Technology Case Study

This work was carried out with funding from the EU Commission under the Framework V programme.( BRPR-CT97-0484)

AET would like to take this opportunity to thank the EU commission for funding this project, all the partners involved in the project consortium and especially the Wageningen Agricultural University for their kind permission for the use of the thermophilic form of the glutamate dehydrogenase.

Due to the expertise of Applied Enzyme Technology Ltd. in the stabilisation of enzymes, AET was required to apply our patented technology to increase the shelf life and operational stability of the enzymes targeted as useful for application into molecular organised thin film sensors.

Our aims were as follows:

  1. Determine the formulation required to stabilise the meso and thermophilic forms of the chosen enzymes. In this case glutamate dehydrogenase (GLDH).
  2. Optimise this formulation for use in thin film biosensors.
  3. Understand the mechanisms behind the stabilisation process. i.e. Can the components required for the stabilisation of an enzyme be predicted.

Stabilisation of Mesophilic GLDH

The optimal conditions for the stabilisation of GLDH were determined by screening a multitude of factors. Enzyme formulations are normally determined in this fashion by investigating, enzyme concentration , buffer type, ionic strength and the stabiliser combinations which give the best longevity of activity.

Figure 1
Figure 1. GLDH activity remaining after 18 days incubation at 45°C in the presence and absence of various stabiliser combinations.

Shown above is the screening of various stabiliser combinations with the optimal combination identified as stabiliser combination 4. Extended thermal degradation of GLDH at this temperature indicated no significant activity loss over 100 days at 45°C (data not shown).

Using the data obtained from the mesophilic enzyme, we investigated the process of stabilisation with the aim of predicting the molecules which would result in increased stability.

Charge Distribution on the Enzyme Surface

Using the PDB database for the meso and thermophilic forms of GLDH to extract the enzyme sequences and thus enzyme structures for the X-ray crystallography data, it was possible to use the GRASP surface charge distribution computation program to determine the overall surface charges on the enzymes of interest. Figures 2a and 2b show the charge distribution data for the mesophilic and thermophilic forms of GLDH respectively. The blue areas indicate positive charge and the red areas negative charge. It is clear that the thermophilic enzyme possesses more negative charge than the mesophilic. Theoretical calculations for the isoelectric points of each enzyme (using the Swiss Prot data base) confirms this observation.

Thermophilic GLDH pI = 5.2
Mesophilic GLDH pI= 7.75

Meso GLDH Thermophilic GLDH
Theoretical calculation of the surface charge distribution on the meso and thermophilic frms of GLDH using GRASP (figures 2a and 2b respectively).

The Prediction of Stabiliser Formulation for GLDH.
The Electrophoretic Analysis of GLDH from Bovine Liver

Figure 3 displays mesophilic GLDH and polyelectrolyte complexes detected by coomassie staining following separation by IEF. In this instance a panel of 5 different polyelectrolytes were examined, all used at a concentration of 0.5%. Unbound GLDH focuses at the normal pI for the protein (untreated samples lanes in the gel as 1,5,9 and 11. The degree of protein complexing with polyelectrolyte is determined by the amount of protein left free to migrate to its normal pI. GLDH binds to polymer 2, 3 and 5 as determined by the loss of free protein. Due to the size of the protein/polyelectrolyte complexes, these are retained at the site of sample application.

Figure 3
Figure 3. The detection of Protein/Polymer Complexes by Isoelectric Focusing.

 1 - Markers   2 - L-GLDH   3 - L-GLDH + 0.5% Polymer 1   4 - L-GLDH + 0.5% Polymer 2   5 - L-GLDH   6 - Markers  
  7- L-GLDH + 0.5% Polymer 3    8- L-GLDH + 0.5% Polymer 4    9- L-GLDH   10- L-GLDH + 0.5% Polymer 5   11- L-GLDH   12- Markers  

By fixing the protein concentration and decreasing the polyelectrolyte concentration it was possible to calculate the stoichiometry of polymer binding to protein from the gel data.

Of the polyelectrolytes tested, 3 were shown to bind mesophilic GLDH.
These polyelectrolytes were screened for binding affinities in the presence the GLDH. The resulting electrophoretic data was scanned densitometrically and summarised in Table 1:

Mesophilic GLDH / polyelectrolyte complex Protein concentration (µM) Binding polyelectrolyte concentration (% w/v) Binding polyelectrolyte concentration (µM) Stoichiometry(protein to polyelectrolyte Molar ratio)
Mesophilic GLDH/polymer 2 1.78 0.006 0.06 30:1
Mesophilic GLDH/polymer 3 1.78 0.01 0.1 18:1
Mesophilic GLDH/polymer 5 1.78 0.02 0.2 9:1

Table 1. Summarised electrophoretic binding data, displaying the limit of polyelectrolyte concentration binding to the mesophilic form of GLDH and the calculated molar ratios of binding (protein to olyelectrolyte).

The Electrophoretic Analysis of Glutamate Dehydrogenase from Pyrococcus furiosus.

This section of the project concentrates on defining the polyelectrolytes which bind the thermophilic GLDH enzyme and determining the stoichiometries of binding. Experimentation with the thermophilic form of GLDH has provided clearer results than with the mesophilic GLDH. This study revealed that polymer 2 does not interact with the thermophilic GLDH (data not shown). Thermophilic GLDH displays a very acidic pI, 5.45. Therefore one would predict binding with positively charged cationic polyelectrolytes.


Figure 4. The separation of Pyrococcus furiosus Glutamate Dehydrogenase by isoelectric focusing in the presence and absence of the polyelectrolytes. Polymer 3 (0.04%-0.006%) (panel A), and polymer 5 (0.08%-0.01%) (Panel B). Free enzyme is detected following treatment with 0.01% polymer 3 and polymer 5 respectively (n=3).

The calculated binding stoichiometries are summarized below in Table 2.

Thermophilic protein /polyelectrolyte complex Protein concentration (µM) Binding polyelectrolyte concentration (% w/v) Binding polyelectrolyte concentration (µM) Stoichiometry (protein to polyelectrolyte Molar ratio)
Thermophilic GLDH/polymer2 2.1 0.5 5 No binding
Thermophilic GLDH/polymer3 2.1 0.02 0.2 10:1
Thermophilic GLDH/polymer5 2.1 0.02 0.2 10:1

Table 2. Summarised electrophoretic binding data, displaying the limit of polyelectrolyte concentration binding to the thermophilic form of GLDH and the calculated molar ratios of binding (protein to polyelectrolyte).

The Investigation of Alternative Polyelectrolyte Molecules for the Stabilisation of Enzymes.

As the stabilisation of enzymes is the basis of AETs expertise and forms the core of our financial turnover, many other projects are being carried out in parallel for industrial clients. Of great interest to this project was the requirement of a client to stabilise a number of alternative dehydrogenases. These included lactate dehydrogenase (LDH), glucose dehydrogenase (GDH) and alcohol dehydrogenase (ADH). LDH in particular showed some interesting preferences for a very highly charged polymer. Interestingly, this enzyme follows the same charge binding predictions as determined for the thermophilic GLDH.

This highly charged polymer was found to bind extremely tightly to the thermophilic form of GLDH. The concentrations observed showed that binding continued down to a concentration of approximately 0.005% w/v. Of course the molar ratios calculated for the stoichiometries of binding varied considerably, depending upon the molecular weight of the polymer under investigation. The binding ratios are summarised below in Table 3.

Thermophilic protein / polyelectrolyte complex Protein concentration (µM) Binding polyelectrolyte concentration (% w/v) Binding polyelectrolyte concentration (µM) Stoichiometry (protein to polyelectrolyte Molar ratio)
GLDH+ cationic polymer 1 2.1 0.005 0.066 32:1
GLDH+ cationic polymer 2 2.1 0.005 1.98 1:1
GLDH+ cationic polymer 3 2.1 0.1 495 1:236
GLDH+ cationic polymer 4 2.1 0.005 1.23 1:2

Table 3. Summarised electrophoretic binding data, displaying the limit of polymer concentration binding to the thermophilic form of GLDH and the calculated molar ratios of binding (protein to polyelectrolyte).

The Stability of Protein/Polymer Complex.

There has been some debate concerning the stability of protein/polymer complexes once formed. Experimentally this has now been determined. By forming enzyme/polymer complexes using high concentrations of enzyme, one can dilute the complex while retaining the ability to detect the enzyme/polymer complex. Upon dilution of the preformed protein/polymer complex no loss of retardation is observed (data not shown). We have the first evidence to suggest that the enzyme/polymer complex, once formed, remains stable, without the requirement for immobilisation.

Measurement of the Protein/Polymer/Polyalcohol Interaction.

There is a great deal of speculation surrounding the mode of stabilisation of polyalcohols (PA). It is generally accepted that the polyalcohol molecules act as a medium of water replacement within the protein molecules with which they interact. The degree of interaction between proteins and polyalcohols is thus far undetermined. Is there any detectable interaction between the polyalcohol molecules and the protein?

Analytical Centrifugation: An Alternative Technique for Determining the Interaction Between Proteins/Polymers and Polyalcohols.

Initial studies using electrophoresis to detect an interaction between protein and sugar meet with little success. It is possible that even forces exerted upon an enzyme/polyalcohol complex during electrophoretic separation may still be too harsh. Thus by introducing an even softer technique we may be able to begin to find evidence for actual interaction. Analytical centrifugation allows for the gradual sedimentation of a complex. The larger the complex formed the faster this will sediment. This is a rather qualitative technique, but the data generated gave us the first indication that measurable interactions between sugar molecules and protein molecules can be measured.

Figures 5a, 5b, and 5c show data from 3 different samples subjected to analytical centrifugation. 6a is the mesophilic GLDH alone, 6b shows the data obtained from GLDH + 0.5% polyelectrolyte+ 10% sugar and 6c displays sedimentation rates of GLDH + 0.5% polyelectrolyte.

Sedimentation of GLDH

dc/dt analysis distribution of sedimentation values


Figure 5a Analytical centrifugation of mesophilic GLDH (1mg/ml) in Tris/HCl buffer


Figure 5b Sedimentation of GLDH + 0.5% polyelectrolyte + 10% sugar


Figure 5c Sedimentation of GLDH + 0.5% polyelectrolyte

dc/dt analysis distribution of sedimentation values Figures 5b & 5c Analytical centrifugation of mesophilic GLDH (1mg/ml) in Tris/HCl buffer plus 0.5% polyelectrolyte+ 10% sugar (5b), plus polyelectrolyte (5c).

As predicted GLDH and 0.5% polyelectrolyte form a huge complex which sediments considerably faster than the native protein alone, which concurs with the gel data.

More interesting is the data obtained from the GLDH/polyelectrolyte/sugar complex. One might predict that this complex would be at least the same size as the enzyme/polymer complex formed. However, the complex examined in figure 5b shows a slower sedimentation rate than the enzyme/polyelectrolyte complex alone, implying that this complex is smaller than the enzyme/polymer complex. Obviously the presence of the polyalcohol changes the stoichiometry of interaction between the polymer and enzyme. This could be the first indication of the underlying mechanism involved in the additive advantage gained in stabilising proteins when a combination of a sugar and a polyelectrolyte are used.

Correlation between stoichiometry of polymer binding and enzyme stability

To date, there has been no direct correlation between polymer concentration and enzyme stability. This has been due to the lack of information on the mode of polymer/polyalcohol interactions with proteins during the stabilisation process.

Figure 6 shows the mesophilic GLDH activity following incubation at 37°C for 24 hours in the presence of various polymers at decreasing concentrations. The critical concentrations for the stoichiometry of binding have been determined from the gel data and these concentrations used in the activity studies. From these data we can determine that the stability conferred by the polymers of choice as determined by gel electrophoresis concur. The best enzyme activity measured for polymers 2, 3 and 4 were shown to be at the point of stoichiometry of binding, suggesting that excess amounts of polymer in the presence of the enzyme complex can be disadvantageous to the overall stability of the enzyme under investigation.

Figure 6
Figure 6. The on line rates of mesophilic GLDH activity measured following incubation at 37°C for 24 hours in Tris/HCl buffer pH 7.6.

From this data we can conclude that the inclusion of polymer with a polyalcohol does indeed confer additional stability to an enzyme compared to incubation with a polyalcohol alone. Furthermore, the concentration of polymer added to the formulation can also play a critical role. When a polymer is in excess this can confer detrimental effects to the enzyme under investigation and cause destabilisation of the enzyme. Finding the stoichiometry of binding for a polymer and protein is necessary to confer the optimal stabilisation formulation to the enzyme in question.

Conclusions

  • Mesophilic GLDH has been stabilised with no loss of activity for more than 100 days at 45°C
  • A combination of surface charge distribution maps and electrophoretic data can be used to predict the polymers which bind to the protein in question
  • Electrophoretic data can be used very accurately to predict the binding stoichiometry between a polymer and a protein
  • The thermophilic form of GLDH as predicted binds tightly to cationic polymers
  • The choice of the right polyalcohol polymer combination is crucial to stability of the enzyme in question
  • Some polyalcohol molecules have been shown to destabilise an enzyme in the presence of certain polymers
  • We now know much more about the underlying mechanisms of stabilisation.
  • Polymers interact electrostatically during the stabilisation process
  • We have shown that polyalcohols also interact directly with a protein during the stabilisation process
  • They also change the manner of interaction between polymer and protein but the mechanisms underlying this have yet to be determined
  • The stoichiometry of protein to polymer binding is indeed crucial to the whole stabilisation process whether in the presence or absence of another stabiliser molecule