An overview of biotechnological production of propionic acid: from upstream to downstream processes

The increasing demand for propionic acid (PA) production and its wide applications in several industries, especially the food industry (as a preservative and satiety inducer), have led to studies on the low-cost biosynthesis of this acid. This paper gives an overview of the biotechnological aspects of PA production and introduces Propionibacterium as the most popular organism for PA production. Moreover, all process variables influencing the production yield, different simple and complex carbon sources, the metabolic pathway of production, engineered mutants with increased productivity, and modified tolerance against high concentrations of acid have been described. Furthermore, possible methods of extraction and analysis of this organic acid, several applied bioreactors, and different culture systems and substrates are introduced. It can be concluded that maximum biomass and PA production may be achieved using metabolically engineered microorganisms and analyzing the most significant factors influencing yield. To date, the maximum reported yield for PA production is 0.973 g·g-1, obtained from Propionibacterium acidipropionici in a three-electrode amperometric culture system in medium containing 0.4 mM cobalt sepulchrate. In addition, the best promising substrate for PA bioproduction may be achieved using glycerol as a carbon source in an extractive continuous fermentation. Simultaneous production of PA and vitamin B12 is suggested, and finally, the limitations of and strategies for competitive microbial production with respect to chemical process from an economical point of view are proposed and presented. Finally, some future trends for bioproduction of PA are suggested.

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Biochemical characterization of three Aspergillus niger ß-galactosidases

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.