%A Devtulya Chander
%A Diksha Koul
%A Shubham Singh
%A Ravi S. Manhas
%A Diljeet Kumar
%A Sanket Shukla
%A Ravi P. N. Mishra
%A Zabeer Ahmed
%A Asha Chaubey
%O Copyright of this data belongs to WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
%J WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
%T Heterologous expression, purification and functional characterization of recombinant serratiopeptidase from Serratia marcescens AD-W2
%X 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 degrees 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 degrees 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.
%N 3
%K <italic>E.coli</italic> K12; serratiopeptidase; Protein refolding; Tangential flow filtration; Anti-inflammatory activity
%V 42
%D 2026
%C VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS
%I SPRINGER
%L open3465