Ramos Margarita D, Miranda Letícia P, Giordano Raquel L C, Fernandez-Lafuente Roberto, Kopp William, Tardioli Paulo W
Dept. de Engenharia Química, PPG-EQ, Univ. Federal de São Carlos (UFSCar), São Carlos, SP, 13565-905, Brazil.
Dept. de Biocatálisis, ICP-CSIC, Campus UAM-CSIC Madrid, Spain.
Biotechnol Prog. 2018 Jul;34(4):910-920. doi: 10.1002/btpr.2636. Epub 2018 May 7.
The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.
系统研究了猪胰脂肪酶交联聚集体（PPL-CLEA）的制备，评估了三种沉淀剂和两种交联剂的影响，以及使用大豆蛋白作为替代供体蛋白对固定化PPL催化性能和稳定性的影响。标准CLEA的总产率（CLEA的观察活性/提供的总活性）低于4%，而添加大豆蛋白（PPL与大豆蛋白的质量比为1:3）后，总产率提高了约五倍。以大豆蛋白作为供体（PPL与大豆蛋白的质量比为1:3）、戊二醛作为交联剂（醛基10 μmol/mg总蛋白）制备的PPL-CLEA活性更高，主要是因为在洗涤步骤中酶的浸出减少。这种CLEA名为PPL-SOY-CLEA，固定化产率约为60%，表达活性约为40%。在大豆油乙醇解反应中，PPL-SOY-CLEA产生的最大脂肪酸乙酯（FAEE）浓度比使用可溶性PPL时高约12倍（在30°C、300 rpm搅拌、大豆油/乙醇摩尔比为1:5的条件下反应34 h），而酶负载量低约2倍（很可能是由于游离酶失活）。PPL-SOY-CLEA在涡流式反应器中进行大豆油乙醇解反应的操作稳定性表明，在24 h的十个反应循环中，FAEE产率高于时50%。对于工业工厂中使用无载体生物催化剂进行生物转化的过程，这种反应器配置可能是传统搅拌反应器的一个有吸引力的替代方案。© 2018美国化学工程师学会生物技术进展，34:910 - 920，2018。
