The Preliminary Study On Purification And Immobilization Of N-carbamoylase

D-amino acid is generally used in many fields including medicine,which is an important mid-product of some raw drugs,such as antimicrobial ,antiviral agent,artificial sweetener,insect killer and quasi-pyrethrins,especially as the raw of side chain radical of semisynthelic antibiotic.With the strengthen of drug resistance of pathogen and the increase of the dose of antibiotic,this will arouse some sideeffect. Modification of the side chain radical of β-lactam class antibiotic is a possible effective way,while the mother nuclide maintain the activity, but the different side chain radical alternating the drug tolerance of bacterium.The 'hydantoinase method' can be used for the production of D-amino acid with 100% theoretical yield.N-Carbamoylase belongs to amidohydrolase,which can been used in the preparation of absolutely D-amino acids starting from N-carbamoyl-D-amino acid . It is the most critical enzyme in the synthesis of optically pure products with 'hydantoinase method',whose activity effect the yield of optically pure products.In this paper, the preliminary study about N-carbamoylase was reported. The following contents included: 1) Purification of the N-carbamoylase;2)Some properties of N-carbamoylase;3)Optimization of the immobilization parameters and some properties of immobilized N-carbamoylase from Burkholderiacepecia Njut01 for the production of optically pure D-amino acids.The results of N-carbamoylase was purified 15.74-fold, with 0.75% overallrecovery from a cell-free extract of Burkholderia cepecia Njut01,which was very low.The reason is that the content of N-carbamoylase of Burkholderiacepecia Njut01 is very little and N-carbamoylase is very unstable. The purified enzyme was homogeneous as judged by SDS/PAGE.The relative molecular mass of the subunit was about 35 KDa..Some properties of N-carbamoyl-D-amino acid amidohydrolase was researched. The enzyme strictly recognized the configuration of the substrate and only theD-enantiomer of the N-carbamoyl amino acid was hydrolyzed.The high concentrationof subsrate decrease the initial velocity of reaction.The Km=10.22mmol.L-1 and Vmax=0.27mmol.L-1.min-1 of the enzyme were obtained by N-carbamoyl-D-phe as thesubstrate.The optimal concentration of subsrate is 1.2mg.mL-1.The optimal pH andtemperature were 7.2 and 52℃ respectively.N-carbamoylase is very unstable in air,which is an important problem to product optically pure D-amino acid. The effectsof metal ions on N-carbamoylase activity were studied. EDTA did not appear to haveany effect on the reaction at up to 20mmol.L-1.It is possible that the the affinity for theion may be low and unrelated to activity. The added K+,Na+ and Li+ ions had littleeffect. The case of the divalent ions was quite different. The rate of reactionaccelarated by adding Ca2+,Mg2+,Ba2+ but it did decrease by adding Cu2+,Hg2+ions.Apart from Mn2+,trasition metal ions Zn2+,Co2+,Ni2+,Fe2+inhibite the rateof reaction.It is likely that sulphydryl groups were necessary for enzyme activity, because both NEM and DTNB were shown to inhibit enzyme activity.The immobilization procedure of N-carbamoylase from Burkholderia cepeciaNjut01 was optimized.Using different methods(carbodiimide,epoxy activated carriers)it was possible to immobilize the N-carbamoylase from Burkholderia cepecia Njut01with a yields of up to 21.4%. Immobilization was carried out in closed containers for 20 h at 4℃ with 2mg.mL-1 protein concentration.500uL EDC should been put in foramino group support.Some properties of immobilizedN-carbamoylase was researched. The pH and temperature optima of the N-carbamoylase were determined to be pH8.5~9.0 and 60℃~65℃.The immobilized NCase showed storage half-life times of approx.six months .The residual activity of immobilized NCase at the 10th was about 50% of the initial value.