De Angelis Maria, Gallo Giovanna, Corbo Maria Rosaria, McSweeney Paul L H, Faccia Michele, Giovine Marinella, Gobbetti Marco
Istituto di Scienze delle Produzioni Alimentari, CNR, Bari, Italy.
Int J Food Microbiol. 2003 Nov 1;87(3):259-70. doi: 10.1016/s0168-1605(03)00072-2.
The phytase activity of 12 species of sourdough lactic acid bacteria was screened. It was intracellular only, largely distributed among the species and strains of Lactobacillus sanfranciscensis possessed the highest levels of activity. A monomeric ca. 50-kDa phytase was purified to homogeneity from L. sanfranciscensis CB1 by three chromatographic steps. L. sanfranciscensis CB1 exhibited the highest hydrolysing activity on Na-phytate after reaching the stationary phase of growth (ca. 12 h). Cells cultivated in the presence of maltose and fructose showed an increase of the phytase activity of ca. 35% with respect to the other carbon sources used. The phytase was optimally active at pH 4.0 and 45 degrees C. The enzyme was strongly inhibited by 2 mM of phenylmethylsulfonyl fluoride (PMSF), and 2 mM Hg(2+) and Fe(2+). It had a pI of ca. 5.0. The substrate specificity was dependent on the type of phosphate ester; a very low activity was detected on alpha-D-glucose-1-phosphate and D-fructose-6- and 1,6-phosphate, while the highest hydrolysis was found towards adenosine-5'-tri-, di- and mono-phosphate. Compared to these substrates, the activity on Na-phytate was also relevant. The enzyme was thermo-stable after exposure to 70 degrees C for 30 min; the D value calculated at 80 degrees C was ca. 10 min. As shown by the Central Composite Design (CCD) applied to study the individual and interactive effects of pH, temperature and NaCl, acidic conditions and elevated temperatures were indispensable for the enzyme adaptation to high NaCl concentrations. L. sanfranciscensis CB1 cells or the correspondent cytoplasmic extract were used to ferment a sourdough for 8 h at 37 degrees C; a marked decreased (64-74%) of the Na-phytate concentration was found compared with the unstarted dough. The sourdough started with L. sanfranciscensis CB1 cells was re-used for several times and the phytase activity was maintained to a considerable level.
对12种酸面团乳酸菌的植酸酶活性进行了筛选。该酶仅存在于细胞内，在不同物种间广泛分布，旧金山乳杆菌的物种和菌株中活性水平最高。通过三步色谱法从旧金山乳杆菌CB1中纯化得到一种约50 kDa的单体植酸酶，使其达到同质。旧金山乳杆菌CB1在生长稳定期（约12小时）后对肌醇六磷酸钠表现出最高的水解活性。在麦芽糖和果糖存在下培养的细胞，其植酸酶活性相对于使用的其他碳源增加了约35%。该植酸酶在pH 4.0和45℃时活性最佳。该酶受到2 mM苯甲基磺酰氟（PMSF）、2 mM Hg(2+)和Fe(2+)的强烈抑制。其pI约为5.0。底物特异性取决于磷酸酯的类型；在α-D-葡萄糖-1-磷酸、D-果糖-6-磷酸和1,6-磷酸上检测到的活性非常低，而对腺苷-5'-三磷酸、二磷酸和单磷酸的水解活性最高。与这些底物相比，对肌醇六磷酸钠的活性也较高。该酶在70℃下暴露30分钟后具有热稳定性；在80℃下计算得到的D值约为10分钟。应用中心复合设计（CCD）研究pH、温度和NaCl的单独及交互作用表明，酸性条件和高温对于酶适应高NaCl浓度是必不可少的。使用旧金山乳杆菌CB1细胞或相应的细胞质提取物在37℃下发酵酸面团8小时；与未发酵面团相比，肌醇六磷酸钠浓度显著降低（64 - 74%）。以旧金山乳杆菌CB1细胞起始的酸面团可重复使用多次，且植酸酶活性维持在相当水平。
