Molecular Modification And Fermentation Optimization Of L-asparaginase From Bacillus Subtilis

L-asparaginase(L-AsnE,E.C.3.5.1.1) can catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Because of the founction to reduce acrylamide effectively which is a potential carcinogenic substance in fried foods by hydrolyzing L-asparagine(acrylamide precursors),L-AsnE comes to be one of the hot spots in the field of food safety. The L-AsnE gene(ansz) from Bacillus subtilis B11-06 was cloned and overexpressed in GRAS B. subtilis 168 and a recombinant L-AsnE was obtained. However, low activity and poor thermostability of L-AsnE appeared to be an obstacle for its application in food industry. Therefore, in order to improve its activity and thermostability and meet the requirement of food industry, site-directed mutagenesis was introduced to engineer L-AsnE. Main conclusions were as follows:(1) Based on sequences alignment and protein structure analysis results, 12 residues near the active sites, Gly and Pro at alpha helix of protein structure and all the His in the amino acid sequence were selected for site-directed mutagenesis and specific activity of five mutants were higher than that of wild-type enzyme. The specific activity of mutant enzymes G107 D, H125 L, E145 A, S299 N and P348 A were improved by 85%, 33%, 45%, 28% and 32%, respectively, as compared with the wild-type enzyme. The optimum temperature of H125 L was 45℃ and that of the other four enzymes were all 40℃ which was consistent with the wild-type enzyme. The half-life at 40℃ of G107 D, H125 L and P348 A were 6 h, 4 h and 10 h, respectively, longer than the wild-type enzyme. Catalytic efficiency of the five mutant enzymes were improved compared with the wild-type enzyme.(2) Residues at 107, 125, 299 and 348 were selected to construct 11 combinational mutations and enzyme purification was conducted. Four mutations with improved activity were obtained, the specific activity of G107D/H125 L, G107D/S299 N, G107D/H125L/S299 N and G107D/H125L/S299N/P348 A were increased by 0.72-, 0.86-, 0.64- and 1.06-fold, respectively, as compared with the wild-type enzyme. The optimum temperatures of G107D/H125 L, G107D/H125L/S299 N and G107D/H125L/S299N/P348 A were improved by 5℃. Optimum pH and pH stability of them were slightly changed compared to the wild-type enzyme. And the values of Km and kcat/Km were higher than that of wild-type enzyme.(3) Residue at 107 was subjected to saturation mutagenesis on the basis of G107D/H125L/S299N/P348 A to construct 18 mutations. Two reasons for this strategy: First, all the four combinational mutations with improved activity contained the mutation of 107 site; Second, the specific activity and thermostability of enzyme G107 D were highly improved as compared to the wild-type enzyme. After purification, enzyme activities were measured. The results showed that the specific activity of G107D/H125L/S299N/P348 A was the highest. Therefore, changing G107 to D107 was the best selection.(4) Mutant strain G107D/H125L/S299N/P348 A was used to produce L-AsnE in 5 L fermenter. The effects of feeding strategy and dissolved oxygen on the production of L-AsnE were studied. It turned out that high-dissolved oxygen was conducive to bacterial growth and shorten the fermentation period. At the speed of 600 r·min-1, extracellular enzyme activity reached its maximum value 22.12 U·m L-1 after 10 hours fermentation, but at 16 h it reduced to zero. Under feeding condition, intracellular enzyme activity accumulated gradually, but extracellular enzyme activity reached maximum value in a short period and dropped to zero rapidly which displayed the same trend with no feeding. According to the comparison of enzyme synthesis level under different agitation speeds, two-stage oxygen control strategy was adopted to optimize the fermentation conditions: at first 10 h, agitation speed was controlled at 600 r·min-1, and then decreased to 450 r·min-1. The results showed that the maximum values of in- and ex-tracellular enzyme activity were 97.57 U·mL-1and 30.89 U·mL-1, respectively, improved by 17.5% and 22.0%, respectively, as compared to that produced at constant agitation speed(600 r·min-1), and the total enzyme activity was 1.76 times higher than that produced at initial condition. Moreover, the extracellular enzyme activity decreased slowly after reached the maximum value and could be detected at 28 h.