Expression, Fermentation Optimization, And Enzymatic Property Research Of The L-asaraginase From Bacillus Subtilis B11-06

L-asparaginase (L-asparaginase amidohydrolase, E.C.3.5.1.1) can catalyze thehydrolysis of L-asparagine to L-aspartic acid and ammonia. L-asparaginase was mainly usedin pharmaceutical and food industry. It was used for treatment of acute lymphoblastic,lymphosarcoma, Hodgkin’s disease, and so on. Also it can reduce acrylamide formation in thefried food. The efficient production of L-asparaginase could be achieved by microbefermentation. The L-asparaginase encoding gene (ansZ) from Bacillus subtilis B11-06wasexpressed efficiently in Bacillus subtilis168, and the culture medium and environmentconditions were optimized. Then the recombinant L-asparaginase was purified andcharacterized. In order to improve the stability of the L-asparaginse, six peptides were fusedto N-terminal domain of L-asparagianse. The main research results were as follows:(1) The L-asparaginase encoding gene from B. subtilis B11-06was amplified by PCRand sequenced. The recombinant expression plasmid pMA5-ansZ was constructed, and thenexpressed in B. subtilis168. The enzyme assay showed that the enzyme activity of therecombinant B. subtilis168/pMA5-ansZ reached9.98U mL-1, which was significantly higherthan that of B. subtilis B11-06.(2) The medium was optimized by one-factor-at-a-time and orthogonal array designmethod. The optimal medium composition (g L-1) was determined to be sucrose,35; tryptone,15; urea,0.8; corn steep liquor,12; K2HPO4,2.61; KH2PO4,2.04; MgSO4·7H2O,1.84; NaCl,5; L-Asn,1. The optimal environment conditions were initial pH7.0, fermentationtemperature of37°C, rotation speed of200r min-1, and inoculation size of4%. Therecombinant B. subtilis168/pMA5-ansZ was cultivated for24h in a5L fermenter, the highestenzyme activity reached89.48U mL-1, which was approximately8.9-fold higher than that ofthe basal medium.(3) The recombinant enzyme was purified by a two-step procedure including ammoniumsulfate fractionation and hydrophobic interaction chromatography. The purified enzymeshowed a specific activity of92.45U mg-1. The optimum pH and temperature of therecombinant enzyme were7.5and40°C, respectively. The enzyme was stable at a pH rangeof6.0-9.0. The enzyme was quite stable below0oC and relatively stable between20oC and30oC. But it was easy to be inactivated when the temperature was higher than50oC. Itexhibited about9%retention of activity following2h incubation at60°C. The enzymeactivity was strongly inhibited by Cu2+、La3+、Fe3+, and slightly inhibited by Na+and EDTA.Results indicated that no metal ions could enhance the enzyme activity. The recombinantenzyme hydrolyzed L-asparagine with a Michaelis constant Kmvalue of0.43mmol L-1and amaximum reaction rate Vmaxof77.51μmol L-1min-1. The acrylamide content was reduced of 75%when the chips were pretreated in6000U L-1L-asparaginase solution for30min prior tofrying. And when the chips were pretreated in8000U L-1L-asparaginase solution for50minthe acrylamide level could decrease about87%.(4) Six peptides with different characteristics were fused to N-terminal of L-asparaginaseand expressed in Escherichia coli BL21. The fusion proteins were purified by Ni2+-NTA.After fusion with peptide4, the specific activity of the fusion protein OP4-ansZ increasedabout1.3-fold than that of the control. The pH stability and thermal stability of OP4-ansZ wasenchanced in comparison with the control.