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BIOTECHNOLOGY LETTERS Volume 16 No.12 (Dec.1994) p.12651268 Received as revked 19th October
OVER EXPRESSION OF PGG GLUCOSE DEHYDROGENASE IN ESCHER/CM% COL/ UNDER HOLO ENZYME FORMING CONmN
Koji SODE’, Arief Budi WlTARTO, Kazumoto WATANABE, Keisuke NODA, Shunsuke IT0 and Wakako TSUGAWA
Department of Biotechnology, Faculty of Technology,Tokyo University of Agriculture 8 Technology, 2-24-l 6 Naka-cho, Koganei-shi, Tokyo 184, JAPAN
KEY WORDS; PQQ glucose dehydrogenase, over expression, Esherichia co/i, thermal stabilii *;The author to whom correspondence should be addressed
SUMMARY
The over expression of PQQ glucose dehydrogenase PQQGDH was investigated. The level of PQQGDH expressed in E.coli PP2418/pGEcl was more than 10 fold when the cultivation was carried out under holo enzyme forming condition in the presence of 800 nM of PGG and 10 mM of MgCl,, compared with those of apo condition. It may be due to the difference in thermal stability of apo and holo PQQGDH.
INTRODUCTION
Glucose dehydrogenases (GDH) possessing pyrroloquinoline quinone (PQQ) have received much
attention as industrial enzymes such as components of enzyme sensors. With the advance of molecular
genetics of PQQ enzymes, various sources of PQQGDHs have been cloned, and their primary structures
elucidated (Cleton-Jansen et a/., 1988a, 1988b, 1990 and 1991). Recently, the authors have reported the
improvement of holo enzyme stability of Escherichia co/i derived PQQGDH by site directed mutagenesis
(Sode and Sano, 1994). In order to advance the protein engineering of PQQGDHs, further improvement
of the method for the PQQGDH production in an E.co/i strain lacking PQQGDH structural gene is essen-
tial.
PQQGDH is produced in Exoli as the apo form, which further requires bivalent metal ion and
PQQ to form the holo enzyme, since E.coli cannot synthesis PQQ. Ameyama ef al. reported that the
thermal stability of holo GDH is much higher than that of apo GDH (Ameyama et a/., 1986). Considering
those observation, over expression of PQQGDH under holo enzyme forming condition might greatly
enhance the production of PQQGDH.
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In this study, using E.coli derived PQQGDH gene cloned in an expression vector, we investigate the
over expression of PQQGDH under holo enzyme forming conditions.
MATERIALS 81 METHODS
1. Chemicals PQQ was obtained from Kanto Chem.(Japan). Isopropyl-f3-D-thiogalactopyranoside (IPTG) was
purchased from Takara (Japan). All other chemicals used were of analytical grade. 2. Bacterial strains and plasmids
Exoli strain used for the host is PP2418 which was kindly obtained from Dr.N.Goosen, Leiden Univ.(the Netherlands). This strain is inactivated PQQGDH structural gene by insertion mutagenesis (Cleton-Jansen ef al., 1990). For the production of PQQGDH, PP2418 harboring plasmid pGEc1 was used. This plasmid is inserted PQQGDH structural gene derived from E.co/i DHSa, under down strem of ffc promoter of an expression vector pTrc99A (Pharmacia, Sweden), as was described in our previous paper. 3. Culture condition
E.&iPP2418/pGEcl was cultured in Luria broth either in L-shaped test tube @ml medium) or in culture flask (150 ml medium). The cells were cultured in a medium with a shaker at 37°C aerobically, and after optical density at 660nm arrived at 0.8, 0.2 mM of IPTG was added for the induction of PQQGDH expression. For the experiment investigating the effect of addition of PQQ and MgCI,, they were added in each medium before inoculation at final concentration described in each figure. 4. Enzyme assay condition
PQQGDH activity was determined using the absorbance decrease of 2,6-dichrolophenolin- dophenol (DCIP) at 600 nm, in the presence of phenazine methosulfate (PMS). In order to examine the level of over expression during the cultivation, PQQGDH activity of whole cells was determined. At an each period, sample was derived from culture. After centrifugation at 185OOxg at 4°C for 3min, cells were washed twice by 1 OmM K-phosphate buffer(pH 7.0). Then, cells were resuspended in a same buffer. Ten micro litter of thus prepared cells was added in a micro test tube, and added 290~1 of 1 OmM K-phosphate buffer(pH 7.0) containing 1 PM PQQ and 1 OmM MgCl , and incubated at 25°C for 10 min. Then, PQQGDH activity was determined as described previous ‘I Sode and Sano, 1994). Protein concentration of cells was determined using DC Protein Assay kit (Bio-Rad, USA), according to the instruction manual.
RESULTS 81 DISCUSSION
Figure 1 and Figure 2 show the time course of PQQGDH over-expression under apo and holo forming
condition, respectively. After the induction by IPTG, the level of PQQGDH gradually increased in both
cases (Fig.1 and Fig.2). The maximum level of expression under apo condition was observed 4 hours
after induction (0.02 U per mg protein, 24 U I-‘). Under holo enzyme forming condition, the maximum
level was achieved at 5 hours after induction, but its level was more than 10 fold of those achieved in apo
condition (0.23 U per mg protein, 300 U I-‘). The holo enzyme forming condition did not effect the growth
of E.co/i PP2418/pGEcl (Fig. 1 and 2). Since the genomic PQQGDH structural gene had been inactivated
by insertion mutagenesis at PP2418 (Cleton-Jansen et al. 1990), the transcriptional level of PQQGDH in
this strain was dependent upon the cloned PQQGDH structural gene controlled under fat promoter of the
expression vector, which should not be affected in the presence or absence of PQQ and/or MgCI,. Con-
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0 2 4 6 8 10 12 lime (hrs)
01 2 4 6 8 10 12 Time (hrs)
Fig.1 Time course of PQQGDH expression.
Cells were cultured in the presence of 6OOnM PQQ and 1 OmM MgCl9.
-O- PQQGDH activity
-+J- Cell growth
Fig.2 Time course of PQQGDH expression. Cells were cultured in the medium without PQQ and additional MgCl9.
-O- PQQGDH activity
-Cl- Cell growth
The arrow indicates the time when IPTG was added.
sidering that the stability of holo PQQGDH is much higher than apo one (Ameyama et a/., 1986), the
increase in production of active PQQGDH achieved under holo enzyme forming condition might be due to
the superior stability of holo enzyme during the production.
In order to confirm this observation in detail, we then examined the effect of the concentration of
each component essential for holo enzyme formation. The experiments were carried out using L-shaped
test tube with 5ml medium. The level of expression was determined after 4 hours of incubation after IPTG
induction. With the increase in PQQ concentration added, the level of PQQGDH also increased. At PQQ
concentration of 600 nM and 6000 nM, around 0.3 U per rng protein were achieved, which was more than
10 fold those achieved without PQQ and additional MgCIJFig3a). Such a drastic difference was not
observed neither by adding PQQ alone nor MgCI, alone (Fig.Sb and 3~). These results suggested that
only under holo enzyme forming condition, can the high level of PQQGDH expression be achieved. The
addition of PQQ and Mg2+ formed stable holo enzyme, consequently resulted in the increase of accumu-
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lated amount of intracellular active PQQGDH. For the over expression of PQQGDH using
PP2418/pGEcl, PQQ concentration of 600 nM together with 10 mfvl additional MgCI, was found to be
adequate.
This study shows the success in the increase of expression level of PQQGDH, by using the cul-
ture condition enables holo enzyme formation.
0.4 I _ a
E 0.3 - z % h 0.2
E ; 0.1
;/-
0.4
b
0.0 I 2 3
0.0 LA&-d=== 10 O 10 ’ 10 10 10 4 O 10 10’ 102. lo3 10 4
PQQ concentration (nM) PQQ concentration (nM)
Fig.3 Effects of PQQ and MgCl 2 concentration in the medium on PQQGDH expression.
a. Effect of PQQ concentration in the presence of 10mM MgC12
b. Effect of PQQ concentration
c. Effect of MgC12 concentration
0 2 4 6 8 10 12 MgCl2 concentration (mM)
REFERENCES
Ameyama, M., Nonobe, M., Shinagawa, E., Matsushita, K., Takimoto, K. and Adachi, 0. (1986). Agric. Viol. Chem., 50(l), 19-57.
Cleton-Jansen, A.-M., Goosen, N., Odle, G. and Van de Pune, P. (1988a). Nucleic Acids Res., 18, 6228.
Cl&on-Jansen, A.-M., Goosen, N., Wenzel, T. T. and Van de Putte, P (1988b). .I. Bacterial., 180,2121-2125
Cleton-Jansen, A.-M., Goosen, N., Fayet, 0. and Van de Pune, P. (1990). J, BacferioL, 172,6308-6315
Cleton-Jansen, A.-M., Dekker, S., Van de Pune, P. and Goosen, N. (1991). Mel .Gen. Genet., 228,206-212
Sode, K. and Sano, H. (1994). Biofechnol. L&f,, 18, 455-460
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