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Exploiting the use of fluorophore in extraction and direct detection of rice’s lipase activity

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  1. Introduction
  2. Extraction of Rice Proteins
  3. Materials and methods
  4. Conclusion

Lipase is the major protein in rice that catalyzes endogenous hydrolysis of lipids. Study on lipase activity is crucial in rice bran utilization, as it causes rancidity, severely affects the product quality and tarnishes its economic value. This experiment aimed to isolate lipase from different rice lines' tissues, detect its activity and determine its molecular weight. Proteins were extracted from grains of Bd192, Kasalath and 2514 rice lines by grinding, buffering and centrifuging. Extracted proteins were separated with SDS-PAGE and lipase activity was detected using 4-MU Butyrate.

Protein bands were visualized with Coomassie staining and molecular weight of lipase was determined by comparison with protein ladder. Bright UV bands were observed in all leaves but not grains samples. The molecular weights of lipase was approximately 36.7kDa. In conclusion, lipase was present in leaves samples of all rice lines but not in grains.

[...] Bright UV bands were observed in all leaves but not grains samples. The molecular weights of lipase was approximately 36.7 kDa. In conclusion, lipase was present in leaves samples of all rice lines but not in grains. INTRODUCTION: Brown rice and milled white rice are staple food for most part of the world's population, especially the Asians (Sugiyama et al. 2003). During rice polishing, the testa and pericarp of grains, which is mainly comprised of bran, are removed. With such enormous demands in standard diet, rice bran are produced abundantly. [...]

[...] Figure Image of Coomassie stained of SDS-PAGE for leaves and grains of different rice lines. Figure Protein ladder bands with molecular weight values. Table Distance migrated by bands, molecular weights and log [distance] of bands. Figure Graph of log [distance migrated] (units) against molecular weight of protein bands (kDa). Calculations: Given that lipase bands for all leaves samples migrated approximately 5.4 cm from initial well, Log [distance]=- 0.0076 (MWlipase)+ 0.9299 log [ 5.4 0.0076 (MWlipase)+ 0.9299 MWlipase = 30.0 kDa Given that E. [...]

[...] RESULTS: Detection of lipase activity From Figure 1 in APPENDIX, moderately bright bands were observed under UV light for Bd192 leaves (lane 2 & less intense fluorescent bands for 2514 leaves (lane and Kasalath leaves (lane & 8). Weakly fluorescent bands were also observed for Escherichia coli positive control and protein ladder, albeit both migrated smaller distance compared to leaves samples. Determination of lipase molecular weight From Figure 1 and 2 in APPENDIX, fluorescent bands were found to have molecular weight of approximately 30kDa for leaves samples for all rice lines (Bd and Kasalath). E. coli's fluorescent band approximately was 54.1 kDa. [...]

[...] Maximizing food utilization thus requires stabilization of rice bran through inactivation of lipase activity. This could be done by destructing active lipases through thermal or chemical treatment (Silva et al. 2006). Identifying and characterizing lipase are important to develop effective strategy to approach issue of rice bran instability. SDS-PAGE is a technique used for separation of proteins in complex mixtures based on size, commonly targets to resolve proteins. SDS is an anionic detergent that denatures the native conformations of proteins, giving them a consistent mass: charge ratio. [...]

[...] DISCUSSION: In Figure detection of fluorescent bands indicated presence of lipase activity. 4-MU butyrate used to stain the gel upon completion of gel electrophoresis acted as a substrate for lipase. The rationale behind this technique was based upon principle where reaction products emitting fluorescent upon hydrolysis. 4-MU butyrate, a non-fluorescent compound liberated one molecule of fluorescent 4-methylumbelliferone and one molecule of butyric acid when lipase hydrolyzed its ester linkage (Gilham & Lehner 2005). In Figure SDS-PAGE separated protein fragments based on their size. [...]

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