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A method for measuring the ethanol concentration in a yeast culture broth was developed using both microtubes and a 96-deepwell microplate. The strategy involved first the solvent extraction of ethanol from the yeast culture broth and measurements of the ethanol concentration using the dichromate oxidation method. Particular focus was made on selecting the extraction solvent as well as determining the measurable range of ethanol concentrations using this solvent extraction-dichromate oxidation method. This method was developed as an assay format in 2.0-ml microtubes and 1.2-ml 96-deepwell microplates, and the ethanol concentration in the batch cultures and fed-batch fermentations was measured. Tri- n-butyl phosphate [non-alcoholic solvent, density = 0.9727, solubility in water = 0.028% (w/v)] was used for solvent extraction when measuring the ethanol concentration from the yeast culture broth. The maximum detectable ethanol concentration was 8% (v/v) when 10 g potassium dichromate in 100 ml of 5 M sulfuric acid was used. The concentrations determined from the solvent extraction-dichromate oxidation methods were remarkably similar to those of gas chromatography in which samples were prepared from seven experiments, such as four batch cultures and three fed-batch fermentations. [ABSTRACT FROM AUTHOR]

In this study, we utilized a catabolite repressor to improve the enzymatic activity of recombinant β-galactosidase inclusion bodies (IBs) produced in Escherichia coli under the araBAD promoter system. Specifically, we employed methyl α- d-glucopyranoside (α-MG) to lower the transcription rate of the β-galactosidase structural gene. In deepwell microtiter plate and lab-scale fermentor culture systems, we demonstrated that the addition of α-MG after induction improved the specific β-galactosidase production, even though β-galactosidase was still produced as an IB. Particularly, the addition of 0.0025% α-MG led to the most significant increase in the specific activity of the β-galactosidase. Interestingly, the β-galactosidase IBs obtained in the presence of 0.0025% α-MG were more loosely packed, as determined by IB solubilization in guanidine hydrochloride solution. We propose that the reduced gene transcription rate was responsible for the increased specific β-galactosidase activity and the loose packing that characterized the IBs produced in the presence of α-MG. This principle could be applied throughout the enzyme bioprocessing industry in order to enhance the activity of aggregate-prone enzymes within IBs. [ABSTRACT FROM AUTHOR]

In this study, a dissolved oxygen (DO)-stat fed-batch process was conducted in a pressurized 75-L bioreactor, resulting in the production of the short version of human leukotactin-1 (shLkn-1) using Pichia pastoris as the host, with control of the DO-stat profile and an extension of the recombinant shLkn-1 production phase. By regulation of the exhaust-gas valve, we were able to maintain the vessel pressure at up to 120 kPa, in order to overcome DO limitations associated with the use of the DO-stat. The lowest DO value was adjusted by varying the feed pump speed, allowing us to control the DO-stat profile. This principle was successfully applied to both glycerol feeding during the growth phase and methanol feeding for the induction of shLkn-1. The extension of the methanol induction phase to a total of 192 h of culture time resulted in a shLkn-1 concentration of 2.5 g/L, and a total of 102 g of cumulative production. During this extended induction period, the C-terminal residue of shLkn-1 was truncated and this was confirmed by both reverse-phase HPLC and mass spectrometry. [ABSTRACT FROM AUTHOR]