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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Purification and characterization of a thermolysin like protease from Thermoactinomyces thalpophilus MCMB-380
Devipriya R Majumder1, Pradnya P Kanekar, Sushama M Gaikwad
1Microbial Sciences Division, MACS' Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India.
Abstract:
The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T50 of the enzyme at 80 °C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80°C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.
Insights
A novel extracellular thermolysin-like protease (TLP) from Thermoactinomyces thalpophilus was purified and characterized. This metalloprotease shows high stability and activity at 80°C, with potential for aspartame production.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Extracellular proteases play crucial roles in various biological processes.
- Thermolysin-like proteases (TLPs) are a class of metalloproteases with significant industrial applications.
- Thermoactinomyces thalpophilus is a source of thermostable enzymes.
Purpose of the Study:
- To purify and characterize the extracellular thermolysin-like protease (TLP) from Thermoactinomyces thalpophilus MCMB-380.
- To evaluate the enzyme's properties, including its stability, activity, and potential for industrial applications.
- To compare its performance with existing enzymes like thermolysin.
Main Methods:
- Purification using ultrafiltration and anion exchange chromatography.
- Characterization of molecular mass, isoelectric point, and metal ion requirements (Ca2+, Zn2+).
- Assay of proteolytic activity, thermostability (T50 at 80°C), and activation energy determination.
Main Results:
- The purified TLP has a molecular mass of 34.4 kDa and an isoelectric point of 9.5.
- The enzyme is a metalloprotease requiring Ca2+ for full activity and thermostability, and contains Zn2+.
- TLP exhibited high stability at 80°C for 1 hour and a faster reaction time for dipeptide production compared to thermolysin.
Conclusions:
- The purified TLP from T. thalpophilus is a robust metalloprotease with significant potential for industrial applications, particularly in aspartame synthesis.
- Its enhanced thermostability and faster reaction kinetics make it a promising alternative to existing enzymes.
- The Zn2+ ion is critical for maintaining the enzyme's active conformation and stability.
