Spray encapsulation as novel method for generation of micro encapsulated biocatalysts and their application in multi step synthesis
Technical Chemistry
Final Report Abstract
In the first part of the present project the spray dryer B-290 (Büchi) was tested to evaluate the spray congealing for enzyme immobilization. Thereby different modifications had to be introduced for realizing the spraying of different carriers and different amounts of enzyme with good yields of up to 80%. For this first evaluation the laccase from Myceliophthora thermophile (MtL) was used, due to its robustness. Different carriers were tested, whereby cetyl alcohol turned out as the most suitable carrier with high retained activities of up to 55.5%. Furthermore a spherical shape of the particles with a main size of 25 – 35 µm could be observed. The re-usage of the particles resulted in a comparable big loss of protein after the first step followed by only small but continuously losses of activities. Measurements of protein content confirmed a high release after the first 30 minutes. In addition a higher amount of MtL resulted in a higher retained activity and during comparison of three different enzymes (MtL, TvL, PLE), the highest activity was found with the MtL, which showed also the highest thermostability. For further optimization of enzyme immobilization via spray congealing a special set up in the group of Prof. Passerini in Bologna, Italy, could be tested. Especially the used wide pneumatic nozzle enabled an easier handling and in addition larger particles of 150 – 250 µm with a spherical shape and yields up to 90%. To increase the retained activity of the PLE further carriers, formulations and an ionic liquid were tested and characterized. No significant difference in particle size by the used water amount could be observed, nevertheless, the addition of an emulsifier (Gelucire 50/14) increased the retained activity of the immobilized enzyme. For the PLE immobilized in Precirol ATO5 an activity of 8% was found, whereby the addition of 20% Gelucire resulted in 22% residual activity. For the recyclability also a positive effect of this additive could be observed, but here 15% Gelucire showed the lowest loss of activity after 5 batches. The tested IL MOAT showed on the one hand a comparable low retained activity of 3%, nevertheless, the retained activity after 5 batches was with almost 30% the highest and demonstrated the promising ability as carrier for the enzyme immobilization. In the third part of the project laccase initiated coupling reaction were investigated to increase the selectivity of these reactions. The TvL with a pH optimum of 4 was used for the coupling reaction of 4-methylcatechol and cyclohexylamine and resulted in a comparable unselective reactions with a larger amount of dimeric and polymeric products. By using a fed batch reactor to ensure the amine excess during the reaction the selectivity with regard to C-N coupling products could be increased significantly. This effect was improved with a lower pumping rate of the substrate flow, due to further raising of amine excess. In addition, different pH values were tested, whereby pH 5 found out to be the best compromise between enzyme activity and the availability of the amine for the coupling reaction. Nevertheless, the usage of the immobilized laccase in a fixed bed reactor resulted in a highly unselective reaction, because with this reaction set up no amine excess could be realized, like with usage of a fed bed reactor. In conclusion, the spray congealing enabled the enzyme immobilization with a good retained activity. Nevertheless, a continuous loss could be observed, due to the nature of the carrier and the physical entrapment only.
Publications
- (2015) Laccase initiated C-C couplings: Various techniques for reaction monitoring. Process Biochemistry 50, 1591-1599
Engelmann, C.; Illner, S.; Kragl, U
(See online at https://doi.org/10.1016/j.procbio.2015.05.011) - (2018) Spray congealing as innovative technique for enzyme encapsulation. Chemical Technology and Biotechnology 93, 191-197
Engelmann, C.; Kragl U.
(See online at https://doi.org/10.1002/jctb.5339)
