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Background <p>Unsaturated polyester resins are currently polymerized with the utilization of cobalt-based siccatives. The imposed health concerns and the rising demand for cobalt make a sustainable alternative favorable. Heterologous peroxidases that are produced via protein secretion in <i>Komagataella phaffii</i> present a promising substitute for cobalt in these applications. This demands strain and bioprocess development with sufficient throughput, placing emphasis on adequate analytics for secreted peroxidase activity and total protein concentration that can cope with sample throughput generated from state-of-the-art micro-cultivation technology.</p> Results <p>In this work, the analytical procedure for the determination of peroxidase activity and protein concentration in supernatant samples from <i>K. phaffii</i> was embedded in an automated workflow. The ABTS assay for peroxidase activity assessment was optimized regarding pipetting schemes and batch-wise sample measurement, not only increasing operator walk-away time, but cutting mean relative standard deviation from 18 to 7&#xa0;%. This enables more precise decision making in strain and process engineering. Additionally, the Bradford assay for the determination of total protein concentration was successfully automated and optimized concerning non-linear standard curve progression and routine application of different sample dilution factors to correct for this. While the automated Bradford assay did not improve in precision through automation, the operator walk-away time of the whole analytical procedure was increased from 41 to 92&#xa0;%. The automated assays were applied to analyze a set of samples from four bioreactor cultivations and allowed for autonomous sample analysis. Even larger sample sets from automated micro-cultivations can be processed, paving the way for accelerated bioprocess development for the production of peroxidases in <i>K. phaffii</i>.</p> Conclusions <p>No a priori prediction for best strain and cultivation parameters is possible for the production of peroxidases with <i>K. phaffii</i>. Therefore, it is necessary to perform a sufficient number of cultivation experiments to find suitable conditions. For this purpose, the presented automated analytical method poses a valuable tool that can autonomously analyze large amounts of samples in a reliable and robust manner to provide sufficient analytical capacity even for larger strain and process development campaigns.</p>

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Automated assays accelerate bioprocess development for the production of peroxidases in Komagataella phaffii

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月13日

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  • Christian Mathis Wagner,
  • Philipp Dante Zinn,
  • Katharina Schwartz,
  • Anton Glieder,
  • Wolfgang Wiechert,
  • Marco Oldiges

摘要

Background

Unsaturated polyester resins are currently polymerized with the utilization of cobalt-based siccatives. The imposed health concerns and the rising demand for cobalt make a sustainable alternative favorable. Heterologous peroxidases that are produced via protein secretion in Komagataella phaffii present a promising substitute for cobalt in these applications. This demands strain and bioprocess development with sufficient throughput, placing emphasis on adequate analytics for secreted peroxidase activity and total protein concentration that can cope with sample throughput generated from state-of-the-art micro-cultivation technology.

Results

In this work, the analytical procedure for the determination of peroxidase activity and protein concentration in supernatant samples from K. phaffii was embedded in an automated workflow. The ABTS assay for peroxidase activity assessment was optimized regarding pipetting schemes and batch-wise sample measurement, not only increasing operator walk-away time, but cutting mean relative standard deviation from 18 to 7 %. This enables more precise decision making in strain and process engineering. Additionally, the Bradford assay for the determination of total protein concentration was successfully automated and optimized concerning non-linear standard curve progression and routine application of different sample dilution factors to correct for this. While the automated Bradford assay did not improve in precision through automation, the operator walk-away time of the whole analytical procedure was increased from 41 to 92 %. The automated assays were applied to analyze a set of samples from four bioreactor cultivations and allowed for autonomous sample analysis. Even larger sample sets from automated micro-cultivations can be processed, paving the way for accelerated bioprocess development for the production of peroxidases in K. phaffii.

Conclusions

No a priori prediction for best strain and cultivation parameters is possible for the production of peroxidases with K. phaffii. Therefore, it is necessary to perform a sufficient number of cultivation experiments to find suitable conditions. For this purpose, the presented automated analytical method poses a valuable tool that can autonomously analyze large amounts of samples in a reliable and robust manner to provide sufficient analytical capacity even for larger strain and process development campaigns.