ABSTRACT
The production of large amounts of paclitaxel for use as an anticancer treatment has been a challenging problem because of the low concentration of the compound in yew trees and its occurrence as part of a mixture of other taxanes. Two novel enzymes were isolated to facilitate the production of 10-deacetylbaccatin III, a precursor used for semisynthesis of paclitaxel and analogs. A strain of Nocardioides albus (SC13911) was isolated from soil and found to produce an extracellular enzyme that specifically removed the C-13 side chain from paclitaxel, cephalomannine, 7-beta-xylosyltaxol, 7-beta-xylosyl-10-deacetyltaxol, and 10-deacetyltaxol. The enzyme was purified to near homogeneity to give a polypeptide with 47,000 M(r) on a sodium dodecyl sulfate gel. A strain of Nocardioides luteus (SC13912) isolated from soil was found to produce an intracellular 10-deacetylase that removed the 10-acetate from baccatin III and paclitaxel. The 10-deacetylase was purified to give a polypeptide with 40,000 M(r) on a sodium dodecyl sulfate gel. Treatment of extracts prepared from a variety of yew cultivars with the C-13-deacylase and C-10-deacetylase converted a complex mixture of taxanes primarily to 10-deacetylbaccatin III and increased the amount of this key precursor by 4-24 times.
Subject(s)
Bridged-Ring Compounds , Carboxylic Ester Hydrolases/metabolism , Paclitaxel/metabolism , Taxoids , Bridged Bicyclo Compounds/chemistry , Bridged Bicyclo Compounds/metabolism , Carboxylic Ester Hydrolases/isolation & purification , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Nocardiaceae/enzymology , Substrate SpecificityABSTRACT
Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.
