Production and characterization of alkaline protease from bacteria strains isolated from cotton field

A number of bacterial strains producing protease enzymes were isolated from the soil of cotton field. Five strains producing high level of extracellular protease with alkaline conditions were selected. Maximum production of 4675 U/ml of protease in 48 h of growth was obtained from strain MW17, while maximum specific activity of 21515U/mg was obtained with strain MW09. pH optimum of 8-10 was observed with different proteases. Protease from strain MW05 showed maximum stability after 24 h of incubation. Strains showed different optimum temperature for protease activity, that is, 30°C (MW08), 40°C (MW05, MW28) and 50°C (MW09, MW17). Mg 2+ , Fe 3+ and Zn 2+ were found to increase the enzyme activity of different strains, while Ca 2+ decreased the activity. Most of proteases were stable in commercial detergent, thereby, indicating the possible application of these proteases in detergent industry. Selected strain MW09 showing required properties was characterized with respect to physiological and biochemical properties.


INTRODUCTION
Enzymes being selective in terms of function and specificity are suitable catalyst as compared to chemical catalysts.Further enzyme controlled reactions can occur in aqueous environment.Enzymes find applications in diagnostic, food and feed, beverages and other biotechnology industry besides application in research and development.The present enzyme industry worldwide is approximately worth $5.8 billion.The demand of enzyme will rise from 6.8% annually to $8.0 billion in 2015 (http://www.freedoniagroup.com/World-Enzymes.html).Among the various enzymes, hydrolytic enzymes have maximum application and commercial values.Enzymes are being isolated from microorganism, plants and animals.
Proteases are group of enzymes that are able to hydrolyze the peptide bonds in proteins and polypeptides and classified as serine protease, cysteine protease, aspartic protease and metallo-protease (Singh et al., 2001).Proteases are used in detergent, food and feed industry, leather industry, photography industry and meat industry.To improve the softness and shining property, proteases are applied on raw silk fibers.They also find applications in oncology, inflammatory conditions, blood rheology control, immune regulation and constitute more that 60% of the total enzyme market.Protease in association with lipase constitute part of lens cleaning solutions for removal of soil particles (Moreira et al., 2002;Najafi et al., 2005;Ray, 2012;Binod et al., 2013;Sathiya, 2013).Wide sources of protease from bacteria, fungi, plant and animal are reported (Shaginian et al., 1990;Wang et al., 2006;Chi et al., 2007;Chu, 2007).Microorganism which is easier to grow under controlled *Corresponding author.E-mail: waliameenu15@gmail.com.
conditions is preferred source of enzymes.As microorganisms can be manipulated to increase production and modify the properties of the enzymes of interest, a lot of work has been reported on microorganisms.Microorganisms have been isolated from various environmental conditions with the aim to have enzyme with special characteristics and applications (Ogawa and Shimizu, 1999;Nigam, 2013).Protease having different pH optimum can be used in variety of applications.Khan (2013) reviewed a number of microbial protease especially alkaline proteases for their industrial applications.The present study involves the isolation, production and characterization of alkaline protease from the microorganisms with possible applications in industry.

Isolation of alkaline protease producing microorganisms
Samples of soil from various fields including cotton soil were collected from different parts of Haryana (India).These samples were suitably diluted and plated on the skim milk agar plates containing peptone (1.5%), malt extract (1%), NaCl (0.5%) and skim milk (1%).The plates were incubated at 40°C for 24 h.A clear zone of skim milk hydrolysis indicated protease producing organism.All the strains producing proteases were transferred to skim milk plates having different pH: 8, 9, 10 and 11.Selected strains were processed for future work.

Production and partial purification of alkaline protease
Production of the protease from the selected strains of bacteria was carried out in a medium containing peptone (1.5%), malt extract (1%) and NaCl (0.5%) at temperature of 40°C and 150 rpm.The pH of the medium was adjusted to 10 with 0.1 N NaOH.Samples were taken out at regular interval of 6 h and estimated for optical density, protein and protease activity.After 48 h, cultures were centrifuged at 10,000 rpm, 4°C and supernatant was collected.Supernatants were subjected to ammonium sulphate precipitation (100% saturation) followed by dialysis against glycine-NaOH buffer (0.1 N; pH 10.0).The resulted products were used as partially purified enzyme for characterization of proteases.

Enzyme assay
Protease activity was estimated as per protocol of Singh et al. (2001) with certain modification.To 100 µl of azocasein (1% w/v) in glycine-NaOH buffer (0.1M, pH 10.0), 100 µl of enzyme was added and incubated for 60 min at 40°C (otherwise specified) followed by addition of 1 ml of 10% (w/v) trichloro acetic acid (TCA).Tubes were kept on ice for 15 min and centrifuged at 10,000 rpm for 30 min.One ml of supernatant was mixed with 500 µl of NaOH (1.0 N).Absorbance was measured at 420 nm using appropriate blanks.
One unit (U) enzyme activity was defined as the amount of enzyme required to produce an increase in absorbance equal to 1.0 in 60 min.

Protein assay
Concentration of protein was estimated by the method of Lowry et Batra and Walia 703 al. (1951).Routine calibration curve from standard BSA solution (0.02-0.2 mg) was used to measure protein concentration.

Effect of pH on enzyme activity and stability
Activity of the enzyme was measured at different pH values: pH 6.0-8.0 (sodium phosphate buffer); pH 9.0-11.0(glycine-NaOH buffer, 0.1 N).Mixtures were incubated at 40°C (otherwise specified) and the activity of the enzyme was measured.Enzyme was diluted in different buffers (pH 6.0-11.0)and incubated at 40°C for 24 h and the relative activity was measured as per assay procedure.

Effect of temperature on enzyme activity and stability
The activity of the enzyme was determined by incubating the reaction mixture at different temperature ranging from 30 to 60°C.
To determine the enzyme stability with changes in temperature, partial purified enzyme was incubated at different temperatures (37, 45, 50 and 60°C) and relative activities were assayed at standard conditions.

Effect of various metal ions on protease activity
The effects of metal ions e.g.Ca 2+ , Fe 3+ , Cu 2+ , Mg 2+ , Na + and Zn 2+ (50 mM) were investigated by adding them into the reaction mixture.Relative protease activity was measured.

Stability of proteases with laundry detergents
The compatibility of partially purified alkaline proteases with local laundry detergents was studied.Detergents used were: Henko (Jyothy Laboratories Ltd); Ariel, Tide (Procter and Gamble, India), Rin detergent, Surf Excel Quickwash, Surf Excel Blue (Hindustan Lever Limited, India).The detergents were diluted in distilled water (1.0%w/v), incubated with protease for 1 h at optimum temperature of each protease.The enzyme activity of a control sample (without detergent) was taken as 100%.

Selection and production of alkaline protease
Thirty nine (39) strains of bacteria producing alkaline protease out of 57 strains were isolated from different soil samples.Based on clear zone obtained and production of protease under alkaline conditions, five strains were selected for further characterization.Strain MW05 and 08 showed maximum activity at pH 9.0 as compared to strains MW17, MW28 at pH 10 and strain MW09 at pH 8.0 (data not shown).These strains were grown in same medium as described in materials and methods, but at pH optimum for respective strains.Strain MW05 showed rapid growth in the initial phase with maximum cell mass (2.12 OD/ml) in 48 h (Figure 1).As compared to this, other strains are slow growing and less cell biomass were observed.Maximum production of 4675 U/ml was  obtained with MW17 within 48 h, however, specific activity of 21515 U/mg protein was maximum with strain MW09.The production of protease was found to be growth associated in all the strains (Figures 1 and 2).

Characterization of enzyme
Partial purified enzymes from different strains were subjected to characterization studies.The result of pH  studies indicated a broad pH activity range of 8.0-11.0(Figure 3).Strain MW05 showed optimum pH of 10.0.This enzyme is sensitive to change in pH.As pH increased or decreased, the activity decreased drastically.However, in the case of MW28, smooth decrease was observed with change in pH.There was a special observation in MW09, there was two optimum at pH 9 and pH 11 with relative activity of 100 and 89%, respectively.At pH 8, about 82% of protease activity was observed as compared to the control.For MW17, pH optimum was at 8 and 11, thereby, indicating the possibility of isoenymes of protease in these organisms.
Protease from MW08 showed optimum activity at pH 9.
The optimum catalytic activity from different proteases reported is in the range of pH 7-11 (Cha et al., 2005;Kocabiyik and Ozdemir, 2006;Miyaji et al., 2006;Vidyasagar et al., 2006).The enzymes obtained from MW08 showed wide stability from pH 6-10 with more than 98% activity retained even after 24 h of incubation (Figure 4).About 87 and 98% protease activity was retained at pH 7 and 11 respectively, for MW05 protease.MW09 enzyme retained 95% of activity at pH 7-9 after 24 h of incubation, while 49% activity was retained at pH 11.MW28 protease had 100% retention in activity at its optimum pH of 10.In Bacillus pumilus, alkaline protease was stable over the pH 8-11 at 50°C (Miyaji et al., 2006).
The protease activity of the crude enzyme was measured at temperatures ranging from 30-60°C at different optimum pH: 8-10.MW05 and MW28 protease have 40°C to be the optimum temperature, while, MW08 strain had heat sensitive protease with optimum at 30°C.MW09 protease had optimum activity at 50°C, while 98 and 89% of relative activity was observed at 55 and 60°C, respectively.Partial purified MW17 protease activity decreased as temperature rose from 50 to 60°C (Figure 5).The optimum catalysis reported is in the range of 30 (Shikha et al., 2007), 37 (Patel et al., 2005) and 55°C (Huang et al., 2003).Our studies (Figure 6) indicated that strain MW09 protease was completely stable at temperature range of 37-50°C.Only 2% loss in activity was observed even after incubation at 55°C.This range of stability is quite broad as compared to some reports for alkaline proteases, where the stability is maximum at 50°C (Chu, 2007) and 55°C (Wang et al., 2006).Similarly, 100%  activity was retained even after incubation at 24 h upto 50°C in MW17 protease.Half life (t 1/2 ) of MW17 protease at 60°C was found to be 24 h.A good detergent protease is expected to be stable in the presence of detergents.Wide variation in the stability of proteases isolated from different strains was observed (Figure 7).Strain MW08 showed complete stability in Tide.Protease MW05 was almost 98% stable in Tide and Henko detergent.MW09 protease had wide stability (67-98%) in varieties of detergents.Different proteases demonstrated variation in stability when applied in the presence of commercial detergents (Gupta et al., 2008;Vijayalakshmi et al., 2011).The effect of various metal ions on protease activity was tested in 50 mM glycine-NaOH buffer.Among the metal ions tested, Fe 3+ acted as strong enhancer of enzyme with enhancement of activity upto 315% (Table 1).Zn 2+ , Mg 2+ , Na + and Ca 2+ enhanced the activity by 217 (MW17), 215 (MW17), 182 (MW05) and 149% (MW05) respectively.Ca 2+ acted as strong inhibitor by decreasing activity to 5% of MW28 protease.As protease MW09 showed maximum desired properties (maximum production, optimum pH of 9 and 11; pH stability of 7-9; temperature optimum 55-60°C; temperature stability 37-55°C and stability in metal ions and detergents), it made it to be suitable for industrial applications.The strain MW09 was characterized with  respect to physiological and biochemical properties and identified using BioLog system.
The BioLog GEN III Microplate (table 2) was used which provided 71 carbon utilization assays, 23 chemical sensitivity assays and two controls (positive and negative).The microplate data was entered and analyzed with MicroLog software.MW09 strain was identified as Gram positive rod shaped Bacillus licheniformis MW09.Further purification and characterization of protease from this strain is being carried out.

Figure 1 .
Figure 1.Growth profile of microorganisms producing alkaline proteases.

Figure 2 .
Figure 2. Enzyme activity of alkaline protease produced by different bacterial strains.

Figure 3 .
Figure 3.Effect of pH on the partially purified alkaline protease activity.

Figure 4 .
Figure 4. pH stability of alkaline protease produced by different bacterial strains.

Figure 5 .Figure 6 .
Figure 5.Effect of incubation temperature on alkaline protease produced by different bacterial strains.

Figure 7 .
Figure 7. Stability of the protease enzyme in commercial detergents.

Table 1 .
Effect of various metal ions on protease activity.

Table 2 .
Layout and results in the GENIII Microplate TM .