Enzymes for industrial acrylation : redesign of candida antarctica lipase B and characterization of a new cutinase from alternaria brassicicola

• Main Author: Liu, Danni
• Other Authors: Schmid, Rolf D. (Prof. Dr.)
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: 2009
• Subjects: Lipasen; Cutinase; Proteindesign; Heterologe Genexpression; Pichia pastoris; Candida antarctica; 570; Acrylierung; Immobilisierung; Charakterisierung; acrylation; immobilization; characterization; Antarc*; Antarctica
• Online Access: https://doi.org/10.18419/opus-1207; http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-46357; http://elib.uni-stuttgart.de/handle/11682/1224

Candida antarctica lipase B (CALB) (EC 3.1.1.3) is a serine hydrolase with an alpha/beta hydrolase fold. BASF have screened some well-known enzymes for transesterification of hydroxypropylcarbamate (hpc: 1hpc, 2hpc, 3hpc and 4hpc) with methyl acrylate. The results showed that the CALB had the highest activity among all the screened enzymes. However, the wild-type CALB does not catalyze full conversion in an acceptable time scale. For industrial purposes it was necessary to increase the activity of the wild-type enzyme. The S-enantiomer of the secondary alcohol was identified as the bottleneck for reaching full conversion. In order to improve the acrylation, the stereospecificity pocket was redesigned using predictions from molecular modeling. Positions 278, 104 and 47 were targeted and a library for two-site saturation mutagenesis at positions 104 and 278 was constructed. For library screening, CALB variants were expressed in Escherichia coli (E. coli). This expression system is more suitable for high-throughput requirements in terms of expression time and number of clones. However, Pichia pastoris (P. pastoris) X-33 was selected as an ideal expression host of CALB for the higher expression level of the recombinant gene. After expression in P. pastoris, CALB variants were characterized with respect to activity in tributyrin hydrolysis, hydroxypropylcarbamates acrylate (hpca) hydrolysis and stereoselectivity in hydrolysis of 1-phenylethyl propionate. With respect of acrylation of hpc with methyl acrylate, lyophilized and immobilized CALB were used as an almost water-free reaction system since the presence of water in reaction can disturb the conversion. Apart from the identification of appropriate mutants, a suitable carrier for the enzyme also plays an important role in the reaction. It was shown that CALB showed only activity towards the substrate after immobilization on hydrophobic carriers, e.g. on Accurel MP1000. Therefore, hydrophobic carriers Accurel MP1000 and Diaion HP20L were prepared for the following ...