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 mol min Ϫ1 mg Ϫ1 at 80°C using [9, 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°C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90°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°C and enthalpy 1.3 ϫ 10 5 Calorie mol Ϫ1 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.Lipases (EC 3.1.1.3.) are versatile enzymes that catalyze the hydrolysis of ester linkages, primarily in neutral lipids such as triglycerides. They hydrolyze the acyl chains either at primary (Ransac et al., 1990; Rogalska et al., 1993) or secondary positions (Candida antarctica lipase, Geotrichum candidum lipase B, lipase from Vernonia anthelmintica, etc.). However, a few lipases do not show any positional specificity (Rollof et al., 1987;Hiromasa et al., 1998). Plant lipases hydrolyze triacylglycerols at much lower rates (usually Ͻ0.5 mol min Ϫ1 mg Ϫ1 ) as compared with animal or microbial lipases. All the members of the lipase gene family have a conserved Ser, which is the nucleophile essential for catalysis. The active site triad of lipases consisting of Ser-His-Asp/Glu is reminiscent of the Ser proteases (Blow, 1990). In addition to triglycerides, lipases are also known to degrade Tween and water-soluble and insoluble esters. There are reports of a few animal and microbial lipases that hydrolyze phospholipids (Durand et al., 1978;Fauvel et al., 1981;Jensen et al., 1982; van Oort et al., 1989; Thirstrup et al., 1994). Enzymatic activity of many lipases has been shown to be modulated by calcium (Rosenstein and Gotz, 2000), ricinoleic acid (Ory et al., 1962), and bile salts (Miled et al., 2000).In plants, the regulation, in some cases the location, and the exact physio...

Genetic control of lutein esterification in wheat (Triticum aestivum L.) grain

“…In earlier work on ester formation in bread wheat (Ahmad et al, 2013) we demonstrated that the constituent involved in esterification is very thermostable. In this context, it is worth noting that a thermostable (phospho) lipase has been reported to be present in rice bran (Bhardwaj et al, 2001). The marker Xgwm295, which co-segregates with Lute, has previously been assigned to a wheat BAC (TaBAC470M18), which was in turn anchored to a position on rice chromosome 6 (Bossolini et al, 2006) based on similarity of sequences within the BAC to the gene LOC_Os06g05000 on rice chromosome 6.…”

Lipid deacylating enzymes in plants: Old activities, new genes

“…SAG101 belongs to a family of 21 genes in Arabidopsis that is not yet characterized [23]. In addition to having TAG lipase activity, a protein isolated from rice bran, with a very low MW of 9.4 kDa, also displayed Ca 2þ -independent phospholipase activity [8] (Table 1).…”

Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

“…A characterization of the inhibition caused by different surfactants was carried out in lipase from rice bran, and the results differed from those obtained with Lip3F9. For rice bran lipase, a 2 mM concentration of SDS caused the greatest inhibitory at 50%, while Triton X-100 had an inhibitory effect at a low concentration of 0.5 mM and restored activity when in a higher concentration of 4 mM [21]. Together with previous studies, the results shown in the present study confirm that the effects of detergents are not homogenous for all enzymes nor are enzymes affected in the same way by all surfactants [22].…”