Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies

J Biotechnol. 2024 Jan 10:379:65-77. doi: 10.1016/j.jbiotec.2023.11.006. Epub 2023 Nov 29.

Abstract

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g-1 h-1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Keywords: Enzyme characterization; Inclusion body; Lactate dehydrogenase; Processing chain; Recombinant production; Refolding dynamics.

MeSH terms

  • Fermentation
  • Inclusion Bodies* / metabolism
  • L-Lactate Dehydrogenase* / genetics
  • L-Lactate Dehydrogenase* / metabolism
  • Recombinant Proteins / chemistry

Substances

  • L-Lactate Dehydrogenase
  • Recombinant Proteins