Laccase Encapsulation in ZIF-8 Metal-Organic Framework Shows Stability Enhancement and Substrate Selectivity

CgL1 laccase from Corynebacterium glutamicum was encapsulated into the metal-organic framework (MOF) ZIF-8 which was synthesized in a rapid enzyme friendly aqueous synthesis, the fastest in situ encapsulation of laccases reported to date. The obtained enzyme/MOF, i. e. laccase@ZIF-8 composite showed enhanced thermal (up to 70 °C) and chemical (N,N-dimethylformamide) stability, resulting in a stable heterogenous catalyst, suitable for high temperature reactions in organic solvents. Furthermore, the defined structure of ZIF-8 produced a size selective substrate specificity, so that substrates larger than the pore size were not accepted. Thereby, 2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was used to verify that the enzyme is immobilized inside the MOF versus the outside surface. The enzyme@MOF composite was analyzed by atomic absorption spectroscopy (ASS) to precisely determine the enzyme loading to 2.1 wt%.     

植物体系中甲酯酶的底物专一性的理论研究

酶对天然底物的高度专一性是酶的特点之一. 然而关于酶是如何对底物具有高度专一性以及识别能力, 我们的理解仍然缺乏. 本文以植物体系中发现的一组甲酯酶(MESs)对一些底物[包括水杨酸甲酯(MeSA), 茉莉酮酸甲酯(MeJA)和吲哚-3-乙酸甲酯(MeIAA)]的催化反应为例, 报道了同源建模和理论计算对茉莉酮酸甲酯酶(AtMES10)和水杨酸结合蛋白2(SABP2)的研究结果. 基于简单的锁-钥匙理论(底物与酶结合时不发生基团的碰撞或严重排斥), 以底物对接到酶的活性部位(即底物中—COO的一部分占据可被催化丝氨酸亲核进攻的位置) 为原则, 可以在空间上为酶对底物的专一性提供解释. 模拟结果表明, SABP2可对MeSA有高活性, 对MeJA和MeIAA有低或无活性; AtMES10可对MeJA有高活性, 而对MeSA和MeIAA有低或无活性, 这与实验结果相一致. 因此, 相关酶的结构预测与计算机模拟对了解酶的底物专一性具有重要的意义.     

Non-Specific GH30_7 Endo-β-1,4-xylanase from Talaromyces leycettanus

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA²Xyl_n. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA²Xyl_2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.     

Intracellular lectins are involved in quality control of glycoproteins

Glycoprotein quality control is categorized into three kinds of reactions; the folding of nascent glycoproteins, ER-associated degradation of misfolded or unassembled glycoproteins, and transport and sorting of correctly folded glycoproteins. In all three processes, N-glycans on the glycoproteins are used as tags that are recognized by intracellular lectins. We analyzed the functions of these intracellular lectins and their sugar-binding specificities. The results clearly showed that the A, B, and C-arms of high mannose-type glycans participate in the folding, transport and sorting, and degradation, respectively, of newly synthesized peptides. After correctly folded glycoproteins are transported to the Golgi apparatus, N-glycans are trimmed into Man₃GlcNAc₂ and then rebuilt into various complex-type glycans in the Golgi, resulting in the addition of diverse sugar structures that allow glycoproteins to play various roles outside of the cells.     

A virtual high throughput screen for high affinity cytochrome P450cam substrates. Implications for in silico prediction of drug metabolism

Structure-based virtual screening techniques require reliable scoring functions to discriminate potential substrates effectively. In this study we compared the performance of GOLD, PMF, DOCK and FlexX scoring functions in FlexX flexible docking to cytochrome P450cam binding site. Crystal structures of protein-substrate complexes were most effectively reproduced by the FlexX/PMF method. On the other hand, the FlexX/GOLD approach provided the best correlation between experimental binding constants and predicted scores. Binding modes selected by the FlexX/PMF approach were rescored by GOLD to obtain a reliable measure of binding energetics. The effectiveness of the FlexX/PMF/GOLD method was demonstrated by the correct classification of 32 out of the 33 experimentally studied compounds and also in a virtual HTS test on a library of 10,000 compounds. Although almost all the available functions were developed to be general, our study on cytochrome P450cam substrates suggests that careful selection or even tailoring the scoring function might increase the prediction power of virtual screens significantly. The FlexX/PMF/GOLD methodology was tested on cytochrome P450 3A4 substrates and inhibitors. This preliminary study revealed that the combined function was able to recognise 334 out of the 345 compounds bound to 3A4.     

微波辐射对酶促合成甘油酯位置选择性的影响

研究了微波辐射和常规加热下脂肪酶Novozyme 435催化甘油与n-辛酸的反应。在2种加热模式下,n-辛酸与甘油反应的初速度随着反应温度(50~75℃)的升高而加快;同样条件下,微波辐射下的反应初速度略高于常规加热条件下的。微波辐射的产物中的2-单甘酯和1,2-二甘酯的含量增加,但仍明显低于1-单甘酯和1,3-二甘酯的含量,即微波辐射并未根本改变脂肪酶的1,3-专一性;但实验条件下微波辐射均削弱了Novozyme 435的1,3-专一性:微波辐射反应产物中1-单甘酯与2-单甘酯的量比由常规加热下的26.9~43.4下降为16.2~40.4,其中1,3-二甘酯与1,2-二甘酯的量比亦由10.5~19.6降为7.6~15.3。     

筋骨草甾酮C的谱学特征

筋骨草甾酮C是分布于植物中的植物甾酮类化合物,具有抗氧化的作用.本文标定了该化合物中所有的碳信号,并通过¹³C-¹H COSY和¹H-¹H COSY技术纠正了文献中氢信号归属的错误.     

花鲈同工酶的组织特异性研究

采用聚丙烯酰胺垂直平板电泳对花鲈的心、肝、眼球、视网膜、肌肉、肾、鳃、胸鳍等8个组织的10种同工酶(LDH、MDH、ME、α-GPDH、ADH、SDH、SOD、G—6—PDH、EST、GDH)进行了分析，结果认为：10种同工酶在各个组织中的表达频率，以肝脏最高，其次为心脏、肌肉、视网膜，较低的为眼球、锶、肾、鳍组织．其中LDH、MDH、及SOD三种同工酶都能在8个组织中得到表达，但表达的强度有所差异．基因座位表达程度，以肝脏、肌肉两组织为最全面．     

金属元素的正电子鉴别技术

测量了20种金属元素的符合多普勒展宽谱,并与理论计算的结果比较．CDB谱的高动量部分反映了正电子与核心电子的湮灭,由于核心电子在原子组成材料时仍然保留了元素特性,CDB谱的高动量部分可以用来鉴别元素．     

Modeling of enzyme–substrate complexes for the metalloproteases MMP-3, ADAM-9 and ADAM-10

The matrix metalloproteases (MMPs) and the ADAMs (A Disintegrin And Metalloprotease domain) are proteolytic enzyme families containing a catalytic zinc ion, that are implicated in a variety of normal and pathological processes involving tissue remodeling and cancer. Synthetic MMP inhibitors have been designed for applications in pathological situations. However, a greater understanding of substrate binding and the catalytic mechanism is required so that more effective and selective inhibitors may be developed for both experimental and clinical purposes. By modeling a natural substrate spanning P4-P4' in complex with the catalytic domains, we aim to compare substrate-specificities between Stromelysin-1 (MMP-3), ADAM-9 and ADAM-10, with the aid of molecular dynamics simulations. Our results show that the substrate retains a favourable antiparallel beta-sheet conformation on the P-side in addition to the well-known orientation of the P'-region of the scissile bond, and that the primary substrate selectivity is dominated by the sidechains in the S1' pocket and the S2/S3 region. ADAM-9 has a hydrophobic residue as the central determinant in the S1' pocket, while ADAM-10 has an amphiphilic residue, which suggests a different primary specificity. The S2/S3 pocket is largely hydrophobic in all three enzymes. Inspired by our molecular dynamics calculations and supported by a large body of literature, we propose a novel, hypothetical, catalytic mechanism where the Zn-ion polarizes the oxygens from the catalytic glutamate to form a nucleophile, leading to a tetrahedral oxyanion anhydride transition state.