| Abstract
|
:
|
The hydroxycinnamyl esterase substrates: FA-Ara; CA-Ara; SA-Ara; Fura-Ara as well as an affinity ligand: 8-amino-octyl-5-deoxy-5-thio-E-coniferyl-usd\alphausd-L-arabinofuranoside were synthesized. Subsequently, three different methods, TLC, UV and HPLC, were developed to assess hydroxycinnamyl esterase activities. An inducible feruloyl esterase has been purified and partially characterized from Aspergillus niger. The purification procedure utilized ammonium sulfate precipitation, hydrophobic interaction and affinity chromatography, which showed that the affinity ligand, which we designed and synthesized, is a new efficient biospecific adsorbent for the isolation and purification of feruloyl esterases. The enzyme had pH and temperature optima of 5.0 and 60C, and the hydrolysis of the three substrates followed Michaelis-Menten kinetics (kcat 1995 min and Km 20 muM for FA-Ara; kcat 2123 min and Km 7 muM for SA-Ara; and kcat 712 min and Km 1230 muM for CA-Ara). The characterization of feruloyl esterases from Phanerochaete chrysosporium showed the hydrolysis of FA-Ara, SA-Ara and CA-Ara also followed Michaelis-Menten kinetics with Km 1524 muM, 3338 muM and 1967 muM; and pH and temperature optima of 5.0 and 55C, respectively. The continuously monitoring hydrolysis of these three substrates by P. chrysosporium showed this fungus probably produced only one hydroxycinnamyl esterase. 3,4-Dinitrophenyl xylooligosaccharide glycosides, usd({\rm Xyl}p\beta(1\to4))\sb{\rm n}{\rm Xyl}p\betausd-3,4-DNP (n = 0 4) have been made. The initial rates of liberation of 3,4-dinitrophenol from the compounds by the xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, have been measured. The usd\rm k\sb{cat}/K\sb{m}usd values increase up to the xylotrioside, and then decrease, suggesting that exocleavage three saccharide units into the chain is the natural mode of action. A series of cellooligosaccharide glycosides, (Glcpusd\beta((1\to4))\sb{\rm n}usdGlcpusd\betausd-3,4-DNP, where n = 0 6, has been chemically synthesized. The initial rates of liberation of 3,4-dinitrophenol from the compounds by the CBH I of Trichoderma reesei, have been measured. The kusd\rm\sb{cat}/K\sb{m}usd values increase up to the cellopentaoside, and then decrease. The kusd\rm\sb{cat}/K\sb{m}usd data confirm five monosaccharide binding sites in CBH I are discerned in the active site tunnel upstream of the proposed cleavage site, but only two downstream, as predicted by Divne et al., (Science, 265, p 524-528, 1994).
|