| L-ASN(L-asparagine, amidohydrolase; EC 3.5.1.1) can catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. In the pharmaceuticals industry, L-ASN is used for treatment of childhood acute lymphoblastic leukemia(ALL), Hodgkin’s illness, pancreatic cancer and other neoplastic diseases. Lately, because of the research on the enzymatic acrylmaid mitigation with L-ASN, L-ASN comes to be one of the hot spots in the food industry. While the wild strains’ s production is low, and recombinant expression mostly uses Escherichia coli or Erwinia. So, by means of genetic engineering, molecular modification and the fermentation optimization realizing the efficent expression of L-ASN in food safety host has important significance. This research based on L-ASN gene ans Z which is derived from Bacillus subtilis 168 and used Bacillus subtilis WB600 as the expression host. Through signal peptide optimization, ARTP mutation and fermentation optimization greatly improved the production of L-ASN. This research also purified L-ASN and studied pure enzyme properties. The whole research has laid a solid foundation for indurstrial production of L-ASN.(1) The expression of L-ASN in B. subtilis WB600The mature L-ASN gene ans Z was amplified from the genomic DNA of B. subtilis 168 with primers ans ZF and ans ZR, digested with Eco R Iand Bam H I, and ligated into plasmide p MA5, and formed the recombinant plasmide p MA5-ans Z. Transformed the p MA5-ans Z into expression host Bacillus subtilis WB600, we obtained recombinant bacteria WB-ans Z. After fermentation, recombinant bacteria WB-ans Z reach an activity of 15.8 U?m L-1 in fermentation supernatant. Cut off the L-ASN n-terminal 19 amino acids(presumably as a signal peptide), the rest named ans Z△(SP), then express ans Z△(SP) on B. subtilis WB600. We didn’t detect any L-ASN activity in fermentation supernatant. Trough the SDS-PAGE analysis, we found the accumulation of L-ASN in cell.(2) Screening efficient signal peptide to enhance scretion of L-ASNThe gene ans Z△(SP) was ligated into plasmide p MA5 with different signal peptide ywb N, wap A, npr E, vpr, ync M, yvg O and opp A. Recombinant plasmide p MA5 M, p MA5 H, p MA5 E, p MA5 G, p MA5 J, p MA5 L, p MA5 F was constructed. Transformed these recombinant plasmide into Bacillus subtilis WB600, WB-M, WB-H, WB-E, WB-G, WB-J, WB-L, WB-F was obtained. Through enzyme activity and SDS-PAGE analysis, wap A was found the best signal peptide which increased the L-ASN production to 32.3 U?m L-1.(3) Enzymatic properties of recombinant L-ASNAfter ammonium sulfate precipitation and one-step purification by phenyl hydrophobic chromato- graphy, purified L-ASN was obtained. Studied on the properties of purified L-ASN, we reached those results following: the optimal catalytic temperature is 60°C, T1/2 is 42.6 h(at 60°C), Tm is 70.2°C(DSC scan), the optimal catalytic p H is 7, Km and kcat are 2.35 mmol?L-1 and 274 min-1, metal irons Cu2+, Al3+, Fe3+, Sn2+ have an serious inhibition on L-ASN catalytic activity, Ca2+ and Mg2+ have some activation on L-ASN catalytic activity, EDTA, Zn2+, Co2+, Ba2+, Ni+, Li+, Na+ and K+ have little impact on L-ASN catalytic activity.(4) Recombinant strain with ARTP mutation and it’s fermentationAtmospheric and room temperature plasma(ARTP) can make the DNA damage. Based on this theory, through ARTP mutation on WB-H, we got a mutant strains WB-H10 which L-ASN production increased 30%.The influence of inoculum, carbon source and nitrogen source on L-ASN production was analyzed. The optimal medium contained(g?L-1): sucrose 65, peptone 20, urea 0.8, corn steep liquor 15, K2HPO4 2.612, KH2PO4 2.041, Mg SO4·7H2O 1.845, Na Cl 3, L-asparagine 1, p H was 7; inoculum was 3%.Finally, the capacity of the WB-H was evaluated with a fed-batch strategy in the 3 L fermenter. The yield of L-ASN reached 99.4 U?m L-1 with a productivity of 1.4 U?m L-1?h-1, which was the highest secretory production of L-ASN in the B. subtilis system. |
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