Structure-Function Relationships of Enzymes Involved in Starch Modification

Starch, a sustainable and abundant energy storage source found in human food and animal feed, plays a crucial role in diverse applications such as biomaterials, biorefineries, and biomass feedstocks for fuel energy. To enhance its properties, starch can be subjected to enzymatic, chemical, or physical treatments through structural engineering. Enzyme treatment using starch-active enzymes is an environmentally friendly and attractive approach, improving thermal properties, digestion resistance, and complexation capacity. Enhancing catalytic efficiency of these enzymes can be achieved through mutations or constructing starch binding domain (SBD) fusions, which increase the affinity of the enzymes for the substrates and consequently improve their catalytic efficiency.

The thesis is divided into 5 chapters.

Chapter 1 is the Introduction, which starts by an overview of the multi-level structure of starch granules. Additionally, it delves into the realm of carbohydrate active enzymes (CAZymes), with a particular emphasis on the enzymes that were studied in this thesis. It is also explored how CAZymes find application in the modification of starch. Furthermore, it is delved into the understanding of SBDs, in terms both of their structural characteristics and functional roles, along with their innovative application through SBD fusions. As the essence of this thesis, we introduce the concept of interfacial catalysis and kinetics of starch granules, and shed light on its significance. To conclude, a comprehensive overview of the fundamental materials, enzymes, and methodologies in this thesis are provided.

Chapter 2 is the Result and divided into 3 subchapters.

Subchapter 2.1 comprises 2 papers (Paper 1 and Paper 2). Paper 1 focused on the impact of SBDs on the interfacial catalysis on granular starches by C-terminally fusing an SBD from either Aspergillus niger glucoamylase (SBD_GA) or Arabidopsis thaliana glucan, water dikinase 3 (SBD_GWD3) to a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23. The Michaelis-Menten (MM) approach is used to determine kinetic parameters for α-amylase hydrolysis of granular starch. This suits soluble substrates having an excess substrate, but is challenging for insoluble starch with undefined molarity and limited enzyme accessibility. To overcome this, we applied interfacial kinetics analysis with enzyme-starch granule adsorption isotherms, inspired by cellulases acting on cellulose, to measure the attack site density (^kinΓ_max) and binding site density (^adsΓ_max) for various types of starch granules. According to the interfacial kinetics analysis, the AHA-SBD fusions increased the density of enzyme attack sites and binding sites on the starch granules by up to 5- and 7-fold, respectively. Paper 2 focused on the impact of an N-terminal SBD fusion on the activities and starch product structure of a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT). The SBDs were the N-terminal tandem domains (SBD_St1 and SBD_St2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2), and the C-terminal domain (SBD_GA) of glucoamylase from Aspergillus niger (AnGA). The results showed that SBD-TuαGT fusions had higher hydrolytic activity than TuαGT and higher affinity for starch granules. Among the StDPE2 SBD-fusions, SBD_St2 significantly outperformed SBD_St1 in enhancing TuαGT activity, substrate binding, and stability.

Subchapter 2.2 includes 1 paper and 2 manuscripts (Paper 3, Manuscripts 1 and 2). Manuscript 1 focused on the impact of SBDs on interfacial catalysis of starch granules by pullulanase. In this manuscript, we identified the function of N-terminal domains (NTDs), including a CBM41 and two domains of unknown function (DUFs) in the pullulanase from Lactobacillus acidophilus NCFM (LaPul) by two recombinantly produced truncated variants, namely ∆41-LaPul (without CBM41) and ∆(41+DUFs)-LaPul (without CBM41 and two DUFs). Through analyzing the unfolding temperature, binding affinity to β-cyclodextrin (β-CD) and starch granules, as well as kinetics on soluble substrates and interfacial kinetics on insoluble starch granules, we established that CBM41 plays a role in substrate binding, while the DUFs contribute to stability. As inspired by Manuscript 1, we hypothesized that the attack site density (^kinΓ_max) for pullulanase on the granular starches can be used to represent the density of branch point on the surface of starch granules since type I pullulanase (PULI) is only active on α-1,6- linkages (Paper 3). In Paper 3, the kinetics analysis of heterogenous catalysis was adapted to enumerate α-1,6-linked branch points hydrolyzed by a commercial Bacillus licheniformis pullulanase (BlPul) on the surface of granules of waxy and normal maize starch (WMS and NMS). To validate this novel method, we also pretreated these granular maize starches using either branching enzyme from Rhodothermus obamensis that (RoBE) catalyzes introduction of new α-1,6 linked branch chains or by TuαGT (produced in Paper 2). The results indicated that WMS showed 1.9-fold higher branch point density on the starch surface than NMS. Besides, the treatment by RoBE increased the branch point density for WMS from 1.7 to 3.3 nmol/g starch granules, while the treatment by TuαGT did not affect the branch point density for the two maize starch granules. Manuscript 2 is a continuous work after Paper 3, where the Sabatier principle was introduced as a tool to understand the enzymatic reaction on starch granules. In Manuscript 2, we used BE and 4αGT to modify three types of maize starches with different amylose content and analyzed the structure of these granular starches. By analyzing the relationship between the relative affinity and reaction rate to BlPul, it was found that the RoBE-modified starches showed higher affinity and lower reaction rate, except for the RoBE-modified waxy maize starch, than unmodified and TuαGT-modified starches. This change in affinity and reaction rate might stem from the granular structure of the starches, including the crystallinity, surface order degree and chain length distribution.

Subchapter 2.3 includes 1 paper (Paper 4) and is different from subchapters 2.1 and 2.2, as it does not involve enzyme discovery and characterization. In Paper 4, a novel super-branched amylopectin was prepared by modifying gelatinized normal maize starch using RoBE and TuαGT. This modified starch was used for co-entrapment of a curcumin-loaded emulsion in alginate beads (ABs). UV stability and in vitro simulated gastrointestinal digestion were evaluated for of all prepared types of ABs, and demonstrated the potential of using enzymatically modified starch and alginate as a versatile vehicle for co-encapsulation to obtained controlled release and targeted delivery of bioactive compounds.

Chapters 3, 4 and 5 are the general discussion, conclusion, and future perspectives of the thesis, respectively.

This thesis provided new knowledge about the function of SBDs in different starch-active enzymes, especially about the interfacial catalysis of granular starches. This interfacial kinetic analysis provided new insights in the understanding the enzymatic degradation and/or modification of starch granules. Besides, we also investigated the application of enzyme modified starches for encapsulation of bioactive compounds within alginate beads.