重组黑曲霉生产果聚糖酶的优化生物工艺。

Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger.

机构信息

Institute of Biochemical Engineering, Technische Universität Braunschweig, Germany.

出版信息

Appl Microbiol Biotechnol. 2010 Aug;87(6):2011-24. doi: 10.1007/s00253-010-2661-9. Epub 2010 May 26.

Abstract

A comprehensive approach of bioprocess design at various levels was used to optimize microbial production of extracellular fructofuranosidase, important as biocatalyst to derive fructooligosaccharides with broad application in food or pharmaceutical industry. For production, the recombinant strain Aspergillus niger SKAn1015 was used, which expresses the fructofuranosidase encoding gene suc1 under control of a strong constitutive promoter. In a first screening towards an optimized medium, glucose, nitrate, Fe(2+), and Mn(2+) were identified as beneficial for production. A minimal medium with optimized concentration of these key nutrients, obtained by central composite design experiments and quadratic modelling, provided a threefold increased fructofuranosidase activity in the culture supernatant (400 U/mL) as compared to the originally described medium. Utilizing the optimized medium, the process was then transferred from shake flask into a fed-batch-operated bioreactor. Hereby, the intended addition of talc microparticles allowed engineering the morphology of A. niger into a highly active mycelial form, which strongly boosted production. Fructofuranosidase production was highly specific as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The secreted enzyme activity of 2,800 U/mL, corresponding to about 3 g/L of fructofuranosidase, achieved by the microparticle-enhanced fed-batch process, is tenfold higher than that of any other process reported so far, so that the presented bioprocess strategy appears as a milestone towards future industrial fructofuranosidase production.

摘要

采用了一种综合的生物过程设计方法,在不同层面上优化了微生物胞外果聚糖酶的生产,该酶作为生物催化剂在食品或制药工业中有广泛的应用。生产中使用了表达果糖呋喃糖苷酶编码基因 suc1 的重组黑曲霉 SKAn1015 菌株,该基因受强组成型启动子的控制。在首次筛选优化培养基的过程中,发现葡萄糖、硝酸盐、Fe(2+)和 Mn(2+)有利于生产。通过中心复合设计实验和二次建模获得的优化浓度的关键营养物质的最小培养基,使培养上清液中的果聚糖酶活性提高了三倍(400 U/mL),与最初描述的培养基相比。利用优化的培养基,然后将该过程从摇瓶转移到分批操作的生物反应器中。在此,滑石粉微颗粒的预期添加允许将黑曲霉的形态工程设计成一种高度活跃的菌丝体形式,从而大大提高了产量。果聚糖酶的生产具有高度特异性,这一点通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析得到了证实。通过微颗粒增强的分批补料过程实现的 2800 U/mL 的分泌酶活性,相当于约 3 g/L 的果聚糖酶,比迄今为止报道的任何其他过程都要高出十倍,因此,所提出的生物过程策略似乎是未来工业果聚糖酶生产的一个里程碑。

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