- Email: [email protected]
Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors
[J. Ferment. Technol., Vol. 66, No. 3, 267-272. 1988]
Properties of Glucose-Dehydrogenase-Poly(Ethylene Glycol)-NAD Conjugate as an NADH-Regeneration Unit in Enzyme Reactors AKIO NAKAMURA,HIROKAZUMINAMI,ITARU URABE*, and HIROSUKEOKADA Department of Fermentation Technology,Faculty of Engineering, Osaka University, 2-1 Yamada-oka,Suita-shi, Osaka 565, Japan
Glucose-dehydrogenase-poly(ethyleneglycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the Vmaxvalue. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26 s-1 (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GIcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s-t (as a tetramer), the recycling rate of the active NAD moiety is 0.54s -1, and the apparent Km value for glucose is 24 raM. KineticaUy, lactate dehydrogenase behaves like a substrate with an apparent Km value of 1.8 units.m1-1 in this coupled reaction system with low coenzyme concentration. L-Lactate was continuously produced from pyrnvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDFI-PEG-NAD and lactate detwdrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.
Several kinds o f c o v a l e n t l y l i n k e d d e h y d r o g e n a s e - N A D conjugates h a v e b e e n prep a r e d , 1-*) a n d these conjugates c a n be used as N A D ( H ) - r e g e n e r a t i o n units for a c o u p l e d s e c o n d - e n z y m e reaction.2,*, 5) S u c h conjugates a r e a t t r a c t i v e c a t a l y t i c units not o n l y because t h e y h a v e p o t e n t i a l s for a p p l i c a t i o n as a n N A D ( H ) - r e g e n e r a t i o n unit, b u t also beeause t h e y p r o v i d e m a n y i n t e r e s t i n g questions a b o u t t h e effects o f fixing a r e a d i l y dissociable c o e n z y m e in the v i c i n i t y of a n enzyme. Glucose d e h y d r o g e n a s e (EC 1.1.1.47) catalyzes the o x i d a t i o n of B-glucose to uglucono-l,5-1actone, w h i c h is s p o n t a n e o u s l y h y d r o l y z e d to g l u c o n i c acid, using N A D or * Corresponding author
N A D P as a coenzyme. As the e q u i l i b r i u m o f the overall r e a c t i o n is m u c h in favor o f N A D ( P ) H f o r m a t i o n , this e n z y m e is useful as a n N A D ( P ) H r e g e n e r a t o r in e n z y m e reactors.6) Glucose d e h y d r o g e n a s e , however, has v e r y low a c t i v i t y for N A D ( P ) d e r i v a t i v e s ; v,s) the r e d u c t i o n r a t e for p o l y ( e t h y l e n e glycol)-bound NAD (PEG-NAD), a macrom o l e c u l a r N A D d e r i v a t i v e for e n z y m e reactors, 9,1°) is o n l y 0 . 0 8 % o f t h a t o f n a t i v e NAD.s) R e c e n t l y we h a v e p r e p a r e d a g l u c o s e - d e h y d r o g e n a s e - p o l y ( e t h y l e n e glycol)NAD conjugate (GlcDH-PEG-NAD) by c o v a l e n t l y l i n k i n g P E G - N A D to glucose d e h y d r o g e n a s e , a n d f o u n d t h a t the c o n j u g a t e has a m u c h h i g h e r r e a c t i o n r a t e t h a n t h a t o f t h e n a t i v e e n z y m e plus P E G - N A D ; the a c t i v i t y o f the c o n j u g a t e is even h i g h e r t h a n
NAKAMURAet al.
268
that of the native enzyme plus NAD.8) In this work, we have investigated the k i n e t i c p r o p e r t i e s o f G l c D H - P E G - N A D as a n N A D H - r e g e n e r a t i o n u n i t . T h i s r e p o r t also shows that the conjugate can be used in a continuous enzyme reactor.
Materials and Methods Materials Bacillus megateriura glucose dehydrogenase, B. stearothermophilus malate dehydrogenase (EC 1.1.1.37), and Thermus thermophilus malate dehydrogenase were generous gifts from Amano Pharmaceutical Co. Ltd. (Nagoya), Unitika Ltd. (Osaka), and Mitsubishi Petrochemical Co. Ltd. (Tokyo), respectively. Yeast alcohol dehydrogenase (EC 1.1.1.1), rabbit muscle lactate dehydrogenase (EC 1.1.1.27), and beef liver glutamate dehydrogenase (EC 1.4.1.3) were purchased from Oriental Yeast Co. Ltd. (Tokyo); horse liver alcohol dehydrogenase and pig heart malate dehydrogenase were from Boehringer (Mannheim); 5-ethylphenazinium ethyl sulfate (PES) was from Nakarai Chemicals (Kyoto); 3-(4',5'-dimethylthiazol2-yl)-2,5-diphenyhetrazolium bromide (MTT) was from Dojindo Laboratories (Kumamoto). NAD was a generous gift from Kojin Co. Ltd. (Tokyo). PEGNAD and GlcDH-PEG-NAD were prepared as described in Refs. 9 and 3, respectively. Protein and nucleotide concentrations The concentrations of NAD(H), their derivatives, and glucose dehydrogenase were measured using the following molar absorption coefficients: NAD, 18000 lk~-1 em -1 at 260 nm; PEG-NAD, 25000 M -1 cm -1 at 266 nm;~) NADH and PEG-NADH, 6300 M - l o r e -1 at 340nm; and the subunit of glucose dehydrogenase, 35000 M -1 cm -1 at 280 rim. s) The concentrations of PEGNAD and the enzyme subunit in GlcDH-PEG-NAD, and the fraction of reducible NAD in conjugate were measured as described previously. 3) The concentrations of glucose dehydrogenase and GlcDH-PEGNAD were expressed as subunit concentrations. Enzyme assay Enzyme reactions were measured at 30°C with a Hitachi 220A spectrophotometer. The reactions were recorded as the increase or decrease in absorbance at 340 nm, or the increase in absorbance at 570rim (a tetrazolium salt method); 2) the concentration of the formazan produced from the reduction of MTT was measured using a molar absorption coefficient of 13000M-Xcm -1 at 570nm. 11) The activity of glucose dehydrogenase and GlcDH-PEGNAD in the presence of exogenous NAD was assayed in 75 m M Tris/HC1 buffer, pH 8.0, containing 0.1 M D-glucose and 2.0 m M NAD, and the reactions were recorded as the increase in absorbanee at 340 nm.
[J. Ferment. Technol.,
When the kinetic constants for exogenous NAD were measured, the concentration of NAD was varied from 0.2 to 1.0 raM. The activity of the enzyme moiety of GIcDH-PEG-NAD toward the bound NAD moiety (internal activity) e) was measured in 20 m M Tris/FICl buffer, pH 8.0, containing 1.0 M NaCI, 0.1 M nglucose, 20raM PES, and 1.7ram MTT, and the reactions were recorded as the increase in absorbance at 570 nm. The components of reaction mixtures for the assay of coenzyme activity and coupled reactions are given in the legends to Table 1 and Fig. 2, respectively. The coupled reaction of GlcDH-PEGNAD with lactate dehydrogenase was followed by the increase in the L-lactate concentration, which was measured enzymically using NAD and lactate dehydrogenase. 12) One unit of lactate dehydrogenase is defined as catalyzing the oxidation of one /zmol of NADH per rain in 80 m M Tris/HC1 buffer, pH 7.2, containing 0.2 M NaCl, 1.6 m M pyruvate, and 0.2 m M NADH. Continuous enzyme reaction GIcDFI-PEGNAD (1.77/tM) and lactate dehydrogenase (22.6 units.ml-x) were kept in a model 8MC ultrafiltration apparatus (Amicon Corp., Lexington, MA) fitted with a PM10 ultrafiltration membrane. Continuous enzyme reactions were done at 30°C by continuous ultra filtration with substrate solution containing 15 m M Tris/HC1 buffer, pH 8.0, 0.75 M NaC1, 50 m M D-glucose, and 5 m M pyruvate as the eluent under N2 pressure; the volume of the reaction mixture was kept at 7 ml. The flow rate of the substrate solution was adjusted to 2.5ml'h -1. The filtrate was collected in fractions at 1-h intervals and the L-lactate concentration and volume of each fraction were measured.
Results
and Discussion
Characterization
of GlcDH-PEG-NAD
P E G - N A D w a s c o v a l e n t l y l i n k e d to g l u c o s e dehydrogenase by the procedures described p r e v i o u s l y , s) G l c D H - P E G - N A D , thus prep a r e d , h a s a l o n g ( a b o u t 25 n m ) , f l e x i b l e , and hydrophilic spacer of poly(ethylene glycol) b e t w e e n the e n z y m e a n d N A D . The average number of NAD moieties bound per molecule of enzyme subunit (NAD c o n t e n t ) 2) is 0.66, w h i c h is a b o u t o n e t h i r d of that of GlcDH-PEG-NAD prepared previously, s) About 80% of the bound NAD m o i e t i e s is r e d u c e d b y its e n z y m e m o i e t y i n t h e p r e s e n c e o f D-glucose. T h i s m e a n s t h a t a tetramer of GlcDH-PEG-NAD has about two molecules of active and covalently linked
Vol. 66, 1988]
NADH-R.egencrafion Unit
NAD. The specific activity of the enzyme moiety of GlcDH-PEG-NAD in the presence of exogenous NAD is about 77% of that of the native enzyme. When the activity of the conjugate was measured at different concentrations of exogenous NAD, a linear reciprocal plot was obtained (data not shown), and the K= and Vmax values were calculated to be 0 . 7 6 m M and 185 kat.mo1-1, respectively. The Km value is similar to that of the native enzyme (0.94mM), and the V=** value is about 70% of that of the native one (260kat.mol-1). These results indicate that the NAD(H) moiety of the conjugate does not compete with exogenous NAD for the coenzyme-binding site of the enzyme moiety, and that the covalent linking causes a slight loss in the activity of the enzyme moiety. Figure 1 shows the effects of p H on the stability of GlcDH-PEG-NAD and the native enzyme. As they have the same pHstability profile, the linking of PEG-NAD does not seem to affect the stability of the enzyme. The stability of the conjugate in the alkaline region is increased by the I
I
I
•
I
I
I
1oo
? 80
addition of NaC1; the enzyme is stable at p H 8.0 in the presence of 1.0 M NaCI (data are not shown). The activity of the enzyme moiety of GlcDH-PEG-NAD toward the bound NAD moiety was measured by the coupled redox system of PES and M T T (internal activity).2,a7 The value of the internal activity is 0.26s-1; this is comparable to that obtained for t h e previous preparation (1.18 s-1) if account is taken of the differences in the NAD content and the specific activity of the enzyme moiety. The coenzyme activity of the NAD moiety of GlcDH-PEG-NAD was measured by the tetrazolium salt method, and the results are shown in Table I. GlcDH-PEG-NAD has similar coenzyme activity to PEG-NAD except for B. stearothermophilus malate dehydrogenase. This means that the enzyme moiety of the conjugate does not affect the activity of the NAD moiety. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. Coupled reaction of GIcDH-PEG-NAD with lactate dehydrogenase The re-
action of glucose dehydrogenase in GlcDHPEG-NAD and that of lactate dehydrogenase were coupled by the recycling of the NAD moiety of the conjugate : lactate dehydrogenase
.>
pyruvate f
~ so c
GlcDH PEG N A D H
"E 40
'~ 20 o
269
D.glucono.
J i 4
,~"~
~ N h " L lactate GIcDH PEG NAD
J
D.glucose
1,5-1actonc 5
6
7
8
9
The couplcd rcaction was followcd by thc increase in the L-lactate concentration. As Fig. 1. Effectsof pH on the stability of GlcDH-PEG- a control systcm, the coupled reaction of NAD. GlcDH-PEG-NAD (O) or native glucose dehydrogenase (Q) was incubated at 25°C for glucose dchydrogcnasc, lactatc dehydroge30min in the following buffer solutions: 0.2 M nase, and N A D was also done. Figure 2 acetate buffer, pH4.0--5.5; 0.21V£ phosphate shows that thc rcactions proceed until almost buffer, pH 6.0-7.5; 0.2 M Tris/HC1 buffer, all pyruvatc is convertcd to L-lactate; this pH 8.0-9.0. The remaining activity was measured mcans that thc cquilibrium of the ovcrall as described uner Materials and Methods. rcaction lics much in favor of L-lactate pH
270
Nnr~a~n~ et al.
[J. Ferment. Technol.,
Table 1. Coenzyme activity of PEG-NAD and GlcDH-PEG-NAD relative to NAD. Relative rate (NAN= 100) . . . . . . . . . PEG-NAD GlcDH-PEG-NAD
Enzyme Alcohol dehydrogenase (yeast) Alcohol dehydrogenase (horse liver) Lactate dehydrogenase (rabbit muscle) Glutamate dehydrogenase (beef liver) Malate dehydrogenase (pig heart) Malate dehydrogenase (B. stearothermophilus) Malate dehydrogenase (7". thermophilus)
47 41 301 21 69 83 83
36 42 358 18 69 26 72
Activity was assayed in 20 mM Tris/HC1 buffer, pH 8.0, containing 1.0 M NaC1, 10 mM PES, 1.7 mM MTT, 0.39/tM coenzyme (NAD, PEG-NAD, or the NAD moiety of GlcDH-PEG-NAD), and the following substrates: for alcohol dehydrogenase, 0.1 M ethanol; for lactate dehydrogenase, 0.1 IV[ D,L-lactate; for glutamate dehydrogenase, 0.1 M L-glutamate; for malate dehydrogenase, 0.1 M L-malate. Values were expressed relative to the reaction rates for native NAD. The rates for native NAD: 10.6, 3.27, 25.3, 17.4, 30.9, 160, and 110 nM's-x for yeast alcohol dehydrogenase, horse liver alcohol dehydrogenase, rabbit muscle lactate dehydrogenase, beef liver glutamate dehydrogenase, pig heart malate dehydrogenase, B. stearothermophilus malate dehydrogenase, and 7". thermophilus malate dehydrogenase, respectively. formation. T h e i n i t i a l r e a c t i o n rate (1.8 raM-h-a) for the system of G l c D H - P E G - N A D is c o m p a r a b l e to that for the control system i
i
1
2
i
i
4
I 5
5
0
3
T i m e (h)
Fig. 2. Coupled reaction of lactate dehydrogenase and glucose dehydrogenase. The coupled reactions were done at 30°C in 15 mM Tris/HC1 buffer, pH 8.0, containing 0.75 M NaC1, 5 mM pyruvate, 50 mM D-glucose, 22.6 units-ml-x lactate dehydrogenase, and 1.77/~M GlcDH-PEG-NAD (O) or the mixture of 1.77/~M glucose dehydrogenase and 1.17/~M NAN (0). The coupled reaction for GlcDH-PEG-NAD was also done in the presence of 50% glycerol (A); in this case, the other conditions were the same as above. Samples (each 0.2 ml) were taken at the indicated time, boiled for 5 min, and the L-lactate concentration was measured enzymically.aS)
( 2 . 3 m M - h - 1 ) . F r o m the results shown i n Fig. 2, the specific activity of the t e t r a m e r of G l c D H - P E G - N A D is calculated to be I. 1 s-1, a n d the recycling rate of the active N A D m o i e t y of the conjugate to be 0.54 s-1. T h e v a l u e of the specific activity is almost the same as that of the i n t e r n a l activity; this m e a n s that the a m o u n t of lactate dehydrogenase is e n o u g h i n this system as will be described later. It was confirmed i n the control system that the r e a c t i o n does not proceed w i t h o u t a d d i n g N A D , a n d it should be noted that the activity of glucose dehydrogenase for P E G - N A D is too low to be detected u n d e r the reaction conditions of the control system. 8) These results i n d i c a t e that the N A D moiety of G l c D H - P E G - N A D is, due to the a n c h i m e r i c assistance, 3) efficiently recycled by the reactions of the active site i n the same conjugate a n d of lactate dehydrogenase. Therefore, G l c D H - P E G - N A D works as a n efficient N A D H - r e g e n e r a t i o n u n i t for a coupled second e n z y m e reaction. F i g u r e 3 shows the effects of glucose concentration. T h e Km v a l u e for the system of G l c D H - P E G - N A D is 24 raM, a n d is a little larger t h a n that for the control system
Vol. 66, 1988]
NADH-Regeneration Unit
271
1.0
2.5
0.S
0.6
2.0
0.4 0.2
'~ 1.5 E
t
I
I
I
I
I
I
1
2
4
6
8
10
12
14
16
Time
> 1.0
(h)
Fig. 4. Continuous conversion of pyruvate to Llactate in an enzyme reactor containing GlcDHPEG-NAD and lactate dehydrogenase.
0.5
I
0
I
I
0.1 11 [ G l u c o s e ]
I
0.2 (mM -l)
Fig. 3. Effects of glucose concentration on the rate of the coupled reaction for the systems of GlcDHPEG-NAD (©) and glucose dehydrogenase+ NAD (0). The components of the reaction mixture were the same as those described in the legend to Fig. 2. The reaction rates were measured by the increase in L-lactate concentration.
(18 raM). T h e effects of lactate dehydrogenase concentration on the reaction rate of the control system were studied, and a linear reciprocal plot was also obtained with an apparent Km value for the lactate dehydrogenase concentration of 1.Sunits.ml-1. This means that kinetically lactate dehydrogenase behaves like a substrate in these coupled reaction systems with low coenzyme concentration, and the lactate dehydrogenase concentration used in the coupled reaction shown in Fig. 2 is a saturation level. These results provide informations for selecting appropriate conditions for the coupled reactions. To demonstrate the usefulness of GlcDHP E G - N A D for enzyme reactors, continuous production of L-lactate from pyruvate was done in a reactor containing G l c D H - P E G N A D and lactate dehydrogenase. Figure 4 shows the results of the continuous operation. In this reactor, we used a PM10 ultrafiltration m e m b r a n e ( M r cut-off, 10000) whose pore
size is much larger than that of YM2 ( M r cut-off, 1000) used for the retention of PEG-NAD,10) and in this experiment we reduced the flow rate by adding 50% glycerol. About 12 h after the start of the reaction, the concentration of L-lactate became constant at about 1.1 m M , but the steady-state concentration of L-lactate was much lower than expected. We found that this low value is due to the presence of 50% glycerol; as shown in Fig. 2, the reaction rate in the presence of glycerol is about 15% of that in the absence of glycerol. These results for continuous reactions are still preliminary, but they show that G l c D H - P E G - N A D is applicable in enzyme reactors as an N A D H regeneration unit having a large molecular size. Acknowledgments This work was supported in part by Grants-in-Aid (62603001 for Scientific Research on Priority Area and 63850191) from the Ministry of Education, Science and Culture, Japan.
References 1) M~nsson, M.-O., Larsson, P.-O., Mosbach, K.: Eur. J. Biochem., 86, 455 (1978). 2) Eguchi, T., Iizuka, T., Kagotani, T., Lee, J . H . , Urabe, I., Okada, H.: Eur. J. Biochem., 155, 415 (1986). 3) Nakamura, A., Urabe, I., Okada, H.: J. Biol. Chem., 261, 16792 (1986). 4) Kato, N., Yamagami, T., Shimao, IV[., Sakazawa, C.: Appl. Microb. Bioteehnol., 25, 415 (1987). 5) M~msson, M.-O., Larsson, P.-O., Mosbach, K.:
272
NAKAMURAet al.
F E B S Lett., 98, 309 (1979). 6) Vandecasteele, J.-P.: Appl. Environ. Microbiol., 39, 327 (1980). 7) Okuda, K., Urabe, I., Okada, H.: Eur. J. Biochem., 147, 249 (1985). 8) Okuda, K., Urabe, I., Okada, H.: Eur. J. Biochem., 151, 33 (1985). 9) Katayama, N., Urabe, I., Okada, H.: Eur. J. Biochem., 132, 403 (1983). 10) Hayakawa, K., Urabe, I., Okada, H.: J. Ferment.
Technol., 63, 245 (1985). 11) Eguchi, T., Kanzald, N., Kagotani, T., Taniguchi, T., Urabe, I., Okada, H.: J. Ferment. Teehnol., 63, 563 (1985). 12) Gutman, I., Wahlefeld, A.W.: Methods o9¢ Enzymatic Analysis, (Bergmayer, H.U.), 2nd ed., Vol. 3, 1454, Academic Press, New York (1974).
(Received January 18, 1988)
Properties of Glucose-Dehydrogenase-Poly(Ethylene Glycol)-NAD Conjugate as an NADH-Regeneration Unit in Enzyme Reactors AKIO NAKAMURA,HIROKAZUMINAMI,ITARU URABE*, and HIROSUKEOKADA Department of Fermentation Technology,Faculty of Engineering, Osaka University, 2-1 Yamada-oka,Suita-shi, Osaka 565, Japan
Glucose-dehydrogenase-poly(ethyleneglycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the Vmaxvalue. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26 s-1 (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GIcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s-t (as a tetramer), the recycling rate of the active NAD moiety is 0.54s -1, and the apparent Km value for glucose is 24 raM. KineticaUy, lactate dehydrogenase behaves like a substrate with an apparent Km value of 1.8 units.m1-1 in this coupled reaction system with low coenzyme concentration. L-Lactate was continuously produced from pyrnvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDFI-PEG-NAD and lactate detwdrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.
Several kinds o f c o v a l e n t l y l i n k e d d e h y d r o g e n a s e - N A D conjugates h a v e b e e n prep a r e d , 1-*) a n d these conjugates c a n be used as N A D ( H ) - r e g e n e r a t i o n units for a c o u p l e d s e c o n d - e n z y m e reaction.2,*, 5) S u c h conjugates a r e a t t r a c t i v e c a t a l y t i c units not o n l y because t h e y h a v e p o t e n t i a l s for a p p l i c a t i o n as a n N A D ( H ) - r e g e n e r a t i o n unit, b u t also beeause t h e y p r o v i d e m a n y i n t e r e s t i n g questions a b o u t t h e effects o f fixing a r e a d i l y dissociable c o e n z y m e in the v i c i n i t y of a n enzyme. Glucose d e h y d r o g e n a s e (EC 1.1.1.47) catalyzes the o x i d a t i o n of B-glucose to uglucono-l,5-1actone, w h i c h is s p o n t a n e o u s l y h y d r o l y z e d to g l u c o n i c acid, using N A D or * Corresponding author
N A D P as a coenzyme. As the e q u i l i b r i u m o f the overall r e a c t i o n is m u c h in favor o f N A D ( P ) H f o r m a t i o n , this e n z y m e is useful as a n N A D ( P ) H r e g e n e r a t o r in e n z y m e reactors.6) Glucose d e h y d r o g e n a s e , however, has v e r y low a c t i v i t y for N A D ( P ) d e r i v a t i v e s ; v,s) the r e d u c t i o n r a t e for p o l y ( e t h y l e n e glycol)-bound NAD (PEG-NAD), a macrom o l e c u l a r N A D d e r i v a t i v e for e n z y m e reactors, 9,1°) is o n l y 0 . 0 8 % o f t h a t o f n a t i v e NAD.s) R e c e n t l y we h a v e p r e p a r e d a g l u c o s e - d e h y d r o g e n a s e - p o l y ( e t h y l e n e glycol)NAD conjugate (GlcDH-PEG-NAD) by c o v a l e n t l y l i n k i n g P E G - N A D to glucose d e h y d r o g e n a s e , a n d f o u n d t h a t the c o n j u g a t e has a m u c h h i g h e r r e a c t i o n r a t e t h a n t h a t o f t h e n a t i v e e n z y m e plus P E G - N A D ; the a c t i v i t y o f the c o n j u g a t e is even h i g h e r t h a n
NAKAMURAet al.
268
that of the native enzyme plus NAD.8) In this work, we have investigated the k i n e t i c p r o p e r t i e s o f G l c D H - P E G - N A D as a n N A D H - r e g e n e r a t i o n u n i t . T h i s r e p o r t also shows that the conjugate can be used in a continuous enzyme reactor.
Materials and Methods Materials Bacillus megateriura glucose dehydrogenase, B. stearothermophilus malate dehydrogenase (EC 1.1.1.37), and Thermus thermophilus malate dehydrogenase were generous gifts from Amano Pharmaceutical Co. Ltd. (Nagoya), Unitika Ltd. (Osaka), and Mitsubishi Petrochemical Co. Ltd. (Tokyo), respectively. Yeast alcohol dehydrogenase (EC 1.1.1.1), rabbit muscle lactate dehydrogenase (EC 1.1.1.27), and beef liver glutamate dehydrogenase (EC 1.4.1.3) were purchased from Oriental Yeast Co. Ltd. (Tokyo); horse liver alcohol dehydrogenase and pig heart malate dehydrogenase were from Boehringer (Mannheim); 5-ethylphenazinium ethyl sulfate (PES) was from Nakarai Chemicals (Kyoto); 3-(4',5'-dimethylthiazol2-yl)-2,5-diphenyhetrazolium bromide (MTT) was from Dojindo Laboratories (Kumamoto). NAD was a generous gift from Kojin Co. Ltd. (Tokyo). PEGNAD and GlcDH-PEG-NAD were prepared as described in Refs. 9 and 3, respectively. Protein and nucleotide concentrations The concentrations of NAD(H), their derivatives, and glucose dehydrogenase were measured using the following molar absorption coefficients: NAD, 18000 lk~-1 em -1 at 260 nm; PEG-NAD, 25000 M -1 cm -1 at 266 nm;~) NADH and PEG-NADH, 6300 M - l o r e -1 at 340nm; and the subunit of glucose dehydrogenase, 35000 M -1 cm -1 at 280 rim. s) The concentrations of PEGNAD and the enzyme subunit in GlcDH-PEG-NAD, and the fraction of reducible NAD in conjugate were measured as described previously. 3) The concentrations of glucose dehydrogenase and GlcDH-PEGNAD were expressed as subunit concentrations. Enzyme assay Enzyme reactions were measured at 30°C with a Hitachi 220A spectrophotometer. The reactions were recorded as the increase or decrease in absorbance at 340 nm, or the increase in absorbance at 570rim (a tetrazolium salt method); 2) the concentration of the formazan produced from the reduction of MTT was measured using a molar absorption coefficient of 13000M-Xcm -1 at 570nm. 11) The activity of glucose dehydrogenase and GlcDH-PEGNAD in the presence of exogenous NAD was assayed in 75 m M Tris/HC1 buffer, pH 8.0, containing 0.1 M D-glucose and 2.0 m M NAD, and the reactions were recorded as the increase in absorbanee at 340 nm.
[J. Ferment. Technol.,
When the kinetic constants for exogenous NAD were measured, the concentration of NAD was varied from 0.2 to 1.0 raM. The activity of the enzyme moiety of GIcDH-PEG-NAD toward the bound NAD moiety (internal activity) e) was measured in 20 m M Tris/FICl buffer, pH 8.0, containing 1.0 M NaCI, 0.1 M nglucose, 20raM PES, and 1.7ram MTT, and the reactions were recorded as the increase in absorbance at 570 nm. The components of reaction mixtures for the assay of coenzyme activity and coupled reactions are given in the legends to Table 1 and Fig. 2, respectively. The coupled reaction of GlcDH-PEGNAD with lactate dehydrogenase was followed by the increase in the L-lactate concentration, which was measured enzymically using NAD and lactate dehydrogenase. 12) One unit of lactate dehydrogenase is defined as catalyzing the oxidation of one /zmol of NADH per rain in 80 m M Tris/HC1 buffer, pH 7.2, containing 0.2 M NaCl, 1.6 m M pyruvate, and 0.2 m M NADH. Continuous enzyme reaction GIcDFI-PEGNAD (1.77/tM) and lactate dehydrogenase (22.6 units.ml-x) were kept in a model 8MC ultrafiltration apparatus (Amicon Corp., Lexington, MA) fitted with a PM10 ultrafiltration membrane. Continuous enzyme reactions were done at 30°C by continuous ultra filtration with substrate solution containing 15 m M Tris/HC1 buffer, pH 8.0, 0.75 M NaC1, 50 m M D-glucose, and 5 m M pyruvate as the eluent under N2 pressure; the volume of the reaction mixture was kept at 7 ml. The flow rate of the substrate solution was adjusted to 2.5ml'h -1. The filtrate was collected in fractions at 1-h intervals and the L-lactate concentration and volume of each fraction were measured.
Results
and Discussion
Characterization
of GlcDH-PEG-NAD
P E G - N A D w a s c o v a l e n t l y l i n k e d to g l u c o s e dehydrogenase by the procedures described p r e v i o u s l y , s) G l c D H - P E G - N A D , thus prep a r e d , h a s a l o n g ( a b o u t 25 n m ) , f l e x i b l e , and hydrophilic spacer of poly(ethylene glycol) b e t w e e n the e n z y m e a n d N A D . The average number of NAD moieties bound per molecule of enzyme subunit (NAD c o n t e n t ) 2) is 0.66, w h i c h is a b o u t o n e t h i r d of that of GlcDH-PEG-NAD prepared previously, s) About 80% of the bound NAD m o i e t i e s is r e d u c e d b y its e n z y m e m o i e t y i n t h e p r e s e n c e o f D-glucose. T h i s m e a n s t h a t a tetramer of GlcDH-PEG-NAD has about two molecules of active and covalently linked
Vol. 66, 1988]
NADH-R.egencrafion Unit
NAD. The specific activity of the enzyme moiety of GlcDH-PEG-NAD in the presence of exogenous NAD is about 77% of that of the native enzyme. When the activity of the conjugate was measured at different concentrations of exogenous NAD, a linear reciprocal plot was obtained (data not shown), and the K= and Vmax values were calculated to be 0 . 7 6 m M and 185 kat.mo1-1, respectively. The Km value is similar to that of the native enzyme (0.94mM), and the V=** value is about 70% of that of the native one (260kat.mol-1). These results indicate that the NAD(H) moiety of the conjugate does not compete with exogenous NAD for the coenzyme-binding site of the enzyme moiety, and that the covalent linking causes a slight loss in the activity of the enzyme moiety. Figure 1 shows the effects of p H on the stability of GlcDH-PEG-NAD and the native enzyme. As they have the same pHstability profile, the linking of PEG-NAD does not seem to affect the stability of the enzyme. The stability of the conjugate in the alkaline region is increased by the I
I
I
•
I
I
I
1oo
? 80
addition of NaC1; the enzyme is stable at p H 8.0 in the presence of 1.0 M NaCI (data are not shown). The activity of the enzyme moiety of GlcDH-PEG-NAD toward the bound NAD moiety was measured by the coupled redox system of PES and M T T (internal activity).2,a7 The value of the internal activity is 0.26s-1; this is comparable to that obtained for t h e previous preparation (1.18 s-1) if account is taken of the differences in the NAD content and the specific activity of the enzyme moiety. The coenzyme activity of the NAD moiety of GlcDH-PEG-NAD was measured by the tetrazolium salt method, and the results are shown in Table I. GlcDH-PEG-NAD has similar coenzyme activity to PEG-NAD except for B. stearothermophilus malate dehydrogenase. This means that the enzyme moiety of the conjugate does not affect the activity of the NAD moiety. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. Coupled reaction of GIcDH-PEG-NAD with lactate dehydrogenase The re-
action of glucose dehydrogenase in GlcDHPEG-NAD and that of lactate dehydrogenase were coupled by the recycling of the NAD moiety of the conjugate : lactate dehydrogenase
.>
pyruvate f
~ so c
GlcDH PEG N A D H
"E 40
'~ 20 o
269
D.glucono.
J i 4
,~"~
~ N h " L lactate GIcDH PEG NAD
J
D.glucose
1,5-1actonc 5
6
7
8
9
The couplcd rcaction was followcd by thc increase in the L-lactate concentration. As Fig. 1. Effectsof pH on the stability of GlcDH-PEG- a control systcm, the coupled reaction of NAD. GlcDH-PEG-NAD (O) or native glucose dehydrogenase (Q) was incubated at 25°C for glucose dchydrogcnasc, lactatc dehydroge30min in the following buffer solutions: 0.2 M nase, and N A D was also done. Figure 2 acetate buffer, pH4.0--5.5; 0.21V£ phosphate shows that thc rcactions proceed until almost buffer, pH 6.0-7.5; 0.2 M Tris/HC1 buffer, all pyruvatc is convertcd to L-lactate; this pH 8.0-9.0. The remaining activity was measured mcans that thc cquilibrium of the ovcrall as described uner Materials and Methods. rcaction lics much in favor of L-lactate pH
270
Nnr~a~n~ et al.
[J. Ferment. Technol.,
Table 1. Coenzyme activity of PEG-NAD and GlcDH-PEG-NAD relative to NAD. Relative rate (NAN= 100) . . . . . . . . . PEG-NAD GlcDH-PEG-NAD
Enzyme Alcohol dehydrogenase (yeast) Alcohol dehydrogenase (horse liver) Lactate dehydrogenase (rabbit muscle) Glutamate dehydrogenase (beef liver) Malate dehydrogenase (pig heart) Malate dehydrogenase (B. stearothermophilus) Malate dehydrogenase (7". thermophilus)
47 41 301 21 69 83 83
36 42 358 18 69 26 72
Activity was assayed in 20 mM Tris/HC1 buffer, pH 8.0, containing 1.0 M NaC1, 10 mM PES, 1.7 mM MTT, 0.39/tM coenzyme (NAD, PEG-NAD, or the NAD moiety of GlcDH-PEG-NAD), and the following substrates: for alcohol dehydrogenase, 0.1 M ethanol; for lactate dehydrogenase, 0.1 IV[ D,L-lactate; for glutamate dehydrogenase, 0.1 M L-glutamate; for malate dehydrogenase, 0.1 M L-malate. Values were expressed relative to the reaction rates for native NAD. The rates for native NAD: 10.6, 3.27, 25.3, 17.4, 30.9, 160, and 110 nM's-x for yeast alcohol dehydrogenase, horse liver alcohol dehydrogenase, rabbit muscle lactate dehydrogenase, beef liver glutamate dehydrogenase, pig heart malate dehydrogenase, B. stearothermophilus malate dehydrogenase, and 7". thermophilus malate dehydrogenase, respectively. formation. T h e i n i t i a l r e a c t i o n rate (1.8 raM-h-a) for the system of G l c D H - P E G - N A D is c o m p a r a b l e to that for the control system i
i
1
2
i
i
4
I 5
5
0
3
T i m e (h)
Fig. 2. Coupled reaction of lactate dehydrogenase and glucose dehydrogenase. The coupled reactions were done at 30°C in 15 mM Tris/HC1 buffer, pH 8.0, containing 0.75 M NaC1, 5 mM pyruvate, 50 mM D-glucose, 22.6 units-ml-x lactate dehydrogenase, and 1.77/~M GlcDH-PEG-NAD (O) or the mixture of 1.77/~M glucose dehydrogenase and 1.17/~M NAN (0). The coupled reaction for GlcDH-PEG-NAD was also done in the presence of 50% glycerol (A); in this case, the other conditions were the same as above. Samples (each 0.2 ml) were taken at the indicated time, boiled for 5 min, and the L-lactate concentration was measured enzymically.aS)
( 2 . 3 m M - h - 1 ) . F r o m the results shown i n Fig. 2, the specific activity of the t e t r a m e r of G l c D H - P E G - N A D is calculated to be I. 1 s-1, a n d the recycling rate of the active N A D m o i e t y of the conjugate to be 0.54 s-1. T h e v a l u e of the specific activity is almost the same as that of the i n t e r n a l activity; this m e a n s that the a m o u n t of lactate dehydrogenase is e n o u g h i n this system as will be described later. It was confirmed i n the control system that the r e a c t i o n does not proceed w i t h o u t a d d i n g N A D , a n d it should be noted that the activity of glucose dehydrogenase for P E G - N A D is too low to be detected u n d e r the reaction conditions of the control system. 8) These results i n d i c a t e that the N A D moiety of G l c D H - P E G - N A D is, due to the a n c h i m e r i c assistance, 3) efficiently recycled by the reactions of the active site i n the same conjugate a n d of lactate dehydrogenase. Therefore, G l c D H - P E G - N A D works as a n efficient N A D H - r e g e n e r a t i o n u n i t for a coupled second e n z y m e reaction. F i g u r e 3 shows the effects of glucose concentration. T h e Km v a l u e for the system of G l c D H - P E G - N A D is 24 raM, a n d is a little larger t h a n that for the control system
Vol. 66, 1988]
NADH-Regeneration Unit
271
1.0
2.5
0.S
0.6
2.0
0.4 0.2
'~ 1.5 E
t
I
I
I
I
I
I
1
2
4
6
8
10
12
14
16
Time
> 1.0
(h)
Fig. 4. Continuous conversion of pyruvate to Llactate in an enzyme reactor containing GlcDHPEG-NAD and lactate dehydrogenase.
0.5
I
0
I
I
0.1 11 [ G l u c o s e ]
I
0.2 (mM -l)
Fig. 3. Effects of glucose concentration on the rate of the coupled reaction for the systems of GlcDHPEG-NAD (©) and glucose dehydrogenase+ NAD (0). The components of the reaction mixture were the same as those described in the legend to Fig. 2. The reaction rates were measured by the increase in L-lactate concentration.
(18 raM). T h e effects of lactate dehydrogenase concentration on the reaction rate of the control system were studied, and a linear reciprocal plot was also obtained with an apparent Km value for the lactate dehydrogenase concentration of 1.Sunits.ml-1. This means that kinetically lactate dehydrogenase behaves like a substrate in these coupled reaction systems with low coenzyme concentration, and the lactate dehydrogenase concentration used in the coupled reaction shown in Fig. 2 is a saturation level. These results provide informations for selecting appropriate conditions for the coupled reactions. To demonstrate the usefulness of GlcDHP E G - N A D for enzyme reactors, continuous production of L-lactate from pyruvate was done in a reactor containing G l c D H - P E G N A D and lactate dehydrogenase. Figure 4 shows the results of the continuous operation. In this reactor, we used a PM10 ultrafiltration m e m b r a n e ( M r cut-off, 10000) whose pore
size is much larger than that of YM2 ( M r cut-off, 1000) used for the retention of PEG-NAD,10) and in this experiment we reduced the flow rate by adding 50% glycerol. About 12 h after the start of the reaction, the concentration of L-lactate became constant at about 1.1 m M , but the steady-state concentration of L-lactate was much lower than expected. We found that this low value is due to the presence of 50% glycerol; as shown in Fig. 2, the reaction rate in the presence of glycerol is about 15% of that in the absence of glycerol. These results for continuous reactions are still preliminary, but they show that G l c D H - P E G - N A D is applicable in enzyme reactors as an N A D H regeneration unit having a large molecular size. Acknowledgments This work was supported in part by Grants-in-Aid (62603001 for Scientific Research on Priority Area and 63850191) from the Ministry of Education, Science and Culture, Japan.
References 1) M~nsson, M.-O., Larsson, P.-O., Mosbach, K.: Eur. J. Biochem., 86, 455 (1978). 2) Eguchi, T., Iizuka, T., Kagotani, T., Lee, J . H . , Urabe, I., Okada, H.: Eur. J. Biochem., 155, 415 (1986). 3) Nakamura, A., Urabe, I., Okada, H.: J. Biol. Chem., 261, 16792 (1986). 4) Kato, N., Yamagami, T., Shimao, IV[., Sakazawa, C.: Appl. Microb. Bioteehnol., 25, 415 (1987). 5) M~msson, M.-O., Larsson, P.-O., Mosbach, K.:
272
NAKAMURAet al.
F E B S Lett., 98, 309 (1979). 6) Vandecasteele, J.-P.: Appl. Environ. Microbiol., 39, 327 (1980). 7) Okuda, K., Urabe, I., Okada, H.: Eur. J. Biochem., 147, 249 (1985). 8) Okuda, K., Urabe, I., Okada, H.: Eur. J. Biochem., 151, 33 (1985). 9) Katayama, N., Urabe, I., Okada, H.: Eur. J. Biochem., 132, 403 (1983). 10) Hayakawa, K., Urabe, I., Okada, H.: J. Ferment.
Technol., 63, 245 (1985). 11) Eguchi, T., Kanzald, N., Kagotani, T., Taniguchi, T., Urabe, I., Okada, H.: J. Ferment. Teehnol., 63, 563 (1985). 12) Gutman, I., Wahlefeld, A.W.: Methods o9¢ Enzymatic Analysis, (Bergmayer, H.U.), 2nd ed., Vol. 3, 1454, Academic Press, New York (1974).
(Received January 18, 1988)