Enhanced stability of newly isolated trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization.

Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde-dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.

Trimeric l-N-carbamoylase from newly isolated Brevibacillus reuszeri HSN1: a potential biocatalyst for production of l-α-amino acids.

l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with Km and Vmax values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn(2+) ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.

Molecular structure of D-hydantoinase from Bacillus sp. AR9: evidence for mercury inhibition.

Stereospecific conversion of hydantoins into their carbamoyl acid derivatives could be achieved by using the enzyme hydantoinase. Specific hydantoinases convert either the D-form or the L-form of the hydantoin and the amino acids responsible for stereospecificity have not been identified. Structural studies on hydantoinases from a few bacterial species were published recently. The structure of a thermostable D-hydantoinase from Bacillus sp. AR9 (bar9HYD) was solved to 2.3 angstroms resolution. The usual modification of carboxylation of the active-site residue Lys150 did not happen in bar9HYD. Two manganese ions were modelled in the active site. Through biochemical studies, it was shown that mercury inhibits the activity of the enzyme. The mercury derivative provided some information about the binding site of the mercuric inhibitors and a possible reason for inhibition is presented.

Caerulomycin A suppresses immunity by inhibiting T cell activity.

BACKGROUND: Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells. METHODS: Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-(3)H] incorporation. RESULTS: CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo. CONCLUSION: This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug.

Crystallization and preliminary X-ray diffraction analysis of a thermostable D-hydantoinase from the mesophilic Bacillus sp. AR9.

D-hydantoinase catalyzes the conversion of DL-hydantoin derivatives to the corresponding optically pure N-carbamoyl amino acids, the first step in the industrial preparation of optically pure amino acids using biological catalysts. A thermostable D-hydantoinase from the mesophilic bacteria Bacillus sp. AR9 has been crystallized in three different crystal forms. The hexagonal faced crystals were the best looking, but did not diffract. One of the crystal forms is star-shaped and appeared to be twinned, but diffracted as a single crystal to a resolution of 2.3 A. These crystals belong to space group P6(4) and have unit-cell parameters a = b = 129.55, c = 102.86 A, alpha = beta = 90, gamma = 120 degrees.