Construciton Of Recombinant Bacillus Subtilis With High Production Of α-acetolactate Decarboxylase And Optimization Of Fermentation Conditions

Alpha acetolactate decarboxylase (α-ALDC, EC.4.1.1.5) can fleetly catalyzeα-acetolactate (the precursor of diacetyl) to acetoin, thus avoiding the formation of diacetyl.Currently, it has a wide range of applications in beer and other food fermentation industr ies.Two α-ALDC production strains were screened from nature. The genes encoding the α-ALDCfrom those two strains were amplified and expressed in E. coli BL21and B. subtilis WB600.Then enzymatic properties were studied using the pure enzyme which was purified fromrecombinant B. subtilis. On the basis of above, fermentation and enzyme productionconditions of recombinant B. subtilis were optimized for the purpose of producing α-ALDCefficiently and making the recombinant B. subtilis more suitable for industrial applications.Eighty V.P test positive strains were screened from nature. After preliminary screeningand shake flask fermentation, fifteen α-ALDC production strains were reserved. At last, tworelatively high-producing strains were obtained after cultivated in low temperature. They wereidentified as S. aureus L3-15and B. subtilis L5-20respectively based on the results of16srRNA identification and physiological biochemical identification.The genes Saald and BsalsD from S. aureus L3-15and B. subtilis L5-20were amplifiedand over expressed in E. coli BL21, respectively. The results showed that the size of Saaldand BsalsD were705bp and768bp, respectively. The recombinant E. coliBL21/pET-28a(+)-Saald produced22.3U/mL α-ALDC, which was53-fold that of thewild-type enzyme from S. aureus L3-15. The recombinant E. coli BL21/pET-28a(+)-BsalsDproduced22.3U/mL α-ALDC, which was54-fold that of the wild-type enzyme from B.subtilis L5-20.After six histidine tags were plugged in the downstream sequence, the α-ALDC genesfrom S. aureus L3-15and B. subtilis L5-20were connected with carrier pMA5, respectively.Then convert pMA5-Saald and pMA5-BsalsD to B. subtilis WB600, respectively. The resultsshowed that both Saald and BsalsD can successful express in B. subtilis WB600. The totalenzyme activity of SaALDC was36.0U/mL, but that was27.0U/mL for BsALDC, whichwas about80-fold that of the wild-type enzyme from S. aureus L3-15and B. subtilis L5-20respectively.The crude enzyme of recombinant B. subtilis was purified by Ni-NTA. The specificenzyme activity of the pure SaALDC was469.5U/mg. The enzyme characterization ofSaALDC were Kmof17.7μmol/L, Vmaxof2.06mmol/(L·min), optimum pH of6.0andoptimum reaction temperature of35°C. The specific enzyme activity of the pure BsALDCwas272.3U/mg. Although the Km(29.04μmol/L), Vmax(2.82mmol/(L·min)) and optimumpH (6.0) of BsALDC were similar to SaALDC. The optimum reaction temperature of BsALDC was lower than that of SaALDC. Compared with SaALDC, BsALDC showed bettertemperature adaptability, that was more prospects for industrial application.Based on the above study, the culture medium and culture conditions of B. subtilisWB600/pMA5-BsalsD were optimized detailedly. The optimal medium formula wereglycerol of52g/L, soy peptone of12g/L, phosphate of25mmol/L, MgSO4·7H2O of4.0mmol/L, carbamide of3g/L, NiSO4·6H2O of1.5mmol/L, corn steep liguor of15g/L andNaCl of5g/L. The optimal culture conditions were culture temperature of37°C, pH of6.5-7.0, inoculation volume of6%and rotation speed of200r/min. when B. subtilisWB600/pMA5-BsalsD were cultivated in a5L fermentor for48h, the intracellular activity,extracellular activity and total activity of α-ALDC achieved54.3U/mL,51.5U/mL and105.8U/mL, respectively.