CAJ | 학술논문

对来源于大肠杆菌的植酸酶基因 appA进行突变获得植酸酶基因突变体 appA-2QN，利用酵母表达系统在摇瓶培养条件下表达后，该植酸酶突变体显示出良好的热稳定性。为提高该植酸酶的表达量，降低植酸酶的生产成本，对表达该酶的重组酵母菌 GS115/appA-2QN进行了高密度发酵研究，通过控制发酵过程中碳源（甘油）的添加使得菌体生长达到一定密度，从而实现高效表达。在诱导蛋白质表达阶段，发酵液中甲醇的含量影响植酸酶的表达量，利用变色酸分光光度法对甲醇含量进行了检测分析。结果表明，在5 L 发酵罐中进行高密度发酵147 h 后，菌体浓度OD600nm达到313，通过将甲醇浓度控制在2.5%左右，经过诱导102 h后，蛋白达到了较高的表达量，为7.06 g/L，酶活性（发酵效价）为2.03×105 U/mL。结果表明，通过高密度发酵提高了产植酸酶 appA-2QN 的酵母工程菌的细胞生长密度和蛋白质表达量，揭示该重组酵母菌株具有良好的表达稳定性。
대래원우대장간균적식산매기인 appA진행돌변획득식산매기인돌변체 appA-2QN，이용효모표체계통재요병배양조건하표체후，해식산매돌변체현시출량호적열은정성。위제고해식산매적표체량，강저식산매적생산성본，대표체해매적중조효모균 GS115/appA-2QN진행료고밀도발효연구，통과공제발효과정중탄원（감유）적첨가사득균체생장체도일정밀도，종이실현고효표체。재유도단백질표체계단，발효액중갑순적함량영향식산매적표체량，이용변색산분광광도법대갑순함량진행료검측분석。결과표명，재5 L 발효관중진행고밀도발효147 h 후，균체농도OD600nm체도313，통과장갑순농도공제재2.5%좌우，경과유도102 h후，단백체도료교고적표체량，위7.06 g/L，매활성（발효효개）위2.03×105 U/mL。결과표명，통과고밀도발효제고료산식산매 appA-2QN 적효모공정균적세포생장밀도화단백질표체량，게시해중조효모균주구유량호적표체은정성。
The gene appA for phytase from Escherchia coli was modified by means of site directed mutagenesis to improve its thermal stability. Using yeast expression system the mutant phytase appA-2QN produced in shaking-flask culture showed an increased thermal stability. To increase its expression level and lower production costs,an approach of the high cell-density fermentation of recombinant yeast GS115/appA-2QN was carried out in this study. During fermentation the concentration of glycerin was controlled to promote the cell growth and achieve the high cell-density fermentation. The method of chromotropic acid spectrophotography was used to determine the concentra-tion of methanol during fermentation. The results showed that the OD600nm of the cell reached 313 after 147 h fermen-tation in a 5 L fermentor. After inducing of 102 h with methanol at a concentration of 2. 5%,the expression level of phytase reached 7. 06 g/L. The phytase activity was 2. 03 × 105 U/mL. These results indicated that the cell density and the expression level of target protein were significantly improved by high density fermentation. The recombinant yeast strain has favorable expression stability.