Purification and characterization of a thermolysin like protease from Thermoactinomyces thalpophilus MCMB-380.
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.
Microbial Sciences Division, MACS' Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India.
Devipriya R
DR
Majumder
Pradnya P
PP
Kanekar
Sushama M
SM
Gaikwad
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D056509:Thermoactinomyces / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification
D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases