Process parameter optimization for hydantoinase-mediated synthesis of optically pure carbamoyl amino acids of industrial value using Pseudomonas aeruginosa resting cells.
Hydantoinase-mediated enzymatic synthesis of optically pure carbamoyl amino acids was investigated as an environmentally friendly, energy-efficient alternative to the otherwise energy-intensive, polluting chemical synthesis. Hydantoinase-producing bacterial strain was identified as Pseudomonas aeruginosa by 16S rRNA gene sequencing and biochemical profiling using the BIOLOG Microbial Identification System. Hydantoinase activity was assessed using hydantoin analogs and 5-monosubstituted hydantoins as substrates in a colorimetric assay. The hydantoinase gene was PCR amplified using gene-specific primers and sequenced on an automated gene analyzer. Hydantoinase gene sequence of P. aeruginosa MCM B-887 revealed maximum homology of only 87 % with proven hydantoinase gene sequences in GenBank. MCM B-887 resting cells converted >99 % of substrate into N-carbamoyl amino acids under optimized condition at 42 °C, pH 8.0, and 100 mM substrate concentration in <120 min. Hydantoin hydrolyzing activity was D-selective and included broad substrate profile of 5-methyl hydantoin, 5-phenyl hydantoin, 5-hydroxyphenyl hydantoin, o-chlorophenyl hydantoin, as well as hydantoin analogs such as allantoin, dihydrouracil, etc. MCM B-887 resting cells may thus be suitable for bio-transformations leading to the synthesis of optically pure, unnatural carbamoyl amino acids of industrial importance.
Microbial Science Division, Agharkar Research Institute, G.G. Agarkar Road, Pune, 411004, India.
Anupama S
AS
Engineer
Anita P
AP
Dhakephalkar
Raghavendra P
RP
Gaikaiwari
Prashant K
PK
Dhakephalkar
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000302:isolation & purification / Q000378:metabolism*; D001711:Biotransformation; D002219:Carbamates / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D006827:Hydantoins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002219:Carbamates; D004269:DNA, Bacterial; D006827:Hydantoins; D000581:Amidohydrolases; C020047:dihydropyrimidinase