Construction and Functional Screening of Metagenomic Libraries for D-Stereospecific Amidases and Their Catalytic Promiscuity

Metagenomic fosmid libraries were prepared from soil and water samples collected from diverse places. Clones in the range of (1.1–2.8) × 10³ were isolated from these samples and used for screening and isolation of D-amidase producer using dyes bromthymol green and phenol red. Maximum number of metagenomic libraries were obtained from soil rich in fishery waste while minimum number of libraries were obtained from tap water genome. Five positive clones after two-stage functional screening for D-alanine amidase were isolated on the basis of change in colour of growth medium due to enzyme production. Selected isolate on the basis of maximum enzyme production (2 U/mL) was cloned and overexpressed. Selected isolate from fishery waste metagenomes was found to be constitutive producer of D-amidase enzyme which after optimization led to 32% increase in enzyme activity. Isolated novel enzyme showed catalytic promiscuity with amides of D-Leu, D-Pro, D-Met and D-Val. Optimization of reaction conditions leading to production of 72% D-Ala and 65% D-Leu from their D-amide salts, respectively, in 8 h were confirmed by HPLC. Isolated homologous and putative genes after genomic screening were confirmed to be matching with Rhodococcus and Pseudomonas aeruginosa amidases. Enzyme also had acyl transferase activity with various amide substrates.