Cloning, expression, purification, crystallization and preliminary X‐ray characterization of allantoinase from Bacillus licheniformis ATCC 14580

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N 1 —C 2 amide bond of the five‐membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin ( R ‐enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C‐terminal His 6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour‐diffusion method using 0.1  M potassium thiocyanate, 20%( w / v ) polyethylene glycol 3350 as a crystallization solution. X‐ray diffraction data were collected to a resolution of 3.5 Å with an R merge of 29.2% from a crystal belonging to space group P 12 1 1, with unit‐cell parameters a = 54.93, b = 164.74, c = 106.89 Å, β = 98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient ( V M = 2.34 Å 3  Da −1 ).