Projects per year
Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however, expected to be more and more combined with enzyme-catalyzed synthesis, resulting in chemo-enzymatic approaches towards peptide synthesis. The racemization that hampers chemical synthesis can be prevented by forming the peptide bond enzymatically.
In the work in this thesis the bioprocess engineering aspects of a specific chemo-enzymatic peptide synthesis route are studied. In this route, an N-protected, C‑terminally activated amino acid is coupled with a C-protected amino acid nucleophile. The coupling step is catalyzed by Alcalase. The initial idea was to also enzymatically catalyze the activation of the amino acid, which is used in the coupling step, and to carry out the coupling and activation steps in one pot. In the work of Chapter 2 lipase B from Candida antarctica (CalB) and Alcalase were used as a model system for such a one-pot chemo-enzymatic peptide synthesis, in order to investigate the (in)compatibility between the two enzymes. The rate of activity loss of native and immobilized CalB in the absence and presence of native and immobilized Alcalase was calculated from the rate of triacetin hydrolysis. It was shown that native Alcalase degrades native CalB under aqueous conditions. Immobilization of both or either CalB or Alcalase onto macroporous beads, however, effectively prevented hydrolysis of CalB by Alcalase.
Due to the current impracticality of the enzyme-catalyzed activation step, the rest of the work in the thesis focuses on the Alcalase-catalyzed coupling step. The non-aqueous coupling in tetrahydrofuran (THF) of phenylalanine amide (Phe-NH2) and the carbamoylmethyl ester of phenylalanine (of which the amino group was benzyloxycarbonyl-protected, Z-Phe-OCam) was used as a model coupling reaction.
In protease-catalyzed peptide synthesis the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. In the work of Chapter 3 the model coupling reaction was catalyzed by cross-linked enzyme aggregates of Alcalase optimized for use in organic media (Alcalase CLEA-OM) at a range of water activity (aw) values, including the coupling in the presence of molecular sieves (i.e. at very low aw values). The rate of peptide synthesis could not be increased by increasing awvalueswithout significantly increasing the rate of hydrolysis, i.e. without significantly decreasing the synthesis / hydrolysis (S/H) ratio. Hydrolysis (in the present system, only the activated substrate, not the dipeptide product, may be hydrolyzed) was found to dominate above aw ≈0.2.To prevent hydrolysis, the presence of molecular sieves was found to be necessary. Nevertheless, the use of molecular sieves over longer periods of time should be carefully considered as they may dehydrate and thereby inactivate the enzyme in time.
In the work of Chapter 4, besides CLEA-OM, also other Alcalase formulations were used to catalyze the model coupling reaction. The reaction was done in the presence of molecular sieves (i.e. under near-dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous THF)of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Without such initial hydration, the enzymes seem to lack the water needed to maintain their catalytically active conformation. Repeated use in the presence of molecular sieves, without intermediate rehydration, led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation, Alcalase immobilized onto dicalite, was only partially reversible by hydration. Alcalase immobilized onto dicalite was found to be initially the most active in dipeptide synthesis. Nevertheless, due to its small particle size and its lack of operational stability, this formulation may not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads (in this thesis abbreviated as Cov) due to its reasonable activity, its seemingly good operational stability, and its practical size and uniform spherical shape.
If, for economic reasons, Cov should be reused repeatedly for dipeptide synthesis in organic media, its operational stability is important and thus its activity should not decrease significantly. The long-term stability and reuse of hydrated Cov in THF was investigated in the work of Chapter 5. Cov was incubated with and without molecular sieves (beads or powder) in anhydrous THF. After different incubation periods in THF, the enzyme activity was determined in an aqueous environment. In addition, Cov was repeatedly recycled in order to examine its reusability. The effect of reuse on the aqueous activity of Cov and on the Cov-catalyzed model coupling reaction in near-anhydrous THF was studied. Without molecular sieve beads, Cov hardly inactivated in THF. Nevertheless, when Cov was incubated with molecular sieve beads in THF in rotating reaction vials, Cov lost activity over time. Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the instability of Cov. In order to reuse Cov for the model coupling reaction in the presence of molecular sieves, it needs to be rehydrated in between the batches. Nevertheless, each intermediate rehydration step also caused a small but significant enzyme activity loss.
In the work of Chapter 6, the coupling kinetics of the model coupling reaction, catalyzed by hydrated Cov, were investigated. Near-anhydrous conditions were maintained by a carefully chosenamount of molecular sieve powder (in contrast to molecular sieve beads, molecular sieve powder does not lead to mechanical damage of Cov). Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. It was found that the kinetics of the coupling can be described well with a two-substrate kinetic model with two inhibitory products. To reduce the effect of the product inhibition on Cov, a reactor should be designed in which at least glycolamide is selectively removed, as it was found to be the strongest inhibitor.
In Chapter 4 it was shown that molecular sieves dehydrate and thereby reversibly inactivate the enzyme. In the work of Chapter 7 the effect of enzyme dehydration by molecular sieves on the Cov-catalyzed model coupling reaction was studied in detail. The dehydration kinetics of Cov by different amounts of molecular sieve powder were determined by incubating Cov with molecular sieve powder for different periods of time. Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder seemed to occur in three phases: (1) an initial, rapid,major dehydration-induced inactivation that takes place during the first activity measurement (1 h), (2) a phase of first-order inactivation (20 h), and (3) a relatively low plateau phase in activity. These dehydration kinetics were incorporated into the reaction kinetics model described in Chapter 6. The resulting model was then used to fit progress curve data of the model coupling reaction in the presence of different amounts of molecular sieve powder. Using the estimated parameter values, the model was used to predict independent data sets and found to work well.
The work of Chapter 8 is a case study about a process design for enzymatic peptide synthesis, which is based on the findings of the previous chapters. The choices with regard to Alcalase formulation, type of reactor, way to control the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amounts of substrate, enzyme and molecular sieves, needed in order to produce a specific demand for peptides. We believe that this case study gives a good impression of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices.
|Qualification||Doctor of Philosophy|
|Award date||5 Dec 2012|
|Place of Publication||[S.l.|
|Publication status||Published - 2012|
- bioprocess engineering
- protein synthesis
- enzyme activity