BIOTECHNOLOGY LETFERS Volume 18 No. 9 (September 1996) pp.997-1002 Received 1 July

STABILIZATION OF PYRROLOQUINOLINE QUINONE GLUCOSE

DEHYDROGENASE BY CROSS-LINKING CHEMICAL MODIFICATION

Koji Sode,* Tomonori Shimakita, Shokichi Ohuchi, and Tomohiko Yamazaki

Department of Biotechnotogy, Faculty of Technology, Tokyo University of Agriculture and

Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184, JAPAN

SUMMARY Cross-linking chemical modification of pyrroloquinoline quinone (PQQ) glucose

dehydrogenase (GDH) by glutaraldehyde was carried out and its stability was analyzed. Although native PQQGDH was inactivated within 30 min at a higher temperature than 50 cross-linked PQQGDH retained more than 40% of initial activity even after 30 min of incubation at 54°C. In addition to the increase in thermal stability, cross-linked PQQGDH gained high EDTA tolerance. The stabilization may be achieved by increased the rigidity of PQQGDH holo enzyme conformation.

INTRODUCTION

Among various glucose oxido-reductases, glucose dehydrogenases (GDHs)

possessing pyrroloquinoline quinone (PQQ) as their prosthetic group are being expected to

be a future promising enzyme glucose sensor constituent (Turner et al., 1987; Yokoyama, et

a/,, 1989; Ye et al,, 1993; Sode et al., 1993), considering their negligible effect on the

presence of oxygen in the reaction mixture. Several PQQGDHs have been purified,

characterized and cloned their structural genes (Clenton-Jansen et al., 1988, 1989, 1990).

Due to their highly hydrophobic properties, their tertial structures have not yet been

elucidated. Focusing that PQQGDH shares high homology within their primary structure

but their enzymatic characteristics are strain specific, the authors have been carried out the

protein engineering approach in order to improve several enzymatic properties of PQQGDHs

(Sode et al., 1994, 1995).

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The improvement of thermal stability of enzymes is one of the ultimate goal of

protein engineering and enzyme engineering researches, which promises highly operational

stability together with long term storage stability. There are several approach in order to

improve protein thermal stability. Among several enzyme engineering approach, stabilization

of enzymes by immobilization was successful if the immobilization resulted the increase in

the rigidity of the enzyme, which was achieved by multipoint covalently binding between

enzyme and support materials and by increasing stabilization of subunit (Kozulic et al., 1987;

Fernandez-Lafuente et al., 1995).

In this paper, we carried out the cross-linking chemical modification of Escherichia

coli PQQGDH by glutaraldehyde, and investigated enzyme thermal stability. In addition to

the increase in the thermal stability by cross-linking modification, chemically modified E. coli

PQQGDH gained highly EDTA tolerance, which has been considered to be improved only

by introducing appropriate metal binding site from Type II PQQGDHs.

MATERIALS AND METHODS Bacterial strain and plasmid

Escherichia coli PP2418, the PQQGDH structural gene (gcd) of which disrupted by insertion mutagenesis, was used as the host strain for the expression of E. coli PQQGDH using an expression vector, pTrc99A (Pharmacia, Upsla, Sweden) with the corresponding structural genes inserted (Sode and Sano, 1994). Enzyme purification and cross-linking chemical modification

Purified PQQGDH was prepared following our previous report (Sode and Sano, 1994). The enzyme was incubated in 10 mM potassium phosphate buffer at pH 7.0 containing 0.2 (w/v)% Triton X-100, 5 ~tM PQQ and 10 mM MgCI 2 at 25°C to form the holo-enzyme. The holo-form PQQGDH was then cross-linked in the presence of 1 (w/v)% glutaraldehyde for 30 min at 25°C. This cross-linked holo-enzyme was dialyzed in 10 mM potassium phosphate buffer containing 0.2 (w/v)% Triton X-100 at pH 7.0 for over night at 4°C in order to remove excess glutaraldehyde. Thermoinactivation of cross-linked PQQGDH

The time course of irreversible thermoinactivation was measured by incubating PQQGDHs before and after glutaraldehyde cross-linking in 10 mM potassium phosphate buffer at pH7.0 containing 0.2 (w/v)% Triton X-100 at each temperature. The remaining PQQGDH activity was analyzed described previously (Sode and Sano, 1994), except using a buffer containing 5 mM PQQ and 1 mM MgCl 2 in order to keep holo enzyme status. EDTA tolerance of cross-linked PQQGDH

PQQGDHs before and after glutaraldehyde cross-linking were incubated in the presence of 10 mM EDTA and time course of the residual PQQGDHs activity were determined. PQQODHs activity measurement was carded out as described previously (Sode and Sano, 1994).

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RESULTS AND DISCUSSION

Effect of cross-linking chemical modification on enzymatic activity

Table 1 summarizes the kinetic parameters of PQQGDH before and after

glutaraldehyde cross-linking. By cross-linking modification, Vmax value decreased about to

30% of native PQQGDH. However Km values and substrate specificity were not affected by

cross-linking modification.

Table 1. Kinetic Parameters of Native and Cross-linked PQQGDHs

Substrate Native PQQGDH

Km a Vmax b Vmax/Km c %

Cross-linked PQQGDH

Km a Vmax b Vmax/Km c %

D-Glucose 1.3 24 18.2 100 1.5 7.7 5.1 100

2-deoxy-D-Glucose 1.7 27 15.7 87 2 9.4 4.7 92

Mannose 28 25 0.9 5 31.3 8.6 0.27 5

D-Xylose 11 16 0.7 4 N.D. d N.D. d N.D. d N.D. d

Maltose 6 14 0.4 2 N.D. d N.D. d N.D. d N.D. d

amM.

bu.mg(protein) -1.

Cu.mg(protein)-l.mM-1.

dnot detected below a substrate concentration of 50 mM.

When the cross-linking modification was carried out with apo-enzyme, the

reconstitution of holo-enzyme after chemical modification was not succeeded (results were

not shown). This was possibly due to the formation of rigid conformation of apo-enzyme by

cross-linking which resulted holo-enzyme formation difficult. Therefore, in the further

experiments, we carried out cross-linking modification of PQQGDH after holo-enzyme

formation.

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Increase in thermal stability

Figure la and lb show the time of thermal inactivation of native (la) and cross-linked

(lb) PQQGDH. Native PQQGDH readily inactivated at higher temperature than 50 °(2

within 30 rain. Native PQQGDH showed less than 5% of initial activity within 10 rain of

inactivation at 54 °C. In contrast, cross-linked PQQGDH showed higher thermal stability

than native PQQGDH. Cross-linked PQQGDH retained more than 40% of initial activity

even after 30 min of incubation at 54 °C. These results suggested that the thermal stability of

PQQGDH was greatly enhanced by cross-linking chemical modification. We assumed that

this thermal stabilization may be achieved by the increase of rigidity via glutaraldehyde cross-

linking.

100 lOO!

80 80

= 60 ~ 60

~ 4 0 ~ < 40

20 20

0 0 0 5 10 15 20 25 30

Time(min)

0 5 10 15 20 25 30 Time (min)

Fig, 1. Time courses for the thermoinactivation of native PQQGDH (a) and crosslinked PQQGDH(b) at each temperature. Enzyme activity was determined at 25°C after 1 min of incubation at 4°C. At: Enzyme activity at each time, Ao: Initial enzyme activity. I , r-l; 48oc: O, O; 50°C: &, A; 52°C: ,e,, <>; 54°c.

EDTA tolerance

Figure 2 shows the time course of PQQGDH inactivation in the presence of 10 mM

EDTA. Escherichia coli PQQGDH is known to be reversibly inactivated in the presence of

EDTA (Clenton-Jansen et al., 1988, 1990). As is shown in Figure 2, native E. coli

PQQGDH readily inactivated in the presence of l0 mM EDTA, and after 15 min of

incubation less than 5% of initial activity was detected. In contrary, cross-linked PQQGDH

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showed high EDTA tolerance. More than 60% of initial activity remained after 15 min of

incubation. Therefore, cross-linked PQQGDH is highly stable toward EDTA treatment.

o

'°° L 80

0 0 0 0 0 0

60 - 0 0 0 0 0 0 0 0

40

21 -wmmwmmm 0 5 10 15 20

Time (rain)

Fig. 2. EDTA tolerance of native and crosslinked PQQGDHs. Enzyme activity was determined at 25 °C. At; Enzyme activity at each time after adding EDTA. Ao; Initial enzyme activity. O; Crosslinked PQQGDH: II Native PQQGDH. EDTA concentration was l0 mM.

We previously reported the elucidation of the region responsible for EDTA tolerance

in Acinetobacter calcoaceticus PQQGDH (Kozulic et al., 1987), which is known to possess

high EDTA tolerance. PQQGDH inactivation procedure in the presence of EDTA is

believed to be due to the chelate of bivalem metals, such as Ca 2+ and Mg 2+, which are

essential for holo-enzyme formation in the presence of PQQ. Therefore, it might be logical

that the increase in co-factor binding ability of E. coli PQQGDH will be achieved by

increasing metal binding ability by appropriate site directed mutagenesis at the region

responsible for EDTA tolerance or by introducing A. calcoaceticus PQQGDH in E. coil

PQQGDH by constructing chimeric enzymes. However by cross-linking chemical

modification, E. coli PQQGDH EDTA tolerance was greatly increased. This result indicated

that the increase in EDTA tolerance will be achieved not only by introducing the region

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responsible for EDTA tolerance bivalent metal binding. Matsushita, et al. reported that

during PQQGDH holo-enzyme formation, a drastical conformational change occurs

(Matsushita et al., 1995). Therefore, the increased EDTA tolerance of E. coli PQQGDH by

cross-linking chemical modification may be achieved by the increase of rigidity of holo-

enzyme conformation.

In conclusion, by cross-linking chemical modification, E. coli PQQGDH gained high

thermal stability together with EDTA tolerance. This success is of both practical and

theoretical interest. Thus prepared modified PQQGDH will be readily utilized for enzyme

glucose sensor constituent with high operational stability. Recently, Ghosh, et al. proposed

that PQQGDH might possess a structural motif, I]-propeller motif, as PQQ methanol

dehydrogenase of which tertial structure was elucidated (Ghosh et al., 1995). Therefore, the

fact that cross-linked PQQGDH gained both thermal stability and EDTA tolerance will

indicate a protein engineering strategy in enhancing the stability of not only PQQGDH but

also other ~i-propeller proteins, by increasing the rigidity of their typical conformations.

REFERENCES

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