Kotik Michael, Stepánek Václav, Kyslík Pavel, Maresová Helena
Laboratory of Enzyme Technology, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, 142 20 Prague 4, Czech Republic.
J Biotechnol. 2007 Oct 15;132(1):8-15. doi: 10.1016/j.jbiotec.2007.08.014. Epub 2007 Aug 14.
The gene encoding an epoxide hydrolase from Aspergillus niger M200 has been cloned and its sequence determined. The gene is interrupted by seven introns, one exon being only nine nucleotides long. The non-coding 5'- and 3'-regions of the mRNA are composed of 47 and 76 nucleotides, respectively. Overexpression of the fungal epoxide hydrolase in E. coli TOP10 has led to a 15-fold increase in specific activity (compared to the wild-type strain). Saturation mutagenesis at codon 217 resulted in the discovery of nine enzyme variants showing in several cases profound differences in activity and enantioselectivity towards various epoxides when compared to the data of the wild-type enzyme. The site 217 is located at the entrance of the tunnel that provides the substrate with access to the active site. The exchange of Ala at this position for Cys has led to a doubled enantioselectivity (E-value of 5.0) towards benzyl glycidyl ether. The same substitution resulted in a threefold-enhanced activity of the enzyme towards allyl glycidyl ether and styrene oxide without affecting enantioselectivity. The variant A217L showed an enhanced enantioselectivity towards tert-butyl glycidyl ether reaching an E-value of 100 (from 60 for the wild-type enzyme). Replacement of A217 by Val has led to higher activity towards allyl glycidyl ether by a factor of six. The substitutions Ala-->Glu and Ala-->Gln increased the enantioselectivity towards allyl glycidyl ether and styrene oxide by over 50% to E-values of 10 and 16, respectively. The study underlines that single amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity of the epoxide hydrolase from A. niger M200.
来自黑曲霉M200的一种环氧化物水解酶的编码基因已被克隆并确定了其序列。该基因被7个内含子打断，一个外显子仅有9个核苷酸长。mRNA的非编码5'和3'区域分别由47个和76个核苷酸组成。该真菌环氧化物水解酶在大肠杆菌TOP10中的过表达导致比活性提高了15倍（与野生型菌株相比）。对第217位密码子进行饱和诱变，发现了9种酶变体，与野生型酶的数据相比，在几种情况下，这些变体对各种环氧化物的活性和对映选择性存在显著差异。第217位位于为底物提供进入活性位点通道的隧道入口处。将该位置的丙氨酸替换为半胱氨酸，导致对苄基缩水甘油醚的对映选择性提高了一倍（E值为5.0）。相同的替换使该酶对烯丙基缩水甘油醚和氧化苯乙烯的活性提高了三倍，而不影响对映选择性。变体A217L对叔丁基缩水甘油醚的对映选择性增强，E值达到100（野生型酶为60）。将A217替换为缬氨酸，使对烯丙基缩水甘油醚的活性提高了6倍。将丙氨酸替换为谷氨酸和将丙氨酸替换为谷氨酰胺分别使对烯丙基缩水甘油醚和氧化苯乙烯的对映选择性提高了50%以上，E值分别达到10和16。该研究强调，底物隧道区域的单个氨基酸替换可显著提高黑曲霉M200环氧化物水解酶的对映选择性和活性。
