No abstract

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.

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Optimization of pilot-scale enzyme production is described for the case of an extracellular protease and an intracellular esterase. Media optimization was conducted to reduce medium costs and to determine the effect of various de®ned ingredients as well as complex nitrogen sources on enzyme production. Fermentation conditions such as inoculum transfer timing, agitation rate, and cultivation temperature were evaluated for their effect on enzyme production. Broths were harvested via micro®l-tration, dia®ltered, and in the case of the extracellular enzyme, lysed via homogenization. An improvement in enzyme titer and reduction in medium costs for the extracellular protease was realized through replacement of Sabouraud dextrose broth medium with more reasonably priced complex nitrogen sources such as N-Z-amine A. An improvement in enzyme titer and reduction in medium costs for the intracellular esterase was realized by decreasing the amount of malt extract and omitting glycerol from the medium. An improvement in the harvest conditions for both enzymes was realized by using largelumen-diameter hollow-®ber membranes (2.7 mm) which seemed wide enough to pass clumps of fungal and ®la-mentous bacterial fermentation broth without clogging. IntroductionOptimization of enzyme production is a key factor for the successful implementation of enzymes for synthetic use. While several published studies exist for shake¯asks [7,10,15,19] and small-scale fermenters [8], fewer results have been published for larger-scale fermenters above 20 L in volume. Some notable exceptions exist [1,5,9,12,21,22]. Some examples from the literature for small-scale fermenters clearly indicate that optimization efforts have focused on media development. Increased OUR (oxygen uptake rate), cell growth, and speci®c production of extracellular glucose oxidase from Aspergillus niger was obtained with the addition of 2 g/L yeast extract to a mineral salts minimal medium in 1±3 L fermentations [11]. Bactopeptone, yeast extract, glucose, and pH control were optimized to improve dextransucrase activity produced by a constitutive mutant of Leuconostoc mesenteroides to 20±25 IU/mL [14]. Lactose and soybean meal concentrations were optimized in conjunction with amino acid supplementation and mid-cycle lactose shots in a 2-L fermentation of pyranose oxidase by a Basidiomycetous fungus to improve total productivity 45-fold and speci®c productivity 4-fold.Other examples from the literature describe optimization efforts of fermentation conditions at the small scale. An optimal agitation rate of 420 rpm (from a range of 280± 420 rpm) and optimal air¯ow rate of 0.25 vvm (from a range of 0.25±0.6 vvm) in a 5-L fermenter resulted in increased enzyme production by 75% in recombinant Saccharomyces cerevisiae to produce glucose oxidase [13]. Raising dissolved oxygen levels from 10 to 15% during a 1-L fermentation of Acinetobacter radioresistens to produce an alkaline lipase produced a two-fold increase in lipase titer (although similar dry cell weights were obtained) [6]. F...