title: Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family.
abstract: A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: K
authors.initials: K
authors.lastname: Bhardwaj
authors.forename: A
authors.initials: A
authors.lastname: Raju
authors.forename: R
authors.initials: R
authors.lastname: Rajasekharan
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010741:Phospholipases A / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D014304:Triolein / Q000378:metabolism
chemical_list: D003902:Detergents; D008244:Lysophosphatidylcholines; D014304:Triolein; D008049:Lipase; D010741:Phospholipases A