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Screening, characterization, and cloning of a solvent-tolerant protease from Serratia marcescens MH6
1College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, China.
Abstract:
A solvent-tolerant bacterium strain MH6 was isolated by hydrophilic organic solvent DMSO enrichment in medium and identified as Serratia marcescens. The extracellular protease with novel organic-solvent-stable properties from strain MH6 was purified and characterized. The molecular weight of the purified protease was estimated to be 52 kDa on SDS-PAGE. The open reading frame (ORF) of the MH6 protease encoded 504 amino acids with 471 amino acid residues in the mature protease. Based on the inhibitory effects of EDTA and 1, 10-phenathroline, the MH6 protease was characterized as a metalloproteinase. The enzyme activity was increased in the presence of Ni2+, Mg2+, and Ca2+. The protease could also be activated by the nonionic surfactants Tween 80 (1.0%) and Triton X-100 (1.0%). The protease showed remarkable solvent stability in the presence of 50% (v/v) solutions of long-chain alkanes and long-chain alcohols. It was also fairly stable in the presence of 25% solutions of hydrophilic organic solvents. Due to its high stability in solvents and surfactants, the MH6 protease is an ideal candidate for applications in organic catalysis and other related fields.
Insights
A novel solvent-stable metalloproteinase was isolated from Serratia marcescens strain MH6. This enzyme exhibits remarkable stability in organic solvents and surfactants, making it ideal for industrial applications.
Area of Science:
- Biochemistry
- Microbiology
- Enzyme Engineering
Background:
- Extracellular proteases are crucial industrial enzymes.
- Many proteases lack stability in organic solvents, limiting their applications.
- There is a need for robust enzymes for use in organic synthesis and catalysis.
Purpose of the Study:
- To isolate and characterize a novel extracellular protease with organic solvent-stable properties.
- To evaluate the potential of this protease for applications in organic catalysis.
Main Methods:
- Isolation of solvent-tolerant bacteria using DMSO enrichment.
- Identification of the bacterial strain using standard microbiological techniques.
- Purification and characterization of the extracellular protease via SDS-PAGE, ORF analysis, and enzyme activity assays.
- Assessment of enzyme stability in various organic solvents and surfactants.
Main Results:
- Serratia marcescens strain MH6 was identified as the source of the protease.
- The purified protease has a molecular weight of 52 kDa and is a metalloproteinase.
- The enzyme showed enhanced activity with Ni2+, Mg2+, Ca2+, Tween 80, and Triton X-100.
- Remarkable stability was observed in 50% long-chain alkanes/alcohols and 25% hydrophilic organic solvents.
Conclusions:
- The MH6 protease is a novel metalloproteinase with significant stability in organic solvents and surfactants.
- Its unique properties make it a promising candidate for biocatalysis and other industrial applications requiring solvent tolerance.
