Extracellular production of Bacillus penicillinase by Escherichia coli carrying pEAP2

Journalof Ir~lJl~,#ll~i~l

Eur J Appl Microbiol Biotechnol (1983) 18:339-343

Microbiology and

Biotechnology

9 Springer-Verlag 1983

Extracellular Production of Bacillus Penicillinase by Escherichia coli Carrying pEAP2 Chiaki K a t o , Toshiaki K u d o , K a z u h o W a t a n a b e , and K o k i H o r i k o s h i The Institute of Physical and Chemical Research, Wakoshi Saitama 351, Japan

Summary. Escherichia coli H B 101 containing the plasmid p E A P 2 with alkalophilic Bacillus no. 170 penicillinase gene was f o u n d to p r o d u c e large a m o u n t s of penicillinase in L B - b r o t h containing 0.2% glycerol. Extracellular p r o d u c t i o n of the e n z y m e was n o t i n d u c e d by lysis of the cells but was strongly stimulated by the addition of N a +, K + or Li +, with a b o u t 90% of total activity being r e c o v e r e d in the culture fluid. W i t h o u t the addition of these ions, h o w e v e r , m o r e than 80% of the e n z y m e activity r e m a i n e d in the cellular fraction. T h e e n z y m e p r o d u c e d was very stable and no decrease in activity was o b s e r v e d after long cultivation.

Materials and Methods Media and Strains Used. The following culture media were used throughout the experiments: LB-broth containing 10 g tryptone (Difco Laboratories, Detroit, Mich.), 5 g yeast extract, 1 g glucose and 10 g NaC1 in 1 1 deionized water; the pH was adjusted to 7.0 with NaOH and the medium solidified if necessary with 20 g agar. A basic medium was the LB-broth minus NaC1. The super-rich medium contained 25 g tryptose, 20g yeast extract (Difco Laboratories), 30 g glucose and 3 g of K2HPO4 (pH 7.0) in 11 deionized water. P-broth is the penicillin assay medium of Difco Laboratories and N-broth is nutrient broth. Tryptose broth and polypeptone broth were modified LB-broths containing tryptose or polypeptone (10 g) instead of tryptone. The alkalophilic Bacillus no. 170 grew well at a pH of 6,5-11.0; the best growth and maximum penicillinase production were observed in an alkaline medium containing 0.2% glycerol of pH 9.5 (Sunaga et al. 1976). Strains and plasmids used are listed in Table 1. E. coli HB101 carrying the plasmid pEAP2 which we constructed was used throughout the experiments (Fig. 1).

Introduction Analysis of Penicillinase Distribution. Fractionation of extracellular, periplasmic and intracellular penicillinase was performed by a

In previous investigations (Horikoshi and A k i b a 1982), m a n y alkalophilic microorganisms which grew well in the p H range 1 0 . 0 - 1 1 . 0 were isolated. T h e s e m i c r o o r g a n i s m s p r o d u c e m a n y interesting extracellular enzymes, such as alkaline proteases, alkaline amylases, alkaline pectinase, penicillinase etc. W e have cloned several genes of alkalophilic microorganisms and expressed t h e m in Escherichia coli and Bacillus subtilis ( K u d o et al. and H o n d a et al. unpublished work). O n e of t h e m , a constitutive penicillinase gene of alkalophilic Bacillus no. 170 was cloned (plasmid p E A P 2 ) by using p M B 9 and expressed in E. coIi. T h e penicillinase was f o u n d to be secreted into the m e d i u m f r o m E. coli cells. This p a p e r deals with the p r o d u c t i o n of extracellular penicillinase by E. coli carrying p E A P 2 and the localization of the penicillinase in the cells. Offprint requests to: Toshiaki Kudo

Apr = ampicillin resistance; Tcr = tetracyclin resistance

Table 1. Strains and vectors used Bacterial strains and plasmid vectors

Genotypes and properties

Origin or reference

Alkalophilic Bacillus No. 170

Produces a penicillinase.

Our laboratory

Escherichia coli HB101

pro, leuB, B1, lacY, hsdR, hsdM, aral4, galkz, xyl5, mtll, supE44, F-, endoI-, recA-

Goldfarb et al. (1982)

pEAP 2

pMB9 plus 2.4kb HindIII fragment of no. 170 DNA, Apr

Our laboratory

pBR 322

Apr, Tcr

Bolivar et al, (1977)

C. Kato et al,: Production of Bacillus Penicillinase by E. coli

340 Hind [lI AI ka[ophil i c Bach [ us No. 170 DNA

I

Lid]ti'on

Hind III restriction

Hi nd g

Fig. 1. Construction of the plasmid pEAP2 having Bacillus penicillinase gene. Further details are to be published elsewhere

Table 2. Media and penicillinase production Media

h

Extracellular

Intracellular

Total activity

LB-broth

24 30

7.1 11.2

3.0 2.8

10.1 14.0

Super-rich

24 30

0.8 0.2

0.2 0.1

1.0 0.3

P-broth

24 30

1.6 6.1

14.6 14.2

16.2 20.3

N-broth

20 30

0.6 0.4

0.2 0.1

0.8 0.5

The values represent units of penicillinase activity per milliliter of the media

31 rag; specific activity 516 U/mg) was used for the following experiments.

Assay of PenicillinaseActivity. Penicillinase was assayed at pH 7.0 modified method of Cornelis et al. (1982). The culture broth was centrifuged at 10,000 g for 10 min and the supernatant fluid was analysed for the extracellular penicillinase fraction. The cells were washed twice in half the volume of culture broth with 0.9% NaC1 and resuspended in the same volume of 25% sucrose. The suspension was shaken for 10 min at room temperature in the presence of 1 mM EDTA. The cells collected by centrifugation (10,000 g for 10 rain) were quickly and vigorously resuspended in the same volume of ice-cold water. After 10 min shaking at 4~ C, the suspension was centrifuged for 10 rain at 15,000 g and the supernatant fluid was removed. The cells were then resuspended in the same volume of 0.05 M phosphate buffer (pH 7.0) followed by sonication (20 KHz, 3 min, 200 W). The extracellular penicillinase was assessed as the sum of the activities found in the supernatant of culture broth, two washes, and the supernatant of the EDTA treatment. The periplasmic penicillinase was the activity found in the supernatant of the cold-water treatment. The cellular penicillinase was in the supernatant fluid of the sonicate. In some experiments, the total activity of periplasmic and cellular enzymes was expressed as intracellular activity.

Purification of the Enzymes. The culture fluid (2,000 ml) was centrifuged at 10,000 g for 10 rain. The supernatant fluid (21 U/ml) was brought to 80% saturation with ammonium sulphate and stored at 5~ overnight. The precipitate was collected by centrifugation (10,000 g for 10 rain) and dissolved in 0.05 M phosphate buffer (pH 7.5). The solution was dialysed overnight against the same buffer at 4~ C. The resultant solution (120 ml) was applied to a DEAE-cellulose column (27 x 400 mm), which had been equilibrated with 0.05 M phosphate buffer (pH 7.5). The enzyme was not adsorbed at all and passed through the column. The penicillinase was precipitated with 80% saturated ammonium sulphate, dissolved in 0.01 M phosphate buffer (pH 6.5), and dialysed overnight against the same buffer at 4~ C. The dialysate (38.5 ml) was adsorbed on a CM-cellulose column (27 x 350 mm) equilibrated with 0.01 M phosphate buffer (pH 6.5). The enzyme was eluted by applying a linear gradient of sodium chloride at a flow rate of 25 ml/h. A linear gradient was obtained by using 300 ml each of 0.01 M phosphate buffer (pH 6.5) and the same buffer containing 0.4 M NaC1; 5-mI fractions were collected and the activity of each fraction was measured. The fractions exhibiting penicillinase activity were combined and concentrated by using Ficol. The concentrate (5 ml; total activity, 16,000 U; total protein,

using the method of Sargent (1968) as modified by Sawai et al. (1978). One unit of penicillinase hydrolyses 1 gmole of benzylpenicillin per minute at 30~ C.

Chemicals. The crystalline benzylpenicillin was purchased from Meijiseika Kaisha Ltd. (Tokyo). All other reagents were from Wako Chemical Industries (Osaka).

Results Culture Conditions f o r PeniciIlinase Production C u l t u r e c o n d i t i o n s for p e n i c i l l i n a s e p r o d u c t i o n w e r e examined in media containing various nutrients and metal ions. E. coli H B 101 c a r r y i n g t h e p l a s m i d p E A P 2 , t h a t is, E. coli H B 101 ( p E A P 2 ) , was i n o c u l a t e d in 5 0 0 - m l flasks c o n t a i n i n g 100 m l of v a r i o u s m e d i a a n d cult u r e d at 37 ~ C o n a r o t a r y s h a k e r . E x t r a c e l l u l a r a n d i n t r a c e l l u l a r e n z y m e activities w e r e a s s a y e d a f t e r 24 h a n d 30 h ( T a b l e 2). It is q u i t e c l e a r t h a t e x t r a c e l l u l a r p e n i c i l l i n a s e was d e t e c t e d o n l y in L B - b r o t h . O t h e r m e d i a t e s t e d w e r e n o t s u i t a b l e for e x t r a c e l l u l a r p r o d u c t i o n of t h e e n z y m e . V a r i o u s c a r b o h y d r a t e s w e r e c o m p a r e d as p o t e n t i a l c a r b o n s o u r c e s for e n z y m e p r o d u c t i o n . A s s h o w n in T a b l e 3, e n z y m e p r o d u c t i o n was i m p r o v e d by the addition of glucose and glycerol or maltose and g l y c e r o l , b u t n o a d d i t i o n a l effect was o b s e r v e d w i t h s t a r c h o r s u c r o s e . T a b l e 4 s h o w s t h e effect of v a r i o u s o r g a n i c n i t r o g e n s o u r c e s o n e n z y m e p r o d u c t i o n in t h e presence or absence of 1% NaCI. No significant d i f f e r e n c e was d e t e c t e d a m o n g t h e o r g a n i c n i t r o g e n c o m p o u n d s so far t e s t e d . H o w e v e r , it is s t r i k i n g t h a t t h e a d d i t i o n of 1 % N a C I r e s u l t e d i n t h e b e s t

C. Kato et al. : Production of Bacillus Penicillinase by E. coli

341

Table 3. Effect of carbohydrates Media

h

Extracellular

Intracellular

Total

LB

24 30

8.3 10.5

3.5 1.9

11.8 12.4

LB + glycerol

24 30 24 30

20.5 18.5 4.3 5.2

1.0 0.5 2.1 1.9

21.5 19.0 6.4 7.1

LB-glucose + glycerol

24 30

5.4 5.2

0.6 1.8

6.0 7.O

LB-glucose + maltose

24 30

7.3 9.8

2.6 2.0

9.9 11.8

LB@ucose + maltose + glycerol

24 30

12.8 13.8

1.5 2.6

14.3 16.4

LB-glucose + sucrose

24 30

5.5 5.3

2.5 1.8

8.0 7.1

LB-glucose + sucrose + glycerol

24 30

7.1 6.5

2.0 4.3

9.1 10.8

LB-glucose + starch

24 30

5.0 4.5

3.0 1.0

8.0 5.5

LB-glucose + starch + glycerol

24 30

6.6 4.9

2.3 3.9

8.9 8.8

LB-glucose

The carbohydrates tested were added to the media at a concentration of 0.2%. The values represent units of the penicillinase activity per ml of the media

Table 4. Effect of organic nitrogen sources

T a b l e

Media

Addition of NaC1

h

Extracellular

Intracellular

Total activity

LB-broth

+ + -

24 30 24 30

14.7 15.6 2.2 3.1

1.9 2.8 8.8 7.7

16.6 18.4 11.0 10.8

+ + -

24 30 24 30

13.7 15.4 1.4 2.3

1.1 1.6 7.5 8.7

14.8 17.0 8.9 11.0

Polypepton + broth +

24 30 24 30

16.4 17.1 1.8 3.4

1.5 0.7 5.7 5.8

17.9 17.8 7.5 9.2

Tryptosebroth

-

-

5. Effect of metal ions

Salts added

None NaC1

Na2SO4 KC1 LiC1 CsC1

Concentration

0.08 0.16 0.32 0.80

M M (1%) M M

0.08 M 0.16 M 0.16 M 0.16 M

Extracellular

Periplasmic

1.2 11.9 20.5 14.1 2.5

0.1

16.3 16.2 16.8 < 0.1

0.2

Cellular

Total

7.8 4.2* 2.5 3.8* 3.5*

9.1 16.1 23.2 17.9 6.0

5.3* 4.8* 3.0* < 0.1"

21.6 21.0 19.8

* Intracellular activities Media used: the basic medium plus 0.2% glycerol with or without salts. The values represent units of penicillinase activity per milliliter of the culture

The values represent units of penicillinase activity per milliliter of the culture

production of extracellular penicillinase, with only 10% of the total enzyme activity remaining in the intracellular fraction. Addition of KC1, LiC1 or Na2SO 4 also stimulated the production of extracellular penicillinase and to the same extent (Table 5). Thereafter pH values of the LB-broth with 0.2% glycerol were adjusted with NaOH and HC1.

Distribution of Penicillinases During Cultivation E. coli HB101 (pEAP2) was inoculated in 500-ml flasks containing 100 ml of LB-broth with a 0.2% glycerol content and cultured at 37~ on a rotary shaker. A 2-ml aliquot of the broth was withdrawn from the culture at 4-h intervals. Cell growth was

342

C. Kato et al.: Production of Bacillus Penicillinase by E. coli

measured at 660 nm, and extracellular and intracellular penicillinases were assayed. As shown in Fig. 2, the bacteria reached the maximum cell concentration at 16 h, and no lysis of the cells was observed up to 48 h (viable counts: 3 x 10 9 at 16 h, 2 x 10 9 at 48 h). The extracellular penicillinase activity increased at about 24h and reached the maximum at 28 h. The enzyme produced was very stable and no significant decrease was observed during prolonged culture. On the other hand, intracellular penicillinase was detected at an early stage of cultivation (8-20 h) and decreased quickly. No intracellular penicillinase activity was observed after 28 h cultivation. These data suggest that the lack of intracellular penicillinase probably indicates that release was not by autolysis, but by secretion from E. coli cells. As a reference

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r~ _(3

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r-

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.Q <

13_

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0.1

8

Some Properties of the ExtraceIIular Penicillinase Produced by E. coli HBIO1 (pEAP2) Effects of p H on Activity and on Stability of the Enzyme. Stability of the enzyme was investigated in buffer solutions of various pH values. The mixture was incubated at 30 ~ C for 45 rain. The enzyme was stable at pH 7-9. The optimum pH value for enzyme action was about 7.0.

20~

//

experiment, the time course and localization of penicillinase activity of E. coli HB101 carrying pBR322 were studied. E. coli HBI01 (pBR322) was cultivated in LB-broth and the enzymatic activities were assayed under the same conditions described above. Figure 3 shows the time course of penicillinase production during cultivation. Less than 10% of enzyme activity was observed in the cell filtrate and most was in the intracellular fraction. These results indicate that the secretion was probably caused by the plasmid constructed by us, and was not a special feature of the strain E. coli HB101.

16

24 32 40 48 Time (hr) Fig. 2. Bacterial growth and penicillinase production by Escherichia coli HB101 (pEAP2). E. coli HB101 (pEAP2) was inoculated in the LB-broth containing 0.2% glycerol and cultured at 37 ~ C on a rotary shaker. Bacterial growth (absorbancy at 660 nm, 0) and penicillinase activities (extracellnlar, 9 intracellular, A; and total, A) were determined

Thermal Stability. The enzyme was dissolved in 0.05 M phosphate buffer of pH 7.0 and the solution were heated at the indicated temperatures for 10 rain, the residual activities being measured at pH 7.0. The enzyme was stable up to 50~ and inactivated at 70~ C. Molecular weight of Sephadex Estimated

Weight of the Enzym e. The molecular the enzyme was estimated by using the gel filtration~ method (Whitaker 1963). molecular weight was about 24,000.

Discussion 40 E

/ ~ / ",, \ E

cO

,U

2.0

3O 5

\\ 9

- . - -

>

1.0 trd

20

0.5

r

~0~ r

oo..._~o__.-%-o-o - ~ 1 7 6

<

0.1

9t 0 1

0

8

I

i

I

I

I

16

24

32

40

48

....

Y_

Time (hr) Fig. 3. Bacterial growth and penicillinase production by E. coli HB101 carrying pBR322. E. coli HB101 (pBR322) was aerobically cultured in the LB-broth containing 0.2% glycerol at 37~ C. Bacterial growth (absorbancy at 660 nm, O) and distribution of penicillinases were determined. Extracellular, (3, intracellular ~, total 9

We have cloned a constitutive penicillinase gene from alkalophilic Bacillus no. 170 in E. coli HB 101 and C 600 by using plasmid vector pMB9. The cloned gene was found to be functionally expressed in E. coli HB 101 and C 600 at neutral pH. Imanaka et al. (1981) expressed B. licheniformis penicillinase gene in E. coli C 600, but the enzymatic activity was very low (less than 0.2 U in our units). Our most striking results are as follows: 1. E. coli HB101 carrying pEAP2 could produce large amounts of extracellular penicillinase in the culture media, and the activities produced were as high as those produced by alkalophilic Bacillus no. 170 (20-30 U/ml). 2. The secretion of the enzyme was strongly stimulated by the addition of Na +, Li + , or K § Without the addition of these ions, more than 80% of total

343

C. Kato et al,: Production of Bacillus Penicillinase by E. coli

penicillinase activities remained in the cellular fraction of the cells. It is clear that the penicillinase gene which we cloned from alkalophilic Bacillus no. 170 is entirely different from other penicillinase genes, such as Amp resistant gene of pBR322, because penicillinase activity initiated by pBR322 could not be detected in the culture broth but was observed mainly in the periplasmic fraction. 3. The enzyme produced by E. coli HB 101 was very stable a n d no significant decrease in activity was observed after long cultivation; it thus differs in stability from the enzyme of alkalophilic Bacillus no. 170. The viable counts of E. coli HB101 (pEAP2) at 16 h was 3 • 109/ml and no decrease was observed in prolonged cultivation. Protein secretion across the cell surface is an important biological phenomenon. Although there is no evidence that the penicillinase gene which we isolated gives the signals for secretion, our results indicate that the introduction of our gene brought about a change in the outer membrane and the penicillinase could be released from the cells in the presence of Na +, Li +, or K +. There is no experimental evidence as to whether the penicillinase is secreted by the cells in the true sense or is leaked from cells. In either case, the great advantage of our process is that it eliminates the necessity for enzyme extraction from the cells. It is well known that the majority of heterologous penicillinase molecules made in Bacillus species are translocated across the membrane and subsequently processed proteolytically to form the exoenzyme. Thus it is of interest to compare the penicillinases of alkalophilic Bacillus no. 170 and E. coli HB 101 (pEAP2). Some enzymatic properties such as optimum pH, stable pH, and thermal stability are almost

the same as those of Bacillus no. 170 penicillinase. Further studies on this point are in progress. Acknowledgement. This work was partially supported by the grant for Life Science Promotion in the Institute of Physical and Chemical Research.

References Bolivar F, Rodriguez RL, Greene P J, Betlach MC, Heineker HE, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113 Cornelis P, Digneffe C, Willemot K (1982) Cloning and expression of a Bacillus coaglans amylase gene in Escherichia coli. Mol Gen Genet 186:507-511 Goldfarb DS, Rodriguez RL, Doi RH (1982) Translation block to expression of the Escherichia coli Tng-derived chloramphenicol-resistance gene in Bacillus subtilis. Proc Natl Acad Sci USA 79 : 5886-5890 Horikoshi K, Akiba T (1982) Alkalophilic Microorganisms. Springer, Berlin Heidelberg New York Imanaka T, Tanaka T, Tsunekawa H, Aiba S (1981) Cloning of the genes for penicillinase, pen P and pen I, of Bacillus lichen# formis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis and Bacillus licheniformis. J Bacteriol 147: 776- 786 Sargent MG (1968) Rapid fixed-time assay for penicillinase. J Bacteriol 95:1493-1494 Sawai T, Takahashi I, Yamagishi S (1978) Iodometric assay method for beta-lactamase with various beta-lactam antibiotics as substrates. Antimicrob Agents Chemother 13:910-913 Sunaga T, Akiba T, Horikoshi K (I976) Production of penicitlinase by an Alkalophilic Bacillus. Agric Biol Chem 40:1363-1367 Whitaker JR (1963) Determination of molecular weights of proteins by gel filtration on Sephadex. Anal Chem 35:1950-1953 Received May 13, 1983