利用雙水相萃取純化分離生物分子及木糖□水解酵素的催化機構與應用研究

Abstract

文獻中報導，天然物質含酵素或中草藥對現代醫學的貢獻良多。尤其，天然物中許多活性成分的陸續被發現，引起了人們對天然物或其生物工程產品極大之興趣。近幾十年來生物工程產品持續快速發展，然而對此產品而言，如何發展更新穎、實用之分離與純化技術是相當重要之課題，因為生物產品就其生產成本而言，分離純化費用常需佔成本之90%以上。因此本論文以木糖□酵素(□-xylosidase)及兩種天然植物(大黃及梔子)為研究對象，利用雙水相萃取技術(Aqueous Two-Phase Extraction)從上述幾種物質中純化分離糖□類化合物、糖□水解酵素，並探討其應用。 本研究共分為幾個部分，其研究內容及結果簡述如下: (1)利用poly(ethylene glycol)(PEG)及sodium dihydrogenphosphate(NaH2PO4)所形成之雙水相系統，從含Trichoderma koningii G-39之發酵液中萃取分離木糖□酵素(□-xylosidase)，當雙水相組成為25% (w/v) PEG，20-25% (w/v) NaH2PO4時，可從salt-rich phase(水相層)中得到高產率、高濃縮之□-xylosidase。實驗中亦探討，當實驗製程放大至1-liter 規模時，□-xylosidase 之純度(purity)與總活性(total activity)可比原未處理之發酵液增加至少33倍與422%以上。 (2)結合雙水相與部分選擇性沉澱技術，從含Trichoderma koningii G-39之發酵液中萃取分離木糖□酵素(□-xylosidase)。實驗研究中發現PE62 (20% ethylene，80% propylene oxide)polymer 在此純化步驟中扮演極為重要之角色。而實驗結果顯示，在短時間內，且不需要任何管柱層析(column chromatography)的協助之下，由SDS-PAGE 分析得知所純化之酵素為單一成份且純化倍率提高8.8倍，產率達100% (3)以氫核磁共振光譜(NMR spectroscopy)探討木糖□酵素(□-xylosidase)水解DNPX(2,4-dinitrophenyl -□-D-xylosides)或PNPX(p-nitrophenyl -□-D-xylosides)等受質時，我們得到組態保留(retention configuration)之產物。實驗結果顯示此催化反應應為兩步驟(two-step)，第一步為xylosylation(形成xylosyl-enzyme 中間體)，第二步進行水解反應，形成dexylosylation中間體而得產物。以受質離去基之pKa對logkcat及logkcat/Km作圖，可得Bronsted plot。由Bronsted plot 結果顯示，pKa 對logkcat 呈單一線性關係(□1g =0)，其斜率為零，表示反應速率決定步驟與離去基之強弱無關，因此dexylosylation step應為催化反應之速率決定步驟(rate-limiting step)。 二級同位素效應(secondary deuterium kinetic isotope effect)之實驗得知，催化反應的進行類似SN2之機制。 (4)本研究利用由PE62、Na2HPO4及C2H5OH所組成之雙水相系統，配合溫度誘導效應，在很短的時間內可將大黃萃取液中之緩瀉成分如sennoside A和B 與此萃取液中可能具藥理毒性之五種游離態□□衍生物如 rhein、emodin、chrysophanol、aloe-emodin及physcion等分離。實驗中顯示最佳萃取分離之雙水相組成為10% (w/v) PE62，5% (w/v) Na2HPO4及20% (v/v) C2H5OH，至少 95%以上之sennoside A和B 分佈至salt rich phase(水層)，而大部分之游離態□□衍生物則分配至polymer rich phase(PE62 層)。將水層再進行簡單處理，可得purity提高1.63倍，回收產率為80.1%之緩瀉成分sennoside A和B。 (5)近年來geniposide神奇的療效陸續被發現後，興起了人們極大之興趣，尤其如何發展快速且簡單之分離純化技術已成為重要課題。本研究利用雙水相萃取分離系統，開發一套快速純化geniposide之方法，實驗中探討各種可能影響分離系統之因素，以了解geniposide在雙水相系統之分配行為外，並由較好的條件中進行純化。實驗結果顯示當在雙水相系統為5% (w/v) PE62、7.5% (w/v) KH2PO4及10% (v/v) C2H5OH時，90%以上之geniposide可分配至salt rich phase，經用95% C2H5OH再純化處理後，可得純度為40.3%之產物，比原出萃液(純度為26.9%)高出許多，而總回收率仍達到90.6%。

Recently, commercial applications of natural molecules have gained a great deal of interest. To obtain a large amount of those molecules become an important issue in bio-industry. In this study we developed a series methods for purifying the target biomolecules with high yield and high purity without column chromatography. In general, the purification strategy involves the partition of bimolecules between two immiscible aqueous phases. An extracellular protein, □-xylosidase, and few glycosides from rhubarb or gardenia fruit were selected as the target molecules for the study. Results on these topics were summarized as follows: (1) Effective extraction of protein from bulk medium is an important technique in bioresearch. In this study, we describe an extracellular □-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly (ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH2PO4) in aqueous solution. □-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20 - 25% (w/v) NaH2PO4 were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated. (2) A rapid process for purification of an extracellular □－xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspened crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% of ethylene oxide and 80% of propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% of PE62 (w/v) and 5% of Na2HPO4 (w/v). The aqueous two-phase extraction was performed with PE62 (10%)/NaH2PO4 (15%) as phase-forming reagent. SDS-PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography. (3) The mechanistic study of the □ -xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy study, the stereochemistry of enzyme catalyzing the hydrolysis of 2,4-dinitrophenyl and p-nitrophenyl-□-xylosides were unequivocally identified as the retention of anomeric configuration. Based on the kcat values of a series of arylxylosides with the leaving group pKas ranging from 4 ~ 10, an extended Bronsted plot was constructed with a slope (□lg) near zero. Enzymatic hydrolysis of aryl-□-D-xylosides in acetate buffer (pH 4.0) containing 3% or 5% methanol showed a constant product ratio (methylxyloside/xylose). Indicate that a common intermediate, most likely the xylosyl-enzyme intermediate, is present. In the presence of DTT, the kcat values of p-cyanophenyl-□-D-xylopyranoside, and p-nitrophenyl-□-D-xylopyranoside increased largely. A two-step mechanism involving the formation and the breakdown of xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of xylosyl-enzyme intermediate. Secondary deuterium kinetic isotope effects (kH/kD) measured for 2,4-dinitrophenyl-□-D-xyloside is 1.02 ±0.01. Suggest that the transition-state for breakdown of the xylosyl-enzyme intermediate be SN2-like. (4) An effective and rapid method for the extraction and purification of sennosides from liquid media via an aqueous two-phase extraction was developed. The optimized performance was found to add PE62, a random copolymer comprising 20% ethylene oxide and 80% propylene oxide, (10%, w/v), Na2HPO4 (5%, w/v), and C2H5OH (20%, v/v) to the medium. After votexing, the resulting mixture was directly incubated at 35-40oC to form two phases, the salt-rich (top) phase and the PE62-rich (bottom) phase. All sennosides was extracted in the salt-rich phase, whereas the more hydrophobic anthraquinones, such as aloe-emodin, rhein, emodin, chrysophanol, and physcion, exhibited a greater affinity for the PE62-rich phase. Substantial amount of salt in the salt-rich phase can be removed by lyophilizing the salt phase to dryness and followed by extraction with 70% of ethanol. For 1 g of dry rhubarb, the final extraction solution contains 12.1 mg of sennosides. The purity was estimated to be 6% of the total dry weight of the ethanol-extract. As compared with the crude water-extract, the purity of sennosides was enhanced 160% with 80% recovery yield. Most importantly, chrysophanol and physcion were not detected in the final ethanol-extract. The rest of anthraquinones including rhein, aloe-emodin, and emodin were removed at least 55%, 87% and 93%, respectively. This study demonstrated a feasible process for selectively extracting sennosides from a medium with a high recovery yield and substantially removing the contamination of anthraquinones. (5) The partition behavior of geniposide from gardenia fruit under aqueous two-phase system was investigated herein. The system was comprised of PE62, a random copolymer composed of 20% ethylene oxide and 80% propylene oxide, and KH2PO4. Various factors were considered, including : the concentration of salt; the concentration of polymer; the sample loading; and the addition of ethanol. The experimental results demonstrated that the increasing salt concentration or decreasing PE62 concentration results in an enhancement of the geniposide partition in the salt-rich phase. The addition of ethanol and the higher sample loading also promoted the partition efficiency of geniposide. Based on the study, an optimized system containing 5% PE62, 7.5 % KH2PO4, and 10% ethanol was developed and shown to be effective for the extraction of geniposide from gardenia fruit. After the aqueous two-phase extraction, the resulting salt-rich phase was lyophilized to dryness, which was then subject to extraction by 95% ethanol to eliminate salt from geniposide. As compared with the crude water extract, the total recovery yield of geniposide is near 90% with 40% purity.