Markošová Kristína, Camattari Andrea, Rosenberg Michal, Glieder Anton, Turner Nicholas J, Rebroš Martin
Faculty of Chemical and Food Technology, Institute of Biotechnology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia.
Biotransformation Innovation Platform (BIP), 61 Biopolis Drive #14-13, Singapore, 138673, Singapore.
Biotechnol Lett. 2018 Jan;40(1):127-133. doi: 10.1007/s10529-017-2450-y. Epub 2017 Oct 10.
To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production.
Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 Mut strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated.
There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.
在毕赤酵母中克隆单胺氧化酶N（其催化胺选择性氧化脱氨或消旋化为亚胺），并证明为其重组生产选择合适表达系统的重要性。
首次克隆了源自黑曲霉并经过定向进化的单胺氧化酶（MAO-N D5），并在毕赤酵母CBS7435 Mut菌株中进行表达。在微尺度水平筛选了各种转化体。将表达活性最高的酶的克隆扩大到1.5升发酵罐，并优化了作为粗酶提取物的MAO-N D5的制备。讨论并证明了在大肠杆菌和毕赤酵母这两种表达系统中生产该酶时遇到的障碍。
比生产率有所提高，在毕赤酵母中的比生产率高83倍，清楚地证明了为特定酶选择合适表达宿主系统的重要性。
