Development of a hydantoin-hydrolysing biocatalyst for the production of optically pure amino acids using Agrobacterium tumefaciens strain RU-ORPN1

by Foster, Ingrid Margaret

A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature.

The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound.

Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method.

Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions.

Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.