2-Azabicyclo[2.2.1]hept-5-en-3-one (gamma-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (-)-gamma-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-gamma-Lactamase can be used for the kinetic resolution of gamma-lactam to obtain (-)-gamma-lactam. In this study, a novel (+)-gamma-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic gamma-lactam with high enantiomeric excess (ee) (> 99 %) and yield (> 49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known gamma-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-gamma-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-gamma-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 A degrees C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni2+ or Co2+. Mh33H4-5540 displayed much higher (+)-gamma-lactamase activity than any other biochemically characterized (+)-gamma-lactamases. Overall, we discovered a novel (+)-gamma-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.