Purification and characterization of a novel extracellular lipase catalyzing hydrolysis of oleyl benzoate from Acinetobacter nov. sp. strain KM109.
A new lipase (OBase) which efficiently hydrolyzes oleyl benzoate (OB) was found in the culture supernatant of Acinetobacter nov. sp. strain KM109, a new isolate growing in a minimum medium containing OB as the sole carbon source. OBase was purified to homogeneity with 213-fold purification and 0.8% yield. The molecular weight was estimated to be 62,000 +/- 1,000 by SDS-PAGE under denatured-reduced conditions and to be 50,000 +/- 1,000 by gel-filtration HPLC under native conditions; these findings indicate that OBase is a monomeric enzyme. The optimum temperature and pH of OBase were about 45 degrees C and pH 8. Temperature and pH stabilities were at or lower than 35 degrees C and in a range of pH 6-8, respectively. Purified OBase preferentially hydrolyzed p-nitrophenyl benzoate (pNPB) over p-nitrophenyl acetate (pNPA) or p-nitrophenyl caproate (pNPC) [pNPB/pNPA = 20 and pNPB/pNPC = 5.4], indicating that OBase has a high affinity for benzoyl esters. Partial amino-acid sequences of OBase fragments obtained after lysyl endopeptidase treatment showed no similarity with known proteins.
Mitsuhashi
K
K
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Yamashita
M
M
Hwan
Y S
YS
Ihara
F
F
Nihira
T
T
Yamada
Y
Y
D000150:Acinetobacter / Q000201:enzymology* / Q000254:growth & development; D001565:Benzoates / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D012988:Soil Microbiology; D013379:Substrate Specificity; D013816:Thermodynamics; D014280:Triglycerides / Q000378:metabolism
D001565:Benzoates; D014280:Triglycerides; C403615:oleyl benzoate; D002265:Carboxylic Ester Hydrolases; C403614:OBase lipase; D008049:Lipase