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ThesisItem Open Access Functional proteomics of rice bran lipase(s)/esterase(s) in Ranjit, a major high yielding rice variety of Assam.(AAU, Jorhat, 2019) Mishra, Udit Nandan; Kandali, R.On milling, the paddy produces brown rice and hulls. Brown rice upon polishing results in the production of white rice and byproducts such as head rice, broken rice, polish and bran. Rice bran, the byproduct of rice milling has been gaining a commercial importance in the world due to the fact that it contains nutritionally superior oil and a number of health beneficial compounds. Although rice bran is a good nutritional food source, rapid rancidity from the activity of lipolytic enzymes such as lipase, esterase and lipoxygenase deteriorate the nutritional values in rice bran and limits the wide usage of rice bran as a food ingredient. The effective utilization of rice bran is possible only by deactivating the lipolytic enzymes responsible for the hydrolytic rancidity which otherwise severely affects the nutritive value and palatability of rice. Lipases are carboxyesterases acting on long chain acylglycerides and classified as serine hydrolase class due to their catalytic triad mechanism of serine protease. In the past few decades lipases gained a significant choice for scientists from the research point of view due to their ubiquitous nature and wide range of distribution across different kingdoms. Currently lipases from microbial sources are exploited commercially. Present research work was undertaken to study the functional proteomics of rice bran lipase (RBL) in a major high yielding variety (HYV) ‘Ranjit’, a popular high yielding variety of Assam. The objective of the present investigation was to extract and purify the major lipolytic enzyme in the bran, study the enzyme kinetics along with inhibition study and immobilization of the enzyme for subsequent industrial use. Some aspects of functional proteomics of enzyme responsible for rancidity were studied.To have an idea about how rancidity affects the products obtained after the milling process, both white refined rice and bran powder were stored for one week at room temperature after which the visible physical change in the color of the product was evident. Subsequently, chemical constituents like moisture (9.83%), ash (10.31%), reducing sugar (4.91%), total carbohydrate (52.31%), crude protein (14.07%), total soluble protein (4.33%), crude fiber (8.18%), mineral composition such as Fe (22.43 mg/100g), P (1090.07 mg/100g), Na (20.39 mg/100g), K (1512.22 mg/100g), Ca (69.10 mg/100g), Zn (5.09 mg/100g), Mg (786.61 mg/100g) on dry weight basis were determined in defatted rice bran. Rice bran yielded 16.04% of oil and the chemical characteristics for the oil such as acid value (1.11 mg/g oil), saponification value (155.40 mg/g oil) and iodine value (88.83 mg/g oil) were determined. Phytochemical compositions like total phenol (513.38 mg/100g), phytate ‘P’ (542.01 mg/100g) and tannin (32.66 mg/100g) were also analyzed. Lipase was extracted from bran following alkaline extraction method. Preliminary assay of crude lipase with the substrate 4-nitrophenyl palmitate showed specific activity of 0.04 U/mg. Subsequently a 9.45-fold purification was obtained with a specific activity of 0.378 U/mg by following ammonium sulfate precipitation, FPLC and RP-HPLC sequentially. The purified rice bran lipase (RBL) was shown to have an approximate molecular weight of 33 kDa in SDS-PAGE by silver staining and a separate fluorescent staining with 4-methylumbelliferyl butyrate. Further, purified RBL showed a preference for natural oils of long chain unsaturated fatty acids with a maximum specific activity (0.452 U/mg) in case of rice bran oil. The purified lipase was immobilized in calcium alginate beads with an efficiency of 65% to that of free lipase activity. Kinetic study revealed both immobilized lipase (IL) and free lipase (FL) had a pH and temperature optima of 7.5 and 300C, respectively, with a maximum catalytic activity at a substrate (4-nitrophenyl palmitate) concentration of 200μM. Non-polar organic solvents like petroleum ether, hexane and surfactant like SDS reduced the activity of both free (FL) and immobilized lipase (IL). On the other hand in presence of lower concentration of metal ions such as Ca2+, Mg2+ and Na+, both FL and IL showed increased activity. Both synthetic (PBA; IC50 0.92mg PBA/mg of lipase) and natural (mango leaf extract; IC50 0.89mg extract/mg of lipase, ground nut shell extract; IC50 0.88mg extract/mg of lipase) inhibitors caused decrease in lipase activity at optimum reaction condition. In order to check the viability of immobilized lipase for commercial exploitation blending of fish oil free fatty acids (FFA) with sunflower oil (SO) was done at different ratios with 12h, 24h and 36h of incubation during the acidolysis reaction. A highly desirable n-6/n-3 ratio of (5.6:1) was observed in the novel structured triglyceride using pure RBL with 1:1 ratio of SO:FFA with 12h incubation during acidolysis reaction. This was followed by another novel trglyceride synthesized from 1:4 ratio of SO:FFA with 36h of incubation during acidolysis reaction which had a desired n-6/n-3 ratio of 3.6:1. Thus immobilized lipase could be used to optimize the fatty acid profile of existing commercial sunflower oil by either replacing or incorporating n-3 fatty acids from marine source to impart better nutritional desirability.