Almeida M C, Venâncio A, Teixeira J A, Aires-Barros M R
Departamento de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, Braga Codex, Portugal.
J Chromatogr B Biomed Sci Appl. 1998 Jun 26;711(1-2):151-9. doi: 10.1016/s0378-4347(97)00680-4.
The partition behaviour of cutinase on poly(ethylene glycol) (PEG)-hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000-Reppal PES 100 [at pH 4.0; 0.5 M (NH4)2SO4], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000-HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.
对角质酶在聚乙二醇（PEG）-羟丙基淀粉双水相体系中的分配行为进行了表征。对于用纯化的羟丙基淀粉（Reppal PES 100）和粗品（HPS）制备的体系，研究了PEG分子量、体系pH值和系线长度对角质酶分配系数以及角质酶在上相中的产率的影响。还研究了不同盐（如氯化钠、硫酸钠和硫酸铵）的存在对角质酶分配的影响。结果得出结论，由PEG和羟丙基淀粉组成的双水相体系在角质酶纯化方面效率不高。在大多数情况下，分配系数非常接近1，pH值是影响角质酶分配的最主要因素。向体系中添加盐后，分配系数显著提高。对于PEG 4000-Reppal PES 100[在pH 4.0；0.5 M硫酸铵条件下]，角质酶的分配系数为3.7，而对于PEG 4000-HPS（在pH 4.0；1 M氯化钠条件下），该值为12。通过用不同盐进行实验得到的结果构建交叉分配图，确定角质酶的等电点（pI）为7.8。
