Abstract:
:Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Gmelch TJ,Sperl JM,Sieber Vdoi
10.1038/s41598-019-48151-ysubject
Has Abstractpub_date
2019-08-13 00:00:00pages
11754issue
1issn
2045-2322pii
10.1038/s41598-019-48151-yjournal_volume
9pub_type
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