Sodium Hydroxide → Acetate

Temperature

5 °C

A fermentation broth containing 109.9 g/L of ammonium lactate (97.8% L-lactate, 2.2% D-lactate), 0.8 g/L acetate, and 2.8 g/L maltose was centrifuged to remove particulate matter, then filtered through a 0.45 mm filter. The concentration of ammonium lactate in the resulting solution was 1.10M (117.6 g/L determined by HPLC analysis). Into a 300-mL EZE-Seal stirred autoclave reactor equipped with Dispersimax Impeller (Autoclave Engineers) was placed 45 mL of the 1.10M filtered fermentation broth, then 55 mL of distilled water was added to produce 100 mL of an aqueous solution containing 0.50M ammonium lactate. To the reactor was then added 6.70 g (wet weight) of recycled Pichia pastoris transformant strain GS115-MSP10 (6.66 IU/mL glycolate oxidase and 11,070 IU/mL catalase) which had been permeabilized by treatment with 0.1% benzalkonium chloride ("BARQUAT" OJ-50), and the mixture adjusted to pH 7.5 with 50% NaOH and cooled to 5° C. The reactor purged with oxygen, then the mixture was stirred at 750 rpm, which bubbled oxygen through the mixture via the action of the turbine impeller, and at 5° C. under 40 psig of oxygen. The reaction was monitored by taking a 0.40 mL aliquot of the reaction mixture at regular intervals, faltering the aliquot using a Millipore Ultrafree-MC 10,000 NMWL Filter Unit, and analyzing the filtrate by HPLC using 0.10M isobutyric acid as internal standard. After 3.0 hours, the HPLC yields of pyruvate and acetate were 94.1% (96.2% based on L-lactate) and 2.8%, respectively, and 2.5% lactate remained. The recovered activities of permeabilized-cell glycolate oxidase and catalase were 101% and 56% of their initial values, respectively.

1. customThe reactor purged with oxygen
2. custombubbled oxygen through the mixture via the action of the turbine impeller
3. customat 5° C.
4. filtrationFilter Unit

ORD record

ord-bfc885ca953c452098fed011dafcaca6

Source dataset

uspto-grants-1996_07

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05538875