生物合成谷胱甘肽及其发酵动力学模型的构建
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摘要
利用碘量法、可见分光光度法和紫外分光光度法这3种方法对谷胱甘肽的分析方法进行了研究，结果表明紫外分光光度法效果最好，该方法的平均相对误差均在3％以下，具有平行性好、稳定和灵敏的特点，适合于性状不稳定的谷胱甘肽含量的测定。
以编号为346的酿酒酵母为出发菌株，通过紫外线和~(60)Coγ射线诱变处理，运用推理育种技术，选育到一株抗氯化锌和抗乙硫氨酸的突变株0.5Eth400-5。该菌株经摇瓶发酵谷胱甘肽产量为165.96mg／L，较出发株提高3.5倍，每克干细胞含谷胱甘肽19.76mg，较出发株提高3.2倍。菌株经10次传代培养，谷胱甘肽产量下降10.7％，是一株性状较稳定可深入开发研究的优良菌株。
应用Plackett-Burman实验设计和响应面分析方法对突变株0.5Eth400-5积累谷胱甘肽的培养条件进行了系统研究和优化，得到了优化的培养条件：葡萄糖1.95％，糖蜜1.95％，蛋白胨3％，Cys·HCl 0.097598％，MgSO_4·7H_2O 0.5％，甲硫氨酸0.048％，生物素2.4×10~(-6)％，肌醇7.5×10~(-3)％，酵母膏0.05％，麦芽汁3％，NH_4H_2PO_4 0.055％,VB_1 8×10~(-4)％，(NH_4)_2SO_4 1.24％，K_2HPO_4 0.3％，FeSO_4 3ppm,ZnSO_4 3ppm,CuSO_4 0.5ppm，250mL三角瓶装30mL培养基，接种量为10％，28℃，培养3d，摇床转速为200r／min。采用此优化工艺，谷胱甘肽产量达235.7mg／L，比优化前提高45.4％。
在优化的培养工艺基础上，在5升发酵罐中，通过分批补料的方式流加培养基，研究了谷胱甘肽的发酵规律，得出发酵36h谷胱甘肽产量达最大值638.91mg／l。
对酿酒酵母0.5Eth400-5发酵生产谷胱甘肽的动力学特性进行研究，建立发酵过程菌体生长、谷胱甘肽形成和基质消耗动力学模型。通过数据回归分析，Logistic方程、Luedeking-Piret方程及与Luedeking-Piret方程相似的基质消耗方程能很好的分别描述菌体生长、谷胱甘肽合成及葡萄糖的消耗过程，菌体生长动力学模型为：C_x(t)=(0.2272e~(0.27255t))／(0.9927+0.007329e~(0.27255t))谷胱甘肽合成动力学模型为：
C，(t)=
1 .1 128‘027255‘
0 .9927+o.007329eo27255‘
+74.9794hi(0.9927+0.00732矢”·27255‘)一1.1128
葡萄糖消耗动力学模型为:c，(t)=32.52359-
0 .02359eo2，255‘
0 .9927+0.007329eo27255‘
o.1797ln(0.9927+o.oo7329e”.2，2，，‘)，三个模型的线性相关系数R值均在0.9790
以上，正确地反映了谷肤甘肤的发酵过程及其动力学机制。
Biosynthesis of Glutathione and its Kinetices Model
The analytical method of glutathione were studied by iodimetry, visible spectroscopy and ultraviolet spectroscopy. The analytical results obtained by ultraviolet spectroscopy gave the best method, which was very fit the assay of glutathione. The ultraviolet spectroscopy is steady and sensitive. The average relative error of the ultraviolet spectroscopy was no more than 3%.
A zinc chloride- and ethionine- resistant mutant 0.5Eth400-5 was obtained from its parent strain Saccharomyces cerevisiae 346 by UV and 60Co r-ray treatment and rational screening. The glutathione productivity of the mutant reached 165.96mg/L by flask culture, which was 350% higher than the parent strain, and the glutathione content in the dried cells reached 19.76mg/g, which was 320% higher than the parent strain. A desecend of only 10.7% in the glutathione yield of the mutant was observed after ten times of subculture. Therefore, the obtained mutant is stable strain that is worthwhile to be studied further.
Systematic studies were carried out on the optimization of composition of medium which accumulated the largest amount of glutathione in the mutant 0.5Eth400-5. This optimizing medium was formulated vas Plackett-Burman design and response surface analysis(RSA).The glutathione yield was235.7mg/L under the optimum condition, which was 45.4% higher than that of non-optimized medium.
In a 5 -liter scale batch bioreactor, the rules of glutathione fermentation were studied by fed-batch operation. The maximum glutathione yield was obtained as well after 36 hours' fermentation, which was 638.91mg/L.
The fermentation kinetics of glutathione with Saccharomyces cerevisiad).5Eth4QQ-5 were studied. The kinetic models were obtained. A simple model was proposed using the Logistic equation for growth, the Luedeking-Piret equation for glutathione and the Luedeking-Piret-like equation for glucose consumption. The cell growth model
was the glutathione biosynthesis model was
ln(0.9927 + 0.007329
the glucose consumption model was Cs(t)=32.52359
coefficients of the three models were no less than 0.979. The kinetic model provided
a reasonable description for biomass, product and substrate variation along with the
fermentation process.
以编号为346的酿酒酵母为出发菌株，通过紫外线和~(60)Coγ射线诱变处理，运用推理育种技术，选育到一株抗氯化锌和抗乙硫氨酸的突变株0.5Eth400-5。该菌株经摇瓶发酵谷胱甘肽产量为165.96mg／L，较出发株提高3.5倍，每克干细胞含谷胱甘肽19.76mg，较出发株提高3.2倍。菌株经10次传代培养，谷胱甘肽产量下降10.7％，是一株性状较稳定可深入开发研究的优良菌株。
应用Plackett-Burman实验设计和响应面分析方法对突变株0.5Eth400-5积累谷胱甘肽的培养条件进行了系统研究和优化，得到了优化的培养条件：葡萄糖1.95％，糖蜜1.95％，蛋白胨3％，Cys·HCl 0.097598％，MgSO_4·7H_2O 0.5％，甲硫氨酸0.048％，生物素2.4×10~(-6)％，肌醇7.5×10~(-3)％，酵母膏0.05％，麦芽汁3％，NH_4H_2PO_4 0.055％,VB_1 8×10~(-4)％，(NH_4)_2SO_4 1.24％，K_2HPO_4 0.3％，FeSO_4 3ppm,ZnSO_4 3ppm,CuSO_4 0.5ppm，250mL三角瓶装30mL培养基，接种量为10％，28℃，培养3d，摇床转速为200r／min。采用此优化工艺，谷胱甘肽产量达235.7mg／L，比优化前提高45.4％。
在优化的培养工艺基础上，在5升发酵罐中，通过分批补料的方式流加培养基，研究了谷胱甘肽的发酵规律，得出发酵36h谷胱甘肽产量达最大值638.91mg／l。
对酿酒酵母0.5Eth400-5发酵生产谷胱甘肽的动力学特性进行研究，建立发酵过程菌体生长、谷胱甘肽形成和基质消耗动力学模型。通过数据回归分析，Logistic方程、Luedeking-Piret方程及与Luedeking-Piret方程相似的基质消耗方程能很好的分别描述菌体生长、谷胱甘肽合成及葡萄糖的消耗过程，菌体生长动力学模型为：C_x(t)=(0.2272e~(0.27255t))／(0.9927+0.007329e~(0.27255t))谷胱甘肽合成动力学模型为：
C，(t)=
1 .1 128‘027255‘
0 .9927+o.007329eo27255‘
+74.9794hi(0.9927+0.00732矢”·27255‘)一1.1128
葡萄糖消耗动力学模型为:c，(t)=32.52359-
0 .02359eo2，255‘
0 .9927+0.007329eo27255‘
o.1797ln(0.9927+o.oo7329e”.2，2，，‘)，三个模型的线性相关系数R值均在0.9790
以上，正确地反映了谷肤甘肤的发酵过程及其动力学机制。
Biosynthesis of Glutathione and its Kinetices Model
The analytical method of glutathione were studied by iodimetry, visible spectroscopy and ultraviolet spectroscopy. The analytical results obtained by ultraviolet spectroscopy gave the best method, which was very fit the assay of glutathione. The ultraviolet spectroscopy is steady and sensitive. The average relative error of the ultraviolet spectroscopy was no more than 3%.
A zinc chloride- and ethionine- resistant mutant 0.5Eth400-5 was obtained from its parent strain Saccharomyces cerevisiae 346 by UV and 60Co r-ray treatment and rational screening. The glutathione productivity of the mutant reached 165.96mg/L by flask culture, which was 350% higher than the parent strain, and the glutathione content in the dried cells reached 19.76mg/g, which was 320% higher than the parent strain. A desecend of only 10.7% in the glutathione yield of the mutant was observed after ten times of subculture. Therefore, the obtained mutant is stable strain that is worthwhile to be studied further.
Systematic studies were carried out on the optimization of composition of medium which accumulated the largest amount of glutathione in the mutant 0.5Eth400-5. This optimizing medium was formulated vas Plackett-Burman design and response surface analysis(RSA).The glutathione yield was235.7mg/L under the optimum condition, which was 45.4% higher than that of non-optimized medium.
In a 5 -liter scale batch bioreactor, the rules of glutathione fermentation were studied by fed-batch operation. The maximum glutathione yield was obtained as well after 36 hours' fermentation, which was 638.91mg/L.
The fermentation kinetics of glutathione with Saccharomyces cerevisiad).5Eth4QQ-5 were studied. The kinetic models were obtained. A simple model was proposed using the Logistic equation for growth, the Luedeking-Piret equation for glutathione and the Luedeking-Piret-like equation for glucose consumption. The cell growth model
was the glutathione biosynthesis model was
ln(0.9927 + 0.007329
the glucose consumption model was Cs(t)=32.52359
coefficients of the three models were no less than 0.979. The kinetic model provided
a reasonable description for biomass, product and substrate variation along with the
fermentation process.
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