Rice Bran Lipase: Extraction, Activity, and Stability

Transcription

1 Biotechnol. Prog. 1999, 15, Rice Bran Lipase: Extraction, Activity, and Stability Anita V. Prabhu, Shreehas P. Tambe, Neena N. Gandhi, Sudhir B. Sawant,* and Jyeshtharaj B. Joshi Department of Chemical Technology, University of Mumbai, Matunga, Mumbai , India A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mm and ph 7, containing 0.5 mm of CaCl 2 ). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability. Introduction Lipases have a vast potential for application in a number of industries, many of which are totally unrelated (1). The most well-known use of lipase-catalyzed synthesis lies in the interesterification of fats to produce synthetic triglycerides with desirable characteristics (2-7). One such application is in the manufacture of cocoa butter equivalent from palm oil (8). Lipase-catalyzed hydrolysis can be used to obtain fatty acids and glycerol. The castor bean lipase-catalyzed hydrolysis, to manufacture fatty acids and glycerol, is already operative on a commercial scale (9). Lipases can be used as digestive aids in the case of lipase deficiency, with its consequences for fat metabolism (10). Lipases constitute components of detergent mixtures, wherein they function in the removal of fat stains from fabrics (11, 12). Lipases can be obtained from plant, animal, and microbial sources. Manufacture of lipases by microbial fermentations constitutes a process that is currently popular (13, 14). A less-explored avenue is that of lipase extraction from natural sources. This study therefore focuses on the isolation of the lipase from a plant source, rice bran. Rice (Oryza sativa) is one of the most important food cereals for the majority of the world s population. For the most part, polished rice is the staple food, thus giving rise to an important and abundant byproduct, rice bran, obtained during milling of the rice. Bran, which comprises the testa and pericarp, is known to be rich in a number of important components such as oil (edible) (15), vitamins, sugars, and proteins. An important potential protein present in the rice bran is lipase. Like some other lipases, rice bran lipase is known to be regioselective (1, 3) and has a preference for low molecular weight substrates (16, 17). These and other biochemical and enzymic properties (18-23) would enable the employment of this lipase for the manufacture of specified products. Most of the work published on this enzyme deals with its deactivation to stabilize the bran for its subsequent * To whom correspondence should be addressed. Fax: sbs@udct.ernet.in. oil extraction (24, 25). Other studies on rice bran lipase deal with the elucidation of its structural and biochemical properties (18, 23, 26, 27). No efforts have been reported to explore the possibility of extraction of lipase from rice bran for commercial use, a result of either the poor yield of the enzyme or the lack of information on the enzyme content of the different varieties of rice. Therefore, the present study was undertaken to develop a simple and systematic procedure for obtaining a high yield of the lipase in a pure and usable form. However, the enzyme was found to be very vulnerable to deactivation even under ambient conditions. For an enzyme to be of commercial value, a degree of stability needs to be conferred on it. It was therefore necessary to ascertain conditions under which the enzyme would be reasonably stable. Materials and Methods Reagents Used. Samples of bran from local rice varieties such as Jini-Kolam, Jaya, Surti-Kolam, R. P., Massuri, and Ratna were procured from a local rice mill. Gum acacia, hexane, sodium hydroxide, phenolphthalein, and methanol were obtained from S. D. Fine Chemicals Ltd., Mumbai, India. Potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate salts, and calcium chloride were obtained from Qualigens Fine Chemicals, Ltd., Mumbai, India. Tributyrin and oxalic acid were procured from Hi Media Laboratories Pvt. Ltd., Mumbai, India. Apparatus and Equipment. A 250 ml glass beaker provided with a stainless steel propeller was used for defatting and extraction of the rice bran. Rice bran lipase assays were carried out in 100 ml conical flasks with agitation via orbital shaking at 30 C. Centrifugation was carried in a CRU-5000 unit. The 0.22 and 0.45 µm polyvinyldifluoride (PVDF) membranes were procured from Millipore (India) Pvt. Ltd., Mumbai, India. The 10 and 100 kd polysulfone membranes and the Minitan-S tangential flow filtration unit were supplied by Millipore Gesselschaft m.b.h., Wein, Austria. The CIS-24 temperature-controlled orbital shaking incubator from Remi Instruments, Mumbai was used during activity assay /bp990122z CCC: $ American Chemical Society and American Institute of Chemical Engineers Published on Web 11/13/1999

2 1084 Biotechnol. Prog., 1999, Vol. 15, No. 6 Defatting of Rice Bran. Surti variety of rice bran was used for all experiments unless mentioned otherwise. For defatting, 10 g of rice bran was stirred for 30 min each in three batches with 30 ml of n-hexane. At the end of every stirring period, the hexane phase was decanted and fresh hexane was added to the rice bran. This procedure was followed in each experiment unless mentioned otherwise. Extraction of Rice Bran Lipase. The defatted bran from above was allowed to air-dry for about 1 h (so that all hexane was practically removed) and then used for extraction. For this purpose potassium phosphate buffer containing calcium chloride was used. Concentrations of the buffer, as well as of the calcium chloride, were varied in the preliminary experiments. The phosphate buffer concentration of 50 mm with 0.5 mm calcium chloride was found to give maximum activity in the extract. The defatted bran was stirred with eight times its weight of 50 mm phosphate buffer of ph 7.0 (containing 0.5 mm CaCl 2 ) at 10 C for 30 min, after which the suspension was centrifuged for 15 min at 50 rps at 4 C for 30 min. The pellet of the bran obtained was resuspended in fresh phosphate buffer, and the above procedure repeated four times. The supernatants thus obtained were collected and pooled to give the crude lipase extract (UCL). Any variations in this procedure are mentioned in the text where appropriate. Lipase Assay. Lipase activity was estimated by its hydrolytic action of tributyrin, which was emulsified in various emulsifying agents such as gum acacia and poly- (vinyl alcohol). Various compositions of assay mixtures were investigated. The following method yielded the highest and reproducible activity for the lipase. A 2 ml portion of 7.5% gum acacia was mixed with 10 ml of enzyme extract. To this was added 1 ml of tributyrin, and the mixture was stirred for 1 min with a magnetic needle to produce a stable emulsion, which was then placed on an orbital shaker at 30 C. Next, 0.5 ml aliquots of this reaction mixture were removed at specified intervals, and the reaction was quenched using 10 ml of methanol. This was then titrated with sodium hydroxide with phenolphthalein as an indicator. The reported values of activity are the average of three different emulsified assay samples. The activity values were within (3% of the average value. Calculation of Lipase Activity. Lipolytic activity is expressed in terms of lipase units (U) per unit weight of rice bran (in kg). One unit is defined as the micromoles of butyric acid liberated by the hydrolysis of tributyrin at 30 C per minute. The amount of acid released can be calculated from the difference in titer values of a sodium hydroxide solution of known molarity. Specific activity was calculated as the units per mg protein. Purification of Lipase Extract. The crude lipase extract was centrifuged at 100 rps for 25 min to separate the bran particles from the enzyme solution. The clarification of the latter was performed by passing the solution through the 0.45 µm PVDF membranes to get a microfiltration extract (MF) and then through polysulfone membranes of molecular weight cutoff 10 and 100 kd to yield an ultrafiltration extract (UF). Lipase Activity on Different Substrates. Gum acacia (5 g), water (10 ml), and oil (20 g) were stirred with a magnetic needle to obtain 35 g of emulsion. An aliquot of emulsion corresponding to1gofoilwasadded to 10 ml of lipase extract and stirred at 30 C. Then, 0.5 ml aliquots of the reaction mixture were removed at specified intervals, and the reaction was quenched using Table 1. Hydrolytic Activity of Rice Bran Lipase on Different Substrates initial reaction rate substrate (mol acid/min) tributyrin 1.17 olive oil 0.26 palm oil 0.12 castor oil 0.09 coconut oil ml of methanol. This was then titrated with sodium hydroxide using phenolphthalein as an indicator. Results and Discussion Effect of Substrate. To test the substrate specificity of rice bran lipase, tributyrin and various natural oils such as coconut oil, olive oil, castor oil, and palm oil were used as substrates. The results are summarized in Table 1. It is clear that the rate of hydrolysis is highest in the case of tributyrin. Among the oils, it is seen that olive oil is hydrolyzed faster than the other oils. Other studies on rice bran lipase and pancreatic lipase have yielded similar results, namely, that shorter chains, especially buttery chains, are hydrolyzed by the lipase enzyme faster than longer chains. In addition, oleyl chains have been reported to be released slightly faster than other long-chain fatty acids (16, 18). This substrate selectivity, along with the reported 1,3 selectivity (16, 17), confers on rice bran lipase the potential for use in the design of structured lipids, i.e., triglycerides with particular fatty acids esterified at specific positions on the glycerol backbone. Defatting of Rice Bran. This constitutes the first step in the extraction process. It was found that attempts to forego this step resulted in rather low activity yields. Also, aqueous extraction of proteins is rendered rather difficult because of the presence of the fatty material. Solvents such as petroleum ether and n-heptane were tested, but n-hexane appeared to give the better results. Also, as a result of high volatility, it makes oil recovery easier. The effect of hexane on the lipase activity was also checked. For this purpose, 10 g of bran was incubated in 30 ml of hexane for 30 min at 10 and 30 C. As seen in Figure 1, the enzyme activity started dwindling within 10 min at 30 C. On the other hand, no deleterious effect was observed at 10 C, at least up to 30 min. The loss of lipase in the hexane was estimated by extraction of the hexane extract with 20 ml of 50 mm phosphate buffer (ph 7) in three batches, with each extraction being of 1 h duration. The aqueous extract was then tested for hydrolytic activity on tributyrin. It was found that the amount of lipase in this extract was negligible when compared to the overall yield. Aqueous Extraction of Rice Bran. Defatted bran, obtained by the procedure optimized above, was stirred with potassium phosphate buffer to extract the lipolytic activity. The procedure was standardized with respect to ph and concentration of phosphate buffer and extraction temperature. ph of the Extracting Buffer. The lipase enzyme was extracted from bran (Surti variety) with phosphate buffers in the range of ph 5-8 at 10 C. Though the ph of the buffer was varied, the ionic strength was always maintained constant at 50 mm. Also, all of the buffers contained 0.5 mm of CaCl 2. Maximum activity of the lipase enzyme was observed at ph (Figure 1). These results are in agreement with those reported by Shastry and Rao (18) and Rajeshwar and Prakash (27) for the optimum ph of the lipase.

3 Biotechnol. Prog., 1999, Vol. 15, No Figure 1. Effect of ph of extraction on the activity of rice bran lipase. Hence, potassium phosphate buffer of ph 7 was used for the extraction of lipase in all further experiments. Extraction Temperature. Extraction of lipase was carried out at two different temperatures, 10 and 30 C, using 10 g of bran (Surti and Jaya varieties) for each study. The bran was extracted twice with 80 ml of phosphate buffer (ph 7, 50 mm) containing 0.5 mm of CaCl 2. After extraction, the two extracts of Surti rice bran lipase were assayed for their hydrolytic activity. In both the cases, practically the same activity was obtained. However, lipase extract obtained by carrying out extraction at 30 C lost activity within a short period of time even though stored at 10 C. However, for extractions carried out at 10 C, 100% activity was retained by the crude extract for 24 h. Similar results were obtained with lipase from Jaya rice bran. In this case, despite the similar activity at both temperatures, the stability of the lipase obtained from the low-temperature extraction was much higher (no deactivation up to 7 days when stored at 10 C). However, the 30 C extract activity dwindled within 10 h even though stored at 10 C. Thus, extractions were carried out at 10 C throughout this work. Number of Extraction Cycles. Extraction of lipase was carried out in six successive extraction cycles. For the first cycle, the defatted bran was stirred with eight times its weight of 50 mm phosphate buffer of ph 7 (containing 0.5 mm CaCl 2 ) at 10 C for 30 min. After this, the suspension was centrifuged (250g) at 4 C for 30 min. To the residue, eight volumes of buffer was added, and the above steps were repeated five times. The data given in Figure 2 show that for complete extraction of lipase six extraction cycles were required for both the bran varieties. However, with distilled water, the activity in the third extract was found to be zero. In addition, the lipase activity in the first extract with distilled water was 281 U/kg compared to 843 U/kg in Figure 2. Effect of number of aqueous extraction cycles on the yield of rice bran lipase. the first extract with phosphate buffer under otherwise identical conditions. Further, the total lipase activity in all the phosphate buffer extracts was five times higher than that obtained with distilled water. Thus phosphate buffer is a better extractant than distilled water for extraction of lipase. Duration of Extraction Cycles. Defatted bran was extracted with eight volumes of buffer at 10 C for varying time periods. It was observed that the extraction yield increased with time up to 30 min (Figure 3). Beyond 60 min, the activity yield declined, possibly as a result of concomitant extraction of lipase inhibitors from the bran. Hence, all extraction batches were restricted to 30 min. Stability of the Rice Bran Lipase. Effect of Temperature on Enzyme Activity. Crude lipase extracts were assayed at different temperatures (10-45 C) for tributyrin hydrolysis as usual. It was found that there was no significant change in activity up to 30 C (Figure 4). However, at higher temperatures, the activity diminished, indicating the thermolability of the lipase preparation. Rajeshwar and Prakash (27) also obtained activity maxima for triacetin hydrolysis using IR-20 rice bran lipase at 30 C, and on either side of that temperature, they obtained a sharp decline in activity. Thus, they obtained a bell-shaped curve for the temperature dependence of lipase activity. In the present case, the left side of the bell was considerably flattened, indicating that even at lower temperatures, activity comparable to that at 30 C was obtainable. Storage Stability. To study the effect of age on lipase activity, various varieties, namely, Massuri, Ratna, Surti, Jini, R.P., and Jaya were tested. The age of each bran was reckoned from the day of milling (day on which milling was done was taken to be day zero). Because different varieties were procured on different days after milling, the day of commencement of the analysis of lipase activity among varieties differed. Thus the age of each variety at the start of its analysis varied.

4 1086 Biotechnol. Prog., 1999, Vol. 15, No. 6 Figure 3. Effect of duration of aqueous extraction cycle on the yield of rice bran lipase. Figure 4. Effect of temperature on activity of rice bran lipase. Although all of the above bran varieties were stored after procurement at 10 C during the entire period of the study, it was seen that the stability of lipase enzyme varied from variety to variety (Figure 5). The lipase in the Massuri and Ratna varieties was found to remain active for a period of about 3 months, which was found to be the maximum among the varieties tested. However, the Surti variety yielded the highest lipase activity among the strains studied. Also in both of these varieties, the activity curve of the lipase showed biphasic behavior, with the activity remaining constant over the first phase and then declining slowly over the second phase. Other varieties showed a continuous decline in activity with time. Thermostability. Stability toward high temperature is perhaps one of the most important aspects considered before application of an enzyme. Also, a thermostable enzyme is often likely to be stable in general toward other Figure 5. Effect of age and variety of rice bran on activity of the lipase. denaturing conditions. To examine the thermal stability, the extract was incubated at different temperatures ranging from 10 to 65 C for 15 min and subjected at 30 C to assay for the residual activity, using tributyrin as the substrate. It was observed that, up to 40 C, the loss in activity is practically negligible. However, incubation at higher temperatures leads to rapid loss of activity (Figure 6). Storage Stability at Low Temperature (10 C). Rice bran (Surti variety) was extracted with phosphate buffer. The extract obtained was divided into 10 ml portions and stored in different test tubes at 10 C. The activity of lipase in the extract was determined for each time using the content of a fresh test tube. It was observed that rice bran lipase activity remains constant for a day, and thereafter a gradual decrease in the activity was observed, with a half-life of about 60 h (Figure 7). Effect of Glycerol on Storage Stability. Polyols have been known to stabilize proteins (28). Because it was found that the extract obtained from rice bran lost almost all of its activity within 1 day of its storage at 30 C, it was decided to elucidate the effect of glycerol on the storage stability of the lipase. Aliquots of glycerol (90%) were added to 50 ml of extract to obtain final glycerol concentrations (%) of 0, 1, 5, 10, 25, and 50. These extracts were then stored at room temperature for a maximum of 5 days and then tested for activity using tributyrin as the substrate. As seen from Figure 8, no stabilizing effect of glycerol was observed below 10% glycerol concentration. With 25% glycerol concentration, the half-life of the lipase extract was about a day, whereas with 50% glycerol concentration, it was about 72 h, at 30 C. In comparison, as reported in the section on extraction temperature above, very little activity was preserved at this temperature without glycerol addition. This stabilizing effect of glycerol may be due to a solvent effect, the presence of the polyol causing a

5 Biotechnol. Prog., 1999, Vol. 15, No Figure 6. Effect of temperature on stability of the rice bran lipase. Extract incubation was 15 min at different temperatures. Figure 8. Effect of glycerol on storage stability of rice bran lipase at 30 C. Figure 7. Storage stability of rice bran lipase at 10 C. reduction in the hydrogen-bond rupturing capacity of the medium. Gerlsma and Stuur (29) proposed that the effect could originate in the intensification of the intramolecular hydrophobic interactions within the protein. Membrane Filtration of the Rice Bran Lipase. This was carried out in two stages, microfiltration and ultrafiltration. Surti rice bran was defatted and then extracted in phosphate buffer (ph 7, 50 mm) containing 0.5 mm CaCl 2 for 30 min at 10 C. This extract (hereby termed first extract ) was then subjected to microfiltration using a 0.45 µm PVDF membrane. The permeate obtained was called the microfiltration extract (MF). Because the molecular weight of the rice bran lipase is 41 kd (18, 26), the MF was initially subjected to ultrafiltration using a 100 kd polysulfone membrane, so as to obtain a permeate containing the lipase free of large, contaminating molecules. This permeate was then ultrafiltered through a 10 kd polysulfone membrane, so as to obtain a purer and concentrated enzyme solution in the retentate. The protein distribution obtained during this purification process is shown in Table 2. Enzyme activity in the MF extract was increased only marginally from to U/ml, but an 18-fold increase in specific activity was obtained by ultrafiltration using the 100 kd membrane. On further purification, activity in the 10 kd retentate went up to U/ml, while the specific activity was also enhanced to give U/mg protein. Thus, the series of purification steps enables a doubling of enzyme activity and a tripling of the specific activity, with concomitant removal of almost three-fourths of nonspecific proteins. The volume of the enzyme solution was also more than halved, allowing for a much more concentrated and pure enzyme. The above results were obtained when starting from the first aqueous extract. When the same bran was subjected to a second cycle of extraction with calciumcontaining buffer for 30 min at 10 C, a second extract was obtained. This was put through similar membrane filtration steps as above. Again, 75% of nonspecific proteins were eliminated in the final 10 kd retentate, and a doubling of enzyme activity from to U/ml obtained (Table 3). Again, the specific activity almost trebled to U/mg protein. Concentration of the enzyme solution was almost 2.5-fold. In both the cases, only a marginal increase in enzyme activity was obtained on 100 kd ultrafiltration, and the increase in specific activity was mainly due to the sharp reduction in total protein content of the 100 kd permeate. Thermostability of the Different Enzyme Preparations. When the crude unclarified enzyme (UCL) from

6 1088 Biotechnol. Prog., 1999, Vol. 15, No. 6 Table 2. Membrane Filtration of the First Aqueous Extract of Rice Bran Lipase enzyme prep volume (ml) absorbance at 280 nm concn of protein (mg/ml) total protein (mg) enzyme activity (U/mL) specific activity (U/mg) MF kd permeate kd retentate kd permeate kd retentate Table 3. Membrane Filtration of the Second Aqueous Extract of Rice Bran Lipase enzyme prep volume (ml) absorbance at 280 nm concn of protein (mg/ml) total protein (mg) enzyme activity (U/ml) specific activity (U/mg) MF kd permeate kd retentate kd permeate kd retentate Surti rice bran was stored at 10 and 30 C, 35% of the original activity was found to remain after 72 h in the former case, whereas in the latter case, this was found to occur within 24 h (Figure 9). When the MF extract was stored at these temperatures, a similar range of activity was obtained after 5 days at 10 C and 2 days at 30 C (Figure 9). Thus, microfiltration has enhanced the thermostability of the enzyme preparation, possibly as a result of the removal of certain molecules that are potential deactivators of the rice bran lipase. To confirm that this trend is maintained with further purification steps, a 60 g sample of defatted bran was extracted with buffer under the usual condition and subjected to membrane filtration. Table 4 shows the results obtained when the various extracts were stored at 10 C. Half-life of the 10 kd retentate was expectedly the highest (7 days), followed by the 100 kd permeate (5 days) and the MF extract (about 4.5 days), and the unclarified crude extract yielded the lowest half-life (2 days) (Table 4). Rice bran is abundantly available and after the extraction of the rice bran oil, it is sold at a very low price. Preliminary cost estimates indicate the raw material cost of rice bran lipase to be around 4000 U/cent. This number can be seen to be less expensive by an order of magnitude as compared with commercially available preparations. Further, the natural enzymes are considered to be environmentally friendly as compared with genetically engineered microbial enzymes. It is known that rice bran lipase catalyses the hydrolysis of rice bran oil to form free fatty acids. Though the rates are much slower than those for the hydrolysis of low molecular substrates such as tributyrin, the rice bran lipase still can be attractive because of the low price. The benefits may become even more enhanced through some additional work. Conclusion A simple and cost-effective procedure for the extraction of the rice bran lipase has been presented. This is found to be less cumbersome than those reported earlier (18, 23, 30). This method entails the use of cheap, readily available materials. Various conditions of extractions have been optimized, including buffer concentration (50 mm), calcium ion concentration (0.5 mm), ph (7), temperatures (10 C), etc. It was found that almost total lipolytic activity could be isolated by stirring defatted rice bran in eight volumes of buffer in five cycles, each lasting 30 min. Defatting was found to be a necessary first step to remove oil and other lipidic substances in the bran. It was found that Figure 9. Thermostability of unclarified and microfiltered lipase extracts: UCL, unclarified lipase extract; MF, microfiltered extract. Table 4. Thermostability of Various Enzyme Preparations storage time at 10 C (days) unclarified extract lipase activity (U/mL) MF 100 kd permeate 10 kd retentate the enzyme was highly susceptible to thermal inactivation under both storage and experimental conditions. A 50% glycerol addition enabled the enhancement of the half-life of the lipase at 30 C from less than 1 day to 3 days.

7 Biotechnol. Prog., 1999, Vol. 15, No Because the main objective was to obtain high yields of the lipase, some of the available varieties were screened for their lipase contents. Maximum activity was obtained from Surti rice bran, although the lipase in the Massuri and Ratna varieties was found to retain activity for as long as 3 months. Further purification of the bran lipase by microfiltration through a 0.45 µm PVDF membrane, followed by a 2-step ultrafiltration through polysulfone membranes of molecular weight cutoff 100 and 10 kd, resulted in improved activity and specific activity. The stability was also enhanced significantly at each step. Acknowledgment A.V.P. and S.P.T. are grateful to the Department of Biotechnology, Government of India, for the award of fellowship and for supporting the research. N.N.G. is grateful to the Council of Scientific and Industrial Research, India, for their fellowship. References and Notes (1) Gandhi, N. N. Applications of Lipase. J. Am. Oil Chem. Soc. 1997, 74, (2) Posorske, L. H. Industrial-Scale Applications of Enzymes to the Fats and Oil Industry. J. Am. Oil Chem. Soc. 1984, 61, (3) Macrae, A. R. Lipase-Catalyzed Interesterification of Oils and Fats. J. Am. Oil Chem. Soc. 1983, 60, (4) Matsuo, T.; Sawamura, N.; Hashimoto, Y.; Hashida, W. The Enzyme and Method for Enzymic Transesterification of a Lipid. Eur. Patent 35,883, (5) Tajima, I.; Kurashige, A. Fats and Oils Modified with Lipase for Margarines and Shortenings. Jpn. Patent 2,219,581, (6) Coleman, M. H.; Macrae, A. R. Rearrangement of Fatty Acid Esters in Fat Reaction Reactants. U.S. Patent 4,275,081, (7) Arnold, R. G.; Shahani, K. M.; Dwivedi, B. K. Applications of Lipolytic Enzymes to Flavor Development in Dairy Products. J. Dairy Sci. 1975, 58, (8) Bloomer, S.; Adlercreutz, P.; Mattiasson, B. Facile Synthesis of Fatty Acid Esters in High Yields. Enzymol. Microb. Technol. 1992, 14, (9) Stirton, A. J. Fat Splitting, Esterification and Interesterification. In Bailey s industrial oil and fat products; Bailey, A. E., Swern, D., Eds.; John Wiley and Sons: New York, 1964; pp (10) Masuda, T. Digestive Agents Containing Amino Acid Hydrochlorides and Enzymes. Jpn. Patent 1,238,538, (11) Andree, H.; Mueller, W. R.; Schmidt, R. D. Lipases as Detergent Components. J. Appl. Biochem. 1980, 2, (12) Fujii, T.; Tatara, T.; Minagawa, M. Studies on Application of Lipolytic Enzymes in Detergency. 1. Effect of Lipase from Candida cylindracea on Removal of Olive Oil from Cotton Fabrics. J. Am. Oil Chem. Soc. 1986, 63, (13) Sugiura, M.; Oikawa, T.; Hirano, K.; Inukai, T. Purification, Crystallization and Preparation of a Triacylglycerol Lipase from P. fluorescens. Biochim. Biophys. Acta 1977, 488, (14) Chen, J.; Ishii, T.; Shimura, S.; Kirimura, K.; Usami, S. Lipase Production by Trichosporon fermentens WU-C12-A Newly Isolated Yeast. J. Ferment. Bioeng. 1992, 73, (15) Zachariassen, B.; Giasotta, V. N. Prospects of Rice Bran Industry in India. Chem. Age India 1964, 15, (16) Aizono, Y.; Funatsu, M.; Sugano, M.; Hayashi, K.; Fujiki, Y. Enzymatic Properties of Rice Bran Lipase. Agric. Biol. Chem. 1973, 37, (17) Noda, M.; Kobayashi, K. Lipids of Leaves and Seeds III Enzymic Hydrolysis of Rice Bran Lipids. Nippon Nogei Kagaku Kaishi 1968, 42, (18) Shastry, B. S.; Rao, M. R. R. Studies on Rice Bran Lipase. Ind. J. Biochem. Biophys. 1971, 8, (19) Shastry, B. S.; Rao, M. R. R. Chemical Studies on Rice Bran Lipase. Cereal Chem. 1976, 53, (20) Aizono, Y.; Funatsu, M. Studies on Active Groups of Rice Bran Lipase. Agric. Biol. Chem. 1978, 42, (21) Aizono, Y.; Funatsu, M.; Fujiki, Y.; Watanabe, M. Purification and Characterization of Rice Bran Lipase II. Agric. Biol. Chem. 1976, 40, (22) Aizono, Y.; Funatsu, M.; Hayashi, K.; Inamasu, M.; Yamaguchi, M. Biochemical Studies on Rice Bran Lipase II. Agric. Biol. Chem. 1971, 35, (23) Funatsu, M.; Aizono, Y.; Hayashi, K.; Watanabe, M.; Eto, M. Biochemical Studies on Rice Bran Lipase I. Agric. Biol. Chem. 1971, 35, (24) Saunders: R. M. Rice Bran: Composition and Potential Food Uses. Food Rev. Int. 1986, 1, (25) Parbhakar, J. V., Venkatesh, K. V. L. A Simple Method for Stabilization of Rice Bran. J. Am. Oil Chem. Soc. 1986, 63, (26) Munshi, S. K.; Bhatia, N.; Sekhon, B. S.; Sukhija, P. S. Inactivation of Rice Bran Lipase with Metal Ions, J. Chem Technol. Biotechnol. 1993, 57, (27) Rajeshwara, A. N.; Prakash, V. Purification and Characterization of Lipase from Rice (Oryza sativa L.) Bran. Nahrung 1995, 39, (28) Gerlsma, S. Y. Reversible Denaturation of Ribonuclease in Aqueous Solutions as Influenced by Polyhydric Alcohols and Some Other Additives. J. Biol. Chem. 1968, 243, (29) Gerlsma, S. Y.; Stuur, E. R. Effects of Combining Two Different Alcohols on the Heat-induced Reversible Denaturation of Ribonuclease. Int. J. Chem. 1972, 6, (30) Aoyagi, Y.; Yamashita, H.; Matsumoto, S.; Obara, T. Rapid Method for Partial Purification of Rice Bran Lipase by Affinity Chromatography. Agric. Biol. Chem. 1979, 43, Accepted August 28, BP990122Z