Production of Bacillus cereus exopenicillinase on a pilot-plant scale

BIOTECHNOLOGY AND BIOENGINEERING VOL. VII, PAGES 517-528 (1965)

Production of Bacillus cereus Exopenicillinase on a Pilot-Plant Scale

G. MILLER,* G. BACH, and 2. AIARKUS, Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel

Summary A substrain of Bacillus cereus 569/II produced above 10,000 units of penicillinase/

ml. when grown in a pilot-plant fermentor using a medium containing Casamino acids techn. (Difco) or N-Z-Amine type B (Sheffield), and salts. Simplified purification and concentration procedures give an overall yield of 5&65% enzyme. The freeze-dried enzyme preparation had a good storage stability in vacuum- sealed ampules kept a t 4, 30, and 37°C. In vials tontaining air in the head space, partial inactivation occurred in two months a t 30 and 37°C. The freeze- dried preparation showed satisfactory performance in the production of yoghurt fermented milk.

INTRODUCTION

The enzyme peiiicillinase was discovered in 1940 by Abraham and Chain.' Kogut et a1.2 have isolated a mutant of Bacillus cereus (designated 596/H) which constitutively produces extracellular penicillinase. These authors have described experiments in a pilot-plant fermentor and have developed procedures for the purification of the enzyme. Citri et al.3 have used Celite for adsorbing the exopenicillinase and have described an iodometric assay for.this enzyme. Two patents deal with production of penicillinase by Actinomyces candidus4 arid by the inducible strain B. cereus NRRL B569.5

l'eiiicillinase is used in dairies for inactivation of penicillin in milk SO as to prevent inhibition of starter cultures,6 in sterility testing of penicillin preparations; in an automatic assay of penicillin,* and in other cases where inactivation of penicillin is desired.

* Permanent address: Israel Institute for Biological Research, Ness-Ziona, Israel.

517

515 MILLER, BACH, AND MARKUS

In view of these applications, it, was decided to investigate the production of penicillinase on a pilot-plant scale.

In this paper, we present a process leading to a freeze-dried penicillinase preparation suitable for laboratory and industrial applications.

MATERIALS, PROCEDURES, AND EQUIPMENT

Organism B. cereus 569/H was obtained from Dr. N. Citri, the Hebrew Uni-

versity-Hadassah Medical School, Jerusalem. A substrain of high productivity was selected by a method similar to that described by Kogut et a1.2 In our case, a single layer of S-agar containing Andrade indicator was used. Colonies producing a strong pink color within 5 min. after the addition of a concentrated sodium benzylpenicillin solution were isolated. A spore stock was prepared by inoculation of 80 ml. of S-broth2e3 and incubation for 40 hr. a t 35°C. on a rotary shaker (New Brunswick Scientific Co., New Brunswick, K. J., Model GlO) at 240 rpm. The culture was heated for 1 hr. at 60"C., and the spores were collected by centrifugation, washed with sterile distilled water, and finally suspended in 20 ml. of distilled water. This suspension was stored at 4°C. and used as a spore stock.

Seed CuItures Seed cultures for shake-flask experiments were grown as above, but

the S-broth was inoculated with 0.1 ml. of spore stock and shaken for only 6 hr. Seed cultures for the fermentor were grown in 2-1. Erlen- meyer flasks, having a side outlet, each flask containing 200 ml. of S- broth. After inoculation with 0.25 ml. of spore stock, the flasks were shaken for 6 hr. under the conditions described above. For inoculation of a 30-1. batch, 600 ml. of seed (2% v/v) was prepared by aseptic pooling of the content of three 2-1. Erlenmeyer flasks, using a sterile rubber hose connector.

Production Cultures Penicillinase production was carried out in shake flasks or in the

fermentor using two modifications of medium2 CH/C, as outlined in Table I.

Solutions B, C, and D (Table I) were added aseptically to solution A. Medium CH/C2 was prepared as CH/Cl, but the Casamino acids

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PRODUCTION OF PENICILLINASE 519

TABLE I Preparation of Production Medium CH/C1

Solution

A

B

C

D

For 1-1. culture

Ingredients

Casamino acids techn. (Difco)

Na-citrat.e. 5.5 H20 Antifoam

KHtP04

FeSOa. 7H20

Seed culture

Volume and g. sterilization

10.00 In 947 ml. of watep; 2.72 pH adjusted to 7.0; 5.88 Steam sterilizationb;

in shake flasks or in fermentor.

0.41 In 16.5 ml. of water.* Separate steam ster- i1ization.b

0.014 In 16.5 mi. of water.* Sterilized by Seitz filter.

20 ml. ___

a Deionized water. b Holding period: 20 min. a t 15 psig. c Explanation in text.

were replaced by N-Z-Amine, type B (Sheffield Chemical, Korwich, N. Y.).

The following antifoams were used in certain shake flask experiments and in all fermentor propagations: Antifoam Emulsion RD (Midland Silicones, U.K.), 300 ppm; or M-8 (Hodag Chemical Corp., Chicago, Ill.), 50 ppm.

Shake-flask experiments were performed in 2-1. Erlenmeyer flasks containing 400 ml. of culture on the above shaker for 24 hr. at 35°C. Pilot-plant propagations were carried out in a stainless steel fermentor (Palbam Ltd., Ein Harod, Israel) having a total capacity of 65 1. (Fig. 1). The fermentation process was carried out under aseptic conditions in batches of 30 or 40 1. Agitation was by the “fully baffled system” at 210 rpm, and 2 1. of air/min. were supplied through the sparger. The process was conducted at atmospheric pressure in the tank. The culture was maintained at 35°C. by circulation of water at this temperature through the fermentor jacket. Samples were withdrawn from a sterilizable bottom valve. Growth was continued for 13-15 hr., when penicillinase activity had reached its maximal level. The


I AIR

Fig. 1. Fermentor design. Dimensions in mm. Total capacity, 65 1. B, four baffles; I, turbine impeller with six vertical blades; S, sparger with 30 holes, 4, 1.5 mm.

culture was cooled to 5OC. by circulation of chilled water through the jacket, and stored a t this temperature.

Processing of Cultures

Bacteria were separated by a motor driven Sharples centrifuge (Model l A , centrifugal force 13,000 X g). The culture was fed to the centrifuge through a flowmeter (Manostat Corp., New York, Model FBI 1045 B) a t 7 l./hr., and the supernatant containing the enzyme was stored at 4°C. Its pH was adjusted to 4.5 by HC1, and pencillinase was adsorbed on Celite 545 (Johns-ManvilIe, New York) using 1 g. of Celite for 3 X lo5 units penicillinase. Preliminary experiments 011 adsorption were carried out with 1 1. of supernatant in

BIOTECHNOLOGY AND BIOENGINEERING, VOL. V I I , ISSUE 4


2-1. Erlenmeyer flasks on the above shaker for 1 hr. at 240 rpm. Pilot-plant batches of 15-40 1. were processed at 448°C. in a jacketed stainless-steel tank (I.D., 38 em.; depth, 50 cm.) having three baffles. Celite was added at the above ratio, and the suspension was agitated by a “Lightnin” portable mixer (Lightnin Mixers Ltd., U.K., iYIodel NDV-1A) with a marine impeller (O.D., 11 cm.) at 300 rpm for about 45 min.

The Celite was separated by a basket centrifuge (International Chemical Centrifuge, Model CH, centrifugal force 850 X g) fitted with filter paper. The Celite obtained from 15-20 1. of culture was transferred with 1-2 1. of deionized water into a glass column (I.D., 8 em. ; height, 50 em.) provided with a layer of glass ~vool at the bottom end. Elution of the enzyme was carried out by 0.1M phosphate buffer, pH 8.0, containing 1M KaC1 and 0.1M Na-citrate (henceforth: phosphate buffer eluant). 100-200 ml. portions of eluant were passed by gravity through the column, and each fraction was tested for pH and enzyme activity. Fractions containing above 15 X lo4 units of enzymelml. (henceforth : U/ml.) were pooled and dialyzed against 50 volumes of 0.1M phosphate buffer, pH 7.0, containing 0.l.U Sa-citrate, for 16 hr. at 4°C. with stirring. The enzyme was diluted to 13 X lo4 U/ml. in the same buffer. In some experiments, soluble egg albumen (Difco) was added at 0.06-0.60 mg./ml. The enzyme solution was sterilized by vacuum filtration through an HA membrane filter, diameter 142 mm. (Millipore Corp. Bedford, Mass.).

Freeze-Drying

The enzyme solution containing now 12 X lo4 U/ml. was dispensed aseptically by a continuous pipetting outfit (Becton, Dickinson, and Co., Rutherford, N. J.) in lot,s of 0.5 ml. into st.erile 6-ml. vials or ampules. The solution was “shell frozen” by rotation in a -25OC. bath. Freeze-drying was carried out in a laboratory chamber dryer (Ley- bold, Cologne, Germany, Model G07) for 6 hr. Vials were closed by sterile rubber stoppers and aluminum caps, leaving air in their head space. Ampules were sealed under vacuum.

Performance of Enzyme in Yoghurt Production

Lots of 10 1. of pasteurized cow’s milk in dairy cans were prewarmed in a water bath t.0 30°C. Sodium benzylpenicillin, 2 U/ml., and freeze-


dried penicillinase, 0.1 U/ml., were added as indicated (see Table VIII). Penicillin inactivation was carried out for 30 min. at 30°C. The temperature was then raised to 48°C. and 4% (v/v) of a yoghurt starter culture was added. Lactic fermentation was allowed to pro- ceed for 4 hr. at 48°C. Samples were taken and examined for ap- pearance, taste, pH, and total acidity.

Analytical Methods

Penicillinuse activity (a-type) was determined by the “timed iodometric assay” of (2it1-i.~ The unit of penicillinase activity was that amount which hydrolyzes one pmole of benzylpenicillin in 1 hr. at 30°C. and at pH 7.0.1°

Turbidity of cultures was measured in the Klett colorirneter using a 660 mp filter.

Oxygen absorption rates (OAR) in shake flasks and in the fermentor were determined by the sulfite method as described by Corman et al.11 Results are expressed in mrnole of oxygen absorbed/l./min.

Total protein was determined by the method of Lowry et a1.,I2 using bovine serum albumin as the standard.

Storage stability of freeze-dried enzyme preparations was determined by keepingvials and ampules at 4,30, and 37°C. for two months. The preparations were then dissolved in water and assayed for penicillinase activity.

Total acidity of yoghurt was determined by titration of 10 ml. of fermented milk with 0.1N NaOH using phenolphthalein as the indicator. Results are expressed as per cent lactic acid.

RESULTS AND DISCUSSION

Fermentation Process

In the past, we studied the production of this enzyme in a simple ~hem0stat . l~ Under optimal conditions for enzyme formation, 3000 U/ml. were obtained, as compared with 10,000 U/ml. by the batch method. Therefore, experiments reported in this paper were carried out by the batch method.

In shake flask cultures (OAR = O . l ) , in absence of antifoam, more than 10,000 U/nil. were obtained (Table 11). Addition of 50 ppm of M-8 depressed enzyme yields by 8-28%. This decline may be explained by a decrease in OAR caused by the antifoam.14 Similar

BIOTECHNOLOGY AND BIOENGINEERING, VOI,. VII, ISSUE 4

PRODUCTION OF PENICILLIPJASE 523

14 - o----O PENICILLINASE

m - TURBIDITY 2

TABLE I1 Penicillinase Production in Shake Flask Cultures*

Penicillinase, Medium M-8, PPm d m l . Li;,

CH/C1 CH/CI CH/C2 CH/C2

- 10,750 100 50 9,920 92 - 10,320 100 50 7,470 72


less expensive medium, CH/C2, is preferred. It is seen (Fig. 2) that growth and enzyme formation proceeded in parallel, while the pH rose gradually. The initial doseof 50 ppm of M-8 depressed foaming during the whole fermentation process. Kogut et a1.,* on the other hand, did not succeed in breaking the foam, and enzyme inactivation occurred. These authors obtained 4700 U/ml. in shake flasks, and lower yields in the fermentor, while other report^^^^ do not indicate penicillinase yields.

In our initial experiments with the original B. cereus 569/H culture, 30004000 U/ml. were found. The higher yields achieved later in this investigation should be attributed to the selection of the substrain, and to the favorable aeration and agitation conditions in the fermentor. The spore stock maintained its high productivity for 3-4 months. When a decline of productivity mas observed, the selection procedure was repeated and a fresh stock prepared.

Processing of Cultures In preliminary adsorption experiments in shake flasks, 90% of

enzyme was taken up by the Celite in 1 hr. Pilot-plant batches were then adsorbed at 300 rpm for 45 min. with 95% efficiency (Table 111).

Elution by an aqueous solution containing 1M KaCl and 0.1M sodium citrate, and adjusted3 to pH 8.5, was not satisfactory, presum- ably because of the lowering of the pH of the eluant below 7. Effec- tive elution was achieved with the above salts dissolved in 0.1M buffers a t pH 8. Phosphate, borate, and Tris buffers were suitable.

TABLE 111 Absorption of Penicillinase on Celite 545

Penicillinase

Time, I n supernatant, Adsorbed, Adsorbed, min. U/ml.b U/ml: 92

0 11,230 0 0 15 1,600 9,630 86 30 1,120 10,110 90 45 600 10,630 95 50 600 10,630 95

a 30 1. supernatant processed a t 300 rpm. b After separation of Celite in sample by centrifugation for 5 min.

By difference. BIOTECHNOLOGY AND BIOENGINEERING, VOL. VII, ISSUE 4


TABLE IV Elution of Penicillinase with Phosphate Buffer Eluant”

Eluted enzyme,

Eluate fraction Penicillinase

NO. ml. pH U/ml. X 103 Units X 106 9% - - - 1 200 4.7

2 200 4.7 0 .2 0.04 - 3 190 4.7 0.6 0.1 0 1

4 170 7.1 168.0 28.6 23.6 5 225 7.6 268.0 60.3 49.6 6 120 7.9 168.0 20.2 16.6

7 90 8.0 84.0 7.6 6 .3 8 95 8.0 48.0 4.6 3 8

Tot,al eluted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,121.4 100 0

a 15 1. of supernatant containing 10,500 U/ml. were adsorbed on Celite and the adsorbate contained 144 X lo6 units of enzyme.

TABLE IT Purification of Penicillinase by Adsorption and Elution

Penicillinase Total

Volume, protein, Unitslpg. Sample nil. pg./ml. U/ml. protein C/Cb

Culture supernatant 70008. 708 10,800 15 5

Eluate fraction “B” 95 2580 297,520 115 37 Eluate fraction “A” 100 2780 270,720 97 31

7 1. of culture supernatant were adsorbed on Celite and eluted with the phos-

The enzyme protein values were calcu- phate buffer eluant.

lated using the factor 312 U/pg. of crystalline enzyme pro tehz b Penicillinase, as yo of total protein.

Table IV shows typical results with the phosphate buffer eluant. As soon as the adsorbate was neutralized (fraction 4), penicillinase was released. Thus, the pH of the eluate may serve as an indicator for the beginning of elution. In this experiment, the pool of fractions 4, 5, and 6 contained 76% of the enzyme in the adsorbate, and 90% of the eluted enzyme. This pool contained 21 X lo4 U/ml., as compared with 1.1 X lo4 U/ml. in the culture supernatant. Thus, by the de-


scribed simple adsorption-elution procedure, a concentrated enzyme solution was obtained.

In Table V, the results of another experiment are given, in which the degree of enzyme purification was studied. A high yield of enzyme, with a 25-28-fold concentration and 6-%fold purification, has been achieved.

Enzyme losses and yields during the different production steps are summarized in Table VI. The overall yield for freeze-dried enzyme was in the range 50-65% of the enzyme in the culture fluid. One may expect that, in routine production, these yields could be further improved.

TABLE VI Enzyme Losses and Yields a t the Main Production Steps

Production step Losses, To Yield, yoa Separation of bacteria Adsorption Elutionb Dialysis Sterile filtration Freezing and drying

Overall yield

Nil 5-10

15-25 Nil 10 lC-15

100 9C-95 75-85

100 90 85-90

5C-65

a yo of the preceding step. b Pool of eluate fractions containing above 15 X lo4 U/ml.

Storage Stability of Freeze-Dried Penicillinase

Although freeze-drying is an accepted procedure for handling enzymes, surprisingly, no publications giving details on the subject were found. Thus, several factors affecting the storage stability of penicillinase were investigated. After a short drying period of 2 hr., some humidity remained in the vials; the preparation was com- pletely inactivated after one week's storage at 37°C. After drying for 6 hr., no humidity was visually found in the vials, and this drying period was adopted. The effect of dialysis, and of the presence of air on the stability of the dry enzyme, is shown in Table VII. In the presence of air (vials), dialysis improved the storage stability. But at 30 and 37"C., partial inactivation took place. Excellent storage stability of the dialyzed preparation was achieved in vacuum-sealed

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VII, ISSUE 4


ampules, even at 37°C. These resu1t.s indicate that, as in the case of freeze-dried bacteria,15 the purified enzyme undergoes an oxidative denaturation. In the case of vials, the addition of reducing agents as enzyme stabilizers, or the closing of the vials in an inert gas, should be investigated. In view of these findings, dried penicillinase in vials prepared by the described procedure should be stored under refrigera- tion. Addition of soluble egg albumen did not improve the storage stability i, presence of air.

TABLE VII Storage Stability of Freeze-Dried Penicillinaseb

~~

yo Inactivation at storage Treatment of eluate Head apace 4°C. 30°C. 37°C.

Nondialyzed Air 0 13 20

Dialyzed Vacuum 0 0 0 Dialyzed Air 0 6 15

a Each result represents an average of three vials or ampules. Storage for two months.

Performance of Penicillinase in Yoghurt Production

It was supposed that dairies represent the major users of penicillinase. Lactic bacteria, used as dairy starters, are sensitive to penicillin concentrations of the order of 0.1 U/m1.I6 Therefore, the dried penicillinase was tested in yoghurt production. It may be seen from

TABLE VIII Performance of Freeze-Dried Penicillinase in Yoghurt Production

Penicillinase Product

Prepara- Peni- Lactic Experi- tion cillin, acid, merit type U/ml.c U/nil: % pH Coagulation

- 1.26 4 . 5 Normal 1 2 0.50 5 .4 None 2

4.5 Normal 3 0.1 2 1.20 4 0.1 2 1.30 4 . 5 Normal

- -

- -

e

b

a Penicillinase prepared by the described procedure. b A commercial penicillinaae preparation for comparison.

Units per ml. of milk.

528 MILLER, BACH, A N 0 MARKUS

Table VIII that normal acidification and coagulation took place either when no penicillin was added or when added penicillin was inactivated by penicillinase. When the ratio of penicillin/penicillinase equals 20, the freeze-dried penicillinase, prepared by the process described, showed the same performance as a commercial preparation.

The authors wish to express their thanks to Mr. J. M. Sudarsky, Wasco, Calif., for a grant which in part supported this investigation.

References 1. Abraham, E. P., and E. Chain, Nature, 146, 837 (1940). 2. Kogut, M., M. R. Pollock, and E. Y. Tridgell, Biochem. J., 62, S9l (1956). 3. Citri, N., N. Garber, and M. Sela, J . Biol. Chem., 235,3454 (1960). 4. Welch, H., U. S. Patent. 2,601,350 (Julie 24, 1952). 5. Riker Laboratories, Inc., Brit.ish Patent 887,408 (Janiiary 17, 1962). 6. Sizer, I. W., Advan. A p p l . Microbiol., 6,207 (1964). 7. Chain, E., H. W. Florey, N. G. Heatley, and 31. A. Jeririitigs, Ilntibiotics,

8. Goodall, R. It., and 13. Roseda, Analyst, 86,326 (1961). 9. Citri, K., Methods Med. Res., 10, 221, (1964).

Vol. 11, Oxford University Press, London, 1949, Chapter on “Penicillinase.”

10. Poliock, M. R., and A. M. Torriaiii, Compt. IZend. Acad. Sci. (Paris), 237, 276 (1953).

11. Corman, J., H. M. Tsuchiya, H. Y. Koepsell, R. G. Beriedict, 8. E. Kelley, V. H. Feger, R. G. Dworschack, and R. W. Jackson, A p p l . Microbiol., 5, 313 (1957).

12. Lowry, 0. H., -1;. J. Roselibrough, A. L. Farr, and It. J. I<andall, J . Uiol. Chem., 193, 265 (1951).

13. Sheinkin, R., and G. Xiller, Israel J . Med. Sci., 1,317 (1965). 14. Rose, A. H., Industrid Microbiology, Butterworths, London, 1961, p. 89. 15. Lion, M. B., and E. 11. Bergmann, J . Gm. Microbiol., 24, 191 (1961). 16. Whitehead, H. R., and D. J. Lane, J . Dairy Res., 23,355 (1956).

Received July 20, 1965

BIOTECHNOLOGY AND BIOENGINEERING. VOL. VII. ISSUE 4

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Production of Bacillus cereus exopenicillinase on a pilot-plant scale