Fernandes S, Johansson G, Hatti-Kaul R
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.
Biotechnol Bioeng. 2001 Jun 20;73(6):465-75. doi: 10.1002/bit.1081.
Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.
研究了通过双水相系统萃取从酿酒酵母培养上清液中纯化重组野生型角质酶的方法。在聚乙二醇（PEG）-磷酸钾系统中，酶在上层相的分配率随PEG分子量降低而增加。分别在洗涤剂、脂肪酸和醇存在的情况下，研究了酶在20% PEG/15%磷酸盐双水相系统中的分配情况。添加0.5%（w/w）丁酸盐可使分配系数从17提高到135，纯化因子从10提高到23。丁酸盐的作用也通过逆流萃取模式得到了证实。通过在较低pH值或较低温度下二次萃取到新的盐相中，可从上层相中回收角质酶。荧光光谱法证明了丁酸盐与酶活性位点之间存在特异性相互作用。尺寸排阻色谱显示角质酶-丁酸盐复合物的大小是游离酶的两倍多。
