Applied Biochemistry and Biotechnology ( IF 3.1 ) Pub Date : 2021-06-11 , DOI: 10.1007/s12010-021-03592-y Pfariso Maumela 1 , Shaunita Rose 2 , Eugéne van Rensburg 1 , Annie Fabian Abel Chimphango 1 , Johann Ferdinand Görgens 1
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Endoinulinase gene was expressed in recombinant Aspergillus niger for selective and high-level expression using an exponential fed-batch fermentation. The effects of the growth rate (μ), glucose feed concentration, nitrogen concentration and fungal morphology on enzyme production were evaluated. A recombinant endoinulinase with a molecular weight of 66 kDa was secreted. Endoinulinase production was growth associated at μ> 0.04 h−1, which is characteristic of the constitutive gpd promoter used for the enzyme production. The highest volumetric activity (670 U/ml) was achieved at a growth rate of 93% of μmax (0.07 h−1), while enzyme activity (506 U/ml) and biomass substrate yield (0.043 gbiomassDW/gglucose) significantly decreased at low μ (0.04 h−1). Increasing the feed concentration resulted in high biomass concentrations and viscosity, which necessitated high agitation to enhance the mixing efficiency and oxygen. However, the high agitation and low DO levels (ca. 8% of saturation) led to pellet disruption and growth in dispersed morphology. Enzyme production profiles, product (Yp/s) and biomass (Yx/s) yield coefficients were not affected by feed concentration and morphological change. The gradual increase in the concentration of nitrogen sources showed that, a nitrogen limited culture was not suitable for endoinulinase production in recombinant A. niger. Moreover, the increase in enzyme volumetric activity was still directly related to an increase in biomass concentration. An increase in nitrogen concentration, from 3.8 to 12 g/L, resulted in volumetric activity increase from 393 to 670 U/ml, but the Yp/s (10053 U/gglucose) and Yx/s (0.049 gbiomasDWs/gglucose) did not significantly change. The data demonstrated the potential of recombinant A. niger and high cell density fermentation for the development of large-scale endoinulinase production system.
Graphical abstract
中文翻译：
从重组黑曲霉中生产高细胞密度内切菊酯酶的生物工艺优化
内切菊糖酶基因在重组黑曲霉中表达，使用指数补料分批发酵进行选择性和高水平表达。评估了生长速率（μ）、葡萄糖补料浓度、氮浓度和真菌形态对酶生产的影响。分泌出分子量为66 kDa的重组内切菊糖酶。内切菊糖酶生产与μ> 0.04 h -1处的生长相关，这是用于酶生产的组成型gpd启动子的特征。在 μ max (0.07 h -1 ) 的 93% 生长速率下实现最高体积活性 (670 U/ml)，同时酶活性 (506 U/ml) 和生物量底物产量 (0.043 g生物量DW /g葡萄糖)在低μ（0.04 h -1 ）时显着降低。增加进料浓度会导致较高的生物质浓度和粘度，这需要高度搅拌以提高混合效率和氧气。然而，高搅拌和低溶解氧水平（约饱和度的 8%）导致颗粒破裂并以分散形态生长。酶生产曲线、产物 (Y p/s ) 和生物量 (Y x/s ) 产率系数不受进料浓度和形态变化的影响。氮源浓度的逐渐增加表明，限氮培养物不适合重组黑曲霉生产内切菊酯酶。此外，酶体积活性的增加仍然与生物量浓度的增加直接相关。氮浓度从 3 增加。8 至 12 g/L，导致体积活性从 393 U/ml 增加至 670 U/ml，但 Y p/s (10053 U/g葡萄糖) 和 Y x/s (0.049 g biomasDWs /g葡萄糖) 没有显着变化。这些数据证明了重组黑曲霉和高细胞密度发酵在开发大规模内胰岛素酶生产系统方面的潜力。
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