Protein engineering,cofactor engineering,and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A

Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole-cell catalytic production of CSA. Using mechanism-based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its T_m and half-life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1-fold. Via cofactor engineering, we designed a dual-cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole-cell catalytic system of CSA production with an 89.5% conversion rate. This whole-cell catalytic process provides a promising method for the industrial production of CSA.     

A Molecular Dynamics Study of the Stereoselective Interaction between an Enantiomeric Amino-Acid Ester and an l-Histidine-Containing Catalyst in the Bilayer Membrane

Molecular dynamics simulations were performed to investigate the stereoselective interaction between an enantiomeric amino acid substrate (N-acylated-l-phenylalanine-p-nitrophenyl ester) and an l-histidine-containing dipeptide catalyst (N-(N-benzyloxycarbonyl-l-leucyl)-l-histidine) in the bilayer of cationic surfactants (N,N-bisdodecyl-N,N-dimethylammonium chloride). We found that a catalyst–substrate complex, which had an interamide hydrogen bond, was formed spontaneously in vacuum at 500 K. This complex was found to be stable both in vacuum and in the bilayer membrane for 100 ps at 300 K. The distances between the hydrophobic side chains in the complex were consistent with experimental results. The interamide hydrogen bond was retained in the hydrophobic core of the membrane. These results suggest that the catalyst–substrate complex found in this work is relevant to the stereoselective hydrolysis of the l-enantiomer of the substrate.     

Synthesis of quinoline-based glycoconjugates: a facile one-pot three-component reaction

A multicomponent one-pot reaction involving propargyl glycosides, methoxy-substituted aromatic aldehydes and aromatic amines using Cu(I) as catalyst is described, which provides an efficient and practical route to synthesize several quinoline-based glycoconjugates in good yield.     

异源D-海因酶和N-氨甲酰水解酶共表达重组枯草芽孢杆菌的构建

【目的】构建异源D-海因酶和N-氨甲酰水解酶共表达的重组枯草芽孢杆菌,探讨其作为全细胞催化剂合成D-对羟基苯甘氨酸的可行性。【方法】采用P_(aco)表达盒表达D-海因酶基因hyd或sd1,采用P_(AE)表达盒表达N-氨甲酰水解酶基因adc。分别以质粒pHP13和pUB110为载体,构建D-海因酶和N-氨甲酰水解酶共表达质粒pHCS、pHCY和pUCS。在受体菌中整合表达了acoR和sigL基因,敲除了skf和sdp基因。将共表达质粒分别转化不同的受体菌,通过测定全细胞催化活性,表征D-海因酶和N-氨甲酰水解酶共表达的效果。【结果】带有质粒pHCY和pHCS的重组菌,全细胞催化活性分别为0.21 U/mL和0.31 U/mL。整合表达acoR和sigL基因以及高拷贝质粒pUCS,使全细胞催化活性达到1.0 U/mL。【结论】异源D-海因酶和N-氨甲酰水解酶在枯草芽孢杆菌中能够正确表达。基因拷贝数、acoR和sigL基因表达水平,及skf和sdp基因缺失对重组菌的催化活性具有显著影响。     

Light induced DNA scission by a luminescent mixed-ligand uranyl complex

Efficient cleavage of supercoiled pBR322 DNA by X-ray crystallographically characterized complex, [UO₂(phen)(aba)(OC₂H₅)] (phen = 1,10-phenanthroline; aba = 4-dimethylamino benzoate) has been observed on irradiation with UV (350 nm) or visible light without any external additives through a mechanistic pathway involving singlet oxygen. This complex having 1,10-phenanthroline as an intercalator/binder to supercoiled DNA and N,N-(dimethylamino)benzoate as a chromophore.     

Platinum-assisted preparation of PS ligands containing chiral centres; X-ray crystal structure of a dithiophosphinite platinum complex

The design and synthesis of some ligands containing P(III) bonded to sulfur (thiophosphinites) and chiral centres is described in this paper.Their complexes with platinum (II), [PtCl₂L], (L = bidentate dithiophosphinite) have been prepared and characterised and it has been shown that in many cases, the coordination to platinum protects these ligands from decomposition processes operated by moisture and oxygen. The first example of X-ray crystal structure of a platinum coordinated dithiophosphinite is described for complex cis-[PtCl₂L], [L = meso-2,3-bis(diphenylthiophosphinito)-dimethyl-succinate], 4a.     

Iron-dependent oxygen free radical generation in plants subjected to environmental stress: toxicity and antioxidant protection

Iron has a pivotal and dual role in free radical chemistry in all organisms. On the one hand, free Fe can participate in Fenton reactions and catalyze (catalytic Fe) the generation of hydroxyl radical and other toxic oxygen species. On the other hand, Fe is a constituent of the antioxidant enzymes catalase, ascorbate peroxidase, guaiacol peroxidase, and ferro-superoxide dismutase. Protein Fe is Fenton inactive but can be released from proteins upon attack by activated oxygen. Healthy, unstressed plants avoid the interaction of catalytic Fe and peroxides by disposing of Fe in vacuoles and apoplast, by sequestering Fe in ferritin, and by having high levels of antioxidant enzymes and metabolites in most subcellular compartments. However, when plants are exposed to a variety of adverse conditions, including chilling, high light, drought and paraquat, oxidative stress ensues due primarily to the decrease in antioxidant defenses but also to the increase in free radical production mediated by catalytic Fe. The latter accumulates in many stressed plant tissues. Oxidative stress may lead to metabolic dysfunction and ultimately to plant cell death, so it needs to be estimated conveniently by quantifying the oxidation products of lipids (malondialdehyde and other cytotoxic aldehydes), proteins (total carbonyls, methionine sulfoxide, 2-oxohistidine), and DNA (8-hydroxyguanine, 5-hydroxycytosine). Protein oxidation appears to be a more sensitive and precocious marker than is lipid peroxidation, and DNA damage may also prove to be a useful marker for stress studies in plants.     

Chemical synthesis of febrifugine and analogues

The quinazolinone-containing 2,3-disubstituted piperidines febrifugine and isofebrifugine have been the subject of significant research efforts since their occurrence in Dichroa febrifuga and their anti-malarial actions were first described in the late 1940s. Subsequently they have also been shown to be present in other plants belonging to the hydrangea family and various analogues of febrifugine have been prepared in attempts to tune biological properties. The most notable analogue is termed halofuginone and a substantial body of work now demonstrates that this compound possesses potent human disease relevant activities. This review focuses on the literature associated with efforts dedicated towards uncovering the structures of febrifugine and isofebrifugine, the development of practical methods for their synthesis and the syntheses of structural analogues.     

Prediction of electrocatalytic activity of some nitrogen-doped polyaromatic hydrocarbons by molecular modelling

Density function theory calculations of minimum energy structure of an oxygen molecule and oxygen atom bonded to the two dimensional molecules, C₂₃NH₁₂, coronene and C₂₁NH₁₄, pentacene doped with nitrogen, indicate the structures are at a minimum on the potential energy surface having no imaginary frequencies. Calculation of the bond dissociation energy (BDE) to remove an oxygen atom from nitrogen-doped pentacene to which is bonded O₂, (C₂₁NH₁₄O₂) shows it is less than that to dissociate O₂. However, this is not the case for nitrogen-doped coronene. This suggests that nitrogen-doped pentacene could be an effective catalyst for the oxygen reduction reaction in fuel cells assuming it is O₂ dissociation. It is also shown that O₂H can bond to nitrogen-doped coronene and pentacene and that the BDE to remove OH is less than that to remove OH from O₂H indicating that N-doped coronene and pentacene could also catalyse this reaction. The calculated adsorption energy for O₂ and O₂H on these molecules is negative indicating they can bond to N-doped coronene and pentacene.     

Ce−ZSM-5: A Highly Capable Catalyst for the Construction of Acridines in Water and Their Computational Study against DNA Gyrase Protein

Cerium doped ZSM-5 (Ce−ZSM-5) as an environmentally benign and reusable catalyst for the construction of acridines in aqueous medium. This method produced corresponding acridines with good yields and shorter reaction times. Also avoids the use of hazardous solvents and involves a simple work-up process. The solid catalyst was prepared by doping Ce ions with ZSM-5 (Zeolite Socony Mobil-5) and confirmed by XRD, BET SA-PSD and SEM. The synthesised acridine derivatives were confirmed by ¹H-NMR, ¹³C-NMR and FT-IR spectroscopic data. The docking studies of the synthesised compounds are performed by the PyRx auto dock tool against DNA gyrase protein. The products 5a and 6d are found to be the best fit ligands against DNA gyrase protein.