Pantolactone hydrolases

Finally, biocatalytic resolution was developed for more efficient production of D-pantolactone. Whereas the resolution of O-acyl pantolactone with lipases or esterases [12] did not lead to an industrially attractive process, the hydrolysis of rac-pantolactone by pantolactone hydrolases enabled development of a technically feasible and economic process. 

For a kinetic resolution process use of both d- or an L-specific pantolactone hydrolase is possible in principle (Figure 6.3.3). If the unwanted L-form is hydrolyzed it might take longer for the remaining D-pantolactone to reach a sufficient enantiomeric excess the process is, however, much more robust, e.g. towards competing spontaneous chemical hydrolysis. 

The enantioselective hydrolysis of pantolactone into (R)-pantoic acid and (S)-pantolactone (Fig. 8.21), in the presence of (R)-pantolactone hydrolase from Fu-sarium oxysporum [110b], offers a better alternative. An alginate-entrapped... 

Contrary to the Fuji process, BASF described the characterization and cloning of an L-specific pantolactone hydrolase from Agrobacterium tumefa-ciens [103,104]. This enzyme exclusively opens up the undesired lactone l-1 12, providing a more direct route to d-1 12 (Scheme 35, right side). In addition, this new process is expected to be much more robust toward the competing spontaneous chemical hydrolysis, which could theoretically cause a diminished optical yield in the Fuji process. The enzymatic resolution of d/l-112 in repeated batches with membrane filtration techniques provided d-1 12 in 50% yield and with 90-95% ee. By immobilization onto Eupergit C it was possible to obtain a stable biocatalyst which was easy to use in repeated batch reactions. 

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. 

The atom efficiency of a kinetic resolution is increased if the starting material is not an ester but a lactone. Indeed, kinetic resolutions of lactones are used on an industrial scale. Fuji/Daiichi Chemicals produces D-pantothenic acid on a multi-ton scale based on such a resolution. D-Pantolactone is hydrolysed at pH 7 by a hydrolase from Fusarium oxysporum yielding D-pantoic acid with an ee of 96% while L-pantoic acid was barely detectable. The immobilized Fusarium oxysporum cells were recycled 180 times and retained 60% of their activity, demonstrating the great stability of this catalytic system [47-50]. 

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