臺灣博碩士論文加值系統

米糠 (rice bran) 為糙米碾磨後的副產物，富含油脂及多種生理活
性物質 (如生育酚、生育三烯酚、米糠醇、植物固醇等等)，然而米
糠亦含有酯酶(carboxylesterase, EC 3.1.1.1) 與脂肪酶(lipase, EC
3.1.1.3)，會水解三酸甘油脂形成游離脂肪酸及甘油，是導致油脂酸敗
的主因。本研究先進行18 種稻米儲藏實驗，觀察酯酶活性與稻米游
離脂肪酸含量的相關性，接著再以水稻台稉9 號米糠為材料，分離純
化其脂肪酶並研究其特性。米糠蛋白質萃取液以硫酸銨沉澱劃分蛋白
質，並以酯酶基質p-nitrophenyl butyrate 與脂肪酶基質p-nitrophenyl
palmitate 進行活性分析。酯酶活性最高出現於30-50% 硫酸銨飽和度
沉澱區間，比活性為2.3×103 U/mg。脂肪酶活性最高則出現於0-30%
飽和度，比活性為8.6 U/mg。然而以蛋白質電泳SDS-PAGE 分析，
酯酶與脂肪酶活性染色的最高活性區，均出現在0-30%硫酸銨飽和度，
取此區間之蛋白質進行酵素純化，發現純化過程導致脂肪酶失去活性，
然而酯酶則不受到影響，可維持高活性的狀態。逐步分析研究發現，
酯酶是親水性高且相當的安定的酵素蛋白質；脂肪酶性質則為高疏水
性蛋白質，分子間疏水性作用強、容易聚集沉澱，不易與水互溶，不
需要二價金屬離子維持或提高活性。脂肪酶酵素安定性低，容易在實
驗過程失活，導致純化困難，故取出電泳分離、具有脂肪酶活性的蛋
白質條帶，利用全系統蛋白質檢定，結果鑑定得到152 條蛋白質，其
中含有14 條熱休克蛋白和2 條油膜蛋白，驗證了0-30%硫酸銨飽和
區的蛋白質，含有高量的輸水性蛋白質，故推測脂肪酶與這些蛋白質
相似，其表面擁有較大面積的疏水性區塊。

Rice bran, rice milling by-product, is rich in lipids and various
bioactive components (such as tocopherols, tocotrienols, γ-oryzanol,
phytosterols, etc). However, carboxylesterase (EC 3.1.1.1) and lipase (EC
3.1.1.3) in rice bran may hydrolyze triacylglycerols releasing glycerol and
free fatty acids which result in rancidity of the bran. This study conducted
rice storage experiments with 18 cultivars to study the relationship
between bran esterase activities and free fatty acid contents. Then rice
cultivar Taikeng 9 (Tk9) was used as material to explore the lipases
activities and their characteristics. Ammonium sulfate precipitation
method was employed to fractionate rice bran proteins. p-Nitrophenyl
butyrate was used as esterase substrate and the highest activity was
observed at the fraction of 30-50% ammonium sulfate with a specific
activity of 2.3×103 U/mg. p-Nitrophenyl palmitate used as lipase
substrate, the highest activity was observed at the fraction of 0-30%
ammonium sulfate with a specific activity of 8.6 U/mg. However,
SDS-PAGE separation showed the highest activities of both enzymes
were at the fraction of 0-30% ammonium sulfate. This fraction of proteins
was further purified and found that the lipase activity lost fast but the
esterase’s remained stable and high. It was found that esterase proteins
were hydrophilic and very stable; the lipase proteins were hydrophobic
and the protein-protein interaction made them prone to aggregation and
precipitation, but need not metal ions to maintain or increase their activity.
Because of lability the lipase proteins were not amenable for traditional
purification procedures. Therefore the lipase proteins with activity
staining on the gel of SDS-PAGE were isolated for LC-MS/MS analysis
to identify their proteins sequences. There were 152 proteins identified.
Of them fourteen were heat-shock proteins and two oleosin protein. It
suggested that the lipase proteins have a similar feature with those
precipitated at the fraction of 0-30% ammonium sulfate, that they bear
significant hydrophobic areas on the protein surface.

第一章 前言…………………………………………………………..1
第二章 文獻回顧……………………………………………………..3
2.1 稻米介紹……………………………………………………...3
2.1.1 稻米性質之分類………………………………………3
2.1.2 稻米結構………………………………………………4
2.1.3 台稉九號……………………………………………….4
2.1.4 米糠組成及應用……………………………………….5
2.1.5 米糠酸敗………………………………………………6
2.2 脂肪酶………………………………………………………...6
2.2.1 脂肪酶種類……………………………………………7
2.2.2 脂肪酶構型……………………………………………8
2.2.3 脂肪酶催化形式………………………………………8
2.2.4 脂肪酶的性質專一性…………………………………9
2.2.5 酯酶…………………………………………………..10
2.2.6 脂肪酶與羰酸酯酶相異之處………………………..10
2.3 米糠脂肪酶之相關研究…………………………………….11
第三章 材料與方法…………………………………………………..13
3.1 實驗材料……………………………………………………..13
3.1.1 儲藏預實驗稻米……………………………………...13
3.1.2 儲藏實驗……………………………………………...14
3.1.3 脂肪酶萃取材料……………………………………...14
3.2 儀器…………………………………………………………..14
3.3 藥品…………………………………………………………..16
3.4 實驗流程…………………………………………………….18
3.4.1 儲藏實驗步驟………………………………………...18
3.4.2 脂肪酶純化分析流程………………………………...19
3.5 儲藏稻米製備………………………………………………..20
3.5.1 材料準備………………………………………………21
3.5.2 酯酶萃取……………………………………………...21
3.6 酯酶活性分析………………………………………………...21
3.6.1 硝基苯酚酯測定法……………………………………21
3.7 成分分析儀…………………………………………………...23
3.8 游離脂肪酸分析……………………………………………...23
3.8.1 脂肪酸萃取……………………………………………23
3.8.2 游離脂肪酸比色分析法………………………………23
3.9 蛋白質電泳分析……………………………………………...24
3.9.1 膠體製備(Native-PAGE gel 及 SDS-PAGE gel)……24
3.9.2 電泳分析(electrophoresis)…………………………….24
3.9.3 酯酶活性染色…………………………………………24
3.9.4 硝酸銀染色法(silver stain)……………………………25
3.10 蛋白質定量………………………………………………….25
3.11 米糠酵素液製備……………………………………………27
3.11.1 米糠製備…………………………………………….27
3.11.2 脂肪酶萃取………………………………………….27
3.11.3 硫酸銨沉澱………………………………………….27
3.12 脂肪酶活性分析…………………………………………….28
3.13 脂肪酶活性染色……………………………………………28
3.14 二維電泳…………………………………………………….29
3.14.1 樣品處理……………………………………………..29
3.14.2 等電點聚焦分離…………………………………….29
3.14.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳…………….30
3.14.4 螢光活性染色……………………………………….31
3.14.5 蛋白質銀染………………………………………….32
3.15 全系統蛋白質檢定………………………………………..32
3.16 脂肪酶性質分析…………………………………………..33
3.17 酵素蛋白質沉澱物性質分析……………………………...33
第四章 結果與討論…………………………………………………...34
4.1 稻米儲藏實驗………………………………………………34
4.2 米糠脂肪酶初步劃分……………………………………….35
4.3 二維電泳……………………………………………………36
4.4 脂肪酶性質分析……………………………………………37
4.4.1 酵素失活……………………………………………...37
4.4.2 去鹽過程與脂肪酶活性喪失的關係………………...37
4.4.3 酵素液通過濾膜的影響……………………………...38
4.4.4 酵素蛋白質沉澱物性質分析………………………...39
4.4.5 NVoy Polymer 對酵素液的效果……………………...40
4.4.6 全系統蛋白質快速檢定……………………………….41
第五章 結論……………………………………………………………43
參考文獻………………………………………………………………..44
附錄……………………………………………………………………..77

表目錄
表1、米糠脂肪酶之相關研究………………………………………..48
表2、硫酸銨劃分之脂肪酶活性分析………………………………..49

圖目錄
圖1、3 種主要性質之稻米…………………………………………..50
圖2、稻米結構圖…………………………………………………….50
圖3、台稉9 號……………………………………………………….51
圖4、米糠脂質水解和氧化路徑…………………………………….51
圖5、脂肪酶催化三酸甘油脂之反應式…………………………….52
圖6、脂肪酶之蓋子(lid)結構……………………………………...52
圖7、Lid 和β5-loop 的構形變化……………………………………53
圖8、米糠酵素蛋白質native-PAGE/SDS-PAGE 電泳分析……….53
圖9、65 種水稻活性分析……………………………………………54
圖10、貯藏過程中糙米蛋白質含量分析…………………………...55
圖11、貯藏過程中精米蛋白質含量分析…………………………...55
圖12、貯藏過程中糙米水分含量分析……………………………...56
圖13、貯藏過程中精米水分含量分析……………………………...56
圖14、貯藏過程中糙米直鏈澱粉含量分析………………………...57
圖15、貯藏過程中精米直鏈澱粉含量分析………………………...57
圖16、貯藏過程中糙米游離脂肪酸含量分析……………………...58
圖17、貯藏過程中酯酶活性分析…………………………………...58
圖18、貯藏過程中糙米游離脂肪酸含量分析……………………...59
圖19、貯藏過程中精米游離脂肪酸含量分析……………………...59
圖20、不同飽和度硫酸銨劃分之米糠蛋白質含量與酯酶活性關係
圖………………………………………………………………………60
圖21、不同飽和度硫酸銨劃分之米糠蛋白質含量與脂肪酶活性關係
圖………………………………………………………………………61
圖22、米糠粗萃液經過硫酸銨劃分之SDS-PAGE 電泳分析酯酶活性
染色……………………………………………………………………62
圖23、米糠粗萃液經過硫酸銨劃分之電泳分析圖………………...63
圖24、米糠脂肪酶二維電泳圖譜…………………………………...64
圖25、硫酸銨(0-30%)沉澱酵素液與以Tris-HCl (10 mM, pH7.5)透析
後之酵素液脂肪酶活性分析…………………………………………65
圖26、硫酸銨(0-30%)沉澱酵素液經過不同處理後之蛋白質含量變
化………………………………………………………………………66
圖27、不同部位酵素液 (0-30%AS) 脂肪酶活性分析……….........67
圖28、硫酸銨(0-30%)沉澱酵素液加入不同濃度Triton X-100 脂肪酶
活性變化………………………………………………………………68
圖29、不同部位酵素液 (0-30%AS) 脂肪酶活性分析與沉澱物加入
Triton X-100 後的活性比較…………………………………………..69
圖30、硫酸銨(0-30%)沉澱酵素液之脂肪酶活性分析其產物吸光值
OD405 與時間關係圖………………………………………………...70
圖31、硫酸銨(0-30%)沉澱酵素液之離心上清液脂肪酶活性分析其產
物OD405 吸光值與時間關係圖……………………………………..71
圖32、硫酸銨(0-30%)沉澱酵素液之離心沉澱物脂肪酶活性分析其產
物OD405 吸光值與時間關係圖……………………………………..72
圖33、硫酸銨(0-30%)沉澱酵素液之離心沉澱物加入Triton X-100 脂
肪酶活性分析其產物OD405 吸光值與時間關係圖………………..73
圖34、硫酸銨(0-30%)沉澱酵素液於不同濃度NVoy Polymer 中離心
後之脂肪酶活性分析…………………………………………………74
圖35、酵素液 (0-30%AS) 在不同濃度NVoy Polymer 中的蛋白質含
量………………………………………………………………………75
圖36、硫酸銨(0-30%)沉澱酵素液透析後離心上清液與沉澱物以NVoy
Polymer 處理之脂肪酶活性比較…………………………………….76

許志聖、宋勳(1994)水稻直播栽培。台中區農業專訊，7: 18-24。
許志聖、楊嘉凌(2009)台中區農業改良場特刊94 號。水稻篇：
40-41。
林文龍(1983)環境與稻米品質。臺中區農業改良場研究彙報。7:
99-107
Prabhakar, J. V., Venkatesh, K. V. L., A simple chemical method for
stabilization of rice bran. J. Am. Oil Chem. Vol 63, pp 644-646
(1986)
Galliard, T. Enzymic degradation. In: Barnes, P. J. (ed) Lipids in cereal
technology. Academic Press, London, pp 111-147 (1983)
Macrae, A. R., Lipase-catalyzed interesterification of oils and fats. J.
Am. Oil. Chem. Vol 60, pp 291-294 (1981)
Okumura, S., Iwai, M., Tsujisaka, Y., Synthesis of various kinds of
esters by four microbial lipases. Biochem. Biophys. Acta.Vol 575, pp
156-165 (1979)
Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.
M., Harel, M., Remington, S. J., Silman, I., Schrag, J., The α/β
hydrolase fold. Protein Eng. Vol 5, pp 197-211 (1992)
Sarda, L., Desnuelle, P., Actions of pancreatic lipase on esters in
emulsions. Biochim. Biophys. Acta. Vol 30, pp 531-521 (1958)
Svendsen, A., Sequence comparisons within the lipase family. In
Woolley, P., and Petersen, S. B. Lipases, Cambridge University
Press, Cambridge, UK , pp 1-21 (1994)
Morgan, P., Watkinson, R. J., Biodegradation of components of
45
petroleum. In Ratledge, C. Biochemistry of microbial degradation,
Kluwer Academic Publishers, Dordrecht, Netherlands, pp 1-31
(1994)
Macrae, A. R., Lipase-catalyzed interesterification of oils and fats. J.
Am. Oil. Chem. Soc.Vol 60, pp 291-294 (1981)
Verger, R., Interfacial activation of lipase: facts and artifacts. Trends
Biotechnol.Vol 15, pp 32-38 (1997)
Fojan, P., Jonson, P. H., Petersen, M. T. N., Petersen, S. B., What
distinguishes an esterase from a lipase: A novel structural approach.
Biochimie.Vol 82, pp 1033-1041 (2000)
Aizono, Y., Funatsu, M., Hayashi, K., Inamasu, M., Yamaguchi, M.,
Biochemical studies on rice bran lipase. part II. chemical properties.
Agri. Biol. Chem. Vol 35, pp 1973-1979 (1971)
Aizono, Y., Funatsu, M., Sugano, M., Hayashi, K., Fujiki, Y.,
Enzymatic properties of rice bran lipase. Agric. Biol. Chem. Vol 37,
pp 2031-2036 (1973)
Aizono, Y., Funatsu, M., Fuziki, Y., Watanabe, M., Purification and
characterization of rice bran lipase II. Agric.Biol.Chem. Vol 40, pp
317-324 (1976)
Ueki, J., Morioka, S., Komari, T., Kumashiro, T., Purification and
characterization of phospholipase D (PLD) from rice (Oryza sativa
L.) and cloning of cDNA for PLD from rice and maize (Zea mays
L.). Plant Cell Physiol. Vol 36, pp 903-914 (1995)
Bhardwaj, K., Raju, A., Rajasekharan, R., Identification, purification,
and characterization of a thermally stable lipase from rice bran. A
new member of the (phospho) lipase family. Plant Physiol. Vol 127,
pp 1728-1738 (2001)
46
Yang, J., Koga, Y., Nakano, H., Yamane, T., Modifying the
chain-length selectivity of the lipase from Burkholderia cepacia
KWI-56 through in vitro combinatorial mutagenesis in the
substrate-binding site. Protein Eng. Vol 15, pp 147-152 (2002)
Lam, H. S., Proctor, A., Rapid methods for milled rice surface total
lipid and free fatty acid determination. Cereal Chem. Vol 78, pp
498-499 (2001)
Walde, P., Nastruzzi, C., Application of a new, simple and economic
colorimetric method for the determination of non-esterified fatty
acids in vegetable oils. Food Chem. Vol 39, pp 249-256 (1991)
Chuang, H. H ., Chen, P. T ., Wang, W. N ., Chen. Y. T ., Shaw. J. F .,
Functional proteomic analysis of rice bran esterases/lipases and
characterization of a novel recombinant esterase dx. Food Chem.
Vol 59, pp 2019-2025 (2011)
Prabhu, A. V., Tambe, S. P., Gandhi, N. N., Sawant, S. B., Joshi, J. B.,
Rice bran lipase: extraction, activity, and stability. Biotechnol. Prog.
Vol 15, pp 1083-1089 (1999)
Devin, J. R., Oscar A. P., A Simple method to measure lipase activity
in wheat and wheat bran as an estimation of storage quality. J. Am.
Oil. Chem. Soc. Vol. 83, no. 5, pp 415-419 (2006)
Lam, H. S., Proctor, A., Jean-Fran&;ccedil;ois M., Free fatty acid formation
and lipid oxidation on milled rice. J. Am. Oil. Chem. Soc. Vol 78, pp
1271-1275 (2001)
Kanchan, B., Aruna R., Ram, R., Identification, purification, and
characterization of a thermally stable lipase from rice bran. A New
Member of the (Phospho) Lipase Family1. Plant Physiol. Vol 127,
pp 1728-1738 (2001)
Mamun, A. M ., Andrew P., Relative FFA formation and lipid
oxidation of commercially milled unseparated, head, and broken rice.
47
J. Am. Oil. Chem. Soc. Vol. 80, no. 12, pp 1183-1186 (2003)
Prim, N., S&;aacute;nchez, M., Ruiz, C., Pastor, F. I. J., Diaz, P., Use of
methylumbeliferyl-derivative substrates for lipase activity
characterization. J. Mol. Catal. B-Enzym. Vol 22, pp 339-346 (2003)
Fatemeh, M., Ramu M. R.,Witoon, P., Wayne E., Marshall, M. W.,
Mohammed, A. Lipase and lipoxygenase activity, functionality, and
nutrient losses in rice bran during storage. Bulletin Number 870
(2000)
Monsoor, M. A., Proctor, A., Effect of water washing on the reduction
of surface total lipids and FFA on milled rice. J. Am. Oil. Chem. Soc.
Vol. 79, no. 9, pp 867-870 (2002)
Juan, P., Esther, M., Maria, J., Juan, F. G., Laura, C. de. Tera’n., Juan,
B., Preparation of a rice bran enzymatic extract with potential use as
functional food. Food Chem. Vol 98, pp 742-748 (2006)
Feliz, D. J ., Proctor, A ., Monsoor, M. A ., Eason, R. L., The effects of
damaged kernel caused by combine harvester settings on milled rice
free fatty acid levels. Food Chem Toxicol. Vol 70,pp C376-C379
(2005)
Barros, M ., Fleuri, L. F ., Macedo, G. A ., Seed lipases: sources,
applications and properties-a review. BRAZIL. J. CHEM. ENG. Vol.
27, no. 01, pp 15-29 (2010)