Feruloyl esterases - Evaluation of their potential for biotechnological applications
Doktorsavhandling, 2019

Owing to the current efforts to find sustainable alternatives to petrochemical based industries and technologies, enzymatic degradation and valorization of plant biomass has been attracting interest. Due to the complexity of plant biomass, an array of enzymes is required to hydrolyze it, including esterases. Among the esterases involved, feruloyl esterases, which are able to release ferulic acid, were of special interest in this work. Industrial processes aim for enzymes to be as efficient as possible in the designed process conditions, i.e. able to perform chemical reactions for as long as possible at the lowest possible cost. Several strategies can be employed to reach these goals, such as (i) finding novel enzymes with the desired properties, (ii) optimizing enzyme production, or (iii) immobilizing enzyme for improved stability or reusability. These strategies were applied in this work to investigate the potential of some feruloyl esterases for industrial applications.

Based on functional annotations, targets originating from microorganisms found in diverse ecological niches were selected. In one study, five putative feruloyl esterases/tannases from two Aspergillus fungi were selected. In another study, two multi-domain enzymes displaying two predicted esterase domains from the polysaccharide utilization loci of bacteria in the Bacteroidetes phylum were investigated. The enzymes displayed differences in their preferred reaction conditions (pH, temperature), molecular weights, predicted isoelectric points, as well as substrate preferences. The impact of the production host on the final enzyme properties was investigated in an additional study. We demonstrated that in the case of glycosylated enzymes, careful selection of the production host is crucial for thermostability. Studying immobilization, data showed that the best immobilization yield and the best immobilized enzyme performance were not achieved in the same conditions for any of the enzyme-support couples tested. Investigations of immobilized enzyme transesterification or hydrolysis activities clearly demonstrated that immobilization does affect the catalytic activity of enzymes. In the current status of our knowledge, the way an enzyme is affected by immobilization is not predictable. Increased knowledge about esterase structures, reaction mechanisms and surface properties may however allow such predictions in the future. This thesis contributes to increasing the available information about esterases, and in particular feruloyl esterases.

multi-domain enzyme

acetyl esterase

enzyme immobilization

N-glycosylation

carbohydrate active enzyme family 1

acetyl xylan esterase

heterologous production

polysaccharide utilization loci

enzyme stability

feruloyl esterase

FB-salen, Fysikgården 4, Chalmers
Opponent: Peter Westh, Technical university of Denmark, Denmark

Författare

Cyrielle Bonzom

Chalmers, Biologi och bioteknik, Industriell bioteknik

Kmezik C, Bonzom C, Olsson L, Mazurkewich S, Larsbrink J. Investigation of multi-domain esterases from soil and gut Bacteroidetes involved in xylan metabolism.

Bonzom C, Thörn C, Anasontzis G, Schild L, Olsson L. Investigation of five putative esterases from Aspergillus glaucus and Aspergillus zonatus.

All living organisms possess enzymes which are essential for life. Enzymes are proteins able to increase the speed at which chemical reactions happen: they catalyze reactions. Without enzymes we would for instance not be able to digest food fast enough to survive. Enzymes are also very selective for the molecules they act on and very specific for the type of reaction they catalyze. Therefore, in order to perform the wide range of reactions needed for survival, an array of enzymes are needed. Taking the example of food digestion, most mammals, including us humans, do not possess the enzymes needed to fully digest what they eat. Instead we rely on microorganisms, such as bacteria and yeast, living in our digestive tracts to help us.

The enzymes that I studied during my thesis work, called feruloyl esterases, are involved in the degradation of plant material. These enzymes can therefore be found in microorganisms living in digestive tracts, as well as in microorganisms living in soil or growing on trees.  Because they can be encountered in very diverse environments and conditions (i.e. pH, temperature) feruloyl esterases with various preferences in term of reaction conditions exist. Because of the reaction they catalyze, feruloyl esterases are of interest to various industries such as bio-refineries, paper mills, or food and feed industries. By modifying the reaction conditions, it is possible to change the reaction direction and make feruloyl esterases link together compounds they otherwise separate. The products of this type of synthetic reaction can be of interest for cosmetic and pharmaceutical industries. In addition, enzyme-based processes are usually conducted at milder temperatures and require less harmful chemicals than the corresponding chemical ones.

Despite the progress made in the past decades, enzymes remain costly to use. In order to decrease the economic impact of using enzymes in industrial processes, several strategies can be used. During my thesis work, I investigated novel esterases, with the aim of finding better ones (e.g. reacting faster, lasting longer). I also looked into the impact of the chosen microorganism for production. In doing so, I observed that some sugar-chain decorations, N‑glycosylation, which some microorganisms add on enzymes during production, are important for the activity and stability of these enzymes. Interestingly, I was able to demonstrate that the length of the sugar-chains changed the preferred reaction temperature of one feruloyl esterase by 10°C.

Another way of reducing the costs of using enzymes is to reuse them. In order to be able to do so we can attach them onto supports, a technique that is called enzyme immobilization. During my project, I used a support that is made of silica and possesses a porous network. The pores of the support allow the immobilization of high amounts of enzyme and can also shelter enzymes. I studied the impact of being immobilized in porous silica support on the activity of some feruloyl esterases. I also used these immobilized feruloyl esterases to evaluate their ability to perform synthetic reactions.

Altogether, these different aspects (picking the right enzyme, producing it efficiently and reusing it after immobilization) will contribute to make the use of enzymes more economically feasible. This will allow for the development of industrial enzyme-based processes that are more respectful of our planet.

Drivkrafter

Hållbar utveckling

Ämneskategorier

Industriell bioteknik

Mikrobiologi

Biokatalys och enzymteknik

Infrastruktur

Chalmers infrastruktur för masspektrometri

Styrkeområden

Energi

Livsvetenskaper och teknik (2010-2018)

Fundament

Grundläggande vetenskaper

ISBN

978-91-7905-156-3

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4623

Utgivare

Chalmers tekniska högskola

FB-salen, Fysikgården 4, Chalmers

Opponent: Peter Westh, Technical university of Denmark, Denmark

Mer information

Senast uppdaterat

2019-10-22