WO2002101033A1

WO2002101033A1 - A process of isolation and utilization of rice bran lipase

Info

Publication number: WO2002101033A1
Application number: PCT/IN2002/000126
Authority: WO
Inventors: Rajasekharan Ram; Kanchan Bhardwaj
Original assignee: Cb Bio Products India Pvt. Ltd.; Indian Institute Of Science
Priority date: 2001-06-08
Filing date: 2002-06-03
Publication date: 2002-12-19

Abstract

Disclosed herein is a novel isolated from nomocotyledon plant sources such as rice bran having the following properties: a) isolated enzyme is a glycoprotein; b) isolated enzyme is stable and catalytically active at high temperatures; c) isolated enzyme is stable and catalytically active at alkaline pH; d) isolated enzyme has molecular weight range of 9 to 12-kDa; e) isolated enzyme is inhibited by inhibitors of serine proteases such as Diisopropylfluorophosphate; f) Isolated enzyme that can utilize both triacylglycerol and phospholipids. This invention also relates to a process of purifying the lipase described above using the following steps: a) delipidation of the plant extract using organic solvents; b) Filtration of the delipidated extract on a suitable matrix; c) Purification of the enzyme using hydrophobic column matrices; d) characterization of the purified product by known methods.

Description

A PROCESS OF ISOLATION AND UTILIZATION OF RICE BRAN LIPASE

This invention relates to a process for the isolation and utilization of Rice Bran Lipase.

Rice bran proteins were solubilized, and then the lipase was purified to homogeneity and characterized enzymatically and biophysically. The purified enzyme was found to be a glycoprotein of 9 to 12-kDa. Enzyme showed maximum activity at 80 °C and in the pH range of 10-11. The protein was found to be stable and biologically active at temperatures as high as 90 °C, as judged by the far- and near-UV circular dichroism spectroscopy and enzymatic assays, respectively. Differential scanning calorimetric studies indicate that the transition temperature was 76 °C and ΔHwas 1.3x105 Cal/mol at Tm 76 °C. The purified enzyme exhibited dual substrate specificity including phospholipase A2 and Upase activities. Tm for triolein was higher than that for phosphatidylcholine. Activity analysis on reverse-phase HPLC, isoelectric focussing and photoaffinity labeling of the purified protein with photoanalogues of triolein and phosphatidylcholine demonstrated that a single enzyme catalyzes the hydrolysis of both the substrates. The enzyme preferred hydrolysis at srι-2 position of phosphatidylcholine whereas it apparently exhibited no positional specificity towards triacylglycerol. Dϋsopropyl fluorophosphate, an irreversible inhibitor of serine esterase and lipase, inhibited lipase and phospholipase activities of the purified enzyme. Substrate competition, irnmunoinhibition and catalytic residue modification studies provide evidences that the enzyme contains a common catalytic site and independent binding sites for neutral lipid and phospholipid substrates. PRIOR ART

Lipases have been purified from animal, plant and microbial sources. These enzymes catalyze ester hydrolysis in aqueous media and a reverse reaction i.e esterification under non-aqueous phase. Enzymatic skiUs of Upases have been exploited in various industries. Microbial Upases have been preferred for commercial appUcations due to their multifold properties and unUmited supply. The interesting properties for which these Upases are used are their thermal and pH stabiUty, activity in organic solvents. ExtraceUular Upase of A. niger, Chromobacterium viscosum and Khizopus sp. are active at acidic pH. An alkaline Upase active at pH 11.0 has been isolated from P. nitroreducens. Lipases from A. niger, Rjaponicus, and C. viscosum are stable at 50 °C, and Upases of thermotolerant H.

lanuginosa and P. sp. nitroreducens are stable at 60 °C and 70 °C, respectively (Liu et al., 1973) The chief producers of commercial lipases are Aspergillus niger, Candida cylinderacea, Humicola lanuginosa, Mucor miehei, Rhizopus arrhizus, R. delemar, R. Japonicus, R, niveus andR. oryzae (Godfredson et al., 1990).

In a dairy industry, Upases are used for cheese ripening, production of the so-called enzyme-modified cheese (EMC) and lipolysis of fat in milk, butter and cream (Falch et al., 1991). Addition of Upases release short-chain fatty acids that leads to the development of sharp and tangy flavor whereas release of medium-chain fatty acids initiate the synthesis of flavor ingredients such as methyl ketones, flavor esters, aceto- acetate etc. Using a range of immobiUzed lipases for hydrolysis, alcoholysis and glycerolysis have been carried out in the oleo chemical industry as it saves energy and avoids a substantial investment in expensive equipment as weU as in expenditure of large amounts of thermal energy (Arbige et. al., 1989; Hoq et al., 1985).

Demand for lipases in the detergent industry has increased since the world-wide trend is towards lower laundering temperatures. Novo Nordisk'e lipolase (Humicoϊa lipase expressed in Aspergillus oryzae) is one of the present formulations (Hoq et al., 1985).

Regioselective and thermally stable Upases have been used for enzymatic synthesis of surfactants which are widely used as industrial detergents and as emulsifiers in food formulations like low-fat spreads, sauces, ice-creams, mayonnaises, etc. (Adelhorst et al., 1990) have carried out esterification of alkyl-glycosides using molten fatty acids and immobiUzed Candida antarctica Upase. A biosurfactant has been synthesized by transesterification of sugar alcohols with natural oils, using Upase from A. terreus (Yadav et al., 1997). Lipases have also been implicated for the synthesis of amphoteric bio- degradable surfactants (Hills et al., 1990; Kloosterman et al., 1988).

Specificities of various lipases have been useful in the synthesis of Upids with high commercial value. A typical example of such a high-value asymmetric triglyceride mixture is cocoa butter (Jandacek et al., 1987). Same approach has been used for the synthesis of many other triglycerides (Soumanou et al., 1997), preparation of nutritionally important products which generaUy contain medium-chain fatty acids (Eibl et al., 1990), modification of oils rich in high-value polyunsaturated fatty acids (Fregapane et al., 1991).

Stereoselectivity of the Upases has been exploited for the synthesis of opticaUy active polymers that are used as absorbents (MargoUn et al., 1987). Monomers can be prepared by Hpase-catalyzed transesterification of alcohols (MargoUn et al., 1991). Another example of the application of Upases in pharmaceuticals and agrochemicals is the resolution of racemic mixtures. Optically pure cardiovascular drug Diltizem is an example of a product obtained using this technology (Bornemann et al., 1989). Lipases from A. Cameus and A. terreus show chemo- and regiospecificity in the hydrolysis of per-acetates of pharmaceutically important polyphenoUc compounds (Parmar et al, 1998). Further, Upases have applications in the synthesis of ingredients for personal care products (Hoq et al., 1985), they have potential in paper manufacture, waste processing of many food industries (West et al., 1987).

SUMMARY OF INVENTION A novel lipase has been isolated from monocotyledon plant sources e.g. rice bran that has the following properties:

The novel Upase is a glycoprotein of size 9-12-kDa that is stable and catalytically active at 80°C. The enzyme can hydrolyze both triacylglycerol and phosphoUpid, and the enzyme activity is inhibitable by serine protease inhibitors such as diisopropyl fluorophospate. The novel Upase isolated from rice bran has been purifed using the foUowing steps: a) delipdation of the rice bran extract with lOg/lOOml of di-ethyl ether and extraction in 10 mM Tris-HCl pH 7.5 and 1.0 mM EDTA for 12 h; b) filtration of the extract through fine layers of cheese cloth followed by centrifugation at 3,000Xg for 30 min; c) purification of the enzyme on octyl-Sepharose pre-equiUbrated with 0.01 M Tris-HCl pH7.5 and eluting the pure protein with a linear gradient of 0-40% methanoL and d) characterization of the purified protein by known methods.

The purification of such a Upase from rice bran is not restricted to this method only. Based on known and estabUshed principles of protem purification and the description provided in this specification it is possible for any skilled in the art to device alternative methods of purification of such Upases.

Novel Upase obtained from rice bran as described in this specification can be used in a variety of industrial processes as in dairy industry, oleo chemical industry, detergent industry, synthesis of surfactants, synthesis of nutritionaUy important Upid products, synthesis of optically active polymers, resolution of racemic mixtures and synthesis of ingredients for personal care products and generate either novel or known commerciaUy important products.

DETAILED DESCRIPTION OF INVENTION

Interesting properties of rice bran Upase include its stabiUty to temperature and alkaline pH. The enzyme does not show positional specificity towards triolein and has a phosphohpase A2 activity. Importantly, the enzyme is extracted from a by-product and its extraction involves a single chromatographic step.

EXAMPLE 1 Rice bran was delipidated with (10 g 100 ml) diethyl ether and stirred for 12 h in 10 mM Tris-HCl, pH 7.5, and 1.0 mM EDTA. The extract was passed through layers of cheesecloth and centrifuged at 3,000 x g for 30 min. The clear supernatant was then loaded onto an octyl-Sepharose column that had been pre-equiUbrated with 0.01 M Tris- HCl, pH 7.5 at a flow rate of 2 ml/min. The column was washed with the same buffer until the effluent showed a negUgible absorbance at 280 nm. The enzyme was eluted with a linear gradient of 0-40 % methanol and fractions of 10 ml were collected. Upon analysis of the fractions on SDS-PAGE, a single protein band was observed in the later fractions, no protein was obtained in the initial ones. The overall purification was 6.8- fold, with an activity yield of 20 %. Protein concentration was determined by the bicmchoninic acid method (Smith et al., 1985) using bovine serum albumin as standard. Two-dimensional gel electrophoresis and analytical reverse phase HPLC confirmed purity. Protein was analyzed on two-dimensional gel using a Bio-Rad mini-protean H two-dimensional gel apparatus. The first dimension of the gel was run in the acidic direction using ampholytes with pH range of 3.0-10.0, The run was performed at 500 V for 3 h. The second dimension was 15% acrylamide SDS-gel as described by LaemmU (1976) and the protein was visualized by sUver staining. Purified Upase was resuspended in 100 μl of water plus 0.1% trifluoroacetic acid (HPLC grade) and loaded onto a C18

reverse phase column (Vydac reversed phase C18 column, 10-μm particle size, 22-mm inner diameter, 25-cm length). Prior to loading, the column was pre-equilibrated with water plus 0.1% trifluoroacetic acid. Peptides were eluted from the column using a linear gradient of 0-70% acetonitrile (HPLC grade) plus 0.1% trifluoroacetic acid and a flow rate of 1.0 ml/min. The elution profile was monitored by absorbance at 210 nm. Fractions were collected in lmin intervals and each fraction was evaluated for the presence of enzyme activity after dialyzing the sample. Analysis of the purified protein on the SDS- PAGE showed a band of apparent molecular weight of 10-kDa. Samples were electrophoresed using the Laemmli discontinuous buffer system (Laemmli et al., 1976) on 15% SDS-PAGE gels (10 x 10 cm) at 100 V and stained with silver (Nesterenko et al., 1994). Molecular weight was further confirmed by MALDI-TOF and was found to be 9.4 kDa. Lipase purification to apparent homogeneity was achieved in a single chromatographic step.

EXAMPLE 2 Purified lipase was concentrated using a Centricon (5-kDa cut-off) and applied onto an analytical Superdex 75 FPLC column fitted with Bio-Rad Biologic low-pressure chromatography system with a buffer consisting of 0.01 M Tris-HCl, pH 7.5 containing 100 mM sodium chloride. Elution was carried out with the same buffer at a flow rate of 1.0 ml/min. Fractions were collected in 1 min intervals. Standard markers were from Amersham Pharmacia Biotech that consisted of blue dextran (2000 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) carbonic anhydrase (29 kDa) and lysozyme (14 kDa). Two peaks, a void volume and an included were obtained and the activity was found to be associated with both the peaks and when run on the SDS-PAGE, the void volume as weU as the included peaks run at the same place, indicating the void volume peak was observed because the enzyme formed large aggregates. Protein showed anomalous behaviour on the SDS-PAGE which is a characteristic of glycosylated proteins therefore we analysed if the protein was post-translationally modified by glycosylation. For carbohydrate analysis by periodic acid-Schiff reagent, purified protein was resolved on SDS-PAGE under reducing conditions. Gel fixed with trichloroacetic acid was treated for 50 min with 1% periodic acid in 3% acetic acid, and washed with several changes of distilled water. The oxidized samples were stained for 50 min in the dark with fresh Schiff s reagent, treated for 30 min with 0.5% sodium meta-bisulfite, washed and stored in the dark (Warren et al., 1958). Concurrent samples were stained for protein.

EXAMPL 3 Enzyme activity was measured using 3H-labeled triolein, labeled at all the fatty acid chains. Substrate was emulsified with 1 % gum Arabic, in a total volume of 100 μl of assay buffer (0.01 M Tris-HCl, pH 7.5, unless otherwise mentioned). The mixture was incubated at 37 °C (unless mentioned) for 1 h before addition of 400 μl of CHCI3/CH3OH (1:2, v/v). Lipids were extracted according to the method of BUgh and Dyer (Bligh et al., 1959). The organic phase was dried, Upids were resuspended in chloroform and resolved on thin-layer siUca gel plates (SiUca Gel 60 F-254, Merck) using petroleum ether/diethyl ether/acetic acid (70:30:1, v/v/v). The lipids were visuaUzed with iodine vapour and spots corresponding to the unreacted substrate and the products were scraped off and quantitated by liquid scintillation counting. Purified lipase showed maximum activity at 80 °C. Enzyme was more stable at lower temperatures but did not loose activity

completely even at temperatures as high as 80 °C. The optimal pH was between 10-11. To determine the positional specificity towards triacylglycerols, a time course for the release of possible intermediates i.e free fatty acids, diacylglycerol and monoacylglycerol, by the enzyme's action was performed. Upon quantitation of the radioactivity associated with the unreacted substrate and the products, accumulation of fatty acids was observed. Diacylglycerol was formed but did not accumulate. Lipase activity was found to be enhanced marginally by Ca²⁺ whereas other divalent cations like Mg²⁺, Zn²⁺, Cu²⁺ showed inhibitory effect.

EXAMPLE 4 Circular Dichroism spectra were recorded on a Jasco J-720 spectrophotometer equipped with a thermostatted cell holder. Spectra were recorded at a protein concentration of 0.5 mg/ml in a 1-cm path length quartz cuvette sealed with a tefrlon stopper. A resolution of 0.1 nm and scanning speed of 20 nm/min with a 2 s response time were employed. For monitoring thermal stabiUty, spectra were recorded at 20 °C, 40 °C, 60 °C and 90 °C. EquiUbration time of 5 min was included at each temperature interval. In order to check the reversibihty, the sample was cooled to 20 °C and rescanned. Spectra revealed a profile characterized by a sharp negative eUipticity at 232 nm. The results show that the enzyme retained more than 90 % of its secondary structure even at 90 °C and there was no change in the near-UV region, indicating conformational stability. Little denaturation observed was found to be reversible upon rescans of the sample after cooling to 20 V. DSC measurements were performed on a VP- DSC microcalorimeter (Microcal Inc., Northampton, MA). Sample solutions for DSC measurements were prepared by dialyzing the protein against 0.01 M phosphate buffer at pH 7.0 exhaustively. The protein concentrations were 0.07 mg/ml. Samples and reference buffers were degassed by stirring gently under vacuum prior to measurements. Protein unfolding events were recorded between 20 and 90 °C with a scan rate of 90 °C. To check the reversibility of the

observed transitions, rescans were performed after slowly cooling to 20 °C. The scans were analyzed after subtraction of an instrument base line recorded with water in both cells using the software ORIGIN from Microcal. The DSC transition corresponding to thermal denaturation was reversible. The denaturation enthalpy ΔH was 1.3x105 Cal/mol

at Tm 76 °C.

EXAMPLE 5 For phosphatidylchoUne hydrolysis, phospholipid mixed micelles composed of phosphatidylcholine:triton X-100 (1:20, mol/mol) to give about 250,000 cpm/assay. PhosphatidylchoUne was 3H-labeUed at sn-2 position. For analyzing the hydrolysis of other phospholipids, the phosphatidylchoUne was replaced with the Upid to be investigated. Assay was performed as described in example 2. Upon enzymatic activity there was a release of free fatty acids and no other intermediates or products were released from phosphatidylcholine. The enzyme did not accept lysophosphatidylcholine or phosphatidic acid as substrates. This confirmed that the enzyme had phosphoUpase A2 activity. Phospholipase A2 activity was also confirmed by using a 32P-labeled phosphatidylcholine. The phosphoUpase activity was protein concentration and time dependent. It did not require calcium for hydrolysis. That both the activities were associated with the same enzyme was confirmed by colocalizing the activities on HPLC and isoelectric focussing gel with a constant specific activity ratio with both the substrates. Further, photoaffinity analogues of triolein and phosphatidylcholine were synthesized. 12-[(4-Azidosalicyl)amino]-dodecanoic acid (ASD) was synthesized from the N -hydroxysuccinimide ester of j?-azidosalicyHc acid (Rajasekharan et al., 1993). 1,2- dipalmitoyl glycerol (lmmol) was then acylated with the synthesized ASD-anhydride (4 mmol) by stirring the mixture for 30 h at room temperature in dry chloroform. N,N- dimethyl-4-aminopyridine (0.5 mmol) was used as catalyst. The reaction flask was flushed with nitrogen and sealed. The residue was redissolved in 2 ml of chloroform and loaded onto a silicic acid (20 g) column that had been pre-equilibrated with chloroform. The column was washed with chloroform and then eluted with mixtures of chloroform/methanol (1:1, v/v). the purity was checked by TLC using chloroform/methanol/water (98:2:0.5, v/v). The yield was approximately 46%. The purified l,2-dipalmitoyl,3-(4-azidosalicyl)-12-amino) dodecanoyl-sra-glycerol was iodinated using Nal25I and chloramine-T (Shin et al., 1985). The iodinated product was purified using reverse-phase column chromatography. The efficiency of iodination was 59-63%. All operations involving azide were carried out under dim safe Ught. The synthesis of azido-PC was achieved by direct acylation of CdCl₂ complex of glycerophosphochoUne with ASD-anhydride (Gupta et al., 1977). The purified product was iodinated as described earlier. The yield was around 52%. These analogues were used to demonstrate a direct specific interaction between the protein and both the analogues. The photolabeling experiments were carried out in a final volume of 50 μl

containing 10 μg of Upase in 0.01 M Tris-HCl, pH 7.5, 0.1 mM 2-mercaptoethanol and the photoprobe, as described earlier. Mixture was preincubated on ice in the dark for 5 min, in a microfuge tube cap and irradiated for 3 min with a hand-held UV-lamp with the filter removed (5000 μW/cm2), model UVG-54, UV products) at a distance of 8 cm.

Radiolabeled photoprobe (0.5 μCi, 0.5 μM) was used in the presence of increasing concentrations of unlabeled triolein or phosphatidylcholine to analyze the crosslinking on the gel. CrossUnked protein was run on SDS-PAGE. Autoradiography of the gel showed that both the analogues were crosslinked with the lipase and the unlabelled substrate competed with the photoprobe as there was reduction.

EXAMPLE 6 When provided the Upase with radiolabeled phosphatidylchoUne as the substrate, unlabeled triolein was able to chase labeled phosphatidylcholine. Radiolabeled substrate was chased more efficiently by phosphatidylchoUne as compared to triolein since the Km for triolein is higher than for phosphatidylcholine. Moreover, there was a comparable inhibition of both the activities in the presence of serine modifier DIPF. These experiments indicated that both the substrates were competing for the same catalytic site and the active site contains a serine residue. However, in the immunoinhibition assays, Upase but not the phospholipase activity was inhibited. This could mean that upon its interaction with the antibody, the amphipathic phosphoUpid but not the neutral substrate is able to interact with the enzyme. Alternatively, in spite of the steric hindrance and or any conformational changes posed by the antibody, binding site for the triacylgh/ceride is masked but not for the phosphoUpid. This indicates that both have independent binding sites. Therefore, our results provide biochemical evidences to demonstrate that the lipase has a common catalytic site but independent binding sites for the neutral Upids and phospholipids.

EXAMPLE 7 Amino acid composition of the purified rice bran lipase.

Amino acid MW pmol mol%

ASX 115.08 95.4 7.03

GLX 129.11 98.5 7.26

SER 87.07 86.2 6.35

GLY 57.05 188.7 13.9

HIS 137.14 28.6 2.11

ARG 156.18 42.1 3.1

THR 101.1 71.4 5.26

ALA 71.07 181.2 13.35

PRO 97.11 71 5.23

TYR 163.17 36 2.65

VAL 99.13 116 8.55

MET 131.19 22.7 1.67

ILE 113.15 54.1 3.99

LEU 113.15 143.6 10.58

PHE 147.17 49.7 3.66

LYS 128.17 72.1 5.31

Cys/2 103.13 0

AIB* 209 totals: 1357.3 100 REFERENCES

1. Falch, E. A. (1991) Biotechnol. Adv. 9, 643-658.

2. Arbige, M. V., and Pitcher, W. H. (1989) Trends Biotechnol. 7, 330-335

3. Hoq, M. M., Yamane, T., Shimizu, S., Funada, T., and Ishida, S. (1985) J. Am. Oil Chem. Soc. 62, 1016-1021.

4. Adelhorst, K., Bjorkling, F., Godtfredsen, S. E. and Kirk, O. (1990) Synthesis 1, 112- 115.

5. Yadav, R. P., Saxena, R. K., Gupta, R., and Davidson, S. (1997) J. Sci. Ind. Res. 56, 479. 6. Httls, M. I, Kiewitt, I., And Mukherjee, K. D. (1990) Biochim. Biophys. Ada. 1042, 237-240.

7. Kloosterman, M. Elferink, V. H. M., Jack van Lersel, J., Roskan, J. H., Meijer, E. M., Hulshof, L. A., and Sheldon, R. A. (1988) Trends Biotechnol 6, 251-256.

8. Jandacek, R., Whiteside, J. A., Holcombe, B. N., Volpenheim, R. A., and Taulbee, J. D. (1987) Am. J. Clin. Nutr. 45, 940-945.

9. Soumanou, M. M., Bomschener, U. T., Menge, U., and Schmid, R. (1997) J. Am. Oil Chem. Soc. 74, 427-433.

10. Eibl, H., andUnger, C. (1990) Can. Treat. Rev. 17, 233-242.

11. Fregapane, G., Sarney, D. B., and Vulfson, E. N. (1991) Enzyme Microb. Technol. 13, 796-800.

12. Margolin, A. L., Crenne, J. Y., KUbanov, A. M. (1987), Tetrahedron lett. 28, 1607 1610. 13. MargoUn, A. L., Fitzpatrick, P. A, Dublin, P. L., and KUbanov, A. M., (1991) J. Am. Oil Chem. Soc. 113, 4693-4694.

14. Bornemann, S., CasseUs, J. M., Combes, C. L., Dordick, J. S. and Hacking, A. J. (1989) Biochim. Biophys. Ada 1265, 25-253. 15. Parmar, V. S., Kumar, A., Poonam, Pati, H. N., Saxena, R. K., Davidson, S., and Gupta, R. (1998) Biochim. Biophys. Ada.

16. Hoq, M. M., and Yamane, T. (1985) J. Am. Oil Chem. Soc. 62, 1016-1021.

17. West, S. (1987) Food Proc. 56, 35-39.

18. Godfredson, S. E. (1990) Microbial enzymes and Biotechnology, Elsevier AppUed Sciences, The Netherlands, pp. 255-273.

19. Liu, W. H., Beepu, T. and Arima, K. (1973) Agric. Biol. Chem. 37, 157-163.

20. Smith P. K, Krohn, R.I., Hermanson, G, T., MaUia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olsen, B. J., and Klenk, D.C. (1985) Anal. Biochem. 150, 76-85. 21. Laemmli, U. K., (1976) Nature 227, 680-685.

22. M. V. Nesterenko et al., (1994) J. Biochem. Biophys. Methods 28, 239-242.

23. Warren, F., Mc Gyckin and Beenard, F. Mc Kenzie (1958) Clinical Chemistry 14, 476-483).

24. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 25. Rajasekharan, R., RusselL C. M., Shockey, J. M., and Kemp, J. D. (1993) Biochemistry 1, 12386-12391. 26. Ji, I., Shin, J., and Ji, T.H. (1985) Ami. Biochem. 151, 348-349. Gupta, C. M., Radhakrishnan, R., and Khorana, G. (1977) Proc. Natl Acad. Sci. USA 74,4315-4319.

Claims

CLAIMS:

1. A novel lipase isolated from monocotyledon plant sources.

2. A novel lipase as claimed in claim 1 that has the following properties: a. isolated enzyme is a glycoprotein; b. isolated enzyme is stable and catalyticaUy active at high temperatures; c. isolated enzyme is stable and catalytically active at alkaline pH; d. isolated enzyme has molecular weight range of 9 to 12-kDa; e. isolated enzyme is inhibited by inhibitors of serine proteases such as Dusopropylfluorophosphate; and f. isolated enzyme that can utilize both triacylglycerol and phosphoUpids.

3. A process of purifying the lipase as claimed in claim 1 and 2 using the following steps: a. deUpidation of the plant extract using organic solvents; b. Filtration of the deUpidated extract on a suitable matrix; c. Purification of the enzyme using hydrophobic column matrices; and d. characterization of the purified product by known methods.

4. A novel Upase from rice bran.

5. A novel lipase from rice bran as claimed in claim 4 that has the foUowing properties: a. a glycoprotein of size 9 to 12-kDa; b. stable and catalytically active at 80°C; c. isolated enzyme is stable and catalytically active even at pH 12.0; d. enzyme activity is inhibitable by serine protease inhibitors such as diisopropyl fluorophospate; and e. enzyme that can hydrolyze both triacylglycerol and phosphoUpids.

6. A process of purifying the lipase as claimed in claim 4 using the following steps: a. deUpidation of the plant extract using organic solvents; b. Filtration of the deUpidated extract on a suitable matrix foUowed by separation of fine insolubles; c. Purification of the enzyme using hydrophobic column matrices; and d. characterization of the purified product by known methods; 7, A process of purifying the Upase as claimed in claim 4 using the foUowing steps : a. deUpdation of the rice bran with lOg/lOOml of diethyl ether and extraction in 10 mM Tris-HCl pH 7.5 and 1.0 mM EDTA for 12 h; b. filtration of the extract through fine layers of cheese cloth foUowed by centrifUgation at 3,000Xg for 30 min; c. purification of the enzyme on octyl-Sepharose pre-equilibrated with 0.01

M Tris-HCl pH 7.5 and eluting the pure protein with a linear gradient of 0-40% methanol; and d. characterization of the purified protein by known methods.

Use of the novel lipase as claimed in claims 1, 2 and 4 in dairy industry, oleo chemical industry, detergent industry, synthesis of surfactants, synthesis of nutritionally important Upid products, synthesis of optically active polymers, resolution of racemic mixtures and synthesis of ingredients for personal care products.