D-Pantothenic acid

D-Pantothenic acid i»

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The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea/EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and enzyme recovery by membrane filtration give d-pantolactone with 50% yield and 90-95% ee over 6 days.

When the kinetics of the hydrolysis of rac-pantolactone by Lph were investigated a decrease in the reaction velocity was observed; this was found to be because of competitive inhibition by D-pantolactone (Eq. 1) [19] and slight product inhibition of Lph. Under the same conditions of pH (7.5) and temperature (30 °C), l-pantolactone was completely converted to L-pantoic acid. This is certainly a disadvantage of Lph-catalyzed kinetic resolution, because space-time yields come to levels as low as 6 g L_1 h_1.

For economic resolution of rac-pantolactone repeated use of the corresponding catalyst is required. Repeated batch conversions of d, L-pantolactone (30% w/v) with crude extracts of E. coli gave D-pantolactone in 50-53% yield with 90-95% ee over 6 days. The membrane filtration used for enzyme recycling from the product is, however, quite a costly separation step. While cell embedding into different types of (coated) alginate beads led to either diffusional limitations or dissolution of the beads, because of the high concentrations of monovalent cations introduced by titration, covalent immobilization of crude Lph to EupergitC (Rohm, Darmstadt, Germany) led to a stable biocatalyst easy to handle in a repeated batch process. The half-life of immobilized Lph was extended to 13 days. In the hydrolytic step the immobilisate was slowly ground to fine particles that tended to clog the filters during product recovery. Use of a lower solids content (<8% w/v) and optimization of the stirring geometry and velocity (while maintaining efficient titration of the pantoic acid synthesized) are expected to further increase the half-life.

After recombinant overexpression and immobilization and classical process engineering, the genetic engineering of an enzyme surely has great potential for improvement of biocatalysts (for a short review of genetic engineering issues see above). Because no structural information is yet available on Lph from Agro-bacterium, it has not been possible to use a rational approach involving molecular modeling and site-directed mutagenesis. Future X-ray crystallization studies of Lph are expected to reveal molecular details of enzymatic L-pantolactone hydrolysis and to enable site-directed manipulations aiming at higher activity and elimination of competitive inhibition by excess D-pantolactone. For directed evolution (Box 20) of Lph a library of 11680 lph mutants has so far been generated by error-prone PCR. Improved variants of Lph were selected by high-throughput screening in micro-

plates with nitrazinyellow as an indicator of pantoic acid synthesis. The activity of mutants F62S, K197D, and F100L was increased 2.3-, 1.7-, and 1.5-fold, respectively.

Although mutant F62S has excellent activity in the standard enzyme assay, its performance under process conditions (high concentrations of D,L-pantolactone) was poor. Because close adaptation of the high throughput screening assay to these conditions is very difficult for reasons of viscosity, this example shows the challenge of assay development for improvement of industrially valuable catalysts. It also shows that the factors of improvement achievable by only one round of random mutagenesis and screening strongly depend on the starting point, because obviously, as in this example, the specific activity of an already good biocatalyst is not as easily improved by factors in the range 10-100 as when the starting point is substantially lower [20]. Resolution of D,L-pantolactone by mutants K197D and F100L gave D-pantolactone with 90.4% ee at 51.8% conversion and 90.2% ee at 50.1% conversion, respectively, after 12 h using only 80% of the biomass compared with the unmutated control (90.5% ee at 52.3% conversion after 15 h). This corresponds to an increase of the productivity of the recombinant biocatalyst from 136 to 170 g g_1 (g D-pantolactone per g biomass) and slight shortening of the reaction time down to 12 h. Despite these great achievements the process has not reached commercial application, yet.

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