T I B T E C H - JUNE 1988 [Vol. 6]

Application of PEG-enzyme and magnetite-PEG-enzyme

conjugates for biotechnological processes Yuji Inada, Katsunobu Takahashi, Takayuki Yoshimoto, Yoh

Kodera, Ayako Matsushima and Yuji Saito

Enzymes can be made soluble and active in organic solvents by chemical modification with an amphipathic macromolecule, polyethylene glycol (PEG). The PEG-enzyme conjugates can also be conjugated to magnetite (Fe304). The magnetic enzymes stably disperse in both organic solvents and aqueous solutions. When lipase is prepared as such a conjugate, it catalyses ester synthesis in organic solvents, and can be readily recovered by magnetic force without loss of enzymic activity. This approach could have a great practical

potential.

Physicochemical and biological pro- perties of proteins can be altered when polyethylene glycol (PEG) is covalently bound to the surface of the molecules. Applications of PEG- protein conjugates to the fields of biomedical and biotechnological processes are potentially numerous (see Box 1 and Refs 1 and 2).

Magnetite PEG-enzyme conjugates For practial use, PEG-enzyme con-

jugates should be readily recover- able. One useful and attractive method is to use magnets to draw out enzymes linked to magnetic materials 43. Magnetic enzymes have been extensively studied in various laboratories: enzymes have been adsorbed to magnetite (Fe304) with glutaraldehyde 44, bound to macro- molecule-coated magnetite 45 and embedded jointly with magnetite to macromolecules 46. In these studies, the average particle sizes were 300- 700 nm, 40-50 nm and 10-100 ~m, respectively. (Particle size dictates how quickly the particles can be recovered.) These magnetic enzyme

The authors are at the Laboratory of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan.

particles 44~6 dispersed in aqueous solution and were recovered by magnetic force.

Recently, a novel magnetic enzyme has been devised which can be dispersed in organic solvents and in aqueous solution. The magnetic enzyme can be recovered from hydrophobic media by magnetic force. Using the same type of magnetic conjugate, protein drugs can be directed to specific sites.

Magnetic lipase (type I) A magnetic PEG-enzyme (MPE)

can be formed by the coprecipitation of ferrous (Fe 2+) and ferric (Fe 3+) ions at pH 8.0-8.5 in the presence of the PEG-lipase conjugates (PE):

P + E---> PE + M ~ MPE (type I)

This produces magnetite particles (M) which are essentially coated with PEG-enzyme conjugates. The magnetite-PEG-lipase conjugate is not dissociated into magnetite and PEG-lipase even by extensive dialy- sis against water, indicating that all three parts of the enzyme are tightly bound. Although the nature of the bonding between magnetite and the PEG-lipase is unclear, there could be coordinate bonds between iron ions

on the surface of magnetite particles and oxygen atoms in PEG. The properties of magnetic lipase are summarized in Table 2.

The magnetic lipase disperses as well in organic solvents such as benzene and 1,1,1-trichloroethane as .it does in aqueous solution. Neither precipitation nor aggregation of magnetic lipase occurred even after a five-day incubation and after centrifugation at 4500 g for 15 rain in benzene. The average particle diameter of magnetic lipase in ben- zene (measured by a submicron particle analyser) was 120 + 60 nm. The size of magnetite was around 2 0 n m as measured by electron micrograph. The magnetic lipase catalysed ester synthetic reactions in organic solvents such as benzene and 1,1,1-trichloroethane. The synthesis of lauryl laurate from lauryl alcohol (0.45 M) and lauric acid (0.45 M) proceeded efficiently in benzene (1.2 ~mol/min/mg protein). In 1,1,1-tri- chloroethane, the rate of reaction was 11.6 gmol/min/mg protein, approxi- mately ten-fold higher than that in benzene.

Recovering the enzyme After the reaction was completed,

magnetic lipase particles could be readily attracted by magnetic force (Fig. 2). No enzymic activity was detected in residual benzene solution after magnetic separation. The mag- netic lipase recovered after dispers- ing these particles had the same ester synthetic activity in benzene as before (Table 3).

Magnetic lipase (type II) Although the magnetic lipase (type

I) disperses very stably in organic solvents, its enzymic activity is relatively low (Table 2). Improved activity can be obtained if the magnetite-PEG conjugate (magnetic modifier, MP) is first synthesized by coprecipitation of ferrous and ferric ions in the presence of 0~,~0- dicarboxymethylpoly(oxyethylene)- PEG 31'32. Free carboxyl groups in the magnetite-PEG conjugate are then activated with N-hydroxysuc- cinimide and dicyclohexylcarbo- dimide 32. The activated magnetic modifier reacts with surface amino groups on enzymes under mild

1988, Elsevier Publications, Cambridge 0167 - 9430/88/$02,00

- - BOX 1

TIBTECH-JUNE 1988 [Vol. 6]

PEG is non-immunogenic and non- toxic. Modification of some proteins (Table la) with PEG reduces their im° munogenicity and immunoreactivity, and prolongs their clearance-time in clinical uses 1. Similarly, PEG-protein conjugates are less susceptible to proteolytic enzymes and to reticulo- endothelial cells than the native en- zymes.

Modification of allergenic protein with PEG also suppresses immuno- globulin E production 1 (Table lb) perhaps because suppressor T cells specific to the antigen are induced. PEG binding also makes enzymes soluble and active in hydrophobic media (Table lc). PEG-lipase and PEG-chymotrypsin are particularly effective: they catalyse reversed hydrolysis reactions to form ester and acid--amide linkages.

The hydrophilic nature of PEG makes it possible to modify enzymes in aqueous solution, and its hydrophobic nature enables modified enzymes to function in a hydrophobic en- vironment. The PEG-enzyme conju- gates are prepared using 2,4-bis(O- methoxypolyethylene glycol)-6-chloro- s-triazine (activated PEG2, Mr 10 000) 1, N-hydroxysuccinimide ester of ~-carboxymet hyl-co-methoxypoly- (oxyethylene) (Mr 4500) 2e and a co- polymer of polyoxyethylene ally- methyldiether and maleic acid an- hydride [poly(PEG-MA anhydride), Mr 13 000] 23.

The advantages of solubilizing en-

Applications of PEG conjugates zyme in hydrophobic media are: (1) the low concentration of water means that reactions can proceed in the reverse direction (not hydrolysis); (2) organic solvent-soluble materials (insoluble in water) can be used as substrates; and (3) since enzymic reactions proceed under quite mild conditions PEG- enzymes can synthesize compounds with double bonds without these being oxidized. For instance, retinyl palmi- tate (or retinyl oleate) synthesized by ester exchange reactions between retinyl acetate and palmitic acid (or oleic acid) using PEG-lipase in ben- zene in air had peroxide values (a measure of oxidation) 5-10% of those of the same compounds produced by organic synthesis under N2 gas 2B. Several new potential applications of PEG-enzymes in organic solvents have recently been reported. The melting temperature of fat and oil can be changed by ester exchange catalysed by PEG-lipase between two triglycerides (with no other organic solvent) 27. With the same enzyme, terpene alcohol esters, used in per- fumery, were obtained (in 81--95% yields) by ester synthesis from terpene

alcohols and fatty acids 2s, while eicosapentaenoyl phosphatidylcholine was prepared by ester exchange from dipaimitoyl phosphatidylcholine and eicosapentaenoic acid 2s. Coupling PEG-cholesterol oxidase and PEG- peroxidase gives the reactions shown in Fig. 1. These took place in trans- parent benzene solution, not an emul- sion. The amount of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phanylenediamine using two PEG- enzymes.

By linking magnetite (Fe304) to PEG- enzymes, the enzymic activity in organic solvents can be recovered by magnetic force (Table ld). Magnetism can also be applied to selectively deliver or target enzymes such as urokinase to affected parts.

Other biologically active substances apart from proteins (Table le) can be conjugated to PEG. This is useful in, for instance prolonging the clearance- time of insulin, sotubilizing heroin and hematoporphyrin in organic solvents and in aqueous solution, and in recovery of ATP and NAD in bio- reactors.

~ 1

holestero1-1- 02 ~ 4-cholesten-3-one -F H202

202 -t- o-phenylenediamine .~ H20 + oxidized o - phenytenediamine

I The coupling of PEG-cholesterol oxidase and PEG.peroxidase.

Table 1. PEG-protein (or bioactive substance) conjugates Substance modified Use Ref.

(a) Reduction of immunoreactivity and increase in dsorance time arginase anti-tumor 3 L-asparaginase anti-tumor 4 batroxobin anti-thrombotic 5 blood coagulation replacement therapy for 6

factors Vlll and IX hemophilia elastase therapy of arteriosclerosis 7 o<-galactosidase enzyme replacement therapy 8 ~-galactosidase enzyme replacement therapy 9 I~-glucoaidase enzyme replacement therapy 8 ~glucuronidaso enzyme replacement therapy 10 hemoglobin artificial blood 11 immunoglobulin G immunogiobulin therapy 12 interleukin 2 ':' anti-tumor, therapy of infectious 13

disease phenylatanine anti-tumor 14

ammonia lyase streptokinase anti-thrombotic 15 superoxide dismutase anti-inflammatory 16 tryptophanasa anti-tumor 17 uricase therapy of hyperuricea and gout 18 u rok inase anti-thrombotic 19

(b) Suppression of IgE production ovalbumin therapy of type I allergy ragweed pollen therapy of type I allergy

20 21

Substance modified Use Ref.

(c) Solubilization in organic solvents catatase decomposition of H202 22 cholesterol oxidase oxidation of cholesterol 23 chymotrypsin formation of acid-amide linkage 24 lipase ester synthesis, ester exchange 25-29 peroxidase oxidation with H202 30

(d) Magnetization L-asparaginase lipase urokinase

targeting to affected parts 31 recovery from organic solvent 31-33 targeting to affected parts 34

(e) Enhancement of physiological activity ATP reusable coenzyme in continuous 35

bioreactor Cys-Pro-Leu-Cys/ rubredoxin model 36

iron(tl) conjugate hemin peroxidase activity 37. 38 histidine enzyme-like catalysis 39 insulin therapy of diabetes mellitus 40 lysine enzyme-like catalysis 39 NAD(H) reusable coenzyme in continuous 41

bioreactor hematoporphyrin photosensitizer 42

TIBTECH - JUNE 1988 [Vol. 6]

conditions (pH 7.0, room temper- ature) to form acid-amide linkages:

M + P ~ M P + E ~ M P E ( t y p e I I )

The conjugate composition was varied by adding different amounts of the activated magnetic modifier (MP) to a constant amount of enzyme (E). The ratio of the two is given as MP/E. The effect of varying MP/E added is shown in Fig. 3. Lauryl laurate synthesis in benzene occurred at 14.8 ~tmol/min/mg protein at MP/E = 20, approximately 12 times higher than with magnetic lipase (type I). This value is almost the same as that obtained with the unmagnetic PEG- lipase conjugate (13.6 btmol/min/mg protein). In the case of the ester hydrolysis of olive oil in the emulsi- fied aqueous system, the catalysis occurred at 1770 ~mol/min/mg pro- tein at MP/E = 1, approximately seven times higher than with the type I magnetic lipase. When MP/E was more than 20, both hydrolytic activity and ester synthetic activity decreased. When magnetic lipases (type II) were dispersed in organic solvents they could be recovered by magnetic forces of only 250 Oe.

The type I magnetic lipase could be quite stably dispersed in benzene but its enzymic activity was relatively low. On the other hand, the type II

Table 2

Characterist ics o f magnet ic l ipase

Composition (% w/w): magnetite (Fe304) 31% polyethylene glycol 44% protein 25%

Dispersed in: benzene, toluene, chloroform, 1,1,1-trichloroethane, aqueous solution, etc.

Stability in benzene: very stable (no precipitate and no aggregation in 5 days)

Particle size: magnetite 20 nm a magnetic enzyme 120 _+ 60 n m b

Enzymic activityC: Lauryl laurate synthesis

in benzene 1.2 #mol/min/mg protein in 1,1,1 -trichloroethane 11.6 ~mol/min/mg protein

Olive oil hydrolysis in emulsified aqueous solution 250 ~mol/min/mg protein

~Measured by electron micrograph, bMeasured by submicron particle analyser. CHydrolytic activity of native lipase and PEG-lipase hybrid: 3000 and 1500 i~mol/min/mg protein, respectively. Ester synthetic activity of PEG-lipase hybrid in benzene: 13.6 #mol/min/mg protein.

Table 3 Magnet ic separat ion o f magnet ic l ipase f rom benzene

Activi ty of enzyme Solution (l~mol/min/ml)

Colloidal solution of magnetic lipase 2.35 Residual solution after magnetic separation 0,01 Magnetic lipase recovered by magnetic separation 2.36

The magnetic separation was carried out by magnetic force of 6000 Oe for 5 rain in benzene (1 ml) containing 7,9 mg of the magnetic lipase. The enzymic activity for ester synthesis was determined using lauryl alcohol (0.45 M) and lauric acid (0.45 M).

- - Fig. 2

Magnetic separation of magnetic lipase from benzene after lauryl laurate synthesis. The magnetic separation was carried out by magnetic force of 6000 Oe for 5 min in benzene (1 ml) containing 7.9 mg of magnetic lipase.

- - Fig. 3

E

e Q_

1500 . _ .E

E

1000 : =L

v

500 ._~

2. o - r

J i i i i i

0 10 20 30 40 50 MP/E

£

~5 5

o 3" UJ

Characteristics of magnetic lipases prepared by varying the ratio of activated magnetic modifier to enzyme (MP/E). Enzymic activities of ester synthesis in benzene (0) and hydrolysis reaction in emulsified aqueous system (©). For reference, the hydrolytic activity of native lipase in aqueous system was 3000 i~mol/min/mg protein.

TIBTECH - JUNE 1988 [Vol, 6]

Fig. 4

bioactive substance / ",, synthetic protein ~ macromolecule ",,, /

inorganic compound

Possible conjugate combinations.

magnet ic l ipase d i spersed in benzene less s tably but exhib i ted h igher enzymic activity. The type II m e t h o d us ing the ac t iva ted magne t ic mod i - fier has the advan tage that the e n z y m e is e n d o w e d wi th magne t ic p rope r ty in one step.

L-Asparaginase 32 and u rok inase 34 were also coup led wi th the magne t ic modif ier . Magnet ic force will be used to se lec t ively del iver the pro te ins to target sites.

F u t u r e p r o s p e c t s Although this r ev i ew descr ibes

PEG-enzyme and magnet i te-PEG- e n z y m e conjugates, o ther combina - t ions could also be cons ide red (Fig. 4). Viewed in this way, the conjugate concep t is l ikely to open m a n y oppor - tuni t ies in med ic ine , p h a r m a c y , t echno logy and agriculture.

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e ~

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