Crooks G E, Rees G D, Robinson B H, Svensson M, Stephenson G R
Chemistry Department, Boston University, Boston, Massachusetts, USA.
Biotechnol Bioeng. 1995 Nov 5;48(3):190-6. doi: 10.1002/bit.260480304.
Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value.
分别从商品制剂Lipolase和Lipozyme中分离得到的疏棉状嗜热丝孢菌脂肪酶（HIL）和米黑根毛霉脂肪酶（RrnL），溶解于由AOT稳定的正庚烷油包水（w/o）微乳液中，并对它们的水解和缩合活性进行了研究。在未缓冲、R = 10的微乳液中，HIL水解活性的温度依赖性与在水乳液测定中Lipolase对三丁酸甘油酯的水解非常接近。测得的表观活化能分别为13±2和15±2 kJ mol⁻¹。然而，缩合活性在温度5℃至37℃范围内基本与温度无关。在研究的所有pH值（6.1、7.2、9.3）和R值（5、7.5、10、20）下，除了高pH值和低R值组合的情况外，HIL在30天内的稳定性都非常好。生产力曲线的线性反映了其出色的稳定性，这有利于系统优化。RmL对对硝基苯基丁酸酯（pNPC(4)）的水解活性的温度依赖性在40℃时出现最大值，根据曲线的线性区域（5℃至40℃）计算得出表观活化能E(act) = 20±2 kJ mol⁻¹。在研究的温度范围（0℃至40℃）内，RmL的酯化活性对温度的依赖性很小，对于辛酸癸酯的合成，测得表观活化能E(act) = 5±1 kJ mol⁻¹。因此，RmL和HIL在基于微乳液的介质中低温生物转化方面都有应用潜力。在含有pH 6.1缓冲液的R = 7.5和R = 10的微乳液中，RmL在30天内的稳定性良好，这反映在它们各自生产力曲线的线性上。然而，在碱性更强的pH值下，RmL的稳定性明显较差，并且被证明对R值的相对较小变化敏感。
