Immobilization of (R)- and (S)-amine transaminases on chitosan support and their application for amine synthesis using isopropylamine as donor.

Transaminases from Aspergillus fumigatus ((R)-selective, AspFum), Ruegeria pomeroyi ((S)-selective, 3HMU) and Rhodobacter sphaeroides 2.4.1 ((S)-selective, 3I5T) were immobilized on chitosan with specific activities of 99, 157, and 163U/g and acceptable yields (54, 21, and 23%, respectively) for glutaraldehyde (GA) immobilization. Besides GA, also divinylsulfone was used as linker molecule leading to a similar efficient immobilization for two enzymes, GibZea and NeoFis, whereas GA was superior in the other cases. Storage of the GA-immobilized enzymes for one month resulted in increased relative activities between 120 and 180%. The thermal stability was improved, especially for the GA-immobilized AspFum compared to the free enzyme after incubation for 4h at 60°C (10% vs. 235% residual activity). Especially after incubation of AspFum (free or immobilized) for 2h at 50°C a strongly increased activity was observed (up to 359% of the initial activity). This effect was studied in more detail, revealing that one heat activation prior and one after immobilization increased the overall immobilization efficiency. Recycling of the immobilized ATAs resulted only in a small reduction of activity after four batches. Asymmetric synthesis of (R)- or (S)-1-methyl-3-phenylpropylamine from the prostereogenic ketone using isopropylamine (IPA) as amino donor was applied with conversions up to 50% (AspFum) or 75% (3HMU). Except for NeoFis, all immobilized ATAs showed higher conversions compared to the free enzyme.

A self-sufficient Baeyer-Villiger biocatalysis system for the synthesis of ɛ-caprolactone from cyclohexanol.

In order to establish a new route for ɛ-caprolactone production from the corresponding cyclohexanol with an internal cofactor recycling for NADPH, a recently redesigned thermostable polyol dehydrogenase (PDH) and the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus were combined. First, the expression of PDH could be improved 4.9-fold using E. coli C41 with co-expression of chaperones. Both enzymes were also successfully co-immobilized on glutaraldehyde-activated support (Relizyme™ HA403). Cyclohexanol could be converted to ɛ-caprolactone (ɛ-CL) with 83% conversion using the free enzymes and with 34% conversion using the co-immobilized catalysts. Additionally, a preparative scale biotransformation of ɛ-caprolactone starting from cyclohexanol was performed using the soluble enzymes. The ɛ-CL could be isolated by simple extraction and evaporation with a yield of 55% and a purity of >99%.

Protein engineering of a thermostable polyol dehydrogenase.

The polyol dehydrogenase PDH-11300 from Deinococcus geothermalis was cloned, functionally expressed in Escherichia coli and biochemically characterized. The enzyme showed the highest activity in the oxidation of xylitol and 1,2-hexanediol and had an optimum temperature of 45 °C. The enzyme exhibited a T6°50-value of 48.3 °C. The T6°50 is the temperature where 50% of the initial activity remains after incubation for 1h. In order to elucidate the structural reasons contributing to thermostability, the substrate-binding loop of PDH-11300 was substituted by the loop-region of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scope of this chimera basically represented the average of both wild-type enzymes, but surprisingly the T6°50 was noticeably increased by 7 °C up to 55.3 °C. Further mutations in the active site led to identification of residues crucial for enzyme activity. The cofactor specificity was successfully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD⁺ binding cleft, without influencing the catalytic properties of the dehydrogenase.