Immobilization Of Hydantoinase By Oxirane Method And Characters Of The Immobilized Biocatalyst

Due to its significant features, D-amino acid is playing an increasingly important role in medicine and foodstuff. D-hydantoinase is a key enzyme, which was used to prepare D-amino acid in large scale cheaply and efficiently, and can be used in industry. Furthermore, immobilized D-hydantoinase is more suitable for continuous operation. In this paper, characters of D-hydantoinase from Burkholderia cepecia njut01, especially immobilized D-hydantoinase were studied. The main content is following:(1) Purification include: prepare bacterium suspension, cellular fragmentation, precipitation with (NH₄)₂SO₄, Sepharose CL-4B HIC, DEAE Sepharose FastFlow negative IEX. The purification multiple is 19.94, and the yield of enzyme activity is 30.95%. The satisfying result of the yield of enzyme activity shows that D-hydantoinase has a good stability during the purification. The molecular weight of protein subunit is 33KD by SDS-PAGE electrophoresis.(2) The optimal catalyze temperature of D-hydantoinase are 40⁴5℃to BH and 50℃to MH. r_max of D-hydantoinase are 0.49 mmol/(L·min) to BH and 0.76 mmol/(L·min) to MH, K_m are 16.08 mmol/L and 22.81 mmol/L, respectively. It indicates that BH has a stronger appetence with D-hydantoinase. Ea of D-hydantoinase are 15.25KJ·mol^-1 to BH and 18.26 KJ·mol^-1 to MH.(3) Eupergit C_250L was used as the carrier of immobilizing D-hydantoinase, and concentration of protein are 0.2,0.5,1.0g/L. The amount of immobilized protein went up with the increase of protein concentration, but the yield of enzyme activity went down.(4) Immobilization time were 24, 48, 72 hours, and the last has an approved operational stability. After 20 batches, the activity remains 60%.(5) K_m of immobilized D-hydantoinase are1.10 mmol/L to BH and 2.70 mmol/L to MH, it shows that the appetence of immobilized D-hydantoinase with substrate is strengthened. r_max of D-hydantoinase are 0.013 mmol/(L·min) to BH and 0.021 mmol/(L·min) to MH. Ea of immobilized D-hydantoinase are 16.14KJ·mol^-1 to BH and 18.14 KJ·mol^-1 to MH, just like free enzyme.(6) Immobilized D-hydantoinase was packed in column (inside diameter is 1.0 cm, the height of bed is 7 cm) to build an Immobilized enzyme Packed-bed Reactor. Results shows...