Oligosaccharide production with thermophilic enzymes

The goal of the research reported in this thesis was to develop a process concept for the tailor made production of oligosaccharides. These specific non-digestible oligosaccharides can be used as prebiotics. They promote the growth of beneficial bacteria in the gastrointestinal (GI) tract. Commercial prebiotic oligosaccharides are often not pure oligosaccharides, but mixtures. In this thesis focus is on the production of oligosaccharides of higher purity.Our main interest was in a production process at elevated temperatures. This can have many advantages, amongst which is the possibility to increase the substrate concentration. We used a thermophilicβ-glycosidase from Pyrococcus furiosus . Enzymes from thermophilic microorganisms have unique characteristics such as high temperature-, chemical- and pH stability. Applications with thermophilic enzymes are summarised in chapter 2. The main advantages of performing processes at higher temperatures are the reduced risk of microbial contamination, lower viscosity, improved transfer rates and improved solubility of substrates. However, co-factors, substrates or products might be unstable or other side reactions may occur.One route of oligosaccharide production is the synthesis from monosaccharides or disaccharides, using glycosidases as a catalyst. Monosaccharides can be condensated to disaccharides and disaccharides can be transglycosylated to trisaccharides. To investigate the potential of this synthesis withβ-glycosidase fromPyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose, lactose, glucose and galactose. The obtained parameters for initial rate measurements of disaccharide conversion were also used for the interpretation of experiments in time. The model for cellobiose gave a good description of the experiments. The enzyme was found to be uncompetitively inhibited by cellobiose and competitively inhibited by glucose. Lactose conversion however, could not be modelled satisfactorily; apparently additional reactions take place. Monosaccharide condensation also yielded oligosaccharides, but much slower. The use of a hyperthermostable enzyme was found to be positive. More substrate could be dissolved at higher temperatures, which benefited all reactions. This research is described in chapter 3.Besides the advantage of higher substrate solubility, temperature also influences enzyme kinetics. In chapter 4, the thermostable Pyrococcus furiosus-glycosidase was applied for oligosaccharide production from lactosein a kinetically controlled reaction. The experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield.However, at reaction temperatures of 80°C and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor 2, compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modelled by a cascade of a 0 thand 1 storder reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino-groups already gives complete inactivation of the enzyme.Reduction of Maillard reactions can be done by altering process conditions or through modification of the enzyme, either chemically or by altering the enzyme structure through genetic modifications. Chemical modification of the enzyme was studied. The enzyme was covalently immobilised on Eupergit. Unfortunately, the immobilisation did not reduce Maillard reactivity.Further reaction optimisation required a down-stream processing method for oligosaccharide separation. This was also essential for the production of a pure oligosaccharide product. Two methods for oligosaccharide purification are described in chapter 5.Oligosaccharides were produced in a condensation reaction using the -glycosidase from Pyrococcus furiosus. With a 60% (w/w) galactose solution as the substrate and oligosaccharide yield of 18% (w/w) was obtained. The feasibility of a Simulated Moving Bed (SMB) for downstream separation was investigated by modelling. The required parameters were determined experimentally with column experiments. The components could be separated with an SMB into a 91% pure product stream and a 99% pure galactose stream. This galactose stream can be recycled to the enzyme reactor. Also nanofiltration can be used for oligosaccharide purification. This system was also modelled and the results were compared to those that can be achieved with SMB.It is also possible to produce transgalacto-oligosaccharides in a more conventional way, with lactose as a substrate. Production is much cheaper when compared to a galactose-based process. Separation of oligosaccharides from this reaction via SMB was also studied.The size of all separation units is still considerably large and further optimisation is necessary to make a process for the production of specific high purity galacto-oligosaccharides cost-effective.Various aspects of the process are discussed further in chapter 6. Emphasis is on the specific influence of temperature on the process and on further optimisation of the downstream processing of oligosaccharides.