Fermentation conditions optimization and purification of fructosyltransferase produced by Aspergillus oryzae
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摘要: 通过单因素实验和均匀设计实验对一株产果糖基转移酶的米曲霉摇瓶发酵条件进行优化,其最佳发酵条件为:发酵温度24℃,转速161r/min,装液量50mL/250mL,起始pH5.0,接种量1%,发酵时间96h。在该条件下,果糖基转移酶酶活力达到33.07U/mL,较优化前提高64.6%。通过Desalting脱盐柱,DEAE FF阴离子柱和SepHacryl S-100 HR凝胶柱等分离纯化步骤,经SDS-PAGE检测,该酶呈现一条带,纯化倍数、酶的比活力和收率分别为29.35、231.14U/mg和48.9%。经薄层层析检测,该酶能够生成蔗糖-6-乙酸酯（s-6-a）,证明该酶为果糖基转移酶。经计算,该酶分子量约为92.8ku。Abstract: The optimal fermentation conditions of fructosyltransferase produced by Aspergillus oryzae was studied by single-factor experiment and uniform design. The optimized conditions was temperature 24℃, rotation rate161r/min, medium volume 50mL/250mL, initial pH5.0 and inoculum size 1%. The highest enzyme activity was33.07U/mL after 96h, 64.6% higher than before. The fructosyltransferase was separated and purified by using a procedure including column chromatography separation by Desalting, DEAE FF and SepHacryl S-100 HR. The final purified fold, specific activity and yield of the purified enzyme were 29.35, 231.14U/mg and 48.9%, respectively. The purified enzyme was confirmed to be homogeneous by SDS-PAGE. Detected by thin layer chromatography ( TLC) , the enzyme was able to generate sucrose- 6- acetate ( s- 6- a) , as proof of fructosyltransferase. Its molecular weight was estimated to be 92.8ku.
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Keywords:
- Aspergillus oryzae /
- fructosyltransferase /
- optimization /
- purification
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