Institute of Biochemical Engineering, Technische Universität Braunschweig, Gaussstrasse 17, Braunschweig, Germany.
Biotechnol Bioeng. 2010 Apr 15;105(6):1058-68. doi: 10.1002/bit.22614.
Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher-value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision-induced disruption of conidia aggregates and probably also the hindrance of new spore-spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by-product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co-expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production.
采用硅酸钠微粒作为新型方法来控制黑曲霉的形态发育，黑曲霉是柠檬酸和高价值酶的主要世界来源，在浸没培养中。通过仔细改变添加的微材料的大小和浓度，可以可重复地产生多种不同的形态形式，包括不同大小的颗粒、自由分散的菌丝体和短的菌丝片段。氧化铝颗粒同样影响形态，表明这种效应在很大程度上独立于化学颗粒组成。在培养过程中对黑曲霉形态发育的图像分析表明，微粒通过碰撞诱导的孢子聚集体的破坏以及可能在过程的早期阶段阻碍新孢子-孢子相互作用来影响形态。以不同的重组黑曲霉菌株为例，通过添加微粒可以强烈增强酶的生产。与自由分散的菌丝体的形成相关，胞内酶（88 U/mL）和分泌到上清液中的果聚糖酶（77 U/mL）的表达量提高了四倍。此外，草酸副产物的积累被抑制了高达 90%。微粒策略可以成功地转移到生物反应器中的果聚糖酶生产中，在生物反应器中可以达到 160 U/mL 的最终产量。通过与绿色荧光蛋白共表达 GA，酶的生产被定位在黑曲霉的细胞聚集体中。对于颗粒生长，只有在颗粒表面的薄层中达到蛋白质生产的最大值，并且在颗粒内部明显降低，而与微粒的相互作用创造了一种具有高活性的生物催化剂，其中主要部分的细胞有助于生产。
