标题: 利用双水相萃取纯化分离生物分子及木糖□水解酵素的催化机构与应用研究
Extraction of biomolecules by aqueous two-phase systems,and mechanistic study and application of beta-xylosidase
作者: 潘一红
I-Horng Pan
李耀坤
Yaw-Kuen Li
应用化学系硕博士班
关键字: 木糖□水解酵素;双水相萃取;催化机构;大黄;栀子;beta-xylosidase;Aqueous Two-Phase Extraction;Mechanistic study;Rhubard;Gardenia jasminoides Ellis
公开日期: 2000
摘要: 文献中报导,天然物质含酵素或中草药对现代医学的贡献良多。尤其,天然物中许多活性成分的陆续被发现,引起了人们对天然物或其生物工程产品极大之兴趣。近几十年来生物工程产品持续快速发展,然而对此产品而言,如何发展更新颖、实用之分离与纯化技术是相当重要之课题,因为生物产品就其生产成本而言,分离纯化费用常需占成本之90%以上。因此本论文以木糖□酵素(□-xylosidase)及两种天然植物(大黄及栀子)为研究对象,利用双水相萃取技术(Aqueous Two-Phase Extraction)从上述几种物质中纯化分离糖□类化合物、糖□水解酵素,并探讨其应用。
本研究共分为几个部分,其研究内容及结果简述如下:
(1)利用poly(ethylene glycol)(PEG)及sodium dihydrogenphosphate(NaH2PO4)所形成之双水相系统,从含Trichoderma koningii G-39之发酵液中萃取分离木糖□酵素(□-xylosidase),当双水相组成为25% (w/v) PEG,20-25% (w/v) NaH2PO4时,可从salt-rich phase(水相层)中得到高产率、高浓缩之□-xylosidase。实验中亦探讨,当实验制程放大至1-liter 规模时,□-xylosidase 之纯度(purity)与总活性(total activity)可比原未处理之发酵液增加至少33倍与422%以上。
(2)结合双水相与部分选择性沉淀技术,从含Trichoderma koningii G-39之发酵液中萃取分离木糖□酵素(□-xylosidase)。实验研究中发现PE62 (20% ethylene,80% propylene oxide)polymer 在此纯化步骤中扮演极为重要之角色。而实验结果显示,在短时间内,且不需要任何管柱层析(column chromatography)的协助之下,由SDS-PAGE 分析得知所纯化之酵素为单一成份且纯化倍率提高8.8倍,产率达100%
(3)以氢核磁共振光谱(NMR spectroscopy)探讨木糖□酵素(□-xylosidase)水解DNPX(2,4-dinitrophenyl -□-D-xylosides)或PNPX(p-nitrophenyl -□-D-xylosides)等受质时,我们得到组态保留(retention configuration)之产物。实验结果显示此催化反应应为两步骤(two-step),第一步为xylosylation(形成xylosyl-enzyme 中间体),第二步进行水解反应,形成dexylosylation中间体而得产物。以受质离去基之pKa对logkcat及logkcat/Km作图,可得Bronsted plot。由Bronsted plot 结果显示,pKa 对logkcat 呈单一线性关系(□1g =0),其斜率为零,表示反应速率决定步骤与离去基之强弱无关,因此dexylosylation step应为催化反应之速率决定步骤(rate-limiting step)。
二级同位素效应(secondary deuterium kinetic isotope effect)之实验得知,催化反应的进行类似SN2之机制。
(4)本研究利用由PE62、Na2HPO4及C2H5OH所组成之双水相系统,配合温度诱导效应,在很短的时间内可将大黄萃取液中之缓泻成分如sennoside A和B 与此萃取液中可能具药理毒性之五种游离态□□衍生物如 rhein、emodin、chrysophanol、aloe-emodin及physcion等分离。实验中显示最佳萃取分离之双水相组成为10% (w/v) PE62,5% (w/v) Na2HPO4及20% (v/v) C2H5OH,至少 95%以上之sennoside A和B 分布至salt rich phase(水层),而大部分之游离态□□衍生物则分配至polymer rich phase(PE62 层)。将水层再进行简单处理,可得purity提高1.63倍,回收产率为80.1%之缓泻成分sennoside A和B。
(5)近年来geniposide神奇的疗效陆续被发现后,兴起了人们极大之兴趣,尤其如何发展快速且简单之分离纯化技术已成为重要课题。本研究利用双水相萃取分离系统,开发一套快速纯化geniposide之方法,实验中探讨各种可能影响分离系统之因素,以了解geniposide在双水相系统之分配行为外,并由较好的条件中进行纯化。实验结果显示当在双水相系统为5% (w/v) PE62、7.5% (w/v) KH2PO4及10% (v/v) C2H5OH时,90%以上之geniposide可分配至salt rich phase,经用95% C2H5OH再纯化处理后,可得纯度为40.3%之产物,比原出萃液(纯度为26.9%)高出许多,而总回收率仍达到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.
URI: http://140.113.39.130/cdrfb3/record/nctu/#NT890500001
http://hdl.handle.net/11536/67617
显示于类别:Thesis