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<p>Serratiopeptidase, a proteolytic enzyme with therapeutic applications, is traditionally produced from the bacterium <i>Serratia marcescens</i>. Recombinant production of serratiopeptidase in <i>Escherichia coli</i> offers a safer alternative to the biosafety concerns of the producer. Present study involves cloning and heterologous expression of thermoactive serratiopeptidase gene from <i>S. marcescens</i> AD-W2 in <i>E. coli</i> K12 in pET28a plasmid. Optimized expression conditions i.e. 37&#xa0;°C, OD<sub>600</sub> 5, 5% L-rhamnose, 5mM IPTG, and 50% dissolved oxygen led to the final yield of 190&#xa0;mg/g serratiopeptidase (4747&#xa0;mg protein/L within 9&#xa0;h in a bioreactor. Purification and refolding of recombinant serratiopeptidase was performed in a single step using Tangential Flow Filtration (TFF) and diafiltration process. The purified recombinant serratiopeptidase exhibited specific activity of 1800 Units/mg protein, with an optimal activity at pH 9.0 and temperature 50&#xa0;°C. The value of kinetic constant K<sub>m</sub> was calculated as 1.38&#xa0;mg/mL for casein. The recombinant serratiopeptidase demonstrated comparable anti-inflammatory activity to the commercially available serratiopeptidase, inhibiting nitric oxide release and pro-inflammatory cytokine production in LPS-stimulated murine macrophage cell line RAW 264.7. The study reveals that the recombinant serratiopeptidase produced in <i>E. coli</i> K12 holds promising source of safe and effective anti-inflammatory agent.</p>

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Heterologous expression, purification and functional characterization of recombinant serratiopeptidase from Serratia marcescens AD-W2

  • 期刊论文

  • 发表日期：

    2026年2月24日

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  • Devtulya Chander,
  • Diksha Koul,
  • Shubham Singh,
  • Ravi S. Manhas,
  • Diljeet Kumar,
  • Sanket Shukla,
  • Ravi P. N. Mishra,
  • Zabeer Ahmed,
  • Asha Chaubey

摘要

Serratiopeptidase, a proteolytic enzyme with therapeutic applications, is traditionally produced from the bacterium Serratia marcescens. Recombinant production of serratiopeptidase in Escherichia coli offers a safer alternative to the biosafety concerns of the producer. Present study involves cloning and heterologous expression of thermoactive serratiopeptidase gene from S. marcescens AD-W2 in E. coli K12 in pET28a plasmid. Optimized expression conditions i.e. 37 °C, OD600 5, 5% L-rhamnose, 5mM IPTG, and 50% dissolved oxygen led to the final yield of 190 mg/g serratiopeptidase (4747 mg protein/L within 9 h in a bioreactor. Purification and refolding of recombinant serratiopeptidase was performed in a single step using Tangential Flow Filtration (TFF) and diafiltration process. The purified recombinant serratiopeptidase exhibited specific activity of 1800 Units/mg protein, with an optimal activity at pH 9.0 and temperature 50 °C. The value of kinetic constant Km was calculated as 1.38 mg/mL for casein. The recombinant serratiopeptidase demonstrated comparable anti-inflammatory activity to the commercially available serratiopeptidase, inhibiting nitric oxide release and pro-inflammatory cytokine production in LPS-stimulated murine macrophage cell line RAW 264.7. The study reveals that the recombinant serratiopeptidase produced in E. coli K12 holds promising source of safe and effective anti-inflammatory agent.