大豆中異黃酮水解酵素之研究： 一、含醣基大豆異黃酮之β-糖苷鍵水解酶之基因選殖及特性分析 二、含丙二醯葡萄糖苷異黃酮之酯解酶之分離與純化

Abstract

大豆(Glycine max L.)是廣泛使用在食品及食品加工的原料，含有多種機能性成分，例如：具有雌激素活性的大豆異黃酮。大豆異黃酮有12種形式，在大豆中含量較高的為malonylglucosides以及glucosides兩種，但此兩種不易被人體吸收，若能經由malonylesterase以及β-glucosidase將此兩種異黃酮轉換為aglycones形式，則可提高大豆異黃酮之生體可用率(bioavailibility)。 在實驗室先前的研究中，從大豆渣分離出Glycine max β-glucosidase (GmBGL)，經由蛋白質N端定序得到十個胺基酸序列。本研究第一部分利用此序列片段，從黃豆選殖GmBGL基因。結果顯示，GmBGL的ORF (open reading frame)由1884個鹼基組成，轉譯成627個胺基酸，分子量為69 kDa，等電點為8.98。利用農桿菌感染水稻(Oryza sativa L.)，以ubiquitin啟動子驅動GmBGL在水稻大量表現。結果顯示，GmBGL之RNA及蛋白質在轉殖水稻中均有表現。轉殖水稻中的重組GmBGL可催化glucosides (genistin和daidzin) 水解為aglycones (genistein和daidzein)，確認GmBGL是具有功能的β-glucosidase基因。與其它植物之β-glucosidase基因進行序列比對，GmBGL屬於第三族醣苷水解酵素，具有第三族醣苷水解酵素在功能區的保守性序列。分析GmBGL在大豆不同組織的表現，發現在根、莖、葉、果莢和成熟種子都有表現，其中又以根部和葉部最多。 第二部分的研究由大豆分離malonylesterase。將大豆加水打成豆漿後，以0.5%氯化鈣沉澱雜蛋白可以在上清液測得最高的esterase比活性。經過超過濾濃縮、硫酸銨沉澱，選取40-50%飽和度硫酸銨沉澱部分，再以陰離子交換管柱層析收集活性區，最後以活性染色分析得到esterase活性條帶。經過分離純化的步驟，malonylesterase活性回收率為0.208%，純化倍率為73倍。研究結果顯示，大豆具有對malonylglucosides有專一性水解能力的esterase。

Soybean (Glycine max L.) is widely consumed in food and food processing. In soybean, it contains many bioactive components including isoflavones which have estrogenic effects. Soy isoflavones are known to occur in 12 conjugation forms. The predominant forms are malonylglucosides and glucosides, but aglycones were absorbed faster and in greater amounts than their glucosides in humans. Therefore, the bioavailability of isoflavones is able to be improved when malonylglucosides are hydrolyed by malonylesterase and β-glucosidase to produce aglycones. In our previous work, a novel Glycine max β-glucosidase (GmBGL) was isolated from soybean okara and ten N-terminal amino acids were determined. In the first part of this thesis, GmBGL gene was cloned from soybean according to Phytozome blast search with this N-terminal partial sequence. The cDNA of GmBGL contained an ORF (open reading frame) of 1884 bp coding for 627 amino acids. Sequence analysis revealed that the gene encodes a 69 kDa enzyme, which has a theoretical isoelectric point at 8.98. The GmBGL was expressed in rice (Oryza sativa L.) under the control of the constitutive ubiquitin promoter by Agrobacterium-mediated transformation. The recombinant GmBGL expressed in rice catalyzed the hydrolysis of glucosides (genistin and daidzin) to produce aglycones (genistein and daidzein). This result confirmed that GmBGL was indeed a fuctional β-glucosidase gene. Based on the alignment analysis of the deduced amino acid sequence of GmBGL and other plant β-glucosidase, GmBGL was assigned to glycosyl hydrolases family 3 and contained the conserved motif which is thought to be the active site in family 3 members. Analysis of gene expression in various tissues of soybean showed that the GmBGL transcript was presented in stem, pod and mature seed and particularly highly accumulated in root and leaf. The second part of this thesis is to isolate the malonylesterase which is capable specificity to hydrolyze malonylglucosides into simple glucosides of isoflavone. Soybeans were soaked and then homogenized to prepare soybean milk. Isolation processes of the malonylesterase involved using calcium chloride to remove the intrinsic storage protein of soybean, ammonium sulfate fractionation, and DEAE anion exchange chromatography. The results indicated that the supernatant from 0.5% (w/v) calcium chloride showed a superior esterase activity. 40-50% saturation interval of ammonium sulfate precipitation possessed the most specific activity of malonylesterase. After further fractionation by DEAE anion exchange chromatography, an active esterase band was detected through Native PAGE and zymographic analysis and moreover, the corresponding protein also showed high specificity to malonylglucosides. In this purification process, the activity recovery of malonylesterase was 0.208% with a 73-fold purification efficiency.