Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation.
Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.
Fuciños
P
P
Area de Bioquímica e Bioloxía Molecular, Facultade de Ciencias de Ourense, Universidade de Vigo, 32004 Ourense, Spain.
Abadín
C M
CM
Sanromán
A
A
Longo
M A
MA
Pastrana
L
L
Rúa
M L
ML
D019840:2-Propanol / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D000431:Ethanol / Q000494:pharmacology; D004986:Ether / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007848:Laurates / Q000737:chemistry; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D008970:Molecular Weight; D020673:Protein Renaturation; D020358:Sodium Cholate / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology; D016963:Thermus thermophilus / Q000201:enzymology* / Q000254:growth & development
D007848:Laurates; D012997:Solvents; D004986:Ether; C050014:4-nitrophenyl laurate; D000431:Ethanol; D002265:Carboxylic Ester Hydrolases; D008049:Lipase; D019840:2-Propanol; D020358:Sodium Cholate