Effect of induction conditions on production and excretion of Aeromonas hydrophila chitinase by recombinant Escherichia coli

JOURNALOF FERMENTATION AND BIOENGINEERING Vol. 84, No. 6, 610-613. 1997

Effect of Induction Conditions on Production and Excretion of Aeromonas hydrophila Chitinase by Recombinant Escherichia coli SHU-JEN CHEN,’ MING-CHUNG

CHANG,* AND CHU-YUAN

CHENG’*

Department of Chemical Engineering1 and Department of Biochemistry, Medical College,* National Cheng Kung University, Tainan, Taiwan, R. 0. C. Received 24 January 1997/Accepted

16 September 1997

The effects of isopropyl-go-thiogahuztopyranoside (IPTG) induction conditions and cell growth rate on the production and excretion of Aeromonas hydrophila chitinase by Escherichia coli were investigated. The most efficient induction condition for chitinase production was obtained by adding IPTG at a final concentration of 0.5 mM to the medium at the fermentation time of 2 h. Specific chitinase production increased with decreasing cell growth rate, which was manipulated by decreasing the aeration level or NH&l concentration. Overexpression of the chitinase gene as well as the decrease in specific cell growth rate resulted in a high percentage of periplasmic enzyme excretion. Comparing the extracellular fractions of alkaline phosphatase (a periplasmic enzyme) to malate dehydrogenase (a cytoplasmic enzyme), the increase in outer membrane permeability is primarily attributed to chitinase excretion. [Key words: chitinase, excretion, recombinant Escherichia coli] space. In addition, an amount of chitinase could even be excreted into the fermentation broth. In this study, therefore, we investigated the effects of induction conditions and cell growth rate on the formation and excretion of A. hydrophila chitinase by E. coli. The inducer used was IPTG, and the cell growth rate was manipulated by aeration level and the concentration of nitrogen source in the medium. E. coli JM109 (14) was used as a host for the expression of the plasmid pCHlOO1 (9). The plasmid had an insert of the A. hydrophila CHl chitinase gene of 4.5 kb. The complex medium (LB medium), used for investigating the effects of induction condition and aeration level, consisted of (g/l): tryptone, 10; yeast extact, 5; and NaCl, 10. The synthetic medium (M9 medium), used for studying the effect of the concentrated nitrogen source, was composed of (g/l): Na2HP04.7H20, 12.8; KH2P04, 3; NaCl, 0.5; glucose, 2; thiamine hydrochloride, 034; MgS04, 0.12; and CaC12, 0.011. Ampicillin was added to both media at a final concentration of 50 ,ug/ml to ensure plasmid stability. Different concentrations of nitrogen source were prepared by adding various amounts of NH&l to the medium. The study of the effect of induction conditions was carried out using 500-ml shaking flasks operating at 1OOrpm. The working volume was 1OOml. The effects of aeration level and NH&l concentration of the medium on chitinase activity were examined in a 2-l fermentor (M-100, Tokyo Rikakikai Co. Ltd., Tokyo), with a working volume of 1 1. The temperature was kept at 37°C and pH was maintained at 7.0 throughout the addition of 1 N HCl or 10% (v/v) NHIOH. The aeration level was manipulated by aeration rate and agitation speed. Cell concentration was measured by the absorbance of the culture broth at 600 nm using a spectrophotometer (model UV-2000, Hitachi Ltd., Tokyo), correlated with dry cell weight. The separation of cells from the culture broth was achieved by centrifuging at 10,000~g for 5 min. The supernatant was analyzed for the extracellular fraction of enzyme. The

Simultaneous formation and excretion of foreign proteins from a recombinant cell is favorable for downstream processing. The excretion of foreign proteins from Escherichia coli has been demonstrated by using the kil gene of bacteriocin plasmid (l-6). Kato and co-workers (l-4) showed that activation of the kil gene of the plasmid pMB9 by the promoter in the alkalophilic gene caused an Bacillus sp. strain 170 penicillinase increase in the permeability of the outer membrane of a recombinant E. coli cell. As a result, most of the penicil-

linase synthesized was extracellular. Hsiung et al. (5) showed that the human growth hormone could be released from E. coli by expressing bacteriocin release protein, which leads to an increase in outer membrane permeability. Tokugawa et al. (6) isolated a collagenase gene from a species of Vibrio and cloned it into E. coli. They found that cells carrying a plasmid containing this fragment could excrete significant amounts of periplasmic ,&lactamase and alkaline phosphatase into the medium. In addition to the use of the kil gene, overexpression of the ,B-lactamase gene induced by isopropyl-P-Dthiogalactopyranoside (IPTG) was found to increase the permeability of the cell outer membranes and results in the excretion of ,&lactamase from a recombinant E. coli (7, 8). Although the excretion of foreign proteins can successfully be achieved by increasing the permeability of cell outer membranes, the factors that influence excretion efficiency have not yet been reported. In addition, it has been reported that the production of foreign proteins is affected by induction conditions (9-ll), and cell growth rate (12). Also, the relationship between production and excretion of recombinant proteins has not been correlated. In a previous study (13), Aeromonas hydrophila chitinase, with its signal peptide, was shown as capable of being translocated across the inner membrane of a recombinant E. coli and secreted into the periplasmic * Corresponding author. 610

NOTES

VOL. 84, 1997

cell paste was washed twice and re-suspended in phosphate buffer (pH 7.0), followed by sonication treatment at 20 kHz for 6min. The supernatant of the distupted cell suspension, obtained by centrifuging at 17,000 x g for 20min, was used for the intracellular enzyme assay. The extent of enzyme excretion was calculated from the ratio of extracellular enzyme activity to total enzyme activity. The chitinase assay followed the turbidometric method of Yabuki et af. (15), in which one unit of chitinase activity was defined as the amount of chitinase that caused a 1% decrease during absorbance at 610nm per minute. Alkaline phosphatase assay was based on the formation of p-nitrophenol in the hydrolysis of p-nitrophenylphosphate (16). One unit of alkaline phosphatase was defined as the amount of enzyme that liberate 1 /*mol of p-nitrophenol in 1 h. Malate dehydrogenase assay was based on the reduction of NADH in the presence of oxaloacetate (17), and one unit of malate dehydrogenase activity was defined as the amount of enzyme required to reduce 1 pmol of NADH per minute. The effect of IPTG concentration and the timing of its addition on chitinase production is shown in Fig. 1. Various concentrations of IPTG were added to the culture at different fermentation times. The cultivation time was set as 12 h, when cell concentration and enzyme activity were found to be at a maximum. It is apparent that chitinase production is affected by either the inducer concentration or the timing of addition. For each inducer concentration, there existed an optimum time for addition to maximize chitinase formation. The optimum timing occurred later as more IPTG was added. Although chitinase production increased with increasing IPTG concentration, no further improvement in chitinase production was observed when IPTG exceeded 0.5 mM. In contrast, IPTG concentrations that were too high retarded cell growth (data not shown). In order to investigate whether chitinase excretion was due to an increase in the permeability of cell outer membranes, the extracellular fractions of alkaline phosphatase (periplasmic enzyme) and malate dehydrogenase (cytoplasmic enzyme) were examined. Figure 2a shows that the extent of chitinase excretion increases with increasing IPTG concentration, and earlier inducer addition results in a higher percentage of chitinase excretion.

611

The extent of alkaline phosphatase excretion, as indicated in Fig. 2b, exhibited a mode similar to chitinase. In other words, the influence of IPTG concentration on different periplasmic enzyme excretions was similar. In contrast, as shown in Fig. 2c, less than 3% of malate dehydrogenase was detected in the medium, indicating that cell lysis did not cause chitinase excretion in these experiments. Therefore, the expression of chitinase gene, which was induced by IPTG, resulted in excretion of periplasmic enzymes. This was apparently due to the increased permeability of the cell outer membrane. Since the addition of 0.5 mM IPTG at the fermentation time of 2 h resulted in the highest chitinase production (Fig. l), the following experiments were based on this condition. The effect of aeration level on the chitinase fermentation is shown in Table 1. In this experiment, three aeration levels were achieved by changing the aeration rate and agitation speed, namely, 0.5 vvm and 1OOrpm (level I), 1.2 vvm and 250 rpm (level II), and 1.5 vvm and 300 rpm (level III). It can be seen that the cell concentration and the specific growth rate increased with increasing aeration level. Although the highest chitinase activity occurred at level II, the highest

0123456

Induction timing (h)

(b)

W 0123456 Induction timing (h) 5.

c

41 3!

:;

O.O’.““’

““‘,““.”

0

1

2

3

4

5

“.I

-() 0 0 t~~~l~~~~~~~I~~~.l~~~~ 0123456 6

Induction timing (h)

FIG. 1. Effect of induction timing and IPTG concentration on the production of chitinase. Symbols: 0, without induction; 0 , 0.05mM IPTG; A, O.lOmM IPTG; A, 0.5 mM IPTG; 0, l.OmM IPTG; n , 2.0 mM IPTG.

Induction timing (h) FIG. 2. Effect of induction timing and IPTG concentration on the fraction of extracellular enzymes. (a) Chitinase; (b) alkaline phosphatase; (c) mafate dehydrogenase. Symbols: 0, without induction; l ,0.05 mM IPTG; A, 0.10 mM IPTG; A, 0.5 mM IPTG; 0 , I .OmM IPTG; n ,2.0 mM IPTG.

612

CHEN ET AL. TABLE 1.

J.

Effect of aeration level on cell growth, chitinase production, and enzyme excretiona

TABLE 2.

Effect of NH&l on cell growth, chitinase production, and enzyme excretiona

Aeration levels 0.5

vvm

1.2vvm

Cell concentration (g/i) Soecific erowth rate (h-l) dhitinase activity (uimlj Specific chitinase activity (U/mg cell) Extent of chitinase excretion (%) Extent of alkaline phosphatase excretion (%) Fraction of extracellular malate dehydrogenase (%)

0.551 0.429b 0.498 0.904 10.4 15.5 4.6

NH&l concentration (g/i)

1.5 vvm

1OOrpm 250rpm 300rpm 1.357 0.723b 0.903 0.685 6.2 8.7

1.642 0.744b 0.791 0.482 2.7 4.0

1.9

0

a Data was taken at the fermentation time of 12 h unless otherwise noted. b Data was taken from maximum values. specific activity was obtained at level I. Aeration level also affected the excretion of enzymes. As indicated in Table 1, the enzyme excretion percentage increased with decreasing specific growth rate (decreasing aeration level). The percentages of extracellular enzyme were 10.4%, 15.5% and 4.6% for chitinase, alkaline phosphatase and malate dehydrogenase, respectively, if using the aeration level I. From checking the fraction of extracellular malate dehydrogenase, it is obvious that cell lysis was not the reason for the increase in the extent of chitinase excretion, Ryan et al. (18) indicated that there was competition between the replication and expression of chromosome DNA and that of the plasmid DNA for limited cellular resources. A reduction in the specific cell growth rate resulting from a decreasing aeration level leads to the increase in allocation of resources for plasmid-related activities. It is therefore expected that the lower the aeration level, which leads to a lower growth rate, the higher will be the plasmid DNA expression. As a result, the highest efficiency of chitinase formation was obtained when the cells were cultivated at the aeration level I. In addition, since plasmid-related activities impose a large metabolic burden on the host cell, the ability of the cell to repair the damage to the cell membrane is reduced, which in turn causes increased excretion of periplasmic enzymes. The improvement of production efficiency of the recombinant chitinase by decreasing the cell growth rate can also be achieved by manipulating the medium composition. Table 2 demonstrates that the efficiency of chitinase formation (shown as total specific chitinase activity) increases with decreasing NH&l concentration, which corresponds to a reduction of cell growth rate. The enzyme excretion also increased with decreasing specific growth rate (decreasing NH&l concentration). Since the fraction of extracellular malate dehydrogenase was much less than that of chitinase and alkaline phosphatase, the excretion of the latter two enzymes was primarily due to an increase in the permeability of the cell outer membranes. It can therefore be concluded that cultivation of cells at a lower growth rate not only results in the allocation of more cellular resources to overexpress the chitinase gene but also increases the permeability of the cell outer membranes. In summary, the induction condition and cell growth rate influence the production and excretion of recombinant proteins. The most efficient induction condition of IPTG for chitinase formation was obtained when

FERMENT.BIOENG.,

Specific growth rate (h-l) Specific chitinase activity (U/mg cell)

Chitinase excretion (%) Extent of alkaline phosphatase excretion (%) Fraction of extracellular malate dehydrogenase (%)

0.2

0.4

2.0

0.270

0.293

0.326

1.882 25 35

1.711 24 26

0.826 18 20

7.8

6.2

5.9

* Data was taken from maximum values. using a final concentration of 0.5 mM to the medium at the fermentation time of 2 h. Excretion of chitinase increased with increasing IPTG concentration, and earlier inducer addition resulted in a higher percentage of chitinase excretion. It was also found that the total specific chitinase activity and chitinase excretion increased with decreasing aeration level or with decreasing NH&l concentration, which corresponds to a reduction of specific growth rate. Both overexpression of chitinase gene and reduction of cell growth rate contributed to the excretion of periplasmic enzymes. Comparing the extracellular fraction of malate dehydrogenase to alkaline phosphatase, an increase in the permeability of cell outer membranes is suggested to be the primary reason for their excretion. This work was supported in part by a Grant-in-Aid (NSC 852214-EOO6-020) for science research from the National Science Council of Taiwan. REFERENCES

1. Kato, C., Kudo, T., Kazodo, W., and Horikoshi, K.: Extracellular production of Bacillus penicillinase by Escherichia coli carrying pEAP2. Eur. J. Appl. Microbial. Biotechnol., 18, 339343 (1983). 2. Kudo, T., Kato, C., and Horikoshi, K.: Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane. J. Bacterial., 156, 949-951 (1983).

3. Kobayashi, Horikoshi,

T., Kato, C., Kudo, T., Kazudo, W., and K.: Excretion of the penicillinase of an alkalophilic

Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9. J. Bacterial., 166, 728-732 (1986). 4. Kato, C., Kobayashi,

T., Kudo, T., Furusato, T., Murnkami, Y., Tanaka, T., Baba, II., Oiihi, T., Ohtsuka, E., Ikehara, M., Yanagida, T., Kato, H., Moriyama, S., and Horikoshi, K.: Construction of an excretion vector and extracellular

production of human growth hormone from Escherichia coli. Gene, 54, 197-202 (1987). 5. Hsiung, H. M., Cat&all,

A., Luirink, J., Oudega, B., Veros, A. J., and Becker, G. W.: Use of bacteriocin release protein

in E. coli for excretion of human growth hormone culture medium. Bio/technol., 7. 267-271 (1989).

into the

6. Tokugawa,

K., Kakitani, M.,.Ishii, T., Nakamura, K., Masaki, H., and Uozumi, T.: A novel protein secretion factor from a

Vibrio species which operates technol., 35, 69-76 (1994).

in Escherichia coli. J. Bio-

7. Georgiou, G., Shuler, M. L., and Wilson, D. B.: Release of periplasmic enzymes and other physiological effects of ,l-lactamase overproduction in Escherichia coli. Biotechnol. Bioeng., 32, 741-748 (1988). 8. Togna, A. P., Shuler, M. L., and Wilson, D. B.: Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of ,%lactamase from Escherichia coli.

VOL. 84, 1997 Biotechnol. Prog., 9, 31-39 (1993). 9. Shimizu, N., Fukuzono, S., Nishimara, N., Odnwara, Y., and Fujiwarn, K.: Cultivation of Escherichia co/i harboring hybrid plasmids. J. Ferment. Technol., 65, 7-10 (1987). 10. Bee, D. J., Chung, B. H., Hwang, Y. B., and Park, Y. H.: Glucose-limited fed-batch culture of Escherichiu coli for production of recombinant human interleukin-1 with the DO stat method. J. Ferment. Bioeng., 74, 196-198 (1992). 11. Sakamoto, S., Terada, I., Iijima, M., Matsuzawa, H., and Ohta, T.: Fermentation conditions for efficient production of thermophilic protease in Escherichiu coli harboring a plasmid. Appl. Microbial. Biotechnol., 42, 569-574 (1994). 12. Hellmoth, K., Korz, D. J., Sanders, E.A., and Deckwer, W.-D.: Effect of growth rate on stability and gene expression of recombinant plasmid during continuous and high cell density cultivation of Escherichia coli TGl. J. Biotechnol.. 32., 289298 (1994). 13. Chen, J. P., Nagayama, F., and Chang, M. C.: Cloning and expression of a chitinase gene from Aeromonas hydrophila in

NOTES

14. 15.

16. 17. 18.

613

Escherichia coli. Appl. Environ. Microbial., 57, 2426-2428 (1991). Yanishi-Perron, C., Vieira, J., and Messing, J.: Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUCl9 vectors. Gene, 26, 101-106 (1985). Yabuki, M., Mizushima, K., Amatatsu, T., Ando, A., Fujii, T., Shimada, M., and Yamashlta, M.: Purification and characterization of chitinase and chitobiase produced by Aeromonas hydrophila subsp. anaerogenes A52. J. Gen. Appl. Microbial., 32, 25-38 (1986). Matamy, M. and Horecker, B. L.: Alkaline phosphatase (crystalline). Methods Enzymol., 9, 639-642 (1966). Kitto, G.B.: Malate dehydrogenase from Escherichiu coli. Methods Enzymol., 13, 145-147 (1970). Ryan, W., Parulekar, S. J., and Stark, B. C.: Expression of 1% lactamase by recombinant Escherichiu coli strains containing plasmids of different sizes: effects of pH, phosphate, and dissolved oxygen. Biotechnol. Bioeng., 34, 309-319 (1989).