McKellar R C, Cholette H
J Dairy Res. 1986 May;53(2):301-12. doi: 10.1017/s0022029900024900.
A method based on the hydrolysis of beta-naphthyl caprylate (beta-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0.02 ml raw SM in a 2.0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 micrograms/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of beta-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 degrees C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 degrees C when the cell density exceeded 10(8) colony forming units/ml. The presence of butterfat (3.5%) in the SM assay inhibited B52 lipase by 97%. The beta-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the beta-NC assay may be useful for determining lipase activity in raw SM.
已开发出一种基于辛酸β-萘酯（β-NC）水解的方法来定量荧光假单胞菌的胞外脂肪酶。该测定法对脱脂乳（SM）极为敏感；在2.0 ml反应混合物中加入低至0.02 ml的生SM会导致脂肪酶活性明显损失50%。当反应混合物中加入胰蛋白酶（50微克/毫升）时，活性提高了3倍。当使用超单纯形优化法确定β-NC、牛磺胆酸钠（NaTC）、SM/脂肪酶混合物和胰蛋白酶的最佳水平以实现最大活性时，发现NaTC对活性并非必需。随后加入15 mM-NaTC导致活性损失80%。另一方面，在存在SM的情况下，天然脂肪酶活性需要NaTC。天然脂肪酶在70℃加热2分钟会被完全抑制，而B52脂肪酶在相同条件下仍保留75%的活性。当细胞密度超过10⁸菌落形成单位/毫升时，该测定法能够检测荧光假单胞菌B52在5℃下于SM中产生的脂肪酶。SM测定中存在3.5%的乳脂肪会使B52脂肪酶受到97%的抑制。β-NC测定法所得结果与使用十种常见乳制品嗜冷菌的无细胞提取物进行的三丁酸甘油酯琼脂扩散测定法相当。结果表明，β-NC测定法可能有助于测定生SM中的脂肪酶活性。
