Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity

Monooxygenase mutants : A minimal and highly enriched CYP102A1 mutant library was constructed by combining five hydrophobic amino acids in two positions. The library was screened with four different terpene substrates. Eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

     

Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide

Cytochrome P450 BM‐3 (EC 1.14.14.1) is a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions. The enzyme is also capable of functioning as a peroxygenase in the same reaction, by utilizing hydrogen peroxide in place of the reductase domain, cofactor and oxygen. As a starting point for developing a practically useful hydroxylation biocatalyst, we compare the activity and regioselectivity of wild‐type P450 BM‐3 and its F87A mutant on various fatty acids. Neither enzyme catalyzes terminal hydroxylation under any of the conditions studied. While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position. The H₂O₂‐driven reactions with either the full‐length BM‐3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O₂.     

糖基化三氮唑鹰嘴豆芽素A衍生物的合成与抗缺氧活性研究

以叠氮糖、炔丙基溴和鹰嘴豆芽素A为原料,利用"Click chemistry"将糖基化三氮唑药效基团与鹰嘴豆芽素A巧妙结合,得到4种新的标题化合物,产物结构经1HNMR、13CNMR、IR、EI-MS和元素分析确认。通过小鼠常压密闭耐缺氧模型对其药理活性进行评价,结果表明:4个目标化合物均不同程度地延长小鼠的存活时间,其中半乳糖苷三氮唑鹰嘴豆芽素A和木糖苷三氮唑鹰嘴豆芽素A的活性优于乙酰唑胺。     

HPLC法测定Mucor sp.产肉桂酸降解酶的活性和动力学分析

建立了高效液相色谱法测定肉桂酸降解酶活性的方法。以肉桂酸为底物、苯乙酮为探针,通过测定苯乙酮的生成速率来计算酶活并进行酶催化动力学分析。色谱柱为Hypersil ODS2柱(250 mm×4.6 mm,5μm),柱温为30℃,流动相为乙腈∶水∶冰乙酸=50∶50∶0.5(体积比),流速为1.0mL·min-1,检测波长为247nm。苯乙酮的线性范围为1.952~156.2μg·mL-1,回收率为99.46%~101.2%,RSD≤1%,检出限为0.45μg·mL-1(S/N=3)。结果表明,该方法稳定可靠,简便,重复性好,不受杂质干扰,可用于肉桂酸降解酶的活性测定和动力学分析。粗酶液和不同浓度的肉桂酸在30℃下反应2h后,HPLC法测定初反应速率,以双倒数作图法计算酶动力学参数,双倒数图呈直线,符合米氏方程规律,米氏常数Km=0.07g·L-1,即0.473μmol·L-1,Vmax=0.058μmol·min-1·mg-1。     

Combining OSMAC Approach and Untargeted Metabolomics for the Identification of New Glycolipids with Potent Antiviral Activity Produced by a Marine Rhodococcus

Natural products of microbial origin have inspired most of the commercial pharmaceuticals, especially those from Actinobacteria. However, the redundancy of molecules in the discovery process represents a serious issue. The untargeted approach, One Strain Many Compounds (OSMAC), is one of the most promising strategies to induce the expression of silent genes, especially when combined with genome mining and advanced metabolomics analysis. In this work, the whole genome of the marine isolate Rhodococcus sp. I2R was sequenced and analyzed by antiSMASH for the identification of biosynthetic gene clusters. The strain was cultivated in 22 different growth media and the generated extracts were subjected to metabolomic analysis and functional screening. Notably, only a single growth condition induced the production of unique compounds, which were partially purified and structurally characterized by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). This strategy led to identifying a bioactive fraction containing >30 new glycolipids holding unusual functional groups. The active fraction showed a potent antiviral effect against enveloped viruses, such as herpes simplex virus and human coronaviruses, and high antiproliferative activity in PC3 prostate cancer cell line. The identified compounds belong to the biosurfactants class, amphiphilic molecules, which play a crucial role in the biotech and biomedical industry.