PubMed · 15407083
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1949-11-19. CHROMATOGRAPHIC analysis.. https://doi.org/10.1038/164857a0
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A protein demonstrating laccase activity and potent inhibitory activity towards human immunodeficiency virus (HIV)-1 reverse transcriptase (IC50 1.2 microM) was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The laccase had a novel N-terminal sequence and a molecular mass of 75 kDa, which is higher than the range (55-56 kDa) reported for most other mushroom laccases. It was isolated by sequential chromatography on DEAE-cellulose and Affi-gel blue gel and adsorption on Con A-Sepharose. Unlike some of the previously isolated laccases, it was adsorbed only on Con A-Sepharose. The enzyme required a pH of 3-5 and a temperature of 70 degrees C to exhibit maximal activity. Minimal activity was detected at pH 6 and 7. Activity was undetectable at pH 8 and 9 and after exposure to 100 degrees C for 10 min.
Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646cm(-1)) and amide II (1545cm(-1)) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Seven new stationary phases with different number of proline units and/or different linkage to silica gel were prepared and evaluated in order to improve the performance of proline chiral stationary phases. The average separation factor achieved with the 53 analytes increases with the number of proline units in the stationary phases. When the proline peptides are directly attached to the 3-methylaminopropyl silica gel without using the 6-methylaminohexanoic acid linker, the stationary phases perform better overall. For decaproline chiral stationary phase 8, the separation also depends on the mobile phase system used. For this stationary phase, the CH2Cl2/hexanes/2-propanol system significantly outperforms the 2-propanol/hexanes system. For the 53 analytes tested, the separation factors achieved with this stationary phase compare well with those for three commercial columns.