The alkyl sulfatase Pisa1 from Pseudomonas sp. DSM 6611 has been shown to accept a large amount of alkyl sulfate esters. Enantioselectivities, measured as E-values, were ranging from very low (4), to excellent (>200). Expansion of the substrate scope, using benzylic sulfate ester moieties, was achieved, including the resolution of a pyridine heterocyclic compound with perfect E-values. During kinetic resolution, we observed erosion of the enantiomeric excess in the control reaction. Investigation of the underlying autohydrolytic mechanism using H218O to follow the incorporation of the 18O into the product, revealed a combination of SN1 and SN2 mechanisms at a ratio of 1:1.6. The SN1 reaction was shown to occur via the formation of a planar resonance-stabilized benzylic carbocation intermediate leading to racemic product. The SN2 pathway follows the classical nucleophilic attack of an [OH-] onto the benzylic carbon atom, resulting in inversion of configuration. Retention ([OH-] attack on sulfur) was found to be a minor event (<3 %). During catalysis, Pisa1 mediates substrate inversion by protonation of the sulfate ester group R-O-SO3-, thereby providing HSO4- as a good leaving group, making a chemically extremely challenging reaction possible. This knowledge was the basis for a "catalophore-search" to obtain targets with similar arrangement of key residues in the active site. Thus we were able to compare the active site architecture of Pisa1 with the protein database, resulting in three promising candidates: the alkaline phosphatase from northern shrimp (AP), a putative metal dependent hydrolase from Lactobacillus casei and the ser/thr phosphatase 5 from Human. Substrate screening revealed that neither AP, nor the putative hydrolase were active on tested substrates. The human enzyme remains a promising target for the hydrolysis of alkyl sulfates.