Pepsin−colloidal gold conjugates were prepared by a simple protein-friendly process and the enzymatic activity of the bioconjugates is reported. The pepsin−gold conjugates are obtained by mixing colloidal gold and protein solutions at pH = 3 and, thereafter, centrifugation, washing, and redispersion of the pepsin−gold conjugate material in water. The bioconjugates in solution were characterized by UV−vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while films of the bioconjugate material obtained by solvent evaporation on suitable substrates were further analyzed by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), transmission electron spectroscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). While TEM and SEM measurements showed aggregates of the enzyme/colloidal gold conjugates, the intactness of secondary and tertiary structures of the enzyme, as determined by FTIR and fluorescence s...

Pepsin-gold colloid conjugates: preparation, characterization, and enzymatic activity

The anthracycline doxorubicin (DOX) is an exceptionally good antineoplastic agent, but its use is limited by formation of metabolites which induce acute and chronic cardiac toxicities. Whereas the acute toxicity is mild, the chronic toxicity can produce a life-threatening cardiomyopathy. Studies in laboratory animals are of limited value in predicting the structure and reactivity of toxic metabolites in humans; therefore, we used an ethically acceptable system which is suitable for exploring DOX metabolism in human myocardium. The system involves cytosolic fractions from myocardial samples obtained during aorto-coronary bypass grafting. After reconstitution with NADPH and DOX, these fractions generate the alcohol metabolite doxorubicinol (DOXol) as well as DOX deoxyaglycone and DOXol hydroxyaglycone, reflecting reduction of the side chain carbonyl group, reductase-type deglycosidation of the anthracycline, and hydrolase-type deglycosidation followed by carbonyl reduction, respectively. The efficiency of each metabolic route has been evaluated at low and high DOX:protein ratios, reproducing acute, single-dose and chronic, multiple-dose regimens, respectively. Low DOX:protein ratios increase the efficiency of formation of DOX deoxyaglycone and DOXol hydroxyaglycone but decrease that of DOXol. Conversely, high DOX:protein ratios facilitate the formation of DOXol but impair reductase- or hydrolase-type deglycosidation and uncouple hydrolysis from carbonyl reduction, making DOXol accumulate at levels higher than those of DOX deoxyaglycone and DOXol hydroxyaglycone. Structure-activity considerations have suggested that aglycones and DOXol may inflict cardiac damage by inducing oxidative stress or by perturbing iron homeostasis, respectively. Having characterized the influence of DOX:protein ratios on deglycosidation or carbonyl reduction, we propose that the benign acute toxicity should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity should be attributed to alterations of iron homeostasis by DOXol. This picture rationalizes the limited protective efficacy of antioxidants against chronic cardiomyopathy vis-a-vis the better protection offered by iron chelators, and forms the basis for developing analogues which produce less DOXol.

Doxorubicin metabolism and toxicity in human myocardium: role of cytoplasmic deglycosidation and carbonyl reduction.

Aims: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors.



Methods and Results:  The level of lipase production and LIP2 induction, measured using an LIP2–LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase.



Significance and Impact of the Study:  Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media.



Conclusions:  This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2004.02190.x

Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica

On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Publisher Summary The removal of low-molecular-weight solutes and concentration of protein solutions are the most frequently performed operations in protein purification and analysis. The highly specific characteristics of the individual protein and the requirements of the experiment must be taken into consideration. In most cases, concentrating and desalting of a protein solution can be achieved without substantial losses or effects on the biological activity of the protein. Electrophoretic techniques, ion-exchange chromatography, and protein sequencing are particularly sensitive to the presence of salts and may additionally require concentrated protein samples as is the case in size-exclusion chromatography. Modern technology has provided membranes for ultra-filtration and dialysis allowing the concentration and desalting of proteins with acceptable yields and little denaturing effect. New and improved supports for conventional chromatography have made gel filtration often the method of choice to change the buffer conditions or composition of a protein solution. The first parameter to be considered in a concentration or desalting experiment is the initial volume of the protein sample and the volume that is desired after the procedure. In many cases, concentration and desalting are interlinked elements between different steps of protein purification. Whenever possible, the order of the individual steps of protein separation should be arranged in a way that concentrating techniques follow those methods leading to dilute protein samples.

Concentration of proteins and removal of solutes.

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Concentration of proteins and removal of solutes.