The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5-0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.
Water-soluble phosphopeptides from cheese were isolated using immobilized metal affinity chromatography (IMAC). Phosphopeptides from aqueous cheese extracts were completely retained on iminodiacetic acid (IDA) Sepharose equilibrated with FeCl3 and subsequently eluted with ammonium dihydrogen phosphate. Peptides in the eluate from the IMAC-Fe(III) column were identified using reversed phase liquid chromatography-electronic spray identification-tandem mass spectrometry. Phosphopeptides from two different cheeses were analyzed using the described method: a 10-month-old semihard Herrgard cheese made with mesophilic starter and a 24-month-old Parmigiano Reggiano cheese made with thermophilic starter. Elution of the IMAC-Fe(III) column with a gradient of ammonium dihydrogen phosphate resulted in three distinct peaks for Herrgard cheese corresponding to peptides carrying one, two, and four phosphorylated serine residues, respectively. Sixty-five different phosphopeptides were identified from the Herrgard, whereas only 9 from the Parmigiano Reggiano.
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