Microbial lipases catalyze a broad spectrum of reactions and are enzymes of considerable biotechnological interest. The focus of this study was the isolation of new lipase genes, intending to discover novel lipases whose products bear interesting biochemical and structural features and may have a potential to act as valuable biocatalysts in industrial applications. A novel lipase gene (lipSm), from a new environmental Stenotrophomonas maltophilia strain, Psi-1, originating from a sludge sample from Psittaleia (Greece), was cloned and sequenced. lipSm was further overexpressed in E. coli BL21(DE3) and the overproduced enzyme LipSm was purified and analyzed in respect to its biochemical and kinetic properties. In silico analysis of LipSm revealed that it is taxonomically related to several uncharacterized lipases from different genera, which constitute a unique clade, markedly different from all other previously described bacterial lipase families. All members of this clade displayed identical, conserved consensus sequence motifs, i.e. the catalytic triad (S, D, H), and an unusual, amongst bacterial lipases, Y-type oxyanion hole. 3D-modeling revealed the presence of a lid domain structure, which allows LipSm to act on small ester substrates without interfacial activation. In addition, the high percentage of alanine residues along with the occurrence of the AXXXA motif nine times in LipSm suggest that it is a thermostable lipase, a feature verified experimentally, since LipSm was still active after heating at 70 °C for 30 min. The phylogenetic analysis of LipSm suggests the establishment of a new bacterial lipase family (XVIII) with LipSm being its first characterized member. Furthermore, LipSm is alkaliphilic, thermostable and lacks the requirement for interfacial activation, when small substrates are used. These properties make LipSm a potential advantageous biocatalyst in industry and biotechnology.

中文翻译：

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嗜麦芽窄食单胞菌Psi-1（一种新的细菌脂肪酶家族（XVIII）的第一个成员）的新型热稳定脂肪酶（LipSm）的分子，生化和动力学分析

微生物脂肪酶催化广泛的反应，是具有重大生物技术意义的酶。这项研究的重点是新脂肪酶基因的分离，目的是发现新型脂肪酶，其产物具有有趣的生化和结构特征，并有可能在工业应用中充当有价值的生物催化剂。克隆并测序了一种新的脂肪酶基因（lipSm），该基因来自一种新的环境嗜麦芽单胞菌嗜麦芽孢杆菌菌株Psi-1，该菌株源自Psittaleia（希腊）的污泥样品。lipSm进一步在大肠杆菌BL21（DE3）中过表达，对过量产生的酶LipSm进行纯化并对其生化和动力学性质进行分析。对LipSm进行的计算机分析表明，它与不同属的几种未表征的脂肪酶在分类学上相关，它们构成独特的进化枝，与之前描述的所有其他细菌脂肪酶家族明显不同。该进化枝的所有成员均显示相同，保守的共有序列基序，即催化三联体（S，D，H），以及细菌脂肪酶中不常见的Y型含氧阴离子孔。3D建模揭示了盖子域结构的存在，这使LipSm可以作用于小的酯底物而无需界面活化。此外，在LipSm中高百分比的丙氨酸残基以及9次出现AXXXA基序表明这是一种热稳定的脂肪酶，这一特性已通过实验验证，因为LipSm在70°C加热30分钟后仍具有活性。LipSm的系统发育分析表明，建立了一个新的细菌脂肪酶家族（XVIII），LipSm是它的第一个特征成员。此外，当使用小底物时，LipSm具有碱性，热稳定性，并且不需要界面活化。这些特性使LipSm成为工业和生物技术领域潜在的有利生物催化剂。