Hofer Michael, Diener Julia, Begander Benjamin, Kourist Robert, Sieber Volker
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Schulgasse 11a, 94315, Straubing, Germany.
Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315, Straubing, Germany.
Appl Microbiol Biotechnol. 2021 Apr;105(8):3159-3167. doi: 10.1007/s00253-021-11239-5. Epub 2021 Apr 12.
Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (-)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (-)-camphor are limited, and the oxidation of precious (-)-borneol would be too costly. Hence, synthesis of (-)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (-)-camphor with an ee of > 99%. KEY POINTS: • Characterization of a novel borneol dehydrogenase (BDH) from P. putida. • Development of enantioselective BDH variants for the reduction of camphor. • Enzymatic kinetic resolution of camphor with borneol dehydrogenase.
到目前为止，已知有数千种不同的萜类化合物结构，其中许多在医药、香料、香精、生物燃料、杀虫剂或精细化学中间体等应用方面具有吸引力。一个突出的例子是樟脑，自古以来它就被用于医学应用。特别是（-）-樟脑在医药应用中越来越受到关注。因此，需要一个商业上可靠的来源。（-）-樟脑的天然来源有限，而珍贵的（-）-冰片醇的氧化成本太高。因此，由可再生的α-蒎烯合成（-）-樟脑将是一种廉价的替代方法。由于目前用于将α-蒎烯转化为樟脑的路线会产生两种对映体的混合物，因此需要优选的催化方法来分离这种外消旋体，以得到对映体纯的樟脑。酶促动力学拆分是解决这一挑战的可持续方法，但需要合适的酶。在本研究中，对来自假单胞菌属ATCC 17453的首个冰片醇脱氢酶进行了研究，该酶能够催化樟脑的立体选择性还原。通过使用靶向酶工程方法，创建了对映选择性酶变体，其E值>100。最佳变体用于樟脑外消旋体的酶促动力学拆分，得到79%的（-）-樟脑，对映体过量值>99%。要点：• 对来自恶臭假单胞菌的新型冰片醇脱氢酶（BDH）进行表征。• 开发用于还原樟脑的对映选择性BDH变体。• 用冰片醇脱氢酶对樟脑进行酶促动力学拆分。
