Preparations and Properties in Organic Solvents of non-Covalent PEG-Enzyme Complexes
Francesco Secundo, Gianluca Ottolina and Giacomo Carrea
Istituto di Biocatalisi e Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131, Milano, Italy
1. Introduction
It is well established that enzymes can be advantageously employed in organic solvents. In fact, there is an enormous number of applications in organic synthesis and numerous examples also in the fields of food-related conversions and analysis. However, enzymes show a lower catalytic efficiency (up to three or four orders of magnitude) when employed in organic solvents rather than in aqueous buffer. It is evident that such behaviour represents an obstacle to exploit at industrial level the advantages which come from using enzymes in organic solvents (1-2). One of the reasons which can be responsible of the lower catalytic activity of enzymes in organic solvents can be ascribed to diffusional limitations (3). Generally, when enzymes are employed in organic solvents, they are used as a suspended powder and the dispersion degree of the powder may represent a critical factor for the expression of the catalytic activity. In fact, the activity showed by an enzyme depends on the number of productive matches which occur between the enzyme molecule and the substrate. Consequently, all methods which may increase the dispersion of the enzyme in organic solvents may be of interest in improving biocatalyst performance in non-aqueous media. Therefore, those methods which allow the dissolution of the enzyme in the organic solvent deserve particular attention because they represent a way to fully disperse the enzyme in the reaction system. Several procedures such as enzyme complexation with ion-pair forming surfactants (4) or synthetic amphipatic lipids which coat the enzyme molecule (5) or covalent linking of the protein to amphipatic polymers such as poly(ethylene glycol) (PEG) (6), have been described. Here, we report on a methodology suitable to prepare subtilisin Carlsberg and lipase from Pseudomonas cepacia (lipase PS) in a form which can be dissolved or highly dispersed in organic solvents, thanks to the formation of stable non-covalent protein-PEG complexes (subtilisin + PEG and lipase PS + PEG). Both enzyme complexes, obtained by the method here described, have been proved (7-8) to have a much higher catalytic activity than that of untreated enzymes (Table 1). Although the higher catalytic activity shown by the enzymes could, in principle, be attributed also to the lyoprotective properties of PEG, in a recent paper (9) we have proved that lipase PS + PEG, dissolves in 1,4-dioxane. This suggests that the increase of activity is likely due to the high dispersion of the enzyme. This holds also for solvents such as carbon tetrachloride, toluene and benzene which give clear solutions when they are employed as reaction media for the enzyme-PEG complexes.
If the performance in organic solvents of enzyme-PEG complexes is compared with that of the same enzymes covalently linked to PEG, it can be seen that in the latter case the activity is slightly higher at least in the case of lipase PS (Table 1). However, enzyme-PEG complexes are by far preferable to the covalently modified enzymes since their preparation is much more simple. It should also be emphasised that the complex can be reutilized for several cycles of conversion following a simple procedure that consist in : a) centrifugation of the reaction medium, b) removal of the solution present in the upper part of the centrifuge tube, c) addition of a fresh substrate solution.
Table 1. Transesterification, hydrolytic and total (transesterification plus hydrolytic) activity of subtilisin and subtilisin + PEG in 1,4-dioxane, and of lipase PS and lipase PS + PEG in 1,4-dioxane and carbon tetrachloride (7,8).
Enzyme a Transesterificati Hydrolytic Total activity
on activity Activity
Subtilisin b 25 0 25
Subtilisin + PEG b 100 0 100
Subtilisin covalently linked to 93 0 93
PEG b
Crude Lipase PS c 3 13 16
Lipase PS + PEG c 39 61 100
Lipase PS covalently linked to 53 93 146
PEG c
Crude Lipase PS d 4 6 10
Lipase PS + PEG d 62 38 100
Lipase PS covalently linked to 88 38 126
PEG d
a The reaction of vinyl butyrate with 1-octanol (or 1-hexanol in the case of subtilisin) was employed as a model and both transesterification (formation of 1-octyl butyrate or 1-hexyl butyrate) and hydrolytic (formation of butyric acid from vinyl butyrate) activities were measured. Total activity is transesterification plus hydrolytic activity. The activities of enzyme + PEG, enzyme covalently linked to PEG and non-treated enzyme refer to the same amount of enzyme protein.
b Activity measured in 1,4-dioxane, aw=0.003. The activity values were normalised respect to the total activity value obtained by subtilisin + PEG in dioxane.
c Activity measured in benzene, aw=0.5. The activity values were normalised respect to the total activity value obtained by lipase PS + PEG in benzene.
d Activity measured in carbon tetrachloride, aw=0.5. The activity values were normalised respect to the total activity value obtained by lipase PS + PEG in carbon tetrachloride.
2. Materials
2.1 Preparation of Subtilisin + PEG
1. Purified subtilisin Carlsberg.
2. Buffer: potassium phosphate 0.05 M, pH 8.
3. Poly(ethylen glycol) Mr 5000.
2.2 Preparation of lipase PS + PEG
1. Purified lipase PS.
2. Buffer: potassium phosphate 0.01 M, pH 7.
3. Poly(ethylen glycol) Mr 5000.
2.3 Activity determination of subtilisin
1. 1-Hexanol.
2. Vinyl butyrate.
3. One of the following organic solvents: 1,4-dioxane, carbon tetrachloride, benzene, toluene.
2.4 Activity determination of lipase PS
1. 1-Octanol.
2. Vinyl butyrate.
3. One of the following organic solvents: 1,4-dioxane, carbon tetrachloride, benzene, toluene.
3. Methods
3.1 Preparation of Subtilisin + PEG
1. Dissolve subtilisin in buffer (0.2 mg/ml).
2. Dissolve PEG in water (30 mg/ml).
3. Mix in a vial the subtilisin and the PEG solution in a 1/1 ratio.
4. Freeze and lyophilize.
3.2 Preparation of lipase PS + PEG
1. Dissolve lipase PS in buffer (0.2 mg/ml).
2. Dissolve PEG in water (30 mg/ml).
3. Mix in a vial the lipase PS and the PEG solution in 1/1 ratio.
4. Freeze and lyophilise.
3.3 Activity determination of subtilisin
1. Add to a proper amount of enzyme a proper volume of the chosen organic solvent.
2. Add vinyl butyrate to obtain a concentration of 0.4 M.
3. Add 1-hexanol to obtain a concentration of 0.8 M.
4. At scheduled times, withdrawn an aliquot of the reaction medium and determine the conversion degree. It can be determined by GLC using a HP-1 Cross-Linked Methyl Silicone Gum 25 m, 0.32 mm ID, 0.52 mm film column; conditions: oven temperature from 35 °C (initial time) to 180 °C (final time) with a heating rate of 15°C/min, H2 as carrier gas.
3.4 Activity determination of lipase PS
1. Add to a proper amount of enzyme a proper volume of the chosen organic solvent.
2. Add vinyl butyrate to obtain a concentration of 0.8 M.
3. Add 1-octanol to obtain a concentration of 0.2 M.
4. At scheduled times, withdrawn an aliquot of the reaction and determine the conversion degree. It can be determined by GLC using a HP-1 Cross-Linked Methyl Silicone Gum 25 m, 0.32 mm ID, 0.52 mm film column; conditions: oven temperature from 35 °C (initial time) to 180 °C (final time) with a heating rate of 15°C/min, H2 as carrier gas.
3.5 Reuse of the enzyme-PEG complexes
1. Centrifuge the reaction medium containing the enzyme-PEG complex at 10000 g 5 min.
2. Remove (paying attention not to shake the sample) the upper part of the centrifuged reaction leaving about 10 % of the initial volume.
3. Add fresh substrate solution and shake.
4. Notes
1. Although the procedure is here described for purified enzymes, there are experimental evidences that it can be profitably adopted also in the case of crude enzymes.
2. The recommended PEG/protein ratio to dissolve the enzyme is 150 w/w. However, beneficial effects on the activity of the enzymes in organic solvents could be obtained also with lower PEG/protein ratios.
3. The concentrations of protein and PEG indicated in the Methods section are the highest tested by us to dissolve lipase PS + PEG in anhydrous 1,4-dioxane.
4. Freezing of the sample must be as fast as possible.
5. The complexes showed good stability in 1,4-dioxane since the enzyme solution remained transparent and active for several days at room temperature.
Acknowledgments
We thank the Biotechnology Programme of the European Commision (BIO5-CT95-0231) and the CNR Target Project on Biotechnology for financial support.
References
1. Carrea, G., Ottolina, G., Riva, S. (1995) Role of solvents in the control of enzyme selectivity in organic media. Trends Biotechnol. 13, 63-70.
2. Koskinen, A.M.P. and Klibanov, A.M. (eds) (1996) Enzymatic reactions in organic media. Blackie Academic & Professional. London.
3. Klibanov, A.M. 1997. Why are enzyme less active in organic solvent than in water? Trends Biotechnol. 15, 97-101.
4. Paradkar,V.M. and Dordick, J.S. (1994) Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles. Biotechnol. Bioeng. 43, 529-540.
5. Okahata, Y., Fujimoto, Y., Ijiro, K. (1995) A lipid-coated lipase as an enantioselective ester synthesis catalyst in homogeneous organic solvents. J. Org. Chem. 60, 2244-2250.
6. Matsushima, A., Kodera, Y., Hiroto, M., Nishimura, H., Inada, Y. (1996) Bioconjugates of proteins and polyethylene glycol: potent tools in biotechnological processes. J. Mol. Catal. B Enzymatic 2, 1-17.
7. Bovara, R., Carrea, G., Gioacchini, A.M., Riva, S., Secundo, F. (1997) Activity, stability, and conformation of methoxypoly(ethylen glycol)-subtilisin at different concentration of water in dioxane. Biotechnol. Bioeng. 54, 50-57.
8. Secundo, F., Spadaro, S., Carrea, G. (1999) Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvent. Biotechnol. Bioeng. In press.
9. Secundo, F., Carrea, G., Vecchio, G., Zambianchi, F., (1999) Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4-dioxane by non-covalent complexation with methoxypoly(ethylen glycol). Biotechnol. Bioeng. Submitted.