Development of dietary soluble fibres by enzymatic synthesis and assessment of their digestibility in in vitro, animal and randomised clinical trial models.

The aim was to develop novel fibres by enzymatic synthesis, to determine their total dietary fibre by AOAC method 2009.01 and to estimate their potential digestibility and assess their digestibility in vivo using glycaemic and insulinaemic responses as markers in mice and randomised clinical trial models. We found that fibre candidates to which α-(1,2) branching was added were resistant to digestion in the mouse model, depending on the amount of branching. These results show that in vivo models are needed to reliably assess the digestibility of α-glycosidic-linked oligomeric dietary fibre candidates, possibly due to absence of brush border α-glucosidase activity in the current in vitro assessment. α-(1,3)-linked and α-(1,6)-linked glucose oligomers were completely digested in humans and mice. In conclusion, it is possible to develop dietary soluble fibres by enzymatic synthesis. Adding α-(1,2) branching increases their resistance to digestion in vivo and can thus improve their suitability as potential fibre candidates. Clinical Trial Registry: ClinicalTrials.gov, NCT02701270.

Industrial use of immobilized enzymes.

Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.

Protein engineering of nitrilase for chemoenzymatic production of glycolic acid.

A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate using a nitrilase derived from Acidovorax facilis 72W. Protein engineering and over-expression of this nitrilase, combined with optimized fermentation of an E. coli transformant were used to increase the enzyme-specific activity up to 15-fold and the biocatalyst-specific activity up to 125-fold. These improvements enabled achievement of the desired volumetric productivity and biocatalyst productivity for the conversion of GLN to ammonium glycolate.

Calcium alginate bead immobilization of cells containing tyrosine ammonia lyase activity for use in the production of p-hydroxycinnamic acid.

An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.

Biocatalysis: applications and potentials for the chemical industry.

The chemical industry is exploring the use of renewable feed stocks to improve sustainability, prompting the exploration of bioprocesses for the production of chemicals. Attractive features of biological systems include versatility, substrate selectivity, regioselectivity, chemoselectivity, enantioselectivity and catalysis at ambient temperatures and pressures. However, a challenge facing bioprocesses is cost competitiveness with chemical processes because capital assets associated with the existing commercial processes are high. The chemical industry will probably use biotechnology with existing feed stocks and processes to extract higher values from feed stocks, process by-products and waste streams. In this decade, bioprocesses that offer either a process or a product advantage over traditional chemical routes will become more widely used.

Recent progress in enzymatic resolution and desymmetrization of pharmaceuticals and their intermediates.

The literature since August 2000 is surveyed for interesting and useful examples of the resolution or desymmetrization of pharmaceutical entities, their intermediates and potential drug precursors. Existing, putative and developmental drugs used in various therapeutic areas are discussed.

Protein engineering of Acidovorax facilis 72W nitrilase for bioprocess development.

Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to chemical production since the reactions are run at ambient temperature, do not require strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion. However, native enzymes often do not perform desired reactions with the efficiency required for commercial applications. Protein engineering was used to significantly increase the specific activity of nitrilase from Acidovorax facilis 72W for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. Overexpression of engineered nitrilase enzymes in Escherichia coli, combined with immobilization of whole cells in alginate beads that can be recycled many times has facilitated the development of a commercially viable bioprocess for production of 3-hydroxyvaleric acid.

Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.

The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.