Eremothecium ashbyii T_(30)发酵产脂肪酶特性的研究

研究了一株核黄素高产突变株E .ashbyiiT3 0 产脂肪酶的条件 ,确定了比较合适的脂肪酶发酵培养基 :豆饼粉 30g/L ,玉米浆 30ml/L ,豆油 5ml/L ,KH2 PO4 5g/L ,MgSO4 ·7H2 O 0 15g/L ,pH消前7 5。T3 0 在此培养基上振荡培养 2 8h ,既定条件下测脂肪酶活力可达 2 32酶活单位 /ml(较原株提高 36 5 % ) ,从一个侧面证实其突变株特性。另外 ,对T3 0 所产脂肪酶的诱导酶属性及脂肪酶在T3 0 核黄素发酵过程中的作用也进行了初步探讨     

Back Cover: Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking (J. Biophotonics 7/2018)

We disclose a theranostic device for performing image‐guided riboflavin/UV‐A corneal cross‐linking. The device determines treatment efficacy by real time monitoring of riboflavin concentration in the corneal stroma. The study shows efficacy of the device in eye bank human donor tissues. Further details can be found in the article by Giuseppe Lombardo et al. ( e201800028 )

     

Multi-spectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin

Riboflavin (RF) plays an important role in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Human serum albumin (HSA) is an important protein involved in the transportation of drugs, hormones, fatty acid and other molecules which determine the biodistribution and physiological fate of these molecules. In this study, we have investigated the interaction of riboflavin RF with HSA under simulative physiological conditions using various biophysical, calorimetric and molecular docking techniques. Results demonstrate the formation of riboflavin–HSA complex with binding constant in the order of 10⁴ M^?1. Fluorescence spectroscopy confirms intermediate strength having a static mode of quenching with stoichiometry of 1:1. Experimental results suggest that the binding site of riboflavin mainly resides in sub-domain IIA of HSA and that ligand interaction increases the α-helical content of HSA. These parameters were further verified by isothermal titration calorimetry ITC which confirms the thermodynamic parameters obtained by fluorescence spectroscopy. Molecular docking was employed to suggest a binding model. Based on thermodynamic, spectroscopic and computational observations it can be concluded that HSA-riboflavin complex is mainly stabilized by various non-covalent forces with binding energy of ?7.2 kcal mol^?1.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Hormonal effect on glycoproteins and glycosaminoglycans in rabbit uteri

The effect of the female hormones on glycoproteins and glycosaminoglycans in uteri has been studied.The uteri taken from the ovariectomized rabbits treated with estrogen, estrogen plus progesterone, and sham administration (control) were incubated in vitro with [U-¹⁴C]glucose. Subsequently, the tissues were digested extensively with pronase, yielding crude glycan fractions. The amount and radioactivity of the crude glycan fraction increased by the treatment with estrogen, but reduced to certain level with progesterone.Separation of glycoproteins and glycosaminoglycans was achieved by stepwise elution from Dowex 1 (X2, chloride form) with increasing concentration of NaCl. The yield and radioactivity, together with the results of chemical, enzymatic, and electrophoretic studies on the resulting fractions indicated that the metabolism of a slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulf ated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulf ate were stimulated remarkably with estrogen, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was, however, found to be different from each other. It was noticed that sulfated glycoproteins were the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of a neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone.     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Enhanced Uptake and Metabolism of Riboflavin in Erythrocytes Infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [¹⁴C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5–8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0–3 h at 37° C in phosphate buffered saline containing MgCl₂, glucose, and [¹⁴C]riboflavin (2.5–7.5 μM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Impedance spectroscopy as a tool for non‐intrusive detection of extracellular mediators in microbial fuel cells

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio‐energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10‐based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling. Biotechnol. Bioeng. 2009; 104: 882–891. © 2009 Wiley Periodicals, Inc.