Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Marlene Etschmann, Peter Gebhart and Dieter Sell

4. Biopulping

Pulp and paper production is an industrial section where the idea of production integrated biotechnology took root relatively early. The first studies on the application of fungi for wood delignification date back to the 1950s. Significant energy savings by fungal treatment in mechanical pulping were first demonstrated in the 1970s, but it took some twenty years more to achieve industrial application.

To make paper, the wood fibres which are “glued” together by lignin have to be separated from each other. This can be done by chemical degradation and removal of the lignin (chemical pulping) or by physically tearing the fibres apart (mechanical pulping).

About 25% of the world’s wood pulp production is produced by mechanical pulping, which has a high yield, but is energy-intensive.

In two research projects, Biopulping Consortium I and II, joint research groups from US universities and industry evaluated the technical and commercial feasibility of using a fungal pretreatment with mechanical pulping to save energy and/or improve paper quality. The assumption that fungal pretreatment would have less environmental impact than chemical pretreatment proved to be right (Akhtar et al. 1995). The fungi alter the wood cell walls, soften the chips and thus substantially reduce the electric energy needs for pulping. The paper quality increases and 30% electric energy can be saved by inoculating the wood chips (Scott 1998). Many strains of fungi were studied, of which Ophiostoma piliferum proved to be one of the most efficient. It is available under the product name Cartapip®, marketed by Agrasol Inc., Charlotte, North Carolina, USA.

5. Bleach cleanup

The textile finishing industry is characterised by high consumption of energy and resources and time-consuming production processes. For these reasons production- integrated biotechnological processes could make a considerable contribution to conserving energy and water, reducing emissions and to shortening the processes and consequently the throughput time.

As a rule, process innovations that "only" relieve the environment are not sufficient incentive to companies to modify their operations. They are at most desirable byproducts. Only economic advantages convince decision-makers in companies to apply ecologically advantageous, innovative processes. The textile finishing industry differs from other branches in that it is scarcely possible to offer unrivalled products and new or significantly improved quality. Thus, the substitution of a process by one that is economically advantageous can make a considerable contribution towards consolidating or improving the position of a company with regard to the competition.

Nowadays hydrogen peroxide is generally used for bleaching textiles. To achieve high quality in the ensuing dyeing process it is necessary to remove bleach residues as completely as possible from the textile.

In conventional processes residual peroxide is removed by repeatedly (at least twice) rinsing the textile in hot water. This method is not only energy and water-intensive, but cannot guarantee the complete removal of residual hydrogen peroxide that is required.

356

Economic benefits of the application of biotechnology - Examples

For this reason an enzymatic process was developed that may now be considered to be established and that has been extensively, but by no means exhaustively, applied in the textile finishing industry. With this new, biotechnological process only one hightemperature rinse is necessary (at 80-95°C, depending on the type of fabric) after the oxidative bleaching. The catalase enzyme is added to the next rinse and allowed to react for approx. 15 minutes at 30-40°C. In the example studied the compound

"KAPPAZYM AP-Neu" (Kapp Chemie GmbH, Miehlen, Germany) was applied. The enzyme degrades residual peroxide into water and oxygen. Then the necessary consecutive steps can be started. The results are of significantly higher quality compared with the conventional process.

As one rinse is omitted, both the water and energy consumption and the process duration are reduced.

5.1. MATERIALS AND METHODS

An analysis of the bleach cleanup process during textile finishing was performed at

Windel Textil GmbH & Co (Bielefeld, Germany), a medium sized textile finishing company, in 1998.

5.1.1. Selection of the production plant

As there are numerous, only slightly differing bleaching processes, a representative process had to be found for each of the two machine types used, namely the beam and the jet dyeing machines. The beam-dyeing machine derives its name from the fact that the textile roll is wrapped around beam-shaped metal cylinders made of perforated steel. The cylinder with the material is then inserted into the machine.

With the jet-dyeing machine, the textile is transported through the dyeing liquid by the action ofjets. Thus the mechanical impact on the fabric is minimised.

In the period under study, May to July 1998, 355 bleaching processes were carried out using the catalase KAPPAZYM AP-Neu. 281 processes, which is approx. 80% of the total, were carried out on the beam dyeing machines, the remaining 20% (74 processes) on the jet dyeing machines.

The calculations shown were made for a beam dyeing machine with a capacity of

5,800 1 of liquid and an average load of 226 kg of knitted fabric containing cotton. The jet dyeing machine chosen holds 157 kg of material and 3,000 1 of liquid per run.

5.1.2. The process

Figure 1 shows the sequence of a representative bleaching process in a beam dye.

After oxidative bleaching with residual peroxide has to be removed so as not to interfere with subsequent steps. The traditional way to clean up bleach is by twice rinsing with hot water. In the new process "Kappazym AP-Neu" is added after the first rinse. This compound contains the catalase enzyme, which converts any remaining peroxide into water and oxygen. Thus, the second rinsing cycle can be omitted (grey area in Fig. 1), as the liquid is clean enough to start the next process step, reductive bleaching.

357

Marlene Etschmann, Peter Gebhart and Dieter Sell

5.1.3. Economic analysis

programmes for the machine control system had to be modified. To determine the economic differences between the old and the new process, a complete analysis of the two was performed. In this, the fixed and proportional costs taken from the current calculation, the current cost for chemicals and resources and the current municipal prices for water and energy were used. Comparability of the economic results over a longer time span was optimised by basing the analysis on a year's production.

5.2. RESULTS

For data protection reasons no absolute figures can be given, but the costs of the new process are given in relation to the traditional process in Table 3.

Besides hydrogen peroxide a whole range of other chemicals are used in bleaching blended fabrics, e.g. stabilisers, common salt and fabric-protective agents. Steam is required to heat the water, cooling water to cool it. The term process water means water that comes into direct contact with the textiles.

358

Economic benefits of the application of biotechnology - Examples

The enzymatic process can achieve savings both with the beam dyeing and the jet dyeing machines. Admittedly costs for chemicals rise by 7% and 11% respectively. This is due to the additional cost of the enzyme. In all other areas the costs drop, some by up to 20%. Moreover it should be borne in mind that the individual cost factors represent different percentages of total costs. As one rinsing cycle is omitted completely, the reduction in process water costs is particularly striking. The enzyme application reduce the bleaching process by one hour. Thus, costs such as those for labour, machinery and electricity, among others, are also reduced. Theses costs are included in “other finishing costs” and contribute substantially to the savings.

The enzymatic process finally turns out to be about 7% and 8% cheaper respectively than the traditional process.

The substitution of multiple rinsing by enzyme application in the bleach-cleanup process at Windel Textil GmbH & Co produced the following advantages:

•natural resources are saved by the reduction in water consumption (as a rinsing agent as well as a coolant for the whole process) and steam (as the source of process energy)

•the environmental impact is reduced both by the decrease in use of resources and the lower production of wastewater

•the process is significantly cheaper for the company. Depending on the machine type used and the fabric to be treated, costs were reduced by 7 to 8%

•none of the machinery had to be modified

These results demonstrate that without big technical or financial investment even a small change in a production process can lead to a significant decrease in use of resources and environmental impact. It is also an effective means of improving a company’s competitiveness.

359

Marlene Etschmann, Peter Gebhart and Dieter Sell

6. Conclusions

Biotechnological processes offer many opportunities for application in different industrial areas. However, an awareness of these possibilities is frequently lacking. This may – at least in part – have to do with the public perception of biotechnology. Most of the time biotechnology is portrayed as a universal technology for fighting human diseases and world hunger, whereas biotechnology in sectors other than pharmaceutical, medical or agricultural rarely hits the headlines.

Entrepreneurs perceive environmental protection negatively as a cost factor. According to studies carried out for the European Commission in 1995, 77% of all entrepreneurs asked stated that environmental protection based on legislation measures resulted in increased costs. It has to be borne in mind that the majority of investments regarding environmental protection are still made for end-of-pipe or add-on technologies, which will never be productivity factors. The situation is different for preventive and integrated measures. They can create competitive advantages and even decrease operational costs (Heiden 1999). As shown previously, biotechnology can be integrated into industrial processes with benefits both from an economical and from an ecological point of view. This applies to many industrial fields, if executives would only recognise biological processes as an equally good alternative. An analysis, which shows their economic benefits, may be a convincing argument for a decision in favour of a biotechnical process.

References

Akhtar M., Kirk T.K., Blanchette R.A. (1995) Biopulping: An overview of consortia research. In: Srebotnik,

E., Messner, K. (eds) Proceedings of the 6th Int. Conference on Biotechnology in the Pulp and Paper

Industry: Advances in Applied and Fundamental Research. Facultas Universitätsverlag. Wien.

Bretzel W., Schurter W., Ludwig ,. B., Kupfer E., Doswald S., Pfister M., Loon van A. P. G. M. (1999).

Commercial riboflavin production by recombinant Bacillus subtilis: down-stream processing and comparison of the composition of riboflavin produced by fermentation or chemical synthesis. Journal of Industrial Microbiology & Biotechnology 22, 19-26.

Eggerdorfer M. et al. (1996). Vitamins. In. Elvers, B., Hawkins, S. (eds) Ullmann's Encyclopedia of

Industrial Chemistry, Vol A 27 443-613.

Heiden S.(1999). Integrierter Umweltschutz - Biotechnologie auf neuen Wegen. In: Industrielle Nutzung von Biokatalysatoren. Heiden S., Bock A.-K., Antranikian G. (eds). Erich Schmidt. Berlin. 3-26.

Kothuis B., Schelleman F. (1996) Environmental Economic Comparison of Biotechnology with Traditional

Alternatives. Institute for Applied Environmental Enonomics (TME), The Hague.

Loon van A. P. G. M., Hohmann H.-P., Bretzel W., Hümbelin M., Pfister M. (1996). Development of a

Fermentation Process for the Manufacture of Riboflavin. Chimia 50 (9), 410-412. OECD (1998) Biotechnology for clean industrial products and processes. 83.

Scott G. M., Akhtar M., Lentz M. J., Kirk K. T., Swaney R. (1998) New technology for papermaking: commercializing biopulping. Tappi Journal 81 (11), 220-225.

Wiesner J. et al. (1995) Production-Integrated Environmental Protection. In: Ullmann’s Encyclopedia of Industrial Chemistry, Vol B 8 213-309

360

Dr. Guido A. Drago and Dr. Tim D. Gibson

stabilisation are also becoming more fully understood. With the combination of expertise in the field of enzyme stabilisation and the advances in the methods used for predicting enzyme structural changes during the degradation processes, it is becoming increasingly easy to predict how to stabilise an enzyme for a specific industrial application.

Protein engineering can be a useful tool for increasing the stability of certain enzymes, for example luciferase, bacterial proteases (e.g. savinase - used in detergents) and carbohydrases, for example -glucosidase. Of course this is only possible so long as structural data is available for the enzyme under examination. The engineering of an enzyme structure can also lead to instability that is not always by design. The practicality of carrying out such time consuming studies on altering enzyme structure in order to improve stability is a matter of some debate. On the one hand one can generate an enzyme with all the attributes required of its application, on the other one cannot always predict the result of a particular mutation. In any case, even when an enzyme has been stabilised in this fashion, practical application of traditional techniques, such as granulation in the presence of stabilising additives are still applied. One very important point is to define what is meant by the term ‘enzyme stability’.

The two main types of stability may be defined as:

•Storage or Shelf Stability

•Operational Stability.

The first relates to the stability of enzymes when stored as a dehydrated preparation, a solution or as an immobilised preparation and is particularly concerned with retention of activity over time. Clearly such considerations are extremely relevant for enzyme producers from the point of manufacture of their products to the supply of the end users. The shelf life of enzyme based products generally depends on the stability of the enzyme in this context. The second generally relates to the retention of activity of an enzyme when in use. This is important for systems using enzymes for biocatalysis or biotransformations and analytical monitoring systems. The retention of activity in this context is often measured in terms of a half-life or (where the deactivation does not follow first order kinetics), referring to the time taken for the amount of enzyme activity to fall to half its original value.

Both types of enzyme stability will be discussed in this paper, using case studies from the analytical field (alkaline phosphatase, alcohol oxidase, acetylcholine-esterase, a recombinant luciferase and enzyme based biosterilisers (peroxidases). In all cases the stabilisation technique described has been restricted to the use of additives to modify the microenvironment of the enzyme under investigation. No other technique will be described in detail, except covalent immobilisation of pre-stabilised enzyme complexes. Initial trials using this system, (marketed as the PolyEnz™Process by AET Ltd) have indicated significantly higher levels of thermal stability for immobilised biocatalysts, glucose oxidase being used as a model enzyme [1]. The potential to produce operationally stable biocatalysts for use in the biosynthesis / biotransformation field will be discussed. Also a brief discussion of the likely factors influencing stability of enzymes has been included.

362

Enzyme Stability and Stabilisation: Applications and Case Studies

2. Materials and methods

Alcohol oxidase from Hansenula polymorpha was prepared in house by the method of Gibson [2]. Acetylcholine-esterase (930 units true cholinesterase, type III from electric eel E.C.3.1.1.7) and -galactosidase were purchased from Sigma. Horseradish peroxidase (HRP -4), glucose oxidase (GO -3) and bovine glutamate dehydrogenase

(GLDH ) were purchased from Biozyme Ltd. Recombinant luciferase was a gift from Celsis Ltd. The Pyrococcus furiosus glutamate dehydrogenase and -glucosidase were a kind gift from Dr. Serve Kenyan at Wageningen Agricultural University, Holland. The polyelectrolytes DEAE -dextran and dextran sulphate were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden) and Gafquat 755N, Gafquat HS-100 were obtained from ISP (Europe) Ltd, Guildford, Surrey, UK.

The techniques used for the stabilisation of enzymes described in this abstract are varied. Detailed protocols are described in the sited literature, except in certain instances as described below.

Alkaline phosphatase was stabilised as part of a UK government award (SMART Award). The actual formulations are available from Applied Enzyme Technology Ltd under license.

Alcohol oxidase and acetylcholine-esterase biosensors were prepared by carbodiimide immobilisation of the enzymes onto carbon electrodes, which were then treated by dip coating in a mixture of stabilisers. The sequence of transducer activation, enzyme immobilisation and subsequent stabilisation using polyelectrolyte-protein complex formation are described in Gibson et al. [3,4] and Rippeth et al. [5].

Solutions of HRP -4 were stabilised using admixtures of polyelectrolytes, polyalcohols and a specific buffer composition (Patent Application Pending). The actual formulation is available from Applied Enzyme Technology Ltd under license.

Solution stabilisation of recombinant luciferase was carried out by incubation of the enzyme with combinations of polyelectrolytes and polyalcohols in an commercial buffer supplied with the enzyme, the composition of which was unknown. The thermal degradation was carried out using the same techniques as described in Gibson [6] and Pierce et al. [7].

Immobilised glucose oxidase and pre-stabilised glucose oxidase-complexes were carried out by the procedure of Appleton et al. [1]. In all cases the biological activity of the enzyme was used as the main parameter to determine the stabilisation effect. Other techniques to determine any molecular and structural modifications occurring have been utilised. These include gel electrophoresis, circular dichroism, fluorescence and turbidimetric measurements. These methods have used to ascertain protein stability, especially where no simple method is available to directly measure biological activity, however in practical terms the results obtained from activity assays are usually sufficient.

The methods used to ascertain the effect of the stabilisers, generally focus upon thermostability as a suitable parameter for the demonstration of enzyme activity retention. This is a well accepted technique, provided the correct controls are evaluated and true long term stability studies are carried out in real time to corroborate short term, elevated temperature degradation’s [8, 9]. The evaluation of biosensor shelf life using

363

Dr. Guido A. Drago and Dr. Tim D. Gibson

the standard techniques described for pharmaceutical protein shelf-life estimations at elevated temperatures has recently been published [10]. Dry stabilisation studies were investigated by incubation of the dehydrated enzyme preparations or biosensors over freshly dried silica gel as desiccant. Early studies used a single temperature as an indication of the stabilisation effect, usually 37°C whereas the later experimental procedures reported in McAteer et al. [10] used a series of different temperatures. The dehydrated preparations were assayed for residual enzyme activity at selected time points throughout the incubation period and the results usually depicted as a time course of enzyme activity retention (Arrhenius plot).

Solution stabilisation studies were carried out using an elevated temperature method, where the activity of the enzyme under investigation decayed to half the original activity within 1 5 - 2 0 minutes, this is described in detail in Pierce et al. [7]. Each enzyme exhibits a characteristic deactivation response to temperature, which is dependant on the buffer used, the pH and the molarity of the solution. The control values of any particular enzyme system were determined using a solution of native enzyme dissolved in a defined buffer system and the effects of potentially stabilising additives were determined by comparison to the control deactivation profiles observed.

The electrophoretic separation of protein-polyelectrolyte complexes was carried out using standard polyacrylamide isoelectric focusing. Samples were pre-incubated in polyelectrolytes for 30-60 minutes prior to electrorhetic separation. Focusing was carried out for 2.5 hours at 1500v. The gel was subsequently fixed for 15 minutes in 5% sulphosalicylic acid and 10% trichloroacetic acid, rinsed with destaining solution (30% methanol, 10% acetic acid, 60% distilled water). The gel was subsequently stained with Coomassie blue for 10 minutes and destained until the background staining was low and the bands appear easily distinguishable. The gel was then dried down onto Gel Bond PAG film (Pharmacia Biotech.).

3.Results

3.1.ALKALINE PHOSPHATASE SOLUTION STABILITY: ENZYME SOURCE

AND BUFFER PARAMETERS

The source of the enzyme can be critical to the native stability of the enzyme and the ability of additives to further stabilise the enzyme in question. This is the case with alkaline phosphatase. Alkaline phosphatase isolated from bovine sources has a different stability profile compared with that isolated from bacteria (Bacillus species). The stabilisation effects observed are more dramatic in the case of the bacterial enzyme compared with the bovine enzyme, figure 1.

Alkaline phosphatase is presented as an example of the effect of buffer choice in the apparent stability of an enzyme in solution, figures 2 and 3. It can be seen from these results that the buffer used can have an effect on the stability of an enzyme, when the pH and concentration are kept constant, figure 2. In this case the most significant effect is seen using HEPES buffer, which is clearly incompatible with this enzyme. In figure 3, the effect of pH is clearly apparent. The thermostability of the enzyme increases as

364

Enzyme Stability and Stabilisation: Applications and Case Studies

the pH is reduced. These type of considerations are often extremely important in downstream processing of enzymes, where the wrong choice of buffer or pH can deactivate the enzyme being purified.

In our experience similar buffer specificity has been obtained with several other proteins including antibody conjugate solutions. The preference for Tris/HCl buffer systems over phosphate buffers by antibody conjugates in solution is measured by increased stability in the presence of Tris buffer. Another case in point is the

365