Novel thermostable amine transferases from hot spring metagenomes
Novel thermostable amine transferases from hot spring metagenomes
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DOI：
10.1007/s00253-017-8228-2
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发表时间：
2017-06-01
影响因子：
5
通讯作者：
Monti, Daniela
中科院分区：
文献类型：
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作者：
Ferrandi, Erica Elisa;Previdi, Alessandra;Monti, Daniela
温泉宏基因组，从温度范围从55至95摄氏度收集的样品制备，提交了一个硅筛选，旨在识别新的胺转氨酶（ATA），有价值的生物催化剂制备光学纯胺。在冰岛和意大利温泉样品的宏基因组中发现了三种新的（S）-选择性ATA，即Is 3-TA、It 6-TA和B3-TA，从相应的宏基因组DNA中克隆并在E.杆菌新型ATAs的功能表征表明，它们都具有嗜热特性，并能够使用广泛的供体和受体底物进行胺转移反应，从而表明了良好的潜在的实际合成应用。特别地，酶B3-TA显示出异常的热稳定性，在80摄氏度下孵育5天后保留85%的活性，在相同条件下孵育2周后保留40%以上的活性。这些结果与约88 A ℃的表观熔化温度的估计一致，使B3-TA成为据我们所知迄今为止描述的最热稳定的天然ATA。该生物催化剂对不同的水混溶性和水不混溶性有机溶剂也表现出良好的耐受性。B3-TA基于同源性的结构模型的详细检查表明，嗜温（S）-选择性ATA的整体活性位点结构主要在该超嗜热同源物中保守。此外，B3-TA-样转氨酶的一个亚家族，大多是未表征的，所有嗜热微生物，被确定和分析的系统发育关系和序列保守性。
Hot spring metagenomes, prepared from samples collected at temperatures ranging from 55 to 95 A degrees C, were submitted to an in silico screening aimed at the identification of novel amine transaminases (ATAs), valuable biocatalysts for the preparation of optically pure amines. Three novel (S)-selective ATAs, namely Is3-TA, It6-TA, and B3-TA, were discovered in the metagenome of samples collected from hot springs in Iceland and in Italy, cloned from the corresponding metagenomic DNAs and overexpressed in recombinant form in E. coli. Functional characterization of the novel ATAs demonstrated that they all possess a thermophilic character and are capable of performing amine transfer reactions using a broad range of donor and acceptor substrates, thus suggesting a good potential for practical synthetic applications. In particular, the enzyme B3-TA revealed to be exceptionally thermostable, retaining 85% of activity after 5 days of incubation at 80 A degrees C and more than 40% after 2 weeks under the same condition. These results, which were in agreement with the estimation of an apparent melting temperature around 88 A degrees C, make B3-TA, to the best of our knowledge, the most thermostable natural ATA described to date. This biocatalyst showed also a good tolerance toward different water-miscible and water-immiscible organic solvents. A detailed inspection of the homology-based structural model of B3-TA showed that the overall active site architecture of mesophilic (S)-selective ATAs was mainly conserved in this hyperthermophilic homolog. Additionally, a subfamily of B3-TA-like transaminases, mostly uncharacterized and all from thermophilic microorganisms, was identified and analyzed in terms of phylogenetic relationships and sequence conservation.