CAJ | 학술논문

目的优化表达重组藤黄微球菌(M.luteus)过氧化氢酶基因工程菌的发酵条件.方法以生物量和目标蛋白表达量为判断标准,利用单因子试验和正交试验在摇瓶水平优化初始pH、IPTG浓度、诱导时机、诱导时间等影响工程菌发酵制备藤黄微球菌过氧化氢酶的发酵条件.结果最佳发酵工艺参数:培养基初始pH 6.5,摇床转速180 r/min,装液量75 mL,接种量6%,培养温度37℃,诱导表达时间5 h.蛋白表达量比优化前提高近30%,重组蛋白经Ni-NTA纯化和透析后,酶活性为166.0 U/(mg·protein),以实验条件相同野生菌酶活100.0 U/(mg·protein)为对照,重组酶的酶活性提高66%.结论工艺优化后,酶表达量及酶活性均显著提高.
목적우화표체중조등황미구균(M.luteus)과양화경매기인공정균적발효조건.방법이생물량화목표단백표체량위판단표준,이용단인자시험화정교시험재요병수평우화초시pH、IPTG농도、유도시궤、유도시간등영향공정균발효제비등황미구균과양화경매적발효조건.결과최가발효공예삼수:배양기초시pH 6.5,요상전속180 r/min,장액량75 mL,접충량6%,배양온도37℃,유도표체시간5 h.단백표체량비우화전제고근30%,중조단백경Ni-NTA순화화투석후,매활성위166.0 U/(mg·protein),이실험조건상동야생균매활100.0 U/(mg·protein)위대조,중조매적매활성제고66%.결론공예우화후,매표체량급매활성균현저제고.
@@@@Objective To optimize the fermentation conditions of engineered E. coli strain expressing Micrococcus luteus catalase gene. Methods With biomass and expression amount of target protein as the indexes, a single factor experiment and a L 9 ( 34 ) design were used to optimize the liquid submerged fermentation conditions such as initial pH, IPTG concentration, induction time. Results The optimum process parameters were as follows: 37℃, initial pH 6.5, rotation speed 180 rpm, liquid volume in flask 75mL, inoculation amount 6% and IPTG induction time 5h. The expression amount of recombinant catalase was increased by almost 30%. After Ni-NTA affinity chromatography and dialysis, the activity of catalase was 166.0 U/mg·protein, up 66% compared with 100.0 U/(mg·protein)of wild-type strain catalase. Conclusion The expression amount and activity of Micrococcus luteus catalase can be markedly increased under the optimized fermentation conditions of engineered E. coli strain.