High performance production of oligosaccharides by using enzymatic membrane reactors

Bioactive oligosaccharides have broad applications in the food and medicine fields due to their unique physicochemical properties. Oligosaccharides of high quality are therefore increasingly demanded on the market. The traditional process of oligosaccharide production involves chemical hydrolysis of polysaccharides and multiple steps of precipitation leading to high energy consumption and generation of wastewater. More importantly, hazardous chemicals are required in the traditional process of producing bioactive oligosaccharides. A sustainable and safer process for the production of oligosaccharides is therefore necessary.

An enzymatic membrane reactor (EMR) is a green technology to replace chemical methods of producing oligosaccharides. The EMR can simplify the production of oligosaccharides through an in-situ bioconversion and separation in which enzymes are applied as catalysts and a membrane serves as a selective barrier. The EMR approach with free enzymes has been studied in previous research. However, due to the random nature of enzyme hydrolysis, the interplay of enzyme reaction and membrane separation needs to be further investigated to optimize the production of the intermediate-sized oligosaccharide products. Moreover, the reusability of enzymes and membrane fouling are the major limitations that hinder the industrial application of an EMR with free enzymes. In this project, enzyme immobilization is proposed that brings several advantages to the EMR to improve the performance of oligosaccharides production. With immobilized enzymes, the enzyme reaction can be restricted to a narrow area near the membrane surface and the production of oligosaccharides can be controlled by adjusting the operating parameters and thus tailor the molecular weight (Mw) of the oligosaccharides products.

First, we used commercial polymeric membranes (PES30) as the carrier for enzyme immobilization.Surface modification (by dopamine (DA) and tannic acid (TA)/3- aminopropyltriethoxysilane (APTES)) was applied on the chosen membrane to introduce functional chemical groups that offered binding sites for enzymes. Dextranase was used as the model enzyme and was immobilized on the modified membrane surface by various methods (i.e. cross-linking, adsorption and covalent binding). The enzyme loading efficiency and hydrolysis behavior of the immobilized dextranase were investigated to identify suitable immobilization strategies for constructing an EMR. The substrate conversion rate of the immobilized dextranase was greatly affected by the methods of enzyme immobilization. Moreover, the type of enzyme activity (the hydrolysis position on the substrates molecules) shifted when different immobilization strategies were applied. Notably, non-cross-linked dextranase immobilized on the hierarchical TA/APTES coated membrane surface via a fouling-induced mode not only preserved the desired hydrolysis behavior but also retained high activity. This immobilization method offers a promising strategy to establish an EMR for the production of intermediate-sized oligodextrans.

Second, we applied similar surface modification methods as described above to porous electrospun fibers followed by enzyme immobilization to further improve the conversion-separation property of the EMR. Compared to the commercial polymeric membrane, the fiber matrix offered a large surface area and higher porosity that enhanced the hydrolysis performance of the immobilized enzyme. By combining the electrospun fibers and a separation membrane, multiple layered EMRs were developed in this work. The porous structure of the electrospun fibers reduced the mass transfer limitation of substrates at the active site of the immobilized enzymes, and thus the immobilized enzymes showed higher activity when electrospun fibers were applied as the carrier. Furthermore, after introducing the electrospun fibers on the membrane, less membrane fouling occurred and high permeability and enzyme activity were therefore simultaneously preserved in the multiple layered EMR. Additionally, negligible membrane fouling was observed on the EMR after enzyme immobilization and oligosaccharides production using hydrophilic membrane and the surface-modified electrospun fibers. The developed EMRs were also used to produce low Mw oligosaccharides. Due to its high activity and selectivity, the multiple layered EMR outperformed the other EMRs in producing intermediate-sized oligosaccharides of desired Mw. The multiple layered EMR is thus a promising strategy for the production of high-quality oligosaccharides.

In summary, this study aimed to establish a high performance EMR that can produce low Mw oligosaccharides. To this end, we investigated various enzyme immobilization strategies and their effect on the immobilized enzyme activity. In this work, the great novelty of the EMR lies in the use of multiple layers that simultaneously enhanced the immobilized enzyme activity and reduced membrane fouling. Suitable methods and materials were further applied to develop EMRs that retained both high enzyme activity and membrane selectivity. The strategies described in this work provide various possibilities for the design of EMR for production of low Mw oligosaccharides.