Directed Evolution of N‐Carbamyl‐d‐amino Acid Amidohydrolase for Simultaneous Improvement of Oxidative and Thermal Stability

Abstract

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

“…7b). Comparing this immobilized d -Carb with the activities of the enzyme purified by Ni Sepharose beads, the resulting specific activities were much higher, indicating an increased thermal as well as oxidative stability of the d -Carb upon immobilization as also reported from other groups (Oh et al 2002; Chiang et al 2008). Until now, immobilization of a carbamoylase via IMAC chemistry is not reported.…”

“…7b). The longer incubation times of the Ni Sepharose protocol may cause a loss in d -Carb activity due to oxidation as well as thermal instability (Oh et al 2002; Chiang et al 2008). These facts highlight the advantages of using functionalized magnetic beads for purification directly from the crude cell extract.…”

Efficient Preparation of Enantiopure D-Phenylalanine through Asymmetric Resolution Using Immobilized Phenylalanine Ammonia-Lyase from Rhodotorula glutinis JN-1 in a Recirculating Packed-Bed Reactor

“…Obviously, the production of three enzymes and the multi-enzyme catalytic process control are more complicated than a single Rg PAL reaction. Thirdly, the stability of N-carbamoylase is found to be low compared to that of D-hydantoinase, which is considered one of the limiting factors in the process [35] . The immobilized Rg PAL showed high stabilities and 80% activity of was retained after 30 reuses.…”

Directed evolution: tailoring biocatalysts for industrial applications

“…Chimeric lipases more stable and active toward 3-(3′,4′-dichlorophenyl) glutarate Suen et al (2004) Escherichia coli galactokinase GalK Broaden the substrate range Hoffmeister et al (2003) D-amino acid amidase from Ochrobactrum anthropi SV3 Enhanced thermostability and activity Komeda and Asano (2003) N-carboamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens Improvement of oxidative stability and thermostability Oh et al (2002) Pseudomonas aeruginosa lipase Enantioselectivity towards 2 methyldecanoate Jaeger et al Staphylococcus aureus lipase Changing activity to phospholipase Kampen and Egmond (2000) Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Serials Unit -Library on 10/22/12…”