| 摘要: |
| 【目的】用醋酸纤维素膜和96孔板2种方法高通量筛选高产谷氨酰胺转胺酶(MTG)的茂原轮枝链霉菌菌株。【方法】利用紫外诱变获得茂原轮枝链霉菌突变体库后，分别利用醋酸纤维素膜和96孔板2种方法高通量初筛产MTG菌株，然后依次用试管和摇瓶发酵法复筛，并对96孔板高通量筛选方法的条件进行了优化。【结果】利用醋酸纤维素膜显色法从3 000株紫外诱变的菌株中筛选出1株高产MTG茂原轮枝链霉菌，其MTG活性为4.9 U/mL，较出发株(MTG活性2.7 U/mL)提高了80%。优化的96孔板高通量筛选方法为：直径为3 mm左右的单菌落接种至150 μL发酵培养基中，30 ℃ 200 r/min培养72 h；利用该法从3 415株紫外诱变的菌株中筛选获得了2株MTG高产株，其MTG活性为5.4 U/mL，较出发株(MTG活性为3.0 U/mL)提高了80%。【结论】利用基于醋酸纤维素膜和96孔板的2种高通量筛选方法分别筛选到1株和2株高产MTG的茂原轮枝链霉菌；相较传统的选育方法，这2种高通量筛选方法都大大降低了经济成本，提高了筛选效率，缩短了选育周期。 |
| 关键词: 茂原轮枝链霉菌 微生物谷氨酰胺转胺酶 高通量筛选 醋酸纤维素膜 96孔板 |
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| 基金项目:国家自然科学基金项目“IL-17A调节REG3A促进伤口愈合的作用机理”(81072422) |
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| Novel high-throughput screening of Streptoverticillium mobaraense with high microbial transglutaminase yield |
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LIU Ying, TIAN Peilin, CHEN Jia,et al
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East China Normal University,et al
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| Abstract: |
| 【Objective】This study aimed to screen Streptoveticillium mobaraense mutants with high-yield of microbial transglutaminase(MTG)using cellulose acetate film(CAF)and 96-well microtiter plate based high-throughput screening(HTS)methods.【Method】CAF and 96-well microtiter plate were applied in high-throughput primary screening of UV treated S.mobaraense.High-yield candidates were further confirmed by stout tube fermentation and shaking flask method.Conditions of 96-well microtiter plate cultivation were optimized.【Result】1 strain with high yield of MTG(MTG activity as 4.9 U/mL)was selected by CAF assay from a library of over 3 000 mutants with the enzyme activity improved by 80% compared to the parent strain(MTG activity as 2.7 U/mL).Optimized conditions of 96-well microtiter plate fermentation were as follows:colonies with a diameter of 3 mm were inoculated to 150 μL/well fermentation medium before being cultivated at 30 ℃ 200 r/min for 72 h for enzyme assay.2 strains were screened from 3 415 mutants with MTG activities of 5.4 U/mL,80% higher than and starting strain with MTG activity of 3.0 U/mL.【Conclusion】1 and 2 high-yield strains were selected using CAF and 96-well microtiter plate,respectively.Both methods developed in this research provided efficient HTS approaches at a low cost in short period. |
| Key words: Streptoverticillium mobaraense microbial transglutaminase(MTG) high-throughput screening(HTS) cellulose acetate film(CAF) 96-well microtiter plate |
