D‐Amino Acid Tolerant Hosts for D‐Hydantoinase Whole Cell Biocatalysts

Abstract

Whole cell biocatalysts which enable the concerted use of D‐hydantoinase, D‐carbamoylase, and racemase enzymes are valuable for the production of D‐amino acids. However, Escherichia coli host strains used for this purpose efficiently degrade D‐amino acids. This work demonstrates that D‐amino acid degradation occurs largely through the concerted action of D‐amino acid dehydrogenase, encoded by the dadA gene, and D‐serine dehydratase, encoded by the dsdA gene. Deletion mutants of E. coli which lack these activities were constructed and compared against wild type strains in D‐amino acid degradation. An E. coli dadA mutant reduced the degradation of D‐methionine by one third, D‐phenylalanine by two‐thirds, and D‐2‐aminobutyric acid nearly completely. Even though the dadA mutant had no effect on D‐serine degradation, a dadA dsdA double mutant of E. coli additionally reduced degradation of D‐serine, as well as D‐phenylalanine, almost entirely. These strains are appropriate hosts for whole cell biosynthesis of D‐amino acids using general approaches such as the hydantoinase system.

Topological modeling of methane hydrate crystallization from low to high water cut emulsion systems

“…This arrangement probably changes the reflective characteristics of the system, changing the measured chord length number by the FBRM Probe. Experiments performed by Turner et al [19] showed that the number of detected particles by the FBRM depends on the particles reflection; consequently, it is possible to infer that this measure will also be influenced by the reflective characteristics of the system (external phase). In the case that oil is the continuous phase ( Figure 6 (c) and Figure 7 (b)); the chord length number does not decrease around the nucleation step, because there is no change in the external phase before methane hydrate formation.…”

Distribution, industrial applications, and enzymatic synthesis of d-amino acids

“…First, the types of D-AAs made using this synthetic method are limited, second, the enzyme activities of natural D-hydantoinase are often low, and third, the substrates of D-hydantoinase possess low solubility. Some efforts have been made to overcome these disadvantages: (a) optimization of the growth and enzymatic reactions of wild strains such as improving the activity of D-hydantoinase from Ochrobactrum anthropi strain 245 up to 10-fold by considering pH and additions of inducer, ammonium, phosphate, heavy metals, and other ions when setting up reactions (Lee and Kim 1998;Pozo et al 2002), (b) coexpressing D-hydantoinase with Dcarbamoylase in Eschericia coli, while deleting the enzymes related to D-AA degradation, such as D-amino acid dehydrogenase and D-serine dehydratase (Grifantini et al 1998;Turner et al 2004), and (c) improving enzyme catalysis properties by directed evolution such as enhancing the thermostability of D-hydantoinase by DNA shuffling to make the substrate more soluble (Kim et al 2000). D-Stereospecif ic amidohydrolase D-Stereospecific amidohydrolases, such as N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and D-peptidase, could be used for kinetic resolution of racemic amino acid amides to yield D -AAs.…”

Use of Enzymes in the Synthesis of Amino Acids

“…It is expected that this will drive the replacement of the wild-type strains by more active and tuned recombinant production strains as well as the implementation of new processes. The availability of production hosts that are optimized for the production of D-amino acids [104,105], the straightforward synthesis of the racemic hydantoins from cheap raw materials via a number of different routes [76] as well as the low side-product formation will certainly stimulate this.…”