Blank Kerstin, Morfill Julia, Gumpp Hermann, Gaub Hermann E
Lehrstuhl für Angewandte Physik & Center for Nanoscience, LMU München, Amalienstrasse 54, 80799 München, Germany.
J Biotechnol. 2006 Oct 1;125(4):474-83. doi: 10.1016/j.jbiotec.2006.04.004. Epub 2006 May 19.
Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants k(M), v(max) and k(cat) were determined using the substrates p-NPB and p-NPL. Almost identical k(cat)/k(M) values (0.423-0.466 min(-1) microM(-1) for p-NPB and 0.068-0.071 min(-1) microM(-1) for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.
南极假丝酵母脂肪酶B（CalB）是生物有机合成中的一种重要催化剂。为了优化其性能，要么改变反应介质，要么对脂肪酶本身进行修饰。在后一种情况下，在大肠杆菌中产生突变体，随后在真菌宿主中表达以进行表征。在此，我们展示了CalB在大肠杆菌周质中的功能性表达。通过逐步缺失CalB信号肽和前肽，我们能够表达并纯化两种不同的CalB变体（成熟的CalB和带有前肽的CalB）。使用N端FLAG标签和C端His标签进行纯化。对于对硝基苯酚丁酸酯（p-NPB）、对硝基苯酚月桂酸酯（p-NPL）和羧基荧光素二乙酸酯（CFDA）等底物，其比活性显示与在米曲霉中表达的CalB相似。使用底物p-NPB和p-NPL测定了动力学常数k(M)、v(max)和k(cat)。从大肠杆菌和米曲霉获得的CalB变体的k(cat)/k(M)值几乎相同（p-NPB为0.423 - 0.466 min(-1) microM(-1)，p-NPL为0.068 - 0.071 min(-1) microM(-1)）。结果清楚地表明，CalB可以在大肠杆菌中功能性表达，并且标签的附着不会改变脂肪酶的性质。
