Process optimization for biosynthesis of Pyruvate Decarboxylase (PDC) and Neuberg's ketol (PAC) from a novel Pichia cecembensis through Response surface methodology

Abstract 1.1. Purpose Phenyl acetyl carbinol (PAC) is an intermediate for the synthesis of active pharmaceutical ingredients (ephedrine, pseudoephedrine, norephedrine etc.) used for the production of antiasthematics and decongestants. An effective and sustainable biosynthesis of PAC through condensation of benzaldehyde and acetaldehyde catalyzed by a solvent intolerant Pyruvate Decarboxylase (PDC) is being reported. Therefore, a process for the biosynthesis of PAC was designed and optimized through Response surface methodology (RSM) in present studies.1.2. Methods The effects of incubation time (8-18hrs), temperature (30-38oC), pH (4-10) and inoculum size (4-10%, v/v) on PAC yield, sugar consumption and PDC activity was determined during fermentation using a newly isolated, highly potent yeast strain of Pichia cecembensis. PAC was quantified spectrophotometerically and through HPLC. PDC produced in 5L flask using molasses as substrate, was exposed to 40mM benzaldehyde as whole cells, crude extract and partialy purified preparation to check its stability against the said solvent and determine its half life as residuel activity. 1.3. Results PDC activity and PAC yield during present studies were found to be 56.27 U/ml and 8.44 g/L, respectively. The yield of PAC (2.22 to 8.44g/L) was increased by 71% after process optimization through RSM with incubation time (13hrs), temperature (33°C) and total sugar (18%,v/v) as significant factors (p-values, 0.902, 0.260 and 0.247, respectively). Process design had Adj R2 value of 0.562, R-squared as 0.770 and Adeq Precision as 4.888 with a uniformly distributed standard error. PDC 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 hrs at 4oC whereas, 33 and 28.5 hrs at 25oC. PAC was purified though HPLC with a purity level of 76.18%. 1.4. ConclusionIncubation time, temperature and sugar concentration were found to be significant factors for PAC biosynthesis. A newly isolated Pichia cecembensis has produced a highly active, solvent and temperature tolerant PDC which has been found far superior than the yeast strains used presently in industries. Hence, this novel yeast specie is a promising candidate for commercial production of PAC and other related APIs owing to its highly stable PDC in the presence of Benzaldehyde.