditions like ph, temperature, incubation time, inoculum size, agitation and others [18] suitable for each strain. In this view, the objective of the present work was to optimize medium for B. pumilus,an alkaliphilic bacterium, isolated for the first time from the sediment samples collected from Pulicat Lake, India. It also includes the optimization of thermo, solvent, detergent stable alkaline serine protease; characterization of properties of alkaline protease and application of the enzyme as an effective additive in various industries. Material and Methods Isolation of Proteolytic Bacteria Sediment samples collected from Pulicat Lake, Chennai, Tamil Nadu were serially diluted and plated on skim milk agar medium containing (grams per litre): peptone 1.0 (w/w), NaCl 5.0 (w/w), skim milk powder (w/w), agar 20.0 (w/w) and ph 7.0. Five different proteolytic strains were selected based on morphological characteristics and zone of clearance around the colonies. Overnight grown cells of 1 % (v/v) from each selected isolates

3 Appl Biochem Biotechnol (2012) 168: were added to the modified fermentation medium (MFM) [19] comprising (grams per litre): casein 10.0 (w/w), K 2 HPO (w/w), KH 2 PO (w/w) and ph 7.0 and incubated at room temperature for 72 h at 200 rpm. The cultures were then centrifuged at 10,000 rpm for 10 min at 4 C; the cell-free supernatant was collected and used as enzyme source. Enzyme Assay Qualitative Gelatine Cup Assay The culture supernatant of bacteria was added to the wells of agar medium emulsified with gelatine 4 % (w/w) ph 7.0. The plates were incubated at room temperature for 24 h and then flooded with mercuric chloride solution (15 g in 20 ml of 6 N HCl). The zone of clearance around the well indicates the extent of protease activity. Quantitative Enzyme Assay Proteolytic activity was determined by a modified method of McDonald and Chen [20]. Briefly, the enzyme solution was added to 1.0 % (w/v) casein solution (dissolved in 50 mm citrate buffer with ph of 7.6) and incubated at 37 C for 30 min. The reaction was terminated with the addition of 1 ml of 10 % (w/v) trichloro acetic acid and was allowed to stand at room temperature for 10 min. The precipitate was removed by centrifugation at 10,000 rpm for 10 min. Aliquots of 0.5 ml supernatant (v/v) were mixed with 2.5 ml (w/v) of 0.5 M Na 2 CO 3 and 0.75 ml of Folin Ciocalteu s Phenol reagent/water (1:3 v/v) and incubated in dark for 20 min at room temperature. The optical density of the solutions was determined at 650 nm and compared against a tyrosine standard curve. Protein content in the culture filtrate was estimated by the dye binding method of Bradford [21] using bovine serum albumin (BSA) as standard. One unit of enzyme activity was defined as the amount of the enzyme that liberates 1 μg of tyrosine per minute per millilitre under the standard assay conditions. From the assay, the highest protease yielding isolate, MCAS8, was selected for further studies. Identification of Proteolytic Isolate The strain MCAS8 was characterized according to the methods described in Bergey s Manual of Determinative Bacteriology [22] and identified on the basis of the 16S rrna gene sequence analysis. Bacterial genomic DNA was isolated by phenol chloroform method, and 16S rrna gene was amplified using the universal 27F and 1492R bacteria-specific primers that were designed to amplify a 1,500-bp segment of the 16S rdna [23]. The amplified DNA fragments were gel-purified using QIAquick Gel Extraction kit and sequenced using an ABI377 sequencer (Applied Biosystems). The deduced sequences were compared with GenBank data using the BlastN search [24]. Optimization of Fermentation Conditions Factors affecting the protease production such as different inducers, carbon and nitrogen sources, ph, temperature and salt concentrations, were optimized by one-variable-at-a-time approach using submerged fermentation system in MFM. Unless otherwise mentioned, the parameters like ph, size of inoculum, incubation temperature, shaking condition and duration of incubation were maintained at 7.0, 1 %, 37 C, 200 rpm and 72 h, respectively.

4 1852 Appl Biochem Biotechnol (2012) 168: Time Course and Agitation The effect of time course and agitation on protease production by MCAS8 isolate was determined. The overnight grown culture was inoculated in 250-ml Erlenmeyer flask containing 100 ml MFM and incubated at shaking and static condition at room temperature for 72 h. The samples were withdrawn every 6 h aseptically, and the enzyme activity was determined under standard assay conditions. Inducers Various inducers like soyabean meal, casein, peptone, corn steep liquor and casamino acid were amended at the concentration of 1 % (w/w) by replacing casein in the MFM. The selected inducer was evaluated for optimum concentration ranging %. Carbon and Nitrogen Sources Effects of various carbon (citric acid, sucrose, fructose, galactose, glycerol, glucose, lactose, maltose, mannose, soluble starch, tri-sodium citrate and xylose), organic nitrogen (tryptone, casein, yeast extract, peptone and casamino acid) and inorganic nitrogen (ammonium sulphate, ammonium nitrate, ammonium chloride and corn steep liquor) sources at concentrations of 1.0 % along with the inducer on protease production by MCAS8 isolate were investigated. In addition, the optimum carbon source concentration ( %) for the enhanced enzyme production was determined. Culture Conditions For maximum protease production, different culture parameters such as ph ( ), temperature (25, 37, 45 and 55 C), salt concentration ( M NaCl) and inoculum size ( %) were optimized in MFM. Partial Purification of Protease The 48-h grown cell-free supernatant of MCAS8 culture was collected by centrifugation, and the enzyme was precipitated by the addition of ammonium sulphate to 80 % (w/w) saturation. The mixture was incubated overnight at 4 C and separated by centrifugation at 15,000 rpm for 30 min. The active fractions were dialysed against the Tris HCl buffer (ph 9.0), and the enzyme activity was determined under standard assay conditions. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) was performed according to the method of Beg and Gupta [2], while the gel was stained with Coomassie Brilliant Blue R-250 and the molecular weight was determined by comparing with the standard protein markers. Characterization of Partially Purified Enzyme Effects of ph and Temperature on Protease Activity and Stability The activity of the partially purified enzyme was screened at various ph and temperature viz with an interval of 0.5 units using 0.1 M of citrate buffer

5 Appl Biochem Biotechnol (2012) 168: (ph ), phosphate buffer (ph ), Tris HCl (ph ) and carbonate buffer (ph ). The ph stability was determined by pre-incubation of the partially purified enzyme for 30 min at room temperature with appropriate buffers (ph ), and the residual activity was measured under standard assay conditions. Temperature optimum for the protease activity was measured ranging from 40 to 90 C. Thermostability was determined by pre-incubating the partially purified enzyme at various temperatures (20 70 C) for 30 min, and the residual activity was determined as described previously. Substrate Specificity The hydrolytic activity of the partially purified protease was examined with various proteinaceous substrates such as [1 % (w/v)] BSA, casein, skim milk, gelatine, azocasein and haemoglobin under the standard assay conditions. Influence of Various Chemicals on Protease Activity The effect of various protease inhibitors (w/v) phenylmethanesulphonyl fluoride (PMSF), dithiothreitol (DTT); surfactants (v/v) Tween 80, Triton X-100; detergents (w/v) SDS; chelators (w/v) ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA); oxidizing agent hydrogen peroxide (v/v) (H 2 O 2 ), metal ions (w/v) (Mg 2+, Fe 3+, Zn 2+, Ba 2+, Sr 2+, Hg 2+ and Ca 2+ ) and urea (w/v) at different concentrations on protease activity was determined. The enzyme was pre-incubated in these chemicals for 30 min at room temperature, and the residual activity was measured under standard conditions. Effect of Solvents on Protease Activity The effect of various solvents (v/v) [methanol, ethanol, acetonitrile, acetone, dimethyl sulphoxide (DMSO), diethyl ether and ethyl acetate] on the stability of partially purified protease was determined by pre-incubating the enzyme 1 h with solvents, and the residual activity was measured under standard conditions. Wash Performance Assay The stain removal efficiency of the protease from B. pumilus MCAS8 was analysed by stain removal test using three pieces of uniform sized cotton fabric. They were stained with goat blood, dried at 60 C and fixed with 1 % (v/v) formaldehyde.the stain fixed fabrics were immersed in the crude enzyme for 10 min at room temperature and examined for stain removal. The same procedure was followed for the control without the enzyme [25]. A negative control without any treatment was maintained. Dehairing of Goat Skin Goat skins of uniform size were treated with crude enzyme and incubated for 14 h at room temperature. The skin was observed virtually for loosening of hair by mechanical means [26].

6 1854 Appl Biochem Biotechnol (2012) 168: Liquefaction of Blood Clot Goat blood clot (100 mg) was incubated with crude alkaline protease at room temperature and monitored at different time intervals for complete hydrolysis [25]. Statistical Analysis All the experiments were carried out in triplicate, and the standard deviation for each experiment was carried out using SPSS The error bars in the experiments indicate standard deviation (n03). Results Screening and Identification of Protease Producing Bacteria Five different bacterial strains isolated from the sediment samples of Pulicat Lake were selected based on the formation of halos around the colonies on skim milk agar. All proteolytic isolates produced appreciable large clear zone on gelatine agar indicating the extent of the extracellular protease activity. Though comparable protease activity was also shown by other isolates, isolate MCAS8 formed large halo and produced significantly higher activity of 610 U/ml (Fig. 1). The isolate was observed to be a fast growing alkaliphilic, gram-positive bacterium which showed positive results for gelatinase, amylase, protease, oxidase, catalase and Voges Proskauer. It also exhibited positivity for utilization of glucose, galactose, fructose, mannose, starch, glycogen, citric acid, trisodium citrate, maltose, sucrose and xylose while negative results were observed against indole production and citrate utilization. Growth of B. pumilus was observed between the medium ph in the range of 6 12 and at the incubation temperature between 25 and 55 C. The organism was also found to tolerate and produce enzyme in the presence of NaCl concentration between 0.05 and 0.25 M. The nucleotide sequence of 16S rrna gene of the isolate MCAS8 showed the highest homology (99 %) with the previously published sequence of B. pumilus. The Fig. 1 Protease production by different isolates. Enzyme activity represents average of mean ± SD of triplicates

7 Appl Biochem Biotechnol (2012) 168: sequence was deposited in NCBI nucleotide sequence data library with the accession number HQ Fermentation Time Course and Agitation The strain B. pumilus MCAS8 exhibited maximum enzyme production at 48th hour of incubation at shaking condition. Prolonged incubation and static condition showed the reduction in the protease production. Optimization of Inducers Effect of inducers on protease production was determined by replacing casein in the MFM with various inducers (Fig. 2). Among the inducers evaluated, soyabean meal showed significant production (990 U/ml) followed by casein (771 U/ml), casamino acid (732 U/ ml), corn steep liquor (710 U/ml) and peptone (680 U/ml). Elevated level of protease production (1,065 U/ml) was obtained when the medium was supplemented with 2 % (w/ w) soyabean meal as an inducer and further increase in the concentration did not show any progress in the production. Effect of Carbon and Nitrogen Sources on Protease Production The extracellular protease production varied with each carbon substrates tested (Fig. 3). Maximum protease production was noticed in the medium supplemented with citric acid (1,594 U/ml) followed by tri-sodium citrate (1,261 U/ml), mannose (1,218 U/ml), glucose (1,198 U/ml), starch (1,142 U/ml), glycerol (1,043 U/ml), galactose (1,040 U/ml) and lactose (1,007 U/ml), whereas fructose, maltose, sucrose and xylose induced moderate production of enzyme. The activity augmented (115.7 %) when optimum concentration citric acid (1.5 %, w/v) was amended in the fermentation medium. Further increase in the substrate concentration has decreased the protease production. Among the various organic and inorganic Fig. 2 Effects of various Inducers on protease production by Bacillus pumilus MCAS8. CSL corn steep liquor, PEP peptone, CAA casamino acid, SBM soyabean meal, CAS casein. Enzyme activity represents average of mean±sd of triplicates

8 1856 Appl Biochem Biotechnol (2012) 168: Fig. 3 Effect of various carbon sources on protease production. Each source (1%) was amended in the production medium along with 2% soybean meal. CIA citric acid, TSC trisodium citrate, MAN mannose, GLU glucose, STA starch, GLY glycogen, GAL galactose, LAC lactose, FRU fructose, MAL maltose, SUC sucrose, XYL xylose. Enzyme activity represents average of mean±sd of triplicates nitrogen sources used (1 %, w/w), none of the sources increased the protease production to an appreciable level (data not shown). The production status for organic nitrogen was in the order yeast extract (89.6 %)>beef extract (66.0 %)>casein (62.0 %)>peptone (44.4 %)> corn steep liquor (39.4 %), and for inorganic nitrogen, the order was ammonium sulphate (70.5 %)>ammonium chloride (69.2 %)>ammonium nitrate (28 %). Effect of ph and Temperature on Protease Production The enzyme production was determined in medium with different ph (Fig. 4). The activity enhanced gradually from ph 6.0 and reached the utmost production at ph 11.0 (2,034 U/ml) while no activity was observed with ph ranging (data not shown) and activity decreased at ph The isolate produced protease at temperature 25, 37, 45 and 55 C (Fig. 4) with maximum production at 37 C (2,214 U/ml); further increase or decrease in temperature reduced protease production. Inoculum volume of 1 % (v/v) (Fig. 4) and 0.2 M NaCl (Fig. 4) was found to be the optimum for maximum protease production. However, NaCl was found to have no influence on enzyme production. Partial Purification of Protease The steps involved in purification of protease produced by the strain MCAS8 are summarized (Table 1). A consistent increase in the fold purification and specific activity at each step was observed. After dialysis, the enzyme resulted in 1.9-fold increase which exhibited 1,960 U specific activity/mg of protein with 44.3 % yield. The molecular weight of the dialyzed protease was determined by SDS PAGE as 36 kda (Fig. 5).

9 Appl Biochem Biotechnol (2012) 168: Fig. 4 Effect of various parameters on enzyme production from B. pumilus MCAS8. Enzyme activity represents average of mean±sd of triplicates Characterization of Partially Purified Protease Effect of ph and Temperature on Activity and Stability The influence of ph on protease activity was determined from ph 3.0 to 11.0 using appropriate buffers at 37 C (Fig. 6a). The enzyme was stable in the ph range of with maximum activity at ph 9.0 (Fig. 6b). The enzyme maintained residual activity above 60 % between ph 6.0 and ph However, the enzyme activity gets reduced nearly to 20 % at ph 5.0 and Table 1 Partial purification of protease from B. pumilus MCAS8 Purification steps Total activity (U/ml) Total protein (mg/ml) Specific activity (U/mg protein) Purification (fold) Yield (%) Culture supernatant 2, , Ammonium sulphates precipitate (80 %) 1, , Dialysis ,

10 1858 Appl Biochem Biotechnol (2012) 168: Fig. 5 SDS PAGE analysis of protease enzyme from B. pumilus MCAS8. Lane 1 crude extract; lane 2 ammonium sulphate precipitation (80%); lane 3 after dialysis; lane 4 standard protein marker Thermal profile of the enzyme was estimated at various temperatures from 40 to 90 C (Fig. 6c). The enzyme exhibited optimum activity at 60 C (2,502 U/ml) followed by 70 C (1,945 U/ml) and 50 C (1,889 U/ml). The protease enzyme showed maximum thermostability at 20 C. The enzyme retained above 50 % of its original activity ranging C (Fig. 6d). Effect of Substrate Specificity Among the different substrates tested, protease exhibited a better hydrolyzing capacity against casein and skim milk (100 and 92 % residual activity, respectively) followed by gelatine, haemoglobin, BSA and azocasein with 84, 71, 55 and 40 % residual activity, respectively (Table 2).

11 Appl Biochem Biotechnol (2012) 168: Fig. 6 Effect of ph and temperature on activity and stability of protease from B. pumilus MCAS8. a Activity of protease on various buffer ph; b stability of protease on various ph ( ); c effect of protease on various temperature (40 90 C); d stability of protease at various temperature (30 70 C). Enzyme and residual activity represents average of mean±sd of triplicates Effect of Various Chemicals The effect of various chemicals on the proteolytic activity of B. pumilus MCAS8 is summarized in Table 3. The protease inhibitor PMSF greatly inhibited the enzyme activity to 15 % and retained about 88 % of its total activity upon treatment with DTT. A marginal enhancement in the activity of the enzyme was observed in the presence of surfactants like Triton X-100 and Tween 80. The enzyme had maximum stability towards SDS retaining residual activity of 96 %. EDTA and EGTA had residual activity of 74 and 72 %, respectively. The enzyme activity was slightly inhibited in the presence of oxidizing agent like H 2 O 2. Table 2 Substrate specificity of B. pumilus MCAS8 protease over selected natural proteinaceous substrates Residual activity represents average of mean±sd of triplicates Natural proteinaceous substrates Residual activity (%) Casein 95±18.3 Skim milk agar 92±22.7 Gelatine 84±13.2 Haemoglobin 71±9.6 BSA 55±15.3 Azocasein 40±27.3

12 1860 Appl Biochem Biotechnol (2012) 168: Table 3 Effect of various inhibitors (1 mm), surfactants (5 %), detergents (5 %), chelators (1 mm), oxidising agents (5 %), solvents (5 %) and metal ions (1 mm) on the activity of alkaline protease from B. pumilus MCAS8 Residual activity represents average of mean±sd of triplicates a Inhibitors b Surfactants c Detergents d Chelators e Oxidising agents f Solvents g Metal ions Residual activity (%) Chemicals Control 100±12.2 PMSF a 15±2.6 DTT a 88±3.7 Triton X-100 b 105±22.7 Tween 80 b 112±15.3 SDS c 120±22.7 EDTA d 74±2.1 EGTA d 72±5.6 e H 2 O 2 88±10.9 Solvents Ethanol f 27.0±18.0 Ethyl acetate f 16.0±27.0 Acetonitrile f 3.0±15.0 Methanol f 6.0±12.0 Diethyl ether f 15.0±7.1 Acetone f 52.0±7.0 Dimethyl sulphoxide f 63.0±6.2 Metal ions g BaCl 2 113±5.3 g CaCl 2 107±3.6 g MgSO 4 107±10.3 g FeCl 3 21±13.1 g ZnSO 4 22±15.1 g SrCl 2 47±3.8 g HgCl 2 20±15.1 Urea g 18±21.3 The metal ions had varied effect on the enzyme activity (Table 3). Among the various metals, Ba 2+,Ca 2+ and Mg 2+ enhanced the proteolytic activity. The enzyme activity was strongly inhibited by Fe 3+,Zn 2+,Sr 2+,Hg 2+ and urea. Effect of Various Solvents Maximum inhibitory effect was observed on the protease activity with solvents such as ethanol methanol, ethyl acetate, acetonitrile and diethyl ether. About 52 and 63 % residual activity was noticed against acetone and DMSO, respectively (Table 3). Wash Performance, Dehairing and Liquefaction Alkaline protease from B. pumilus has removed the stain completely from the fabrics without the aid of any of the detergents after 10 min of incubation at room temperature (Fig. 7a c). The enzyme removed hairs from goat skin effectively after 14 h of incubation. Loosening of hair followed by complete removal by simple scraping was noted with no

13 Appl Biochem Biotechnol (2012) 168: Fig. 7 Efficiency of alkaline protease from B. pumilus MCAS8 on the digestion of natural proteins. Wash performance assay of alkaline protease: a stain removed from fabric after 10 min of incubation at room temperature without the aid of detergents; against the cotton stained with blood. b Control. c Negative control. Dehairing efficiency of protease tested against goat skin. d Control. e Goat skin incubated after 8 h of incubation at room temperature: f goat skin incubated with enzyme for 14 h of incubation at room temperature. Blood clot removal by alkaline protease: g after 6 h of incubation with alkaline protease at room temperature. h Control observable damage on the collagen showing proper complete skin depilation on direct observation whereas in control, the hair remains intact with the skin (Fig. 7d f). The enzyme also hydrolyzed the blood clot to liquid state completely within 6 h at room temperature (Fig. 7g, h). Discussion The need for industrially important enzymes has kept on increasing; hence, it is mandatory to look into the novel enzymes with desired properties. Since it has been reported that the recombinants produced by the gene manipulation may not be stable [27], it is worthier to isolate the microbes by exploiting natural diversity [28]. It was reported that the proteases are unstable in organic solvents [16]. Hence, the need for organic solvent stable and thermostable protease is of much concerned as it could withstand at harsh industrial process conditions [29]. In the present study, a protease producing B. pumilus MCAS8 strain was isolated from Pulicat Lake, India. Though protease production by B. pumilus are used industrially and well studied [7, 13], still there is a continuous thrust to isolate efficient proteolytic strain with potent characteristics such as solvent tolerant and detergent stability. Moreover, in industrial

14 1862 Appl Biochem Biotechnol (2012) 168: scale, about 40 % cost is being consumed by the substrates in the enzyme production; therefore, it is necessary to formulate media with cost-effective components [18, 30]. Proteases derived from the Bacillus have been exploited by various optimization studies [7, 8, 12, 13]. However, to the best of our knowledge, this would be the first paper which deals with the optimization studies of protease production with wild B. pumilus strain isolated from natural environment. Among the various inducers examined, soyabean meal has showed the optimum activity. Results of the present study showed that the enzyme production increased up to 93 % when the cells were supplemented with 2.0 % soyabean meal in the production medium. Earlier studies reported soyabean meal could act as best nitrogen source and inducer for protease production [30 32]. Owing to the large availability and remarkable nutritional values, usage of soy protein may serve as a cost-effective and alternate source of substrate in the enzyme production. Optimization of carbon and nitrogen sources in the media plays vital role in determining the cost of the substrate and enzyme production [32]. For B. pumilus MCAS8, citric acid served as the best carbon source for maximum production of protease. The results were in concordance with the previous studies [27]. In contrast, the MCAS8 strain produced considerably less protease enzyme in the presence of other substrates. However, glucose, fructose [32, 33], lactose [27, 30], sucrose [34], glycerol [35] and starch [36] were also reported to increase the protease concentration. The results pertaining to galactose and mannitol agree with the earlier report of Abidi et al. [37]. The requirement for a specific nitrogen supplement differs from organism to organism for protease production [4]. However, in the present study, the addition of various organic and inorganic nitrogen sources failed to enhance the protease production. The result corroborates with the earlier reports of Kanekar et al. and Shanmughapriya et al. [38, 39] which indicate that the supplementation of nitrogen sources may not have beneficial effects on the production of protease. The production of protease by the strain B. pumilus MCAS8 in the absence of nitrogen source can be probably explained due to the presence of soyabean meal in the medium. Chu [31] showed the ineffectiveness of nitrogen source in the presence of the soyabean meal. Therefore, it can be considered that soyabean meal may be a possible candidate in the culture medium for the cost-effective production of an extracellular protease. There was a gradual increase in the protease production in the medium from ph 6 with a peak at ph 11. This indicates the importance of growth ph in metabolic reactions which lead to the alkaline protease production in this bacterial strain. Kumar and Takagi [4] showed the production of protease by B. pumilus at ph The ability of the strain to produce the maximum protease at ph 11.0 also indicated the alkaliphilic nature of the strain. Optimum growth for protease production was observed at 37 C which is in concordance with the earlier studies [6, 40, 41]. Effect of inoculum size on the production of enzyme was studied. Inoculum size of 1 % from overnight grown B. pumilus induced maximum protease production which is in contrast with the report of Kanekar et al. [38] with Arthrobacter ramosus and Bacillus alcalophilus. In general, the molecular mass of the alkaline protease varies from 15 to 36 kda [2]. In the present study also, for the partially purified serine alkaline protease, the molecular mass was found to be 36 kda. The optimum ph for the partially purified protease activity was determined at different ph ranging from 3.5 to The enzyme showed maximum activity at ph 9.0 which corroborates with the earlier reports in Escherichia coli [42] and Bacillus cereus MCM B- 326 [43]. However, ph was also reported earlier [27, 44, 45]. This distinctive optimum activity at ph 9.0 is a notable characteristic feature of alkaline proteases [27]. Generally, the commercial proteases from microorganisms have maximum activity in the alkaline ph range of [46]. The protease from the strain B. pumilus MCAS8 was

15 Appl Biochem Biotechnol (2012) 168: found to be stable over a broad range of ph from 6.0 to 11.0 with an optimum stability at ph 9.0. This stability at high alkaline ph indicates the potential use of the enzyme in detergent manufacturing and tannery industries. The percentage yield and fold purification obtained for the B. pumilus MCAS8 during the ammonium sulphate precipitation is similar to that of the earlier report for the B. pumilus [15]. The protease from the strain B. pumilus MCAS8 showed high activity at 60 C which explained that the enzyme may possess thermostable protein with distinct structural features which allowed retaining its activity even at elevated temperatures [47]. The results coincide with the earlier report for Bacillus sp. [44, 45]. The thermal stability of the purified protease showed that the enzyme activity was stable with residual activity above 50 % between 20 and 70 C. However, the protease thermostability between 30 and 70 C was also reported earlier with half-life of 30 min [15]. This thermostability can accelerate the enzyme use in many biotechnological processes to avoid the contamination by common mesophilic microorganisms. Wide range of substrate specificity of the protease in the current study and its maximum hydrolytic activity observed against the substrate casein is similar to the earlier report of Beg and Gupta [2]. The enzyme was strongly inhibited by the serine protease inhibitor (PMSF) indicating that the enzyme might belong to the serine protease family. The observation corroborates with the early report of Kazan et al. [48]. The reducing agent DTT has a mild inhibitory effect on the enzyme activity which might be due to the reduction of intra-molecular disulphide bonds required to maintain the activity and stability of the enzyme [49]. The B. pumilus MCAS8 enzyme had residual activity above 70 % in the presence of EDTA and EGTA indicating no requirement for metal co-factor. The stability of the enzyme in presence of chelating agents is advantageous for use of enzyme as detergent additive. This is because detergents contain high amounts of chelating agents, which function as water softeners and also assist in stain removal [38]. Besides ph and temperature stability, a good protease could also be stable in the presence of various commercially available bleaching agents, detergents and surfactants [2]. The alkaline protease from the strain B. pumilus MCAS8 had increased activity and showed greater stability towards surfactants like Tween 80 and Triton X-100 which might be due to the effect of surfactants on unfolding of substrate moiety [50]. The results were in consistent with those reported for alkaline proteases from various Bacillus sp. [49]. The enzyme also showed striking stability towards SDS and H 2 O 2. This property of stability towards oxidizing agents and detergent is important because oxidation- and SDSstable enzymes from wild-type microorganisms are not generally known except for few reports from Bacillus sp. [40, 51]. The increase in the protease activity with Ca 2+,Mg 2+ and Ba 2+ indicated the protection of the enzyme by the metal ions against thermal denaturation thereby playing a vital role in maintaining the active confirmation of the enzyme at higher temperatures [4]. The effect of various organic solvents on the stability of the partially purified protease is shown in Table 3. In general, the organic solvents scavenge the essential water molecules from the enzyme and result in the loss of catalytic activity [9, 41]. The current study showed that DMSO exhibited 63 % of its residual activity which proves that it could be used as an ideal catalyst for kinetic- and equilibrium-controlled synthesis [41], while acetone showed 52 % of its residual activity. The reason might be due to stabilization of the enzyme structure even after the removal of water molecules by the organic solvents [42]. The wash performance test showed that the enzyme has the efficiency to remove blood stain completely from the fabric piece without the aid of any of the detergents. Similar observation has been made earlier for Pseudomonas aeruginosa and Bacillus circulans [6]. The alkaline proteases from alkaliphilic bacteria, B. pumilus MCAS8, thus could be used as

16 1864 Appl Biochem Biotechnol (2012) 168: an additive in the detergent formulation as it withstands at the harsh washing conditions such as high alkaline ph, temperature and high concentrations of bleach and surfactants as described by Jaouadi et al. [10]. Enzymatic dehairing plays vital role as an alternate to traditional chemical-based method thereby reducing the hazardous effects on the environment [41]. The dehairing test results revealed the intact nature of the goat skin treated with enzyme and effective removal of hairs completely within 12 h which showed the ecofriendly nature of the enzyme. The results were in concordance with the reports on dehairing efficiency of Bacillus strain [17, 26]. B. pumilus MCAS8 protease with ph stability under alkaline condition especially at ph 9 revealed the robustness of the enzyme towards the leather [44]. The ability of the bacterial protease to digest different natural substrates like blood clot has also been reported earlier [25]. Thus, for the newly isolated B. pumilus MCAS8 strain, a chemically defined medium was formulated; its enzyme characterization and applications were studied thoroughly. The costeffective medium ingredients include (grams per litre): citric acid, 15.0; soyabean meal, 20.0; K 2 HPO 4, 0.5 and KH 2 PO 4, 0.5 at ph 11.0 incubated in a shaker of 37 C at 200 rpm for 48 h. The SDS PAGE study showed the molecular mass of dialysed protease was 36 kda, and the further characterization showed that the alkaline protease requires ph and temperature of 9.0 and 60 C, respectively, for the optimum activity. The study also depicted that the enzyme belongs to serine alkaline protease family. Its remarkable activity against EDTA, EGTA, SDS, Tween-80 and Triton X 100 showed that it could be used as a stain remover and this characteristic was further proved by the wash performance and clot removal applications. It also showed activity against the natural substrates such as casein, skim milk agar, gelatine, haemoglobin, BSA and azocasein. Further, the thermal and organic solvent stable proved that it could be used as a suitable candidate for the enzyme-mediated synthesis reactions. The characteristic features of the strain show that it is alkaliphilic in nature and the strain produces the enzyme with of alkaline serine protease type with special potency to be used in various industrial applications. Acknowledgments The authors thank the Council for Scientific and Industrial Research, Government of India, New Delhi, India for financial assistance through a research project under Coastal Hazard Preparedness [No. 23 (0010)/07/EMR-11]. The authors also thank Ms. S. Devilakshmi, research scholar, Department of Biotechnology, IIT Madras, Chennai for her assistance in enzyme purification and zymogram analysis. References 1. Kalisz, H. M. (1988). Microbial proteinases. Advances in Biochemical Engineering/Biotechnology, 36, Beg, Q. K., & Gupta, R. (2003). Purification and characterization of an oxidation-stable, thioldependent serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology, 32, Outtrup, H., & Boyce, C. O. L. (1990). Microbial proteinases and biotechnology. In C. T. Fogarty & K. Kelly (Eds.), Microbial enzymes and biotechnology (pp ). London: Elsevier. 4. Kumar, C. G., & Takagi, H. (1999). Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnology Advances, 17, Genckal, H., & Tari, C. (2006). Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme Microbial Technology, 39, Jaouadi, B., Ellouz-Chaabouni, S., Ali, M. B., Messaoud, E. B., Naili, B., Dhouib, A., et al. (2009). Excellent laundry detergent compatibility and high dehairing ability of the Bacillus pumilus CBS alkaline proteinase (SAPB). Biotechnology and Bioprocess Engineering, 14,

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