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在基于聚合物的控释微量滴定板中用地衣芽孢杆菌表达蛋白酶的补料分批工艺的建立。

Establishing a Fed-Batch Process for Protease Expression with Bacillus licheniformis in Polymer-Based Controlled-Release Microtiter Plates.

机构信息

AVT-Biochemical Engineering, RWTH Aachen University, Aachen, 52074, Germany.

White Biotechnology Research Unit, BASF SE, Ludwigshafen am Rhein, 67063, Germany.

出版信息

Biotechnol J. 2020 Feb;15(2):e1900088. doi: 10.1002/biot.201900088. Epub 2019 Sep 19.

Abstract

Introducing fed-batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed-batch fermentation processes. Therefore, cost efficient and easy to use small-scale fed-batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer-based controlled-release fed-batch microtiter plate is described. In this work, the polymer-based controlled-release fed-batch microtiter plate is used to investigate fed-batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online-monitored within each well of the polymer-based controlled-release fed-batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer-based controlled-release fed-batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed-batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed-batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale-up criterion, microtiter plate- and shake flask-based fed-batch cultivations are highly comparable. On basis of the small-scale fed-batch cultivations, a mechanistic model is established and validated. Model-based simulations coincide well with the experimentally acquired data.

摘要

在开发项目的早期引入分批补料模式对于建立与工业分批补料发酵过程相当的条件至关重要。因此,需要能够与现有实验室设备和工作流程集成的、经济高效且易于使用的小规模分批补料系统。最近,描述了一种新型基于聚合物的控释分批补料微孔板。在这项工作中,使用基于聚合物的控释分批补料微孔板来研究产蛋白酶地衣芽孢杆菌培养物的分批补料培养。因此,使用 µRAMOS 设备在线监测聚合物基控释分批补料微孔板中每个孔的氧传递率(OTR)。两个生产批次的五个独立的聚合物基控释分批补料微孔板的培养显示出良好的重现性,平均变异系数为 9.2%。降低初始生物量浓度会延长批次阶段,同时推迟分批补料阶段。初始液体填充体积会影响体积释放速率,这会直接转化为分批补料阶段的不同 OTR 水平。矿物培养基中初始渗透压的增加会降低葡萄糖释放和蛋白酶产率。以体积葡萄糖释放速率为放大标准,微孔板和摇瓶分批补料培养非常相似。基于小规模分批补料培养,建立并验证了一个机理模型。基于模型的模拟与实验获得的数据非常吻合。

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