The Comparison Of Molecular Biological And Physico-biochemical Properties Of Two Strains Producing Hydantoinase

D-amino acids are widely used in several fields such as pharmaceutics, food additives, pesticides and cosmetics etc. D-amino acids are usually produced by enzymatic or chemo-enzymatic method. In bioconversion of a D-amino acid and its derivative, the substrate, 5'-monosubstituted hydantoin is firstly transferred into an intermediate, D-carbamyl amino acid by D-hydantoinase, and in turn into an optically active D-amino acid by either D-carbamoylase or chemical, NaNO2. Nowadays, the bioconversion of D-amino acids has been industrialized by enzymes, so called one -step process. However, the yield of D-amino acids has been tempered by some limited factors from strain per se resulting in the shortages of that goods in the markets. Therefore, scientists are trying to substitute engineered strains for natural strains in the process described above.During cloning hydantoinase gene from the original strain SS-ori, we occasionally picked up a colony(YZ-II6) which occurred a high hydantoinase activity. A series of experiments were performed to confirm whether this strain was the same as the original one. 1 .The heredity of strain YZ-II6 is quite stable because there is no any change for hydantoniase activity, even after passing the generations for 8 times. 2.The strain can grow on YCG medium supplemented with Amp, up to the concentration of 400ug/mL, whilst the original one cannot grow on the same medium even at 50ug/mL of Amp. 3.Its shown from the results of DNA digestion, hydantoinase gene amplification, RAPD, ERIC-PCR etc, that the electrophoretic patterns of the products are obviously different form the original one. 4.Compared with enzymatic activity, hydantoinase activity(0.72U/mL) for YZ-II6 is higher than that of SS-ori(0.20U/mL),on the contrary, D-carbamoylase activity of the former (0.04U/mL) is obviously lower than that of the latter (0.45U/mL). 5.The morphology of the two strains also shows that the flagella of SS-ori are around the cells, whilst the strain YZ-II6 has only a single flagellum grown at the polar of cells besides the different of cell size, though they both are bacillus and Gram-negative. 6.It is also indicated from the results of physico-chemical properties that oxidase and catalase reaction are both positive and the test for glucose oxidation is the same kind of acid-producing type for both strains. We consider from the above data that YZ-II6 could belong to Pseudomonas sp. and SS-ori to Agrobacteria sp.. Finally, in view of 16S rDNA sequence analysis, strain YZ-II6 is classified to Pseudomonas sp. and strain SS-ori is Sinorhizobium sp. according to the "Blast" data.In addition, a hydantoinase gene (1440bp) from YZ-II6 was amplified by PCR with genomic DNA as the template. The recombinant harboring the enzyme gene was transferred into E.coli BL21 and the engineered strain was expressed in LB medium by the induction of IPTG. The results point out that not only the target protein can be expressed in soluble form, but also the bioactivity increase 6 times compared with that of strain YZ-II6.As far as the enzymatic activity is concerned, on the one hand, strain YZ-II6 has higher D-hydantoinase activity and lower D-carbamoylase activity so as not to be suitable for one-step bioconversion of D-amino acids, on the other hand, the higher hydantoinase activity, engineered strain in particular, may be convenient to be as a biocatalyst to produce N-carbamyl D-amino acids which hard to find in the markets. In conclusion, the results mentioned above really provided us some significant data and useful material for realizing our goal-bioconversion of D-amino acids by engineered strain in future.