期刊
ACS CATALYSIS
卷 9, 期 9, 页码 7769-7778出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.9b02548
关键词
protein engineering; iterative saturation mutagenesis; stereoselectivity; reduced amino acid alphabets; ee-screening; oversampling algorithms
资金
- Max-Planck-Society
- CAS Pioneer Hundred Talents Program [2016-053]
- Biological Resources Programme, Chinese Academy of Sciences [KFJ-BRP-009]
- Key Research Program of the Chinese Academy of Sciences [KFZD-SW-215]
- National Natural Science Foundation of China [21702052]
- State Key Laboratory of Biocatalysis and Enzyme Engineering
Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (SM) at sites lining the enzyme's binding pocket has emerged as a particularly viable approach to control selectivity and activity. Traditionally, NNK codon degeneracy encoding all 20 canonical amino acids is used, but as the size of the randomization site increases beyond a single residue, oversampling of transformants needed to ensure >= 95% library coverage rapidly reaches astronomical dimensions, impossible to screen in a practical manner. Therefore, many groups have been content with screening only a small segment of the designed protein sequence space, but this means that the best mutants will be missed. Alternatively, it has been shown that the use of highly reduced amino acid alphabets allows the generation of small and smart libraries requiring less screening. Here we address the question of which approach is more efficient. Two different enzyme types serve as model systems in stereo-selective reactions, limonene epoxide hydrolase and P450-BM3. Equal numbers of transformants were screened for differently sized SM libraries that were constructed using NNK codon degeneracy and other codons corresponding to only one or just a few amino acids. Conversion as a rough indication of activity was used as the parameter in primary screening followed by GC-based ee-determination. Statistical analyses clearly show that it is more efficient to opt for rationally designed reduced amino acid alphabets because this approach results in a distinctly higher frequency of active mutants, stereoselectivity also being notably higher.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据