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|Title: ||Stability and stabilization of industrial biocatalysts|
|Authors: ||Fattor, Diana|
|Supervisor/Tutor: ||Gardossi, Lucia|
|Issue Date: ||26-Mar-2012|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||Catalytic potential of enzymes is not fully exploited at industrial level and in chemistry due to technical difficulties and long time required for development of new processes. The main issues are:
1) The choice of the biocatalyst and the planning of reaction conditions still relies largely on empirical approaches, leading to long experimental studies;
2) There is still a short knowledge about molecular phenomena occurring in the microenvironment surrounding the enzyme and affecting biocatalyst efficiency;
3) The experimental systems are very complex and influenced by a wide number of experimental variables that cannot be monitored nor measured. This is particularly true when immobilized enzymes are considered.
4) Enzymatic preparations available on the market are not homogeneous in their components and protein content.
The present study concerned the stability of both native and immobilized enzymes in aqueous media, aqueous/organic solvent mixture and low water media (either organic solvent or neat substrates). Moreover, thermal stability and effect of microwave radiations was also considered. The enzymes taken into consideration were lipases (hydrolases EC 184.108.40.206), and laccases (oxydoreductases EC 220.127.116.11).
Aiming at overcoming the above mentioned limitations, this research was focused on the combination of experimental and computational approaches to:
a) analyze enzyme stability under potentially denaturing conditions (polar solvents, temperature, microwave radiation) trying to identify by molecular descriptors for constructing correlation models (chapters 1 and 3);
b) stabilize biocatalysts through immobilization while preserving catalytic activity (chapter 2);
c) investigate experimentally the impact on immobilized biocatalysts of stabilizers and additives present in native crude enzyme preparations (chapter 4).
The computational methods used for this study are Molecular Dynamic simulations (MD) that, together with experimental data, tried to explain changes in the protein structure and thus evaluate their stability in a given environment to better understand the behaviour of a biocatalyst.
The study of the stability of native lipases in water-solvent monophasic systems has pointed out how the three lipases considered (Candida antarctica Lipase B, Pseudomonas cepacia Lipase and Rhizopus oryzae Lipase) behave very differently. Organic solvents for some extent can even mimic more efficiently the physiological environment of lipases, since in nature they are not working on soluble substrates in diluted aqueous solutions.
When another class of enzymes, such as laccases, were taken into account, the stability of the different proteins (Laccase from Basidiomycetous Panus tigrinus, Lentinus strigosus and Steccherinum ochraceum) resulted to be strongly dependent on the extent of glycosylation and not only on the protein structure. Again, the heterogeneity of the glycosylation pathways makes the construction of any rational model, based on enzyme structures, quite a formidable task.
These observations suggest that when working with native enzymes each protein must be studied separately even if belonging to the same class. Therefore, general conclusions and models are hardly applicable when planning stabilization strategies in biocatalysis.
This is also important when designing immobilization protocols aiming at stabilizing enzymes.
Furthermore, when these proteins are immobilized, not only structural features of the enzymes must be considered but also the formulation of the native biocatalyst resulted to play a key role in the performances of the resulting immobilized protein. Additives and stabilizers are often the predominant components in commercial enzymatic preparations which most often are produced for different scopes than biocatalysis (e.g. formulation of detergents) and inevitably severely affect the efficiency of immobilization strategies.
Although, the purification of the protein would be desirable for avoiding the interference of non-enzymatic components. It must be underlined that the use of very crude enzymatic preparations is generally mandatory at industrial level when the cost of the immobilized biocatalyst has a major impact on the economic sustainability of the process. Therefore, enzymes to be applied in biocatalysis ideally should be fermented and processed according to tailored and optimized protocols, which enable the full exploitation of the catalytic potential of the enzyme upon immobilization.
In conclusion, a larger and most efficient exploitation of enzymes in novel biotransformations will be feasible only through a strict integration of all the technological steps leading to the development of effective and economically competitive biocatalysts.|
|PhD cycle: ||XXIV Ciclo|
|PhD programme: ||SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE E TECNOLOGIE CHIMICHE E FARMACEUTICHE|
|Main language of document: ||en|
|Type: ||Tesi di dottorato|
|Scientific-educational field: ||CHIM/06 CHIMICA ORGANICA|
|Appears in Collections:||Scienze chimiche|
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