Preparation of a highly active form of nisin from Streptococcus lactis

F. J. BAILEY AND A. HURST Unilever Research Laboratory, Sharnbrook, Bedford, E r ~ ~ l a ~ d

and Research Laboratories of'the Food and Drug Directorate, Depat.tment of Nationcrl Health arzd We f i r e ,

Ottawa, Canada

Received July 15, 1970

BAILEY, F. J., and A. H u ~ s r . 1971. Preparation of a highly active form of nisinfro~n Streptococcus lactis. Can. J. Microbiol. 17: 61-67.

Cells of Streptococcrrs lactis (354107) synthesized and retained nisin when grown in a complex medium with 2.57, glucose at a constant pH of 6.7. Nisin was extracted from cells by a previously used method with hot 0.05 N HCl but milder methods of extraction from whole and broken cells using a variety of solvents were also tested. In the preferred method broken cells were extracted with 0.05 N HCI at 2°C. The C1- ions of the extract were exchanged for acetate on columns of the resin Amberlite CG 4B and the eluate was concentrated by acetone precipitation at -lg°C. The nisin was finally purified by pH gradient elution from CM cellulose columns. Three peaks with antibiotic activity were found, two of the peaks were minor and represented less than 57, of the nisin. The main peak gave a single band on electrophoresis. Electrophoresis of the material from the CM cellulose peaks revealed about 44 bands of basic proteins. Nisin made by the hot or cold HCI extraction behaved similarly in electrophoresis and CM cellulose chromatography but the antibiotic activity of the material isolated from the cold extract was nine times greater than that of the material isolated from the hot extract.

The antibiotic nisin is produced by certain strains of Streptococcus lactis (13). Although the antibiotic contains 'non-protein' amino acids in- cluding lanthionine, methyllanthionine (I), and dehydroalanine (3), nevertheless the biosynthetic pathway appears to be similar to that of normal protein synthesis (5, 8). The molecular weight of nisin is somewhat controversial; Cheeseman and Berridge (2) reported it to be 7000 and this was confirmed by Ingram et al. (9). However, a mo- lecular weight of 3500 has also been reported (3, 4); Jarvis et al. (10) found both 3500 and 7000 mol. wt. species to be present. The species having a lower molecular weight was present only in samples which had been boiled in 1 M HCl for 10 min.

Although nisin has been regarded as an acid- stable substance (14) and the antibiotic is being used as a preservative in the canned food industry (1 I), the present work suggests that the physiolog- ically active form of nisin may be unstable in acid solutions. Earlier reports describe its ex- traction with hot dilute HCl, and in this paper, we describe a milder purification scheme which starts with the producer cells and finishes with a product which has 9 times the antibiotic activity of the material prepared by the hot dilute HCI method.

Methods Nisin-prod~rcing Organisni, Media, and Culture Con-

ditions Streptococcus lactis 354107 (NCDO 497) was used for

nisin production (5). It was subcultured daily and grown in LTB medium at 30°C; the medium contained (% wlv) meat extract (LabLemco) 1 ; yeast extract (Difco) 1 ; tryptone (Difco) 1 ; glucose 1; NaCl 0.5; Na2HP04 0.2; the final pH was 6.8. The organism was stored on slopes prepared by adding 1.5% (w/v) agar to theabove medium.

Fermentatioris The organism was freshly started from slopes 3 days

before each fermentation and subcultured daily. The medium used was the same LTB medium but the glucose concentration was increased to 2.5% w/v. Cultures were grown in 20-1 batches in a glass bottle and inoculated with 1-1 of overnight culture. The bottles were in a 30°C water bath stirred by a magnetic stirrer through the bottom of the water bath; this avoided aeration and the surface of the medium was under a stream of 02-free N2. The pH of the fermentation was automatically controlled to 6.7 by addition of 5 N NaOH. Stationary phase was reached in 10-11 h and the nisin formed was retained by the cells (7). The cells were recovered in a Sharples super- centrifuge and the medium was discarded. The usual yield was 6 g dry weight cells per liter of medium. The cells contained up to 5% of their dry weight as nisin as measured by bioassay.

Estimations Assays-Nisin was estimated as previously using the

same standard and the same turbidimetric method of bio- assay (5). Nisin was also estimated spectrophotometri-

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62 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

cally at 210mp (16). Protein solutions at 2 pg/ml have an absorbance of about 0.04 when the light path is 1 cm. Absorption at 210 mp was used instead of the more usual 280 mp because nisin contains no aromatic amino acids and has no absorbance at the latter wavelength; the ultraviolet absorption of nisin is shown in Fig. 1. Ab- sorption at 210mp is, at least in part, due to peptide bonds and is therefore very sensitive. However, many solvents also absorb at this wavelength and for this reason our preferred solvent was dilute HCI.

Total proteitt-This was determined by the biuret method with bovine serum albumin as standard (12).

Electrophoresis-Electrophoresis in polyacrylamide gels was done as described previously (6).

Extractiorz of Nisi12 fro111 Cells Hot HCI extracts-These were prepared by mixing 1 g

of wet weight cells with 10 ml 0.05 N HCI with a M.S.E. top drive macerator and treating the suspension in a boiling water-bath for 5 min. After the suspension was centrifuged at 5000gfor 10 min the supernatant was used.

Cold HCI extracts-These were made by overnight extraction of broken cells with 0.05 N HCI at 2'C.

Cell breakage-Preliminary experiments were done to find the best way of cell breakage. The French Press (American Instrument Co., Silver Springs, U.S.A.), the Biox Press (Nacka, Sweden), and a Braun disintegrator (Canlab) were used. The last was the most convenient for large-scale work, and the following was the preferred procediire: 10 g wet weight cells and 10 g ballotini beads were dispersed in 60 m14 m M phosphate buffer, pH 7.2, in a M.S.E. top drive macerator for 2 min at O°C. After the foam collapsed, this suspension was treated in a Braun disintegrator at full speed for three 30-s periods with brief cooling between the treatments.

The precipitate was recovered by centrifuging at 10000 g for lOmin at 2'C, and extracted overnight at 2°C with lOOml of 0.05 N HC:. This supernatant was used for further purification.

WAVELENGTH MILLIMICRONS

FIG. 1. The ultraviolet absorption of nisin. Solid line is 10-1 Mnisin. Broken line is solvent control (0.05 N HCI).

Preparation of Colritrlns Anion exchange resin CB-4B (Amberlite resin, B.D.H.)

was used for exchanging C1- ions for acetate ions. The amount of resin required depended on the volume of extract: 30-50 g resin was sufficient to raise the p H of 1 liter of 0.05 N HCI extract (pH 1.8) to about 4.0

CM-cellirlose colirn~r~ chrotnatograp/zy-This technique was based on that of Philips and Johns (15), which they developed for isolation of nucleohistones. We used a continuous pH gradient with dilute HCI, which enabled us to monitor the eRluent at 210 mp.

DIRECTION OF ELECTROPHORESIS + ANODE CATHODE

1 MARKER

FIG. 2. Electrophoretogram of cold and hot extracts of cells. Leading bands are nisin. S. Iactis was grown to stationary phase at 30°C at a constant pH of 6.7. One gram wet weight of cells was either extracted with 10 ml 0.05 N HCI in a boiling water bath for 5 min (hot extract) or suspended in 4 mM, pH 7.0, phosphate buffer and broken with Ballotini beads in a Braun cell disintegrator; the pellet was extracted overnight with 10 ml of 0.05 N HCI at 2°C (cold extract). Polyacrylamide gel electro- phoresis was performed as previously described (6).

' O r PROTEIN

, 2 4 6 NO. OF PRESSINGS

FIG. 3. Solubilization of nisin from cell structures by repeated cell breakage. Cells were grown and suspended in phosphate buffer as in Fig. 2. They were broken in a Biox press as described in the text.

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BAILEY AND HURST: PRODUCTION OF NISIN FROM STREPTOCOCCUS LACTIS 63

Whatman CM-cellulose microgranular (150g) in its Acid Solvents for the Extraction of Nisin from Na+ form was treated with 500 ml of 0.1 N NaOH. It was Broken Cells filtered and washed in water before it was suspended in 500 m10.5 N HCI and then washed with water to remove One gram of cells in 10 m10.05 N HCI, broken excess acid. The resin was suspended in 0.001 N HC1 in the Braun disintegrator and left gently shaking and packed into a glasscolumn (2.5 X 45 cm). Ascending overnight at 2"C, yielded only about 5y0 of the and descending chromatography were equally effective. n i~ in of the original cells. When the cells were The volume of sample loaded on the column could vary first suspended in 4 m~ phosphate buffer, p~ from 1 to 100 ml but the pH had to be close to 3 for a successf~~I run. 7.0, broken, and then extracted in the cold with

A near linear gradient of increasing HC1 concentration 0.05 N HCI, 90% of the nisin was found in the was used. Generally 1500 ml of the gradient HCI was supernatant by bioassay, and there was no loss of enough to elute nisin and basic proteins. At the end of the bioassayable activity. It was apparently necessary run loo m1 of 0.5 HC1 was any re- to carry out cell breakage at neutral pH and the maining material and at the same time regenerate the column. The column was then equilibrated with p~ 2.45 subsequent cold extraction in 0.05 N HC1 re- H C ~ . covered most of the nisin; cold 5y0 acetic acid or

Isolation of itisin affer CM-celliilose chro~natograplgv- 0.05 N H2S04 could not replace cold HCI. Acetic The nisin-containing fractions were pooled and adjusted acid extracted only about 3y0 the total nisin, to about pH 5.0 with solid sodium acetate. Nisin was then precipitated with volumes of acetone at -190C. but the rest could be recovered with hot 0.05 N This precipitate was centrifuged and washed with cold HCI. Extraction with H2SO4 appeared to destroy acetone to yield a white powder. the biological activity of nisin irreversibly.

Occasionally, the pooled nisin solution was freeze-dried An electrophoretogram of the cold HCI ex- without pH adjustment. Preparation No. 2 in Table 4 tract is shown in Fig. 2. For comparison an was made in this way, the others by acetone precipitation.

Precipitation of proteins-In the steps between the electro~horetogram of the hot HCI extract of the Amberlite 4B and CM-cellulose chromatography, pro- cells is also shown; there appears to be more con- teins were precipitated at -19°C with 7 volumes of taminating material in the hot than the cold HC1 acetone. extracts but the two nisin bands migrated the

Results same distance from the origin.

Acid Solvents for the Extraction of Nisin from Eflect of Degree of Disruption of Cells on Solu- W/~ole Cells bilization of Nisin

Cells (1 g) were extracted at 2OC with 10 ml of The Biox press was convenient for this experi- 0.05 N HCI, 5y0 and 10yo acetic acid, 0.5 N per- ment because the same frozen cell suspension chloric acid, and acid methanol, acid ethanol, could be passed repeatedly through the press. A and acid propanol (alcohol 4:l of 0.25 N HCI lOy0 w/v cell suspension in 4 m M phosphate v/v). Acetic acid gave variable results but the buffer, pH 7.0, was divided into six equal por- other solvents extracted less than 10% of the tions. These portions were used for up to six nisin compared to hot HCI extraction, which was passages through the press at -25°C. The frozen, taken to be 100yo. broken blocks of cells were thawed at 2"C, ex-

TABLE 1 Comparison of four different methods of cell breakage for the extraction of nisin*

- --

Protein, Nisin, Total Ratio, Treatment mg/ml ug /n~ l nis~n ppt./supern.

One passage in? Supern. 1.12 75 675 8 .O Biox press Ppt. 600

Three passages in? Supern. 2.00 151 621 3.1 Biox press Ppt. 470

One vassarret Silvern. 1 .80 160 630 3 .O ~ r e n c h press ppt. 470

Braun disintegrators Supern. 1.80 71 791 10.1 Ppt. 720

* 1 0 4 ~ w / v cell suspension in pH 7.0, 4 m M phosphate buffer was divided into four equal portions and treated as described In the other footnotes.

tDisruprion at -25'C. $Disruption at 2°C at 10 tons pressure. §The machine was cooled to about P C with liquid COz. The cup contained equal weights of cclls and Ballotini

beads.

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64 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

tracted overnight at 2OC with an equal volume of with equal volumes of cold 0.05 N HCI. The 0.05 N HCI, and the protein and nisin contents of Braun disintegrator gave the most favorable the supernatant and pellet were estimated. results: this treatment solubilized about as much

Figure 3 shows that with increasing number of protein as the others, but 91y0 of the nisin was pressings the total nisin content did not change retained by the cells (precipitate). The recovery of but that the protein and nisin contents of the total nisin was also slightly higher by this supernatants increased with the number of press- method. Phase contrast and electron microscopic ings.

Comparison of Different Mechanical Methods of Cell Breakage

A 10y0 w/v cell suspension in 4 mM, pH 7.0, phosphate buffer was divided into four equal por- tions. These were treated as shown in Table 1. The precipitates were extracted overnight at 2OC

A N O D E ORIGIN C A T H O D E

TABLE 2 Chromatography of cold HCI cell extract

on Amberlite resin CG-4B* - --

Fraction pH of Nisin, mg per vol., ml effluent fraction

Total = 42.5

NOTE: The cells were extracted and column prepared as described in methods; 50 ml o f cell extract, pH 1.8, was applied to the column 5 X 5 cm and eluted with 5y0 v / v acetic acid. Total nisin applied was 40.75 mg. recovery o f 104%.

FIG. 5. Electrophoretic pattern of the material isolated from the peaks in Fig. 4. The fractions were concentrated by rotary evaporation and subjected to polyacrylamide gel electrophoresis as previously described (6).

TABLE 3

2.5

0.7

~ 0 . 6

0 N

0.5-

J - ., Percentage recovery of nisin by bioassay*

Experiment No.

o 0.4 - e

. 0 0.2- 0 J

0.1 -

50 100 150 200 TUBE NO.

O/o recovery

-

- -

CG-4B eluate 85 117 97 102 Redissolved acetone

precipitate 62 78 77 104 Nisin peak from

CM-cellulose 48 83 70 85

'Percentage recovery calculated on the original HCl extract o f cells, which was assumed to be 100%.

- '..,

FIG. 4. CM-cellulose pH gradient chromatography of acetone-precipitated material from cold-extracted cells. Cells were extracted and treated as described in Methods. pH gradient, broken line. Absorption at 210 mp, solid line.

7 -

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BAILEY AND HURST: PRODUCTION OF NlSlN FROM STREPTOCOCCUS LACTlS 65

examination showed that these cells retained their shape perfectly. A small portion of the cell wall was knocked off but more than goy0 of the cells were damaged in this way.

Extraction of Nisin with NaCl To avoid acid solvents uns~~ccessful attempts

were made to extract nisin from broken cells with NaCl (up to 5 M). The nisin and protein contents of the extracts were similar; 0.75 M NaCl ex- tracted most material which was up to 15y0 of that available.

Ion Exchange Chromatography on Amberlite Resin CG-4B of Cold HCI Cell Extracts

Table 2 shows complete recovery of nisin from the resin (104y0). In separate experiments, re- coveries lay between 85 and 117y0.

CM-Cellulose Chromatography of the Acetone Precipitates

A typical experiment is shown in Fig. 4. The nisin peaks a, b, and c were identified by bio-

assay, the other peaks contained no antibiotic activity, although they were basic proteins.

Most of the fractions shown in Fig. 4 were isolated and concentrated by rotary evaporation and further characterized by electrophoresis (Fig. 5). Fractions a, c, and e were omitted be- cause of insufficient material from these peaks. The main nisin peak (b) moved as a single com- ponent; most of the other peaks, however, con- tained a number of bands. Altogether there are about 44 bands in Fig. 5; using our previously developed considerations of relating molecular weight to migration from origin (9), the molecu- lar weight of the proteins in these bands ranged from 7000 to 55 000. In similar separations on CM-cellulose, nisin was eluted at pH values be- tween 2.12 and 2.05 so that monitoring of the effluent pH could not be used as a method of identifying the nisin peak. The proteins them- selves tended to neutralize the HCI in the eluate, causing unexpected pH fluctuations. The frac- tionation pattern, however, was so distinctive and

TABLE 4

Recovery of nisin from its peak in CM-cellulose chromatography: comparison of optical and bioassay methods of estimation. Starting material:

cold extract of cells*

Prep. No.

1 2 3 4 5

Proteins (mg) in nisin peak by absorption at 210 mp 5.5 12.4 21.3 18.4

Dry weight (mg) found 3 35 5 17 12 Dry weight as a yo of the peak 55 40 80 65 Nisin (mg) in nisin peak by

bioassay 55 489 110 160 138 Nisin as a Yo of the protein by

bioassay 1000 Ca. 1400 890 750 750

'See methods for preparation of extract.

TABLE 5

Recovery of nisin from its peak in CM-cellulose chron~atography: comparison of optical and bioassay methods of estimation. Starting material:

hot extract of cells*

Prep. No.

Proteins (mg) in nisin peak by absorption at 210 mp 0.8 20.7 4.8 6.4 4.2

Nisin (mg) in nisin peak by bioassay 0.7 14.9 4.5 4.5 4.5

Nisin as a % of the proteins by bioassay 88 72 93 70 107

*See methods for preparation of extract.

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66 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 17, 1971

reproducible that we had no difficulty in identify- Our standard was the same as that used before ing the nisin peak, and bioassay became unneces- (5), but we rechecked its purity. First, we found sary. that the plot of O.D. against concentration was

linear up to 50 pglml. Because absorption and S'mma'~ of the Pr@redProced'ref~' P'rifca- protein content were directly related, our stan-

tion of Nisin dard nisin did not contain salts or other sub- Growth of overnight in LTB medium stances which do not absorb at 210 mp. Secondly,

with 2.5% Iv glucose with pH at 6.7 gel electrophoresis showed that the standard I nisin contained only a single band so that the Recovery of cells by centrifuging

I standard was at least 90y0 pure nisin. Conse- J. quently the mean specific activity of our prepara-

(2) extraction of broken with tions was higher than that of the standard, pas- HCl, extraction at pH about 1.8

I sibly because of the mild method of extraction J.

Amberlite resin CG 4B I

(3) Anions exchanged for acetate and pH raised to about 4.0

I Concentration by precipitation with

7 vol. acetone at - 19O I

pH gradient chromatography on CM-cellulose I

(4) Recovery of nisin from its characteristic peak in an electrophoretically pure form

Recoveries of Nisin Extracted with Cold HCI A summary of the yields of nisin as measured

by bioassay is shown in Table 3. The cold HCl extract was assumed to have 100yo activity and the average recovery of nisin after the final purifi- cation was 71 yo.

However, the amount of material which should have been recovered by weighing did not agree with the amount of material anticipated by bio- assay. Only about 5y0 of the weight anticipated from bioassay was actually recovered although, as shown in Table 3, each of the major steps of purification had been found to be effective. Using the spectropl~otometric method of Tombs et al. (16), we calculated the protein content of the nisin peaks of the four CM-cellulose runs shown in Table 3, and these data, as well as an addi- tional run, are shown in Table 4. By ultraviolet absorption, we obtained reasonable agreement with the weighing data; the weight of recovered material was 4040% of the protein of the nisin peak. By bioassay, however, there was more nisin than total protein in the peak, suggesting that the material we were isolating had considerably higher bioassay activity than the standard.

from the cells.

Recoveries of Nisin Extracted with Hot HCI The effect of the method of extraction on the

apparent recovery of nisin was tested by carrying out our preferred method of purification using hot HCI extracts of cells as our starting material. Table 5 shows that the total protein of the nisin peak in CM-cellulose chromatography (estimat- ed by ultraviolet absorption) agreed closely with the bioassay estimations. This agrees with the finding in Fig. 5 that this peak is almost pure nisin.

Discussion

Using mild methods of extraction and purifica- tion, we obtained a nisin preparation which had nine times the bioassay activity of nisin extracted from cells with hot dilute HCl (Tables 4 and 5). Based on bioassay, nisin of cold HCl extracts represents up to 5y0 of the dry weight of the cell. If it is assumed that hot HCI extraction removes 100% of the nisin, cold HCI extraction removes 90%. Only another 10% can be recovered by hot HCl extraction from the residual pellet. However, since the material of the cold extraction has nine times the specific activity of the hot extract, the cold extraction contains only 10yo of the weight of nisin. Clearly, because the biological activity of nisin depends on the method of extraction, the bioassay method does not lend itself to calcula- tion of the weight contribution of nisin to the cell.

Despite this difference in specific activity the hot and cold extracted nisins had similar electro- phoretic mobilities (Fig. 2); the hot HCl yielded patterns in the pH gradient chromatography on CM cellulose identical with that shown in Fig. 4. It seems that a very small change in the charge

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BAILEY AND HURST: PRODUCTION OF NISIN FROM STREPTOCOCCUS LACTIS 67

and molecular weight, not detectable by the techniques used, is sufficient to cause a very large change in the biological activity.

It is not known what this biological activity depends on; Gross and Morel1 (3) suggested that activity depended on the dehydroalanine con- tent. This is an unstable substance and it may well be partially destroyed during hot HC1 ex- traction. There is also, now, little doubt that nisin is retained on cell structures (17). The bind- ing of nisin to cellular matter is shown by the in- creasing solubility of nisin with increasing de- struction of cell structure (Fig. 3).

1. BERRIDGE, N. J., G. G. F. NEWTON, and E. P. ABRAHAM. 1952. Purification and nature of the anti- biotic nisin. Biochem. J. 52: 529-535.

2. CHEESEMAN, G. C., and N. J. BERRIDGE. 1959. Ob- servations on the molecular weight and chemical composition of nisin A. Biochem. J. 71: 185-195.

3. GROSS, E., and J. L. MORELL. 1967. The presence of dehydroalanine in the antibiotic nisin and its re- lationship to activity. J. Amer. Chem. Soc. 89: 2791- 2792.

4. GROSS, E., J. L. MORELL, and L. C. CRAIG. 1969. Dehydroalanyllysine: Identical COOH-terminal structures in the peptide antibiotics nisin and sub- tilin. Proc. Nat. Acad. Sci. 62: 952-956.

5. HURST, A. 1966. Biosynthesis of the antibiotic nisin by whole Sfrepfococclrs lactis organisms. J. Gen. Microbiol. 44: 209-220.

6. HURST, A. 1966. Biosynthesis of the antibiotic nisin and other basic peptides by Streptococcrrs lacfis

grown in batch culture. J. Gen. Microbiol. 45: 503- 513.

7. HURST, A., and G. J. DRING. 1968. The relation of the length of the lag-phase of growth to the synthesis of nisin and other basic proteins by Streptococcus lacris grown under different cultural conditions. J. Gen. Microbiol. 50: 383-390.

8. INGRAM, L. C. 1969. Synthesis of the antibiotic nisin: Formation of lanthionine and a-methyl- lanthionine. Biochem. Biophys. Acta, 184: 216-219.

9. INGRAM, L. C., M. P. TOMBS, and A. HURST. 1967. Mobility - molecular weight relationships of small proteins and peptides in acrylamide-gel electrophore- sis. Anal. Biochem. 20: 24-29.

10. JARVIS, B., J. JEFFCOAT, and G. C. CHEESEMAN. 1968. Molecular weight distribution of nisin. Biochim. Biophys. Acta, 168: 153-155.

11. JARVIS, B., and M. D. M O R ~ S E ~ I . 1969. The use of antibiotics in food preservation. Int. Biodeterioration Bull. 5: 39-61.

12. LAYNE, E. 1957. Spectrophotometric and turbidi- metric methods for measuring proteins. Methods Enzymol. 3: 450451.

13. M A ~ C K , A. T. R., and A. HIRSCH. 1944. A powerful inhibitory substance produced by group N strepto- cocci. Nature, 154: 551.

14. M A ~ C K , A. T. R., and A. HIRSCH. 1947. Further observations on an inhibitory substance (nisin) from lactic streptococci. Lancet, 1947 (2): 5-8.

15. PHILLIPS, D. M. P., and E. W. JOHNS. 1965. A fractionation of the histones of group F2a from calf thymus. Biochem. J. 94: 127-130.

16. TOMBS, M. P., F. SOUTER, and N. F. MACLAGAN. 1959. The spectrophotometric determination of pro- tein at 210 mp. Biochem. J. 73: 167-171.

17. WHITE, R. J., and A. HURST. 1968. The location of nisin in the producer organism, Sfrepfococc~rs lacfis. J. Gen. Microbiol. 53: 171-179.

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