Chapter 7. CHARACTERIZATION OF PURIFIED ALKALINE PROTEASE FROM MUTANT BACILLUS LICHENIFORMIS (Bl8)

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1 Chapter 7 CHARACTERIZATION OF PURIFIED ALKALINE PROTEASE FROM MUTANT BACILLUS LICHENIFORMIS (Bl8)

2 The properties of purified extracelluar proteases of mutant B.licheniformis were studied and the enzyme was characterized. 7.1 MATERIAL AND METHODS Effect of ph on purified alkaline protease activity The influence of ph on the alkaline protease activity was determined by measuring the enzyme activity at varying ph values ranging form 7 to 12 at 40 0 C for 15 min using different buffers viz., 0.05 M Potassium phosphate-naoh buffer (ph 7.0, 8.0) and Glycine NaOH buffer (ph 9.0, 10, 11, 12). The results were shown in fig 20 and Table: XXIII Effect of ph on the stability of alkaline protease activity To determine the stability of enzyme at different ph levels, the purified enzyme was incubated in buffers with different ph for 24 h at 40 0 C. The buffers used were acetate (ph 5 and 6), phosphate (ph 7 and 8) Glycine NaOH (ph 9 and 10) and phosphate NaOH (ph 11 and 12). The relative activity was determined before and after incubation. The results were shown in fig: 21 and Table: XXIV. The percentage of activity remaining was calculated Effect of temperature on the activity of alkaline protease The influence of temperature on the activity of alkaline protease was determined by measuring the enzyme activity at various temperatures ranging form 30 0 to 70 0 C under the standard assay conditions, using Glycine NaOH buffer 0.2M (ph 10.0) and using casein as substrate. The results were shown in fig: 22 and Table: XXV

3 7.1.4 Effect of temperature on the stability of purified alkaline protease The effect of temperature on the stability of the enzyme was determined both in the absence and presence of calcium chloride. The enzyme was incubated at different temperatures for 30 min in 0.2M glycine NaOH buffer with and without calcium chloride (5mM). After heat treatment the enzyme solutions were cooled quickly. Enzyme activities were determined under the standard assay conditions. Results were shown in fig: 23 and Table: XXVI.The percentage of activity remaining after the heat treatments was calculated Effect of inhibitors on the activity of alkaline protease The effects of inhibitors on the activity of the enzyme were studied. The enzyme was incubated with various inhibitors at 5 mm concentrations for 15min at 40 0 C and the residual activities were determined. Inhibitors used were PMSF, P- CMB, Idoaceticacid and EDTA. The results were shown in fig: 24, Table-XXVII Effect of metal ions on the activity of alkaline protease The effect of various metal ions on the enzyme activity was studied. The enzyme was incubated with various metal ion sources.(10mm) viz, Ca 2+, Mg 2+, Co +2, Cd +2, Fe +3, Na +, Zn 2+ and Cu 2+ for 30min at 40 0 C and relative protease activities were measured. The results are shown in fig: 25 and Table: XXVIII Effect of different substrates on the alkaline protease activity Protease activity was measured with various substrates including bovine serum albumin, casein, egg albumin and gelatin. 1ml enzyme was incubated with different protein substrates as described earlier and the activities measured. The results were shown in fig: 26 and Table: XXIX To determine the digestion of natural proteins 2ml of the enzyme was incubated with human blood clot, coagulated egg white and chicken skin in glycine-naoh buffer (ph 9.5) at 37 0 C for 10h and the results were shown in Plate

4 7.1.9 To determine the detergent stability of the enzyme The compatibility of purified alkaline protease with local laundry detergents was studied in the presence of 10mM CaCl 2 and 1M glycine.the detergents used were Ariel, Surf excel, Rin, Nirma and Wheal. The detergents were diluted in distilled water (0.5% wt/vol), incubated with protease for 3 h at 40 0 C, and the residual activity determined. The enzyme activity of a control sample was taken as 100%. The results were shown in fig: 27 and Table: XXX Determination of destaining property of purified alkaline protease: The destaining property was studied by dipping two pieces of cloth artificially stained with blood either in detergent solution or detergent solution supplemented with enzyme followed by incubation for 10 min at 40 0 C. Results were shown in Plate Effect of substrate concentration on activity of the enzyme Effect of substrate concentration on the activity of purified alkaline protease was studied, by incubating the 1ml of purified protease with different concentrations of casein (substrate) viz, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg respectively and the activity of enzyme was measured. From this km results value of the enzyme was calculated. Results were shown in fig:

5 7.2 RESULTS AND DISCUSSION Effect of ph on purified enzyme activity: For the determination of the ph optimum, phosphate (ph 7.0, 8.0) and glycine NaOH (ph ) buffers were used. The highest protease activity was found to be at ph10 using glycine NaOH buffer. The results are shown in Table: XXIII and fig: 24. The maximum activity shown has been taken as 100%. Durham et al., (1987) reported that alkaline proteases generally have broad ph optima in the range of ph These findings are also in accordance with several earlier reports showing ph optima of for protease from Bacillus sp. Thermos aquaticus, Xanthomonas maltophila and Vibrio metscnikovii (Durham, 1987 and Kwon et a1.1994). The important detergent enzymes subtilisin Carlsberg and subtilisn NOVO or BPN also showed maximum activity at ph 10.5 as was reported by Horikoshi et al., (1995, 1996) Effect of ph on the stability of enzyme: The stability of purified protease was also determined by pre incubation of the enzyme in various buffers with different ph values. The enzyme was stable over a broad range of ph 7 to 10. The results were shown in fig: 21 and Table: XXIV. In the present study enzyme could retain 70% of its original activity after incubation in Glycine NaOH buffer with ph 12.0 at 40 0 C for 24h. This was comparable with the stability shown by the highly alkali stable proteases such as those obtained from Bacillus sp. GX 6638 (Durham et al., 1987). Bacillus subtilis RM 615 (Moon et al., 1994) and Vibrio metschnikovil RH530 (Kwon et al., 1994). Alkaline protease from these bacteria was retaining 88% 85% and 80% activity after incubation under more or less similar condition. The ph stability was also shown by subtilisin Carlsberg ( Durham et al., 1987)

6 Table XXIII Effect of ph on purified alkaline protease activity ph range Relative alkaline protease activity (%)

7 Fig 20 Effect of ph on the activity of purified alkaline protease Relative alkaline protase activity (%) ph

8 Table XXIV Effect of ph on the stability of alkaline protease activity ph range Relative alkaline protease activity (%)

9 Fig 21 Effect of ph on the stability of purified alkaline protease Relative alkaline protase activity (%) ph

10 7.2.3 Effect of temperature on activity of alkaline protease The activity of the purified enzyme was determined at different temperatures ranging from 30 0 C to 70 0 C using Glycine NaOH buffer (ph: 10.0) for 1 hr. The optimum temperature for caseinolysis was recorded was at 50 0 C.Results were shown in fig: 22 and Table: XXV. Alkaline proteases of Bacillus with similar temperature optima have been reported by Durham et al.,(1987), Takil et al.,(1990), Kobayashi et al.,(1995,1996) and Ferrero et al(1996) Effect of temperature on stability of purified alkaline protease The thermal stability of the purified protease was tested at different temperatures ranging from 30 0 C to 70 0 C for 30 min in Glycine NaOH buffer, with and without 5mM calcium chloride, was studied. The percentage of activity remaining after heat treatment at different temperatures is shown in fig 23 and Table: XXVI. The enzyme was stable with about 100% activity at 30 0, 40 0 & 50 0 C for 30 min. Reduction in activity could be observed after incubation at 60C and above. The presence of calcium chloride was found to improve the thermo stability of the enzyme. The thermo stability shown by this enzyme was comparable with or better than that reported for the alkaline proteases from different Bacillus species (Tobe et al., 1975; Durham et al., 1987; Takil et al., 1990); Kobayashi et al., (1995, 1996) Effect of inhibitors on the activity of alkaline Protease Effect of different inhibitors on the activity of the enzyme was shown in Table: XXVIII and fig: 24.The enzyme was strongly inhibited by PMSF, and slightly by EDTA. A complete loss of enzyme activity in the presence of PMSF was reported by Gold et al. (1964). Slight inhibition of enzyme activity by EDTA has been reported in alkaline serine protease from bacterial sources such as Bacillus licheniformis (strongin et al., 1979; Ferrero et al., 1996). Bacillus thermoruber (Manchini et al., 1985). Bacillus sp. (Tobe et al., 1983). Halobacterium halobium (Izotova et al., 1983), Pseudomonas sp. (Kato et al., 1974) Myxococcus viriscens (Gnoss pelius, 1978) and Streptonyces sp. (Yum et al., 1994). In the present study also the enzyme purified from mutant B.licheniformis (Bl8) showed similar inhibition by PMSF and EDTA as reported by various researchers. Among the other inhibitors studied, slight inhibition was observed with iodoacetate, PCMB

11 Table XXV Effect of Temperature on activity of alkaline protease Temperature 0 C Relative enzyme activity (%)

12 Fig 22 Effect of temperature on the activity of purified alkaline protease Relative alkaline protease activity (%) Temperature 0 c

13 Table XXVI Effective of temperature on the stability of enzyme activity Temperature 0 C Relative alkaline protease activity (%) with CaCl 2 without CaCl

14 Fig 23 Effect of temperature on the stbility of purified alkaline protease

15 Table XXVII Effect of protease inhibitors on the alkaline protease activity Inhibitor (5mM) (%) Relative enzyme activity Control 100 PMSF (Phenyl methyl sulphonyl fluoride) 00 PCMB (Para chloro mercuric benzoate) 95 Idoacetic acid 90 EDTA (Ethelene Diamine tetra acetic acid)

16 Fig 24 Effect of inhibitors on the activity of purified alkaline protease Relative alkaline protease activity (%) Control PMSF PCMB Idoacetic acid Inhibitors EDTA

17 7.2.6 Effect of metal ions on activity of alkaline protease Effect of various metal ions on the activity of the enzyme is shown in Table XXVIII and: Fig: 25. Activities are expressed relative to the control. Activity of the control was taken as 100%. None of the metal ions showed considerable enhancing effect on the activity of the protease. Ca 2+ & Mn +2 had a slight enhancing effect on the activity of the enzyme. Of the different metal ions tested Zn +2 showed the maximum inhibition. Mn+2 has been reported as enhancing the activity of alkaline serine proteases from some bacterial sources such as Bacillus sterothermophilus F1 (Rahman et al., 1994) and Nocardiopsis derssonvillei (Tsujibo et al., 1990). Its mechanism of action is not well understood. The inhibition of bacterial alkaline proteases by cations such as Fe 2+ (Chang et al., 1988, Rattray et al., 1995) Co 2+ (Rahman et al., 1994), Cu 2+ (Tsujibo et al., 1990) and Zn 2+ (Rattray et al., 1995) have also been reported Effect of different substrates on the alkaline proteases activity Effect of different substrates on enzyme activity was studied. High level the hydrolytic activity was shown by casein and with poor to moderated hydrolysis of BSA and egg albumin. However the hydrolysis was hardly observed with gelatin. The results were shown in Table: XXIX and. Fig: To determine the digestion of natural proteins The ability of purified protease to digest some natural proteins was tested. The results were shown in Plate 2. These results showed that enzyme can convert the insoluble forms of human clot and coagulated white egg to soluble form. The enzyme was also able to digest chicken skin after incubation for a long time with it. The results suggest usefulness of this enzyme for different application such as, extraction of collagen from skin for collagen replacement therapy, waste treatment and others

18 Table XXVIII Effect of metal ions on the activity of alkaline protease Metal ion Source Relative activity (%) (10mM) Control 100 CaCl 2 (Ca 2+ ) MgCl 2 (Mg 2+ ) 101 CoCl 2 (Co 2+ ) 90 CdCl 2 (Cd 2+ ) 90 FeCl 3 (Fe 3+ ) 80 NaCl (Na + ) 98 ZnCl 2 (Zn +2 ) 82 CuCl 2 (Cu +2 )

19 Fig 25 Effect of metal ions on the activity of purified alkaline protease

20 Table XXIX Effect of different substrates on the alkaline protease activity Substrate (1 ml) Relative enzyme activity (%) Casein 100 Bovine serum albumin 54 Egg albumin 32 Gelatin

21 Fig 26 Effect of substrates on the activity of purified alkaline protease Relative alkaline protease activity (%) Casein Bovine serum albumin Egg albumin Gelatin Substrates

22 Plate-2 A B C T T C C Control Test Digestion of natural proteins: A. Blood clot B. White egg C. Chicken skin

23 7.2.9 To determine detergent stability of alkaline protease activity Enzyme activity and stability in the presence of some available commercial detergents was studied with a view to exploit the enzyme in detergent industry. A good protease is expected to be stable in the presence of commercial detergents. In the present study, the protease showed excellent stability and compatibility in the presence of locally available detergents (wheel, Ariel, Surf excel, Rin, Nirma) at 40 0 C in the presence of CaCl 2 and glycine as stabilisers. The proteases showed good stability and compatibility in the presence of Nirma, Surf excel and wheel. The results were shown in Fig: 27 and Table:XXX. Enzyme activity and stability in the presence of detergents was reported by Madan et al. (2002). They observed that enzyme retains 84.5% of activity in the presence of Vim and more than 40% in the presence of Nirma super, Wheel and Nirma. Interestingly, in the present study, the enzyme retained around 20-40% activity with most of the detergents tested even after 3 hours incubation. Phadatare et al. (1993) studied the compatibility of alkaline protease of Condiobolus cornatus (Ncl ) with commercial detergents. They observed the enzyme protease retains more than 80 percent of its activity in the presence of detergents viz, snow white, Nirma, Revel and more than 56 percent of its activity in the presence of wheel and surf when incubated for 1h. Similarly among the three proteases isolated from Tritirachium album limber, proteinase R and T were reported to retain 90 and 89 percent activity respectively up to 1h in the presence of detergents like ERA plus and Dyname. BPN was highly unstable in all the detergents and retained just 4 percent activity even after 10 min as reported by samal et al., (1990) Determination of destaining property of purified alkaline protease The results of distaining experiment showed the complete removal of stain in detergent solution supplemented with enzyme where as blood stain was not completely removal from cloth dipped in detergent solution alone (Plate 3).This suggested usefulness of this enzyme in detergent industry. From this study the protease could be recommended as an additive in detergents to improve their washing performance of the detergent

24 Table XXX Detergent stability of the enzyme Detergen t (%) Retention of enzyme activity Time 0 hr 0.5 hr Control Wheel Ariel Surf excel Rin Nirma

25 Fig 27 Effect of detergents on the stability of purified alkaline protease (%)Retention of enzyme activity Time (min) Control Wheel Arieal Surfexcel Rin Nirma

26 Plate - 3 (A) (B) (C) Slide 3:- Washing performance of alkaline protease from mutant Bacillus licheniformis (Bl8) (A):- Cloth stained with Blood. (B):- Blood stained cloth washed with detergent only. (C):- Blood stained cloth washed with detergent and enzyme

27 The above results showed that the enzyme isolated from mutant strain of Bacillus licheniformis (Bl8) was stable at high ph and temperature and in the presence of detergents and may be exploited as an additive in the detergent industry Effect of substrate concentration on alkaline protease activity Effect of substrate concentration on enzyme activity was studied. Results were shown in fig 28.The results showed that the enzyme followed a typical Michalis- Menten kinetics from a double reciprocal plot. The apparent Km of the enzyme for casein was found to be 3.2 mg/ml.the km values of 0.4 and 1.3 towards casein (mg ml -1 ) have been reported for alkaline proteases of Bacillus alcalophilus var. halodurans (Takil et al., 1990) and Brevibacterium linens (Juhasz and Skarka, 1996) the higher values 3.7, 7.4 and 9.4 mg ml -1 have been reported for the proteases of Bacillus polymyxa ( kaur et al., 1998) Halomonas sp. Es10 (Kim et al., 1992) and Bacillus licheniformis N3 (Sinha and Satyanarayana, 1991). respectively. The lower km value of 3.2 mg ml -1 in the present study indicating lower affinity of the enzyme from mutant B.licheniformis(Bl8). Menu madan et al.,(2002) reported that apparent km of the enzyme for casein was found to be 2.9 mg ml -1 for mutant Bacillus polymyxa. It has lower km value than the wild B.polymyxa protease

28 Fig 28 Effect of substrate concentration on purified alkaline protease (Line weaver-burk plot)