BIOTECHNOLOGY TECHNIQUES Volume 6 No.2 march~april 1992 pp.133-13 Received 13th January The use of ElYrA or Polymyxin with Lysozyme for the Recovery of Intracellular Products from E.scher/ch~ co/i C.R. Dean and O.P. Ward, Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Summary Escherichia coli cells taken from exponential and late stationary (or decline) phases of culture were very susceptible to lysis by EDTA/lysozyme. Log phase cells were most susceptible to lysis by polymyxin/lysozyme. Treatment of E. coli with EDTA and lysozyme compared favourably with sonication as a method for release of intracellular protein. Concentration ranges for optimal lysis were 100-800 #g/ml for EDTA and 25-50 #g/ml for lysozyme. Introduction Enzymes which lyse cell walls have been used as an alternative to homogenisation for recovery of intracellular products from yeast and Gram positive bacteria (White and Marcus, 1988). The peptidoglycan layer of Gram positive bacteria is attacked by egg and microbial Iysozymes (Andrews and Asenjo, 1987; Suzuki et al., 1985). The peptidoglycan layer of gram negative bacteria is protected from enzymatic attack and the latter bacteria are more resistant to cell lysis. A lytic enzyme complex from Bacillus subtilis contained proteases, bacterial lysozymes and an outer membrane disrupting agent (Takahara et aI., 1976). Lysis of Escherichia coli by Bacillus culture filtrates was attributed to a combination of outer membrane disruption by heat and proteolytic hydrolysis of the peptidoglycan layer (Dean and Ward, 1991). Combined lysozyme chemical treatments are used to lyse E. coli for the isolation ofplasmid DNA and for spheroplast formation (Mukhopadjyay and Mandel, 1983; Saha et aI., 1989). EDTA, polymyxin B and Triton X-100 have also been shown to disrupt the outer membrane (Andrews and Asenjo, 1987). We have investigated E. coli lysis by EDTA/lysozyme and EDTA/polymyxin combinations as a possible downstream processing technique. Experimental Methods Preparation of E. coli for lysis investigations: E. coli cells, obtained from stock cultures incubated at 37"C, were inoculated into a culture tube containing 5 ml of the medium to be used for subsequent cell production, and incubated for 12 h at 37"C. This culture (0.5 ml) was used to inoculate 100 ml medium contained in duplicate 500 ml baffled flasks. Flasks were incubated at 37"C on an orbital shaker set at 200 rpm. Cells were harvested by centrifugation at 1500 g for 20 min and twice washed by resuspension in 0.05 M Tris-HC1 buffer, pH 8.0 (buffer A). Culture media: E. coli was cultivated in Nutrient Broth (Difco), LB Broth (Gibco) and 133
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`FG-Medium (Fraser and Jerrel, 1953). FG-Medium contains 0.3 g/1 MgSO 4 and the other media were supplemented with magnesium as indicated in the text. Cell lysis. Cells were suspended in buffer A to give a spectrophotometric absorbance of approximately 0.90 at 540 nm in a 1 cm light path. This suspension, 2 ml, was added to a cuvette containing buffer A (1 ml) supplemented lysozyme and EDTA or polymyxin B sulphate as indicated. Unless otherwise stated, lysis temperature was 20"C. The decrease in absorbance at 540 nm in a 1 cm light path was monitored. Lysis determinations were carried out in triplicate. One lysis unit produces a decrease in absorbance of 0.001 per min. Sonication: A 70 ml volume of cells, suspended in buffer A was cooled to 4"C and sonicated using a Braun-Sonic 2000 with a large probe at a setting of 180 for 7 to 13 min in one min cycles interspersed with cooling cycles. Cell debris was removed by centrifugation at 12000 g and 4~ for 1 h. Relative cell surface hydrophobicity: E. coli cells, twice washed as described above, were resuspended in buffer A to give an absorbance of 0.86 at 540 rim. To this suspension (1 ml), 2 ml of buffer A containing EDTA and 0.4 ml of carbon tetrachloride was added. Following vortexing, the organic and aqueous layers were allowed to separate for 1 h and the absorbance at 540 nm of the aqueous layer was compared with that of the original suspension. The values are expressed as a percentage of control absorbance at 540 nm. B-Galactosidase: The reaction mixture contained 0.5 ml o-nitrophenyl B-D- galactopyranoside, 4 mg/ml, dissolved in 0.25 M sodium phosphate, pH 7.0 and 0.5 ml of enzyme solution at 40~ for 30 min. Following incubation, the reaction was terminated by addition of 2 ml 1M Na2CO 3. The yellow colour was measured spectrophotometrically at 420 nm and related to equivalents of o-nitrophenol using a standard curve. 1 Unit of 13-galactosidase liberates 1/~mol of o-nitrophenol from o- nitrophenyl 13-D-galactopyranoside per min. Results The relationship between lysozyme concentration and lysis of E. coli ATCC 9723e for different stages in the growth curve is presented in Figure 1. Cell lysis increased sharply with lysozyme concentration reaching a rate maximum at 25 or 50 #g/ml lysozyme. Cells were extremely susceptible to lysis during the logarithmic phase and late stationary phase. The pattern of cell lysis observed with the E. coli sample recovered after a 55 h incubation from this experiment is illustrated in Figure 2. The effect of EDTA concentration, used in combination with lysozyme 25 #g/ml, on the rate of cell lysis (incubated for 45 h in FG-Medium) was investigated. Lysis rate increased rapidly with increasing EDTA concentration to 100/zg/ml. E. coli cells were incubated for 45h in FG-Medium and in Nutrient Broth (Difco) and LB Broth (Gibco) with and without 0.3 mg/ml MgSO4 and the susceptibility of cells to lysis by EDTA (800 #g/ml)lysozyme (25 /zg/ml) was investigated (Table 1). Cells grown in Mg-supplemented media had increased sensitivity to lysis by EDTA/Iysozyme. Cells grown in LB Broth with Mg manifested high sensitivity to EDTA/lysozyme. Cells incubated for 24 h in LB Broth had also reached the stationary phase and magnesium caused a similar sensitivity to EDTA/ lysozyme. The effect of Mg concentration in LB Broth on lysis susceptibility of washed stationary phase E. coli ceils is illustrated in Figure 3. The effect of stage of E. coli growth in this Mg-supplemented medium on lysis susceptibility to EDTA/lysozyme is 134
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`2S00, 0.7- ~000 (cid:12)9 1500- o~ 1000. $00- Media MgSO4 Incub. Lysis .3 mg/ml time h units LB broth 45 733 LB broth + 45 2783 Nutrient broth 45 416 Nutrient broth + 45 1133 FG Medium 45 1500 LB broth + 24 3100 Lysis conditions: EDTA, 800//g/ml; lysozyme, 25/~g/ml C~lti~*n Log cell time h ntunl~r D 12 9Je (cid:12)9 26 9~0 O 36 10.15 AT/ 9.'~ 0~ 0 2.5 50 75 100 12S 150 175 200 225 Lysozyme (/tg/ml) v o G t~ C: r~ ~ O.G' ~0 '--0-- EDTAJlysozyme 0.5' 0.4 " \ -"'0"-- ConlrOI (cid:12)9 EDTA alone t 0.3 " ~OI - lysozyme alone 0.2- oa- -o----o 0.0 , i 0.0 0.5 1.0 1.5 Time (rain) Fig. 2 Lysis curve for E. coli grown for 55 h in FG Medium using EDTA/lysozyme. (Lysis conditions: EDTA, 800/~g/ml; lysozyme, 25 #g/ml) 2000. 3OOO 2500, 1,'500. 1000 0.0 ,. . | - (cid:12)9 . . , - 0.2 0.4 0.6 0.8 MgS04 (mg/ml) Table 1 Effect of media on lysis of E. coli using EDTA/lysozyme Fig. 1 Effect of lysozyme concentration on lysis rate of E. coli grown for various times at 37 ~ C in FG Medium. (Lysis conditions: EDTA, 800/tg/ml) Fig. 3 Effect of magnesium concentration in the growth medium on subsequent lysis susceptibility of E. coli to lysis. (Lysis conditions: EDTA, 800 /~g/ml; lysozyme, 25/~g/ml) 135
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`presented in Figure 4. In Table 2 the lysjs rates of various strains ofE. coli, incubated for 24 h in LB Broth supplemented with 0.3 mg/ml MgSO4 are compared. E. coli ATCC 23231 and ATCC 23227 were resistant to the EDTA/lysozyme treatment. 4O00 2000 " 1000 " 5 10 15 ;~0 25 Cultivation time Ca) Table 2 Lysis of E.coli strains with EDTA/lysozyme ATCC Lysis strain (units) 23717 400 9723e 2300 23716 1866 10978 2100 23231 0 23227 100 Fig. 4 Effect of growth stage of E. coli in LB broth supplemented with MgSO, (0.3 mg/ml) on lysis susceptibility to EDTA/lysozyme. (Lysis conditions: EDTA, 800 #g/ml; Lysozyme, 25 #g/ml) Lysis conditions: EDTA, 800 #g/ml; Lysozyme, 25/~g/ml Log phase E. coli cells were also lysed by polymyxin used in combination with lysozyme. The effect of polymyxin concentration used with lysozyme (25 #g/ml) on lysis of log and stationary phase cells cultivated in LB Broth, supplemented with 0.3 mg/ml MgSO4, is illustrated in Figure 5. Polymyxin concentrations of 27 #g/ml and 5 #g/ml were optimal for lysis of log and stationary phase cells, respectively. However, at the polymyxin concentrations corresponding to optimal lysis, the rate of lysis of log phase cells was 10 times greater than that of stationary phase cells. The relationship between growth phase and lysis susceptibility by polymyxin (27/~g/ml)- lysozyme (25 #g/ml) is illustrated in Figure 6. Temperature optima for E. coIi lysis in EDTA/lysozyme and polymyxin/lysozyme systems were found to be 58"C and 45"C respectively. The effect of EDTA concentration on the extent of outer membrane disruption (measured by hydrophobicity) was compared with cell lysis susceptibility (% of cells lysed in 1 min; lysozyme, 25 #g/ml; EDTA varied) in Figure 7. As EDTA concentration increases, hydrophobicity of the cell surface increases (resulting in a decrease in absorbance in the aqueous phase) to a constant maximum as does percentage cell lysis. The effect of EDTA/lysozyme on protein release by lysis of cell suspensions of increasing cell concentration was investigated using an extended lysis reaction time of 25 rain. A nearly linear relationship is observed between cell concentration and amount of protein released (Fig. 8). Cell suspensions having an absorbance at 540 nm of 15.8 (1 cm light path) released a maximum of 1140 and 1390 #g protein per ml when treated with EDTA/lysozyme and sonication respectively. Specific activities of B-galaetosidase in the soluble protein fractions were 0.40 and 0.38 u/mg respectively for lysed and sonicated cells. 136
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`8OOO r 6000 ' 2000 ' 2o ,.O 8'o Polymyxin (#g/ml) 80 Fig. 5 Effect of concentration of polymyxin on cell lysis of log phase ( (cid:12)9 ) and stationary phase ( (cid:12)9 ) E. coli cells, when used in combination with lysozyme. (Lys/s conditions: lysozyme, 25 #g/ml) ~ = ~= t/] < 20000 15000' 10000. 5000- 0 J J 5 10 15 Cultivation time (h) -8 ,i 89 5 O o 3~ 1 (cid:12)9 Fig. 6 Relation~hlp between growth phase of E. coli and susceptibility of lysis by polymyxin (27 //g/ml) and lysozyme (25 #g/ml). 100 ~ 80 (cid:1)84 v ~g 80 (cid:12)9 ~ ~ $i,0. ~.~ =,5" oe 20- 0 20 40 60 80 100 EDTA (mg/ml) Fig. 7 Effect of EDTA concentration on cell surface hydrophobicity and lysis of E. coli. (Lysis conditions: lysozyme, 25 #g/ml) 800' 600 - 400 (cid:1)84 200 (cid:12)9 i ~ (cid:12)9 ' i 5 10 15 Cell concentration (absorbance 540 rim) Fig. 8 Effect of cell concentration on protein release from E. coli using EDTA/lysozyme. (Lysls conditions: EDTA, 800 #g/ml; lysozyme, 25/.tg/ml) 137
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`Discussion The correlation between increasing hydrophobicity of the ceils and increased EDTA concentration and lysis susceptibility suggests that removal of the lipopolysaccharide (LPS) component with exposure of the hydrophobic phospholipid layer renders the cells susceptible to lysozyme. Leive (1974) observed that outer membrane disruption could be enhanced by addition of magnesium to the growth medium by ErDA and our experiments show that this approach can be used to promote lysis for recovery of intracellular products. Stationary phase cells were most susceptible to lysis by EDTA/lysozyme whereas polymyxin/lysozyme had an optimal lytic effect on log-phase ceils, suggesting EDTA and polymyxin had different mechanisms in promoting lysis. The amphipathic nature of polymyxin allows the cationic end of this molecule to react with negatively charged constituents of the outer membrane while the aliphatie chain penetrates the hydrophobic domain of the membrane (Katsu et al., 1984). The varying responses of different E. coIi strains to lysis by EDTA/lysozyme may be due to differences in LPS composition. LPS deficient E. coli mutants manifested increased sensitivity to lysozyme (De Pacheco et aL, 1985). Increase in lysis rate with temperature is probably due to a combination of factors. Temperature optimum for lysozyme activity is 35~ (Godfrey and Reichelt, 1963). In addition, the outer membrane of gram-negative bacteria is disrupted at higher temperatures while the peptidoglycan layer remains intact (Hitchener and Egan, 1977). E. coli cells were lysis sensitive to added protease when treated at 45" C but not at 37*C (Dean and Ward, 1991). Treatment ofE. coli with EDTA and lysozyme for release of intracellular protein compared favourably with sonication. EDTA and polymyxin have potential as complementary lyric agents as each is effective at different stages of growth. Acknowledgement Support for this research by the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. Technical assistance from Josephine Lau is appreciated. References Andrews, B.A., and Asenjo, J.A. (1987). Trends Biotech. 5, 273-277. Dean, C.R., and Ward, O.P. (1991). Appl. Environ. Microbiol. 57, 1893-1898. Dc Pachcco, C.S., Good.son, M., Rossouw, F., and Rowbury, RJ. (1985). E.rperientia 41, 133-136. Fraser, D., and Jcrrd, E. (1953). L Biol. Chem. 205, 291. Godfrey, T., and Rcichclt, J. (1983). Industrial Enzymology, Now York, Nature Press. Hitchencr, BJ., and Egan, A.F. (1977). Can. Y. MicrobioL 23, 311-318. Ismacd, N., Furr, J.R., and Russell, A.D. (1986). I. Appl. Bactetiol. 61, 373-381. Katsu, T., Yoshimura, S., Tsuchlyeb T., and Fujita, Y. (1984). /. Biochem. 95, 1645-1653. Leive, L (1974). Ann. N.Y. Acad. Sci. 235 (6), 109-129. Mukhopadhyay, M., and Mandel, N.C. (1983). Anal. Biochem. 133, 265-270. Saha, B., Saha, D., Niyogl, S., and Bal, M. (1989). Analyt. Biochem. 176, 344-349. Suzuki, IC, Uyeda, M., and Shibata, M. (1985). Agile. Biol. Chem. 49, 1719-1726. Takahara, Y., Hirose, Y., Yasuda, N., Mitsugj, K. and Murao, S. (1976). Agdc. BioL Chem. 40, 1901-1904. White, M., and Marcus, D. (1988). In: Downstream Processin~ A. Mizrahi, ed. pp.51-96. New York: A.R. Liss. 138
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