Chapter PURIFICATION OF ALKALINE PROTEASES

Chapter PURIFICATION OF ALKALINE PROTEASES

E /xtracellular alkaline proteases produced by Bacillus sp. K 25 and bacillus pumilus K 242, were purified and the homogeneity was examined by electrophoresis. Section A and B of this chapter deals with the purification of alkaline proteases from Bacilus sp. K 25 and Bacillus pumilus K 242 respectively. Section A PURIFICATION OF ALKALINE PROTEASE FROM THE CULTURE SUPERNATANT OF BACILLUS SP. K 25 MATERIALS AND METHODS The extracellular alkaline protease of Bacillus sp. K 25 was produced by submerged fermentation providing the optimum conditions. The culture supernatant was obtained by centrifugation of culture broth at 10,000 r.p.rn. for 15 min. Ammonium sulphate was added into the culture supernatant and the precipitate obtained at 75% saturation was collected. The precipitate was dissolved in 0.01 M potassium phosphate buffer, ph 7.5 (Buffer 1) and was dialyzed against the same buffer overnight. The dialyzed material was then applied to DEAE cellulose (sigma) column (2.5 x 30 cm) equilibrated with buffer 1. The unbound proteins were eluted with the same buffer. 10 rnl fractions were collected. The fractions with alkaline protease activity were pooled, concentrated and dialyzed against 0.01 M potassium phosphate buffer, ph 6.8 (Buffer 2). The dialysate was then applied to CM cellulose (sigma) column (2.5 x 30 cm) equilibrated with buffer 2. Elution was performed first with buffer 2 and then with buffer 2 containing increasing concentrations of KC1 (0.01-0.5M). 10 rnl fractions were collected. A final

elution with buffer 2 containing 1 M KC1 was also performed. Fractions with protease activity were pooled, concentrated and applied on Sephadex G-100 (Pharmacia) column (4 x 60 crn) equilibrated with buffer 2. Elution was accomplished with buffer 2. 10 ml fractions were collected. Filtrate fractions showing the enzyme activity were pooled, concentrated, dialyzed against distilled water, lyophilied and stored at 20 C. After each step in the purification procedure, specific activities (u mg-' protein) were determined. The protein estimation was done following the method of Lowry eta/, (1951). The homogeneity of the purified enzyme preparation was examined by discontinuous disc electrophoretic analysis following the method of Laemmli (1970), in 10% native polyacrylamide gels. Gels were run at ph 8.8 at a constant current of 2 rna per gel. Two gels were run simultaneously, one for staining and the other for detecting the activity. Gel was stained with 1% Coomassie brilliant blue R-250 in 45% methanol - 10% acetic acid solution for 4 h; and destained in the same solvents for 4 h. The other gel was cut into 5 mm pieces, homogenized in 0.2 M glycine-naoh buffer (ph 10.0) and the alkaline protease activity was determined. RESULTS The alkaline protease in the culture supernatant of Baciflus sp. K 25 could be precipitated by ammonium sulphate at 75% saturation. The precipitate obtained was dissolved, dialyzed and applied to DEAE cellulose column. Alkaline protease did not adsorb to the column. Eluate containing unadsorbed proteins was then subjected to CM cellulose chromatography. The elution profiles of alkaline protease from the CM cellulose column are shown in Figure 5.

Fraction No. Figure 5. Elution profile of alkaline protease of &cjlfus sp. K 25 from CM cellulose column

In the chromatogram alkaline protease activity could be seen in a single peak which was eluted with 0.01 M potassium phosphate buffer, ph 6.8 (buffer 2), containing 0.04-0.11 M KCI. The final elution performed with the buffer 2 containing 1 M KC1 did not yield any protease activity. Further purification of protease was performed by gel filtration on Sephadex G-100. The elution profile is shown in Figure 6. The gel filtrate fractions containing protease were pooled, dialyzed against distilled water, lyophilied and stored at 2O"C. When tested for homogeneity by native PAGE, the protein in the sample migrated as a single band (Plate 1) with alkaline protease activity. The summary of purification of extracellular alkaline protease of Bacillus sp. K 25 is given in Table 26. Table 26 Purification of extracellular alkaline protease of Bacillus sp. K 25 Purification Total enzyme Total protein Specific activiiy Purification Yield step activity (u mg-' fold (%I (4 (mg) protein) Culture supernatant 6310 1602 3.94 1 100 Ammonium sulphate 5252 precipitation 359 14.63 3.71 83.23 - DEAE cellulose 4246 202 21.02 5.34 67.29 CM cellulose 3044 91 33.45 8.49 48.24 Sephadex G-100 2542 64 39.72 10.08 40.29

20 30 40 50 60 70 50 Fraction No. Rgure 6. Hution profile of alkaline protease of Badllus sp. K 25 from Sephadex G- 100 column

As a result of purification there was ten-fold increase in the specific activity. The recovery of the enzyme was 40.29%. DISCUSSION Alkaline protease in the culture supernatant of BaciMus sp. K 25 could be purified by procedures involving conventional methods such as ammonium sulphate precipitation, negative adsorption by DEAE-cellulose, CM cellulose chromatography and gel filtration on Sephadex G-100. Alkaline protease in the culture supernatant was found to be precipitated by ammonium sulphate at 75% saturation. The acidic proteins in the dissolved precipitate could be removed by negative adsorption on the anion exchanger DEAE cellulose. Further purification was done by CM cellulose chromatography and gel filtration on Sephadex G-100 column. The specific activity of the purified enzyme towards casein when tested at 45 C and ph 10.0 was 39.72 u mg-' protein, i.e. ten-fold of the specific activity of the culture supernatant. The purified enzyme preparation was found to be homogenous when tested by native PAGE. The high recovery of the enzyme (40%) indicates the efficacy of the scheme of purification employed.

Section B PURIFICATION 'F ALKALINE PROTEASE FROM THE BACTERIAL BRAN EXTRACT OF BACILLUS PUMILUS K 242 MATERIALS AND METHODS The extracellular alkaline protease of Bacillus pumilus K 242 was produced by solid state fermentation, providing the optimum conditions and the enzyme was extracted as described under Section B of Chapter 3. To the supernatant of bacterial bran extract obtained after centrifugation at 10,000 r.p.m. for 15 min, ammonium sulphate was added. The precipitate obtained at 80% saturation was dissolved in 20 mm Tris-HCV2 mm CaC12 buffer, ph 9.0 (Buffer 1). It was then dialyzed against the same buffer overnight and the dialyzed material was applied to DEAE Sephadex A-50 (Pharmacia) column (2.5 x 30 cm) equilibrated with the buffer 1. Elution was performed first with the buffer 1 and then with the same buffer containing increasing concentrations of NaCl(O.O1-0.5 m). A final elution with the buffer containing 1 M NaCl was also performed. 10 ml fractions were collected. In chromatogram, protease activity could be seen in two peaks. Pooled fractions forming the major peak was concentrated and dialyzed against 0.02 M phosphate buffer, ph 7.5 (Buffer 2). The dialyzed material was further purified by gel filtration on Sephadex G-100 (Pharmacia) column (4 x 60 an) equilibrated with buffer 2. Elution was done with buffer 2. 10 ml fractions were collected. Filtrate fractions with the enzyme activity were pooled, concentrated, dialyzed against distilled water, lyophilied and stored at ZWC. After each step in the purification procedure, specific activities (u mg-' protein)

were determined. Protein concentrations were estimated according to Lowry etd (1951). The homogeneity of the purified enzyme preparation was tested by native PAGE as described under section A of this chapter. RESULTS The proteases in the supernatant of bacterial bran extract were precipitated by ammonium sulphate at 80% saturation. Mer dialysis it was subjected to DEAE Sephadex A-50 column chromatography. The elution profiles are shown in Figure 7. Proteases were eluted from DEAE Sephadex A-50 column in two peaks. The peak 1 was the major peak and was obtained by elution with the buffer containing 0.23-0.33 M NaCI. Peak 2, the minor peak was obtained by elution with 0.39-0.45 M NaCI. A final elution with buffer containing 1 M NaCl did not yield any protease activity. The specific activities of the pooled fractions in peak 1 and 2 were 48.18 and 12.91 u mg-' protein respectively. Fractions forming the major peak were pooled and further purified by gel filtration on Sephadex G-100. The elution profile is shown in Figure 8. Gel filtrate fractions with alkaline protease activity were pooled, lyophilued and stored at 20 C. On testing the homogeneity of the preparation, by native PAGE, protein was found to be migrating as a single band (PLate 2) with alkaline protease activity.

Fraction No. Figure 7. Elution profife of proteases of Bacillus pumihs K 242 from D EE Sephadex A-50 colu - p\ -'"'.

Fraction No. Figuro 8. Elution profile of alkaline protease of Badlus puumil K 242 from Sephadex G- 100 column

The summary of purification of extracellular alkaline protease of Bacillus pumilus K 242 is given in Table 27. Table 27 Purification of extracellular alkaline protease from the bacterial bran extract of Bacilluspumilus K 242 Total Specific Purification protein activity Purification Yield step activity (U mg-' fold (mg) (4 protein) Bacterial bran exbact 9008 944 9.54 1 100 Ammonium sulphate 6435 precipitation 308 20.89 2.19 71.44 DEAE Sephadex A-50 Peak 1 4292 95 45.18 4.74 47.64 Peak 2 297 23 12.91 - - Gel filtration (of peak 1 from DEAE 3635 72 50.49 5.29 40.35 Sephadex A-50) on Sephadex G-100 As a result of purification there was more than five-fold increase in the specific activity. The specific activity of the purified enzyme towards casein was 50.49 u mg-' protein, when assayed at ph 10.0 and 45 C. The recovery of the enzyme was 40.35%.

DISCUSSION Alkaline protease of Bacillus pumilus K 242, produced by solid state fermentation was purified from the bacterial bran extract by procedures involving ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The proteins precipitated by ammonium sulphate at 80% saturation were dissolved, dialyzed and subjected to DEAESephadex A-50 column chromatography. In chromatogram two peaks (peak 1 and 2) with protease activity could be seen. Of these, peak 1 was the major peak. Pooled fractions in this peak showed high specific activity (45.18 u mg-' protein). It was further purified by gel filtration on Sephadex G-100 column. Peak 2 being a minor peak with low specific activity (12.91 u mg-' protein) was not further purified. When the purified enzyme preparation was subjected to native PAGE, the protein was found to be migrating as a single band with alkaline protease activity indicating the homogeneity of the preparation. The recovery of the enzyme was more than 40%. As a result of purification, there was only five-fold increase in the specific activity. The reason for getting the electrophoretically pure enzyme even with five-fold increase in specific activity, was the presence in high concentration of alkaline protease in the bacterial bran extract, which in turn can be attributed to the very high yielding nature of the shin. Purification of alkaline protease of a high yielding bacterium Bacillus sp. AJ 3368, to the electrophoretically pure state, with only 4.66-fold increase in specific activity, was reported by Tobe et a/. (1975). Fujiwara and Yamarnoto (1987) who purified alkaline protease from the culture supernatant of the high yielding Bacillus sp. B21-2, could obtain the enzyme in the electrophoretically pure form at 8.5-fold purification.