Kazuyoshi Yajima

Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.

Immobilization of thermotolerant N-carbamyl-D-amino acid amidohydrolase.

Abstract N-Carbamyl- d -amino acid amidohydrolase (DCase), in which amino acid residues were substituted by mutation, followed by the selection based on thermotolerance, showed improved thermostability, by 5° or 10°C, compared to the native DCase. These DCases were immobilized on a macroporous phenol formaldehyde resin, Duolite A-568, and the immobilized thermotolerant enzymes showed higher activity than the immobilized native DCase. From the results of repeated batch reactions, the half-lives of the activities of immobilized thermotolerant DCase, in which Leu was substituted for Pro 203, and immobilized native DCase were 104 and 58 times, respectively. It was revealed that the higher thermotolerance enabled the immobilized enzymes to be more stable in reactions. A reductant, dithiothreitol, also stabilized the enzymes in reactions. Compared with soluble DCase, immobilized DCase was somewhat stable, and its activity was optimum at a lower pH.

Cloning and characterization of decaprenyl diphosphate synthase from three different fungi

Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ10 in addition to endogenous CoQ8. Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombedps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ10, indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ10 in other organisms.