Process optimization for biosynthesis of pyruvate decarboxylase (PDC) and Neuberg’s ketol (PAC) from a novel Pichia cecembensis through response surface methodology

Abstract

Abstract Purpose Phenylacetylcarbinol (PAC) is an intermediate for the synthesis of several active pharmaceutical ingredients (ephedrine, pseudoephedrine, norephedrine, etc.) used for the production of antiasthematics and decongestants. An efficient biosynthesis of PAC through condensation of benzaldehyde and acetaldehyde catalyzed by a solvent tolerant pyruvate decarboxylase (PDC) is being reported. A process for the biosynthesis of PAC was designed and optimized through response surface methodology (RSM) in the present study. Methods The effects of incubation time (8–18 h), incubation temperature (30–38 °C), medium pH (4–10), and inoculum size (4–10%) on PAC yield, sugar consumption, and PDC activity were determined through submerged fermentation using a newly isolated potent yeast strain of Pichia cecembensis. PAC was quantified spectrophotometerically and through HPLC. PDC produced was exposed to 40 mM benzaldehyde as whole cells, crude extract, and partialy purified preparation to check its stability against the said solvent. Results The highest PDC activity and PAC yield during present study were found to be 56.27 U/ml and 8.44 g/l, respectively. The yield of PAC was increased by 71% (2.22 to 8.44 g/l) after process optimization through RSM with incubation time of 13 h, incubation temperature of 33 °C, and 18% total sugar as significant factors (P-values, 0.902, 0.260, and 0.247, respectively). R-squared value of 0.770 and Adeq Precision value of 4.888 show the goodness of fit of the process design. PDC is used in the form of Pichia cecembensis whole cells revealed higher stability towards benzaldehyde and elevated temperature as compared to partially purified PDC. Whole cells and partially purified PDC showed half-lives of 240 and 72 h at 4 °C, whereas 33 and 28.5 h at 25 °C. PAC was purified though HPLC with a purity level of 76.18%. Conclusion Incubation time, temperature, and sugar concentration were found to be significant factors for the biosynthesis of PAC. A newly isolated Pichia cecembensis produced a highly active, solvent, and temperature-tolerant pyruvate decarboxylase (PDC) which is superior to its counterpart being presently used in the industry. Hence, this novel yeast species is a promising candidate for commercial production of PAC and other related APIs owing to its highly stable PDC.