An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50؇C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg 2؉ and Ca 2؉ activated the proteinase, as did NaCl; however, Hg 2؉ , Fe 2؉ , and Zn 2؉ caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH 2-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Ser-Gln-Pro-Gly. The surface microflora of various smear surface-ripened cheeses, such as Limburger, Gruyère, Münster, Brick, Appenzeller, and Tilsiter, includes yeasts, moulds, and bacteria (17, 19). Low-molecular-weight compounds produced at the surface through the action of various enzymes from the smear microflora diffuse into the interior of each cheese and contribute to the characteristic qualities of these cheeses; the extracellular enzymes are much too large to diffuse into the cheeses (20, 22). The growth of Brevibacterium linens on the surface is an essential prerequisite for the development of the characteristic flavors, colors, aromas, and textures of smear surface-ripened cheeses (1, 3, 15). Extracellular enzyme activities reported for B. linens include proteinase (7, 13, 16), aminopeptidase (7-11, 14), and esterase (7). The bacterium possesses intracellular peptidases (5, 7, 23, 26) and esterases (4, 7); cell-bound lipase and esterase activities have also been reported (24). B. linens ATCC 9174 was obtained from the Microbiology Department, University College, Cork, Ireland. The organism was precultured in 250 ml of medium (1.0% trypticase peptone [wt/vol], 0.3% Bacto Peptone [wt/vol], 0.25% yeast extract [wt/ vol], 0.5% glucose [wt/vol], 0.25% K 2 HPO 4 [wt/vol], 0.02% MgSO 4 ⅐ 7H 2 O [wt/vol]) in a 500-ml flask on an orbital shaker at 170 cycles per min and 23ЊC. After 48 h, 100 ml of preculture was used to inoculate 5 liters of the same medium in a 10-liter fermentor (B. Braun Diessel Biotech GmbH., Melsungen, Germany). The temperature was maintained at 23ЊC, with 10% O 2 saturation; the pH was not regulated. After cultivation for 70 h, i.e., at late log phase, cells were removed by centrifugation at 14,500 ϫ g for 25 min at 4ЊC. The cell-free supernatant was concentrated from 5 liters to 250 ml by ultrafiltration with a Sartocon Mini SM 17521 unit, which was fitted with 20-kDa cutoff membranes (Sartorius AG, Goettingen, Germany). Proteinase activity was measured by the method described by Twinning et al. (27); 40 l of fluorescein isothiocyanatelabelled casein (0.5% [wt/vol] in H 2 O) was added to 40 l of