Robertson Dan E, Chaplin Jennifer A, DeSantis Grace, Podar Mircea, Madden Mark, Chi Ellen, Richardson Toby, Milan Aileen, Miller Mark, Weiner David P, Wong Kelvin, McQuaid Jeff, Farwell Bob, Preston Lori A, Tan Xuqiu, Snead Marjory A, Keller Martin, Mathur Eric, Kretz Patricia L, Burk Mark J, Short Jay M
Diversa Corporation, San Diego, California 92121, USA.
Appl Environ Microbiol. 2004 Apr;70(4):2429-36. doi: 10.1128/AEM.70.4.2429-2436.2004.
Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.
腈水解酶在生物圈中十分重要，它们参与天然存在的以及外源衍生的腈类的合成和降解途径。由于其固有的对映选择性，腈水解酶作为精细化学合成中用于构建手性中心的温和、选择性催化剂也颇具吸引力。不幸的是，科学和专利文献中报道的腈水解酶不到20种，并且由于稳定性或特异性方面的缺陷，它们的实用性在很大程度上尚未得到实现。在本研究中，对从筛选600多个生物群落特异性环境DNA（eDNA）文库中发现的137种独特的腈水解酶进行了表征。采用不依赖培养的方法，从整个生物群落中捕获了系统发育多样的基因组，并在一个共同的克隆宿主中对其基因进行了异源表达。在每个eDNA文库中包含10⁶至10¹⁰个成员的克隆群体的基于选择的表达测定中，将腈水解酶基因作为目标。对发现的新序列进行系统发育分析，结果显示腈水解酶亚科内至少存在五个主要序列分支。使用三种因其在手性药物合成中的潜力而被靶向的腈底物，对这些酶的底物特异性和立体特异性进行了表征。在序列分支与这些腈水解酶的选择性特性之间发现了许多重要的相关性。这些通过高通量、不依赖培养的方法发现的酶，为各种羧酸衍生物的对映体特异性合成提供了一个催化工具箱，同时也为进化和结构分析提供了一个有趣的文库。
