Improving Productivity and Enzyme Stability Through Process Design: Lipase-catalysed Synthesis of Epoxides and Esters

Interest in sustainable development has increased throughout society during the past decades, and the chemical industry is no exception. The appeal for renewable raw materials has consequently increased and has paved the way for the implementation of industrial biotechnology. Since enzymes are used to convert biomass in nature, they may also be a good choice in the chemical industry. However, to identify if the use of enzymes is the best alternative for a particular application they must be evaluated. Investigations can be carried out on the enzyme itself, the reaction, the reactor design or the complete process. Analyses of the environmental impact and the economics of the process are also important. The focus of the interdisciplinary research programme Greenchem at Lund University is to develop speciality chemicals from renewable raw materials using enzymes as catalysts. The work presented in this thesis was focused on the reactor and the process, rather than the catalyst itself, or a specific reaction, as those have been covered in other projects within Greenchem. Lipases are among the most extensively studied enzymes. Candida antarctica lipase B (CalB), which can be used to catalyse several different reactions, is one example. This enzyme was most frequently used in the work described in this thesis, usually in an immobilised preparation, Novozym® 435 (N435). In the initial study presented in this thesis, four wax esters were produced and tested regarding their applicability as wood coatings. Unfortunately, even the best of these waxes showed too low resistance to grease stains. Towards the end of this work a new study was initiated; acrylation of polymers to be used as the main component in lacquers, with the hope of achieving better resistance than that achieved with the wax esters. These acrylates were produced on litre-scale via lipase-catalysed trans-esterification between ethyl acrylate and polyesters and polyethers, respectively, and were found to be comparable to the commercial alternatives available today. A biolubricant, trimethylolpropane (TMP) oleate, was produced via esterification using the same lipase preparation, as well as epoxidised rapeseed methyl ester (RME) through chemo-enzymatic epoxidation. Several parameters must be considered when setting up a biocatalytic process. Water activity is one of them, and a system for water activity control was developed using wax ester synthesis as a model reaction. The system consisted of a sensor, a control unit and the facility to bubble air through the reactor, either dry or saturated with water, to decrease or increase the humidity in the reactor. The optimum water activity for esterification using N435 was determined and found to be close to zero. For comparison, an immobilised lipase from Candida rugosa was used, and this preparation was found to work best at higher water activities, i.e. above 0.5. By-product removal is another important parameter, and this was accomplished by evaporation through bubbling air through the reactor in wax ester synthesis, or by vacuum in the synthesis of acrylates and TMP oleate. The stability of the enzyme and the performance of the processes were investigated by varying different parameters. In the acrylation reaction half-lives of up to 320 h were achieved for two different CalB preparations, which resulted in reasonable catalyst costs for the process. However, when using N435 for TMP oleate synthesis, the process was not viable due to the combination of the long reaction times required at moderate temperatures and the relatively low stability of the enzyme (~90 h half-life). The stability of the biocatalyst was also found to be too low for an economically feasible production of epoxidised RME, although it could be enhanced by appropriate process design. Productivity and enzyme stability were investigated in different reactor designs, and it was found to be crucial to maintain the hydrogen peroxide concentration at a low, controlled level in order to prolong the life-time of the biocatalyst. The work presented in this thesis shows the direct applicability of biocatalysis to some processes such as the acrylation reaction, which has been proven to be competitive with traditional chemical production methods. In other cases, further development regarding choice of biocatalyst, reaction medium and process design are needed. One of the most important factors in investigations regarding industrial biotechnology is the enzyme stability, as this determines the possibility of implementing the process on an industrial scale