Metabolic production of a novel polymer feedstock, 3-carboxy muconate, from vanillin.

Vanillin can be produced on a commercial scale by depolymerising renewable lignin. One product of microbial metabolism of vanillin by common soil microbes, such as Acinetobacter baylyi, is a tricarboxylic acid with a butadiene backbone known as 3-carboxy muconate (3CM). Three enzymes, 4-hydroxy benzaldehyde dehydrogenase, vanillate monooxygenase and protocatechuate 3,4-dioxygenase, catalyse the biotransformation of vanillin to 3CM. These three enzymes were metabolically engineered into an Escherichia coli host, giving a biocatalyst that converted vanillin into 3CM. The biocatalyst was found to give 100% yield of 3CM from 1 mM of vanillin after 39 h. The rate-limiting reaction was identified as the conversion of vanillate to 3,4-dihydroxybenzoate catalysed by vanillate monooxygenase. Low expression of the reductase subunit of this enzyme was identified as contributing to the reduced rate of this reaction. Proof of principle of a novel application for 3CM was demonstrated when it was converted into a trimethyl ester derivative and copolymerised with styrene.

Effect of the substrate concentration and water activity on the yield and rate of the transfer reaction of ß-galactosidase from Bacillus circulans.

Prebiotic galactosyl oligosaccharides (GOS) are produced from lactose by the enzyme ß-galactosidase. It is widely reported that the highest GOS levels are achieved when the initial lactose concentration is as high as possible; however, little evidence has been presented to explain this phenomenon. Using a system composed of the commercial ß-galactosidase derived from Bacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availability were assessed. Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constant lactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose, while initial reaction rates for transfer increased but remained constant for hydrolysis. This suggests that the high oligosaccharide levels achieved at the raised initial saccharide concentration are due to increases in reactions that form oligosaccharides rather than decreases in concurrent reactions, which degrade oligosaccharides. There were different effects from changing the initial concentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be more important for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosyl acceptors.

Facile pretreatment of Bacillus circulans beta-galactosidase increases the yield of galactosyl oligosaccharides in milk and lactose reaction systems.

The commercially available preparation of beta-galactosidase from Bacillus circulans , known as Biolacta FN5, has been extensively used in the production of prebiotic galactooligosaccharides (GOS). This study focuses on characterizing the production of GOS in two reaction systems: 10% lactose (w/v) in buffer and skim milk. Analysis of the temperature dependence of the GOS yield along with the relative rates of GOS synthesis and degradation leads to the finding that GOS degradation activity was selectively decreased in Biolacta FN5 above 40 degrees C. Facile heat treatment of Biolacta FN5 solution prior to use allowed for GOS yields to be significantly increased in both reaction systems.