Very high activity biocatalysts for low water systems: Propanol rinsed enzyme preparations (PREPs)

Barry D. Moore, Johann Partridge and Peter J. Halling

Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, U.K.

Correspondence details:

Barry D. Moore

Department Pure & Applied Chemistry,,

Thomas Graham Building,

University of Strathclyde, Glasgow G1 1XL, U.K.

Tel. (+44) 141 548 2301

FAX (+44) 141 548 4822



Depending on the preparation method used enzymes under low water conditions can exhibit differences in catalytic activity that vary by several orders of magnitude. Since this can make the difference between whether a particular biotransformation is practically useful or not it is important to control enzyme history carefully. The basic problem is simple: how to transfer the enzyme from an aqueous environment to one where it is dehydrated whilst ensuring firstly it remains in a native conformation and secondly the active site residues are in the correct protonation state. Although the importance of retaining native structure is well recognised potential detrimental changes to protonation state are often not considered. Usually it is assumed that the enzyme will exhibit 'pH memory' of the previous aqueous solution. As discussed in detail in chapter ?? Partidge this is not always a valid assumption.

Removal of water from proteins is most often carried out by lyophilisation because it is thought to minimise the extent of irreversible denaturation. Excipients are added to stabilise the protein during the dehydration process and to provide a convenient powder for storage. Despite these precautions lyophilisation commonly results in a considerable degree of reversible denaturation. This is of little consequence where the protein is to be redissolved back into water but is of vital importance when native enzyme is required for use as a biocatalyst under low water conditions .

Because of this problem many alternative forms of enzyme preparation have been explored, some of which are described in other chapters of this book (e.g. chapters ??,??,??). Here we describe a simple practical procedure for dehydrating enzymes which results in preparations with 1000 fold higher catalytic activities than obtained with conventional lyophilised powders [Partridge 1998a]. The advantage of this method is that it is rapid and inexpensive and can be implemented in a conventional organic preparative laboratory without any specialised pieces of equipment.


Figure 1 illustrates the process involved in the production of PREPs. The initial step is to immobilise the enzyme on a support material in aqueous buffered solution. This could be via covalent attachment or by an adsorption process. With subtilisin Carlsberg and a-chymotrypsin we have adopted a very simple method of adsorbing the protein onto chromatography grade silica gel. For other enzymes this may not be the best support and materials such as celite, polypropylene, alumina or ion-exchange resins may give better results. The main requirement is for the enzyme to bind efficiently to the support material from aqueous solution without denaturing.

Figure 1. Production of propanol rinsed enzyme preparations. a The particles should be kept wetted and not dried b the water content can be matched to the water activity required in the subsequent reaction mixture

It should be noted that the aqueous immobilisation step does not need to be carried out at the pH optimum for enzyme activity as the protonation state can be altered later, under low water conditions (see chapter ??Partridge). This may be useful if the pI of the protein is such that good adsorption to the support is not possible at the pH for maximal activity or if autolysis is a problem.

Following immobilisation most of the aqueous solution should be decanted off from the supported enzyme. However, it is important to ensure the particles remain fully wetted and do not come into prolonged contact with the air. This is because when supported enzyme is dried by conventional means there is a high risk that a significant proportion of activity expressed under low water conditions will be lost. This is certainly the case with silica-enzyme which when dried in air or vacuum has activity no better than a lyophilised powder.

The next and key step is to carry out a rapid dehydration by rinsing the supported enzyme with a suitable water miscible organic solvent. A particularly good solvent for this process is 1-propanol but comparable results can be obtained with other solvents such as ethanol [Partidge 1996]. It is notable that methanol and ethylene glycol give poor results indicative of irreversible enzyme denaturation.

The dehydration step is best carried out using solvent containing low levels of water (0.5-10 % v/v) in order to ensure essential water molecules remain bound to the protein.. For convenience the level can be chosen to give the same water activity, aw, as required in the subsequent reaction mixture. Control and measurement of aw is described in detail in chapter ??Halling. The number of solvent rinses is not critical but to reach a particular fixed water level at least three rinses of the volume of the original aqueous solution should be used. Karl Fisher titration can be used to ensure the water content of the rinsing solvent is the same before and after contact with the PREP.

It is convenient to carry out the rinsing step in eppendorf tubes. Following each addition of solvent the sample is shaken, allowed to settle (or centrifuged) and the solvent removed using a pipette. If a larger scale preparation is required samples can be prepared in stoppered glass flasks, left to settle under gravity between rinses and the solvent removed by decanting or partial filtration. This means PREPs can easily be prepared in a normal synthetic laboratory since no specialised equipment is required.

If the PREPs are to be stored prior to use they should be kept in suspension in propanol containing > 1.5 % water. Their stability will depend upon the enzyme and support material used. When required for use in a different reaction solvent the PREPs may be rinsed once or twice with solvent to remove propanol. In some polar solvents (e.g acetonitrile) PREPs have lower stability than in propanol and so following transfer to this type of solvent it is best for the reaction to be carried out immediately.


Subtilisin Carlsberg Protease, Type VIII from Bacillus licheniformis, 13.5 units/ mg solid (P5380) and a-chymotrypsin, Type II from Bovine Pancreas, 52 units/ mg solid (C-4129) can be purchased from Sigma Chemical Co. (Poole, U. K.). N-acetyl-L-tyrosine ethyl ester (A-6751) and silica gel (S-0507) can also obtained from Sigma. Anhydrous solvents are available from Merck Ltd (Poole, U.K.).

Enzyme adsorption onto silica

The enzyme is firstly dissolved in a suitable buffer. For subtilisin Carlsberg we have used concentrations of 2mg/ml and either 20 mM sodium phosphate buffer, pH 7.8, or 20mM sodium pyrophosphate, pH 5.7. With a-chymotrypsin good results are obtained with 4 mg/ml enzyme in 25mM Tris/HCl containing 10mM CaCl2 (pH 7.8).

20 ml of the above enzyme solutions held at 4 C are then mixed with 1g of untreated chromotography grade silica gel (Sigma S-0507) and the mixture shaken for 1-4 hr. A total protein assay (Biorad) of the supernatant can be used to monitor the amount of enzyme adsorbed onto the support as a function of time. Using concentrations of 2-4 mg/ml protein and 50 mg/ml of silica quite high loadings can be achieved with these enzymes. For subtilisin typically >90% and >80% of protein adsorbs at pH values of 7.8 and 5.7 respectively. For chymotrypsin adsoption as high as 99% can be obtained. Following rinsing with buffer the suspension of immobilised enzyme can be stored for weeks at 4 oC in aqueous buffer containing azide.

Propanol rinsing of the immobilised enzyme

The stock aqueous mixture of immobilised enzyme is agitated to obtain a homogeneous suspension and a 1 ml aliquot removed using a pipette, transferred to an eppendorf tube and allowed to settle out for a few minutes (1 ml ~ 50 mg of silica-enzyme). The excess aqueous buffer over the precipitate can then be removed carefully with the pipette ensuring the silica-enzyme particles are not disturbed. The precipitate is now ready for the key dehydration step using propanol. This is achieved by rapidly adding 1 ml of propanol containing a measured amount of water to the immobilised enzyme and agitating to make a suspension.

Dehydration occurs because most of the water bound to the enzyme is rinsed off and dissolves in the propanol. Having some water in the rinsing solvent ensures a fraction of essential water molecules remain bound to the enzyme. The most appropriate water level to use depends upon what water activity, aw, the subsequent reaction is going to be carried out at (see Table 1). For example if the reaction is to be carried out at aw = 0.44 it can be seen from Table 1 the water content of the rinsing propanol should be 3.4 v/v%.

After the first rinse the enzyme is allowed to settle again and the excess propanol removed. The rinsing process is then repeated at least twice using further 1 ml aliquots of propanol containing the relevant v/v % water. On the final rinse the water content of the rinsing solvent ought to be unchanged after contacting the supported enzyme. By application of this procedure PREPs can be rapidly prepared for reaction at any particular water activity.

  water activity                       water content % v/v                     

        aw              1-propanol          acetonitrile       tetrahydrofuran   

       0.11                0.77                 0.5                  0.2         

       0.22                 1.6                 1.0                  0.5         

       0.44                 3.4                 2.7                  1.2         

       0.55                 4.7                 4.5                  1.6         

       0.76                 9.8                 13.0                 3.5         

Table 1. Water content required to attain selected water activities in three solvents. Details on how to calculate values for other solvents can be obtained from [Bell 19997]

Transferring the PREPs to another solvent

Excess propanol is removed from the PREP and 1 ml of the desired reaction solvent containing a certain level of water added. The % v/v water required will depend upon the solvent and the desired water activity of the reaction mixture. As an example we consider transfer to THF for a reaction at aw = 0.44. It can be seen from Table 1 that the water content required for aw = 0.44 is 1.2% in THF compared to 3.4% in propanol. Following agitation and then settling excess THF is removed and the process repeated The PREP is now ready for biocatalysis. PREPs of subtilisin have been found to give good results in solvents with a wide range of polarity.


It has been reported that commercially available cross-linked enzyme crystals(CLECs) exhibit very high activities and good stability in organic solvents compared to most other preparations [Lalonde 1995, Persichetti 1996] It has also been shown by x-ray crystallography that cross-linked crystals of subtilisin Carlsberg retain native protein structure in dry acetonitrile and dioxane [Schmitke 1997]. CLECs therefore provide a useful bench-mark to compare other preparations with.

It is often difficult to make accurate comparisons between results obtained in different laboratories because of variations in the protocols used for assaying. We have therefore directly compared the activities of PREPs, CLECs and lyophilised powders of subtilisin Carlsberg under exactly the same reaction conditions. The reaction studied was a simple model transesterification of N-acetyl-L-tyrosine ethyl ester with propan-1-ol in acetonitrile.

Typical protocol for assaying activity

N-acetyl-L-tyrosine ethyl ester was dissolved in anhydrous propan-1-ol and added to a suspension of PREP/CLEC/powder in 10ml of acetonitrile to give 10mM ester and 1M propanol. The acetonitrile also contained fixed amounts of water chosen to give particular water activities as in Table 1. After brief mixing, the zero time sample was removed. The mixture was then incubated at 24oC with constant reciprocal shaking (150 min-1). Samples from the reaction mixture were taken at regular intervals and analysed by HPLC on a Gilson 715 equipped with an ODS2 reverse phase column (Hichrom). The mobile phase consisted of 40% acetonitrile mixed with an aqueous phase adjusted to pH 2 with orthophosphoric acid. The retention times of the acid, ethyl ester and propyl ester were 2.1, 2.9 and 3.9 min respectively. The reaction rates are obtained by dividing the number of nanomoles per ml of ester converted per minute by the enzyme concentration in mg per ml, giving convenient units of nmol min-1 mg-1. Using a discontinuous HPLC sampling procedure the highest rate that can effectively be followed with a 10 mM ester concentration is 50 mM min-1. With 1 mg/ml of enzyme this corresponds to 50 nmol min-1 mg-1 and our rates with PREPs and CLECs are generally higher than this. We therefore routinely use enzyme concentrations of less than 0.2 mg/ml in our assays. For the PREPs the weight of subtilisin in the reaction mixture is calculated from the known loading of enzyme on the silica support.

Comparison of activities

It can be seen from Table 2 that PREPs of subtilisin Carlsberg are over 1000 times more active than freeze-dried powde and exhibit comparable activities to cross-linked enzyme crystals. Rate enhancements of the same order have also been observed with a-chymotrypsin PREPs, an enzyme for which CLECs are not commercially available [Partridge 1998a]. The activity measurements suggest the propanol rinsing procedure is much less damaging to protein tertiary structure than conventional lyophilisation. In fact, the similarity in rates of PREPs to that of the cross-linked crystals is consistent with high retention of native enzyme conformation during the dehydration.

 enzyme preparation                  Rate (nmol mg-1 min-1)a                 

                         0% H2Oc       1% H2O c     2.7% H2O c     4.5% H2O c   

 freeze-dried powder      <0.01          0.13           --            0.28      

cross-linked crystal       278           204           214            156       

   propanol rinsed         0.82          288           543            530       

Table 2. Variation of activity in acetonitrile with water content for preparations of subtilisin Carlsberg.

a Initial transesterification rates b As described in [Partidge 1996] CLECS exhibit an initial 5 minute higher burst of activity. c Water content of acetonitrile in v/v %.

A practical operational advantage of PREPs over CLECs is that at any particular water activity lower levels of hydrolysis are obtained. For example, using acetonitrile containing 2.7 v/v % for the above transesterification the CLEC gives 19 % hydrolysis and the PREP 1% hydrolysis. Hydrolysis side reactions reduce product yield and can lead to substantial changes in catalytic rates because acidic or basic side-products change the enzyme protonation state in the solvent (see chapter ??Partridge). The cause of the increased hydrolysis with CLECS is not clear. It could be that there is higher density of strong binding sites for water in the crystal lattice resulting in a higher effective water concentration near the enzyme active site.

The above studies have been carried out with model enzyme systems where the protein is readily available in high purity. Here the relative merits of PREPs versus CLECs are not clear-cut. They depend on the availability and relative cost or ease of preparation of the cross-linked crystals. For example, CLECs of subtilisin have now become much cheaper and are of comparable cost to other sources of the protein. On the other hand CLECs of chymotrypsin are not currently available.

By contrast where the enzyme of interest is only available in small quantities or in a partially purified form then production of cross-linked crystals is not an option. Here PREPs offer a major advantage over most other methods of preparing enzymes for low water systems. They can be made rapidly, on a small scale using simple equipment. Furthermore if rate enhancements comparable to above are obtained 1 mg of PREP will have comparable activity to 1 g lyophilised powder. PREPs are therefore attractive options for applications such as enzyme screening and combinatorial biocatalysis [Michels 1998].

Effect of water activity on PREPs

The variation in catalytic activity of PREPs as a function of water availability is very different to that observed for lyophilised powders. With PREPs the rate rapidly increases from aw= 0 to aw = 0.22 and the activity reaches a plateau from aw = 0.44 upwards [Partidge 1998a]. By comparison lyophilised subtilisin generally show very little activity below aw = 0.44 but exhibit rapid increase from thereon upwards [Parker 1995, Partridge 1998b].The practical effect of these differences is that even at quite low water activites PREPs of subtilisin behave as efficient catalysts. This is of direct interest in applications where hydrolysis is an undesirable side-reaction such as peptide synthesis.


G. Bell, A. E. M. Janssen, and P. J. Halling (1997). "Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities". Enzyme and Microbial Technology. 20:471-477

K. Griebenow, and A. M. Klibanov (1997). "Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?". Biotechnology and Bioengineering. 53:351-362.J. Lalonde (1995). "The Preparation of Homochiral Drugs and Peptides Using Cross-Linked Enzyme Crystals". Chimica Oggi-Chemistry Today. 13:31-35.P. C. Michels, Y. L. Khmelnitsky, J. S. Dordick, and D. S. Clark (1998). "Combinatorial biocatalysis: a natural approach to drug discovery". Trends in Biotechnology. 16:210-215.M. C. Parker, B. D. Moore, and A. J. Blacker (1995). "Measuring enzyme hydration in non-polar organic solvents using NMR". Biotechnol.Bioeng. 46:452-458.J. Partridge, G. A. Hutcheon, B. D. Moore, and P. J. Halling (1996). "Exploiting hydration hysteresis for high activity of cross-linked subtilisin crystals in acetonitrile". Journal of the American Chemical Society. 118:12873-12877.J. Partridge, P. J. Halling, and B. D. Moore (1998a). "Practical route to high activity enzyme preparations for synthesis in organic media". Chemical Communications:841-842.J. Partidge, P. R. Dennison, B. D. Moore, and P. J. Halling (1998b). "Activity and mobility of subtilisin in low water organic media: hydration is more important than solvent dielectric". Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology. 1386:79-89.R. A. Persichetti, J. J. Lalonde, C. P. Govardhan, N. K. Khalaf, and A. L. Margolin (1996). "Candida-Rugosa Lipase - Enantioselectivity Enhancements in Organic- Solvents". Tetrahedron Letters. 37:6507-6510.J. L. Schmitke, L. J. Stern, and A. M. Klibanov (1997). "The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile". Proceedings of the National Academy of Sciences of the United States of America. 94:4250-4255.