| Summary: | 右旋-乙內醯脲酶（D-Hydantoinase, DHTase）是一重要生化觸媒，能將D-5-substituted hydantoing水解成N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG)，之後再利用N-carbamoyl-D-amino acid amidohydrolase (DCase)水解成D-p-Hydroxyphenylglycine (D-p-HGP)。D-p-HGP能與六青黴素酸（6-aminopenicillanic acid, 6-APA）反應生成合成Amoxicillin，Amoxicillin、Cefadroxil、Cefatrizine、Cefaparole和Cefaperazon屬於β-lactam抗生素。在全球巿場，每年有數千噸β-lactam抗生素需求量應用於治療方面。 脂肪分解酶能催化油脂水解成甘油和長鏈脂肪酸，而且當其與脂質表面接觸時能因酵素構型改變而增加其活性。Candida antarctica lipase B (CalB)因為具有廣泛的基質通用性、高有機溶劑的耐受性、立體選擇性與光學選擇性而被廣泛應用。因此，CalB 在動力學分析、胺解、酯化、與轉酯化等工業應用上被認為是一種廣泛使用的生物催化劑。 近年來純化技術發展，固定化金屬親和薄膜（immobilized metal affinity membranes, IMAM）己被應用於酵素純化領域，因為它較一般填充床系統無顆粒內部擴散問題，且具有短擴散途徑、低的壓力降和易規格化等優點。此外，由於不同酵素皆具有其獨特的特性，如何有效利用固定化技術來克服酵素在工業生物催化上可能遭遇的問題，例如:酵素的回收率、穩定性、可利用性與保存性等則變成相當重要的焦點，一個適當的固定化程序將可大大提升酵素在業界成功使用的可行性。 本研究共分四個部分，第一到三部分探討如何製備親水性固定金屬親和薄膜來進行右旋-乙內醯脲酶 (DHTase)的純化與固定化，第四部分則探討如何製備疏水性固定化金屬親合薄膜固定化脂肪分解酵素(CalB)。 第一部份探討不同金屬離子對DHTase活性與在IMAM系統純化效率的影響性。在批次的吸附實驗中可以發現當選用銅離子當ligands時擁有DHTase的最大吸附量(1.513 ± 0.12 mg)；選用鈷離子當ligands時擁有DHTase最小吸附量但卻在薄膜上展現出最高活性。在DHTase粗酵素液中添加錳、鈷、鎳、二價鐵與三價鐵離子時對DHTase的活性有增強作用，但若將銅離子添加於經nickel ions chelated IMAM (Ni-IMAM)純化後的DHTase，卻反而會抑制其活性。cobalt ions chelated IMAM (Co-IMAM)、Ni-IMAM與zinc ions chelated IMAM (Zn-IMAM) 對DHTase的純化倍率約6-7倍，此外，從電泳圖中也可以發現經IMAM純化後的DHTase只具有單一分子量 ，這表示IMAM純化系統對DHTase而言有高度專一性，純化效果佳。若在不同金屬離子當ligands的IMAM純化系統下，所得到的純DHTase中添加相對應的金屬離子，則DHTase的活性也會隨之增加，其中又以添加錳離子在用Mn-IMAM純化後的DHTase與添加鈷離子在Co-IMAM 純化後的DHTase活性增加最多(約9-10倍)。 第二部份藉由化學藥劑改質再生纖維素薄膜並用鎳離子當ligands來建構IMAM用來固定化DHTase。固定化DHTase的最適化條件如下:製備完成之IMAM膜有高的鎳離子吸附量155.5±5 μmol Ni2+/disc ，使用0.1 M 、pH質為8並添加0.8M氯化鈉的Tris–HCl緩衝液來固定化DHTase ，所需時間為14小時，且膜上DHTase 活性達4.2±0.3 U/disc。經過固定化後的DHTase比粗酵素液對pH值與熱的耐受性較大，在經過15次的重複使用下仍有99%活性，且其保存在4℃、pH8 、0.1M Tris-HCl的緩衝溶液下經過7週仍有99% 活性 第三部份利用改質後再生纖維素薄膜，再以鈷、鎳、銅、鋅四種金屬離子分別當 ligands製備成不同種類的IMAM，其吸附等溫線與動力學參數(Vmax, Km) 也分別被計算與比較，其結果發現Co-IMAM 有最高的比活性，Co- IMAM其吸附鈷離子能力為161.4±4.7 μmol Co2+/disc且其膜上DHTase活性達4.1±0.1 U/disc。因為Co-IMAM膜上活性與Ni-IMAM相近，且只有0.08 mg/disc 的蛋白質量在其上，因此Co-IMAM的比活性為51.9 U/mg，是Ni-IMAM的4.9倍。Co-IMAM 與Ni-IMAM一樣都具有pH與熱的耐受性，在經過7次的重複使用下仍有99%活性，且其保存在4℃、pH8 、0.1M Tris-HCl的緩衝溶液下經過4週只有一點活性的損失。這是所有文獻中第一次使用鈷離子來當ligands且能同步純化與固定化酵素的操作方式。 第四部分利用3-Glycidoxypropyltrimethoxysilane (GPTMS) 與RC膜作用生成網狀疏水性固定化親和薄膜。當 GPTMS 與鹽酸作用時，比使用氫氧化鈉更容易使epoxy 開環，但是鹽酸濃度愈高，反而會造成RC膜因酸水解而損傷，導致薄膜太脆弱而限制其使用。在最適化反應條件: 一片RC膜浸泡於24 ml、 18.75 ％ HCl及1ml溶液，以24 °C、150 rpm 反應12 h進行製備，並使用IDA當螯合劑來鍵結鎳離子，製備完成之IMAM膜有高吸附71.5±1.5 μmol Ni2+/disc與高活性 6.74 ± 0.2U CalB/disc之量。此外，在經過10次重複使用下仍有95％活性，且其在保存性的測試上經過4週仍有97％活性。 D-Hydantoinase (DHTase) is an important bioenzyme, which could hydrolysis D-5-substituted hydantoin to N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG). Then, an-other enzyme N-carbamoyl-D-amino acid amidohydrolase (DCase) would hydrolysis Nca-HPG to D-p-Hydroxyphenylglycine (D-p-HGP). D-p-HGP could react with 6-aminopenicillanic acid (6-APA) and become to Amoxicillin. Amoxicillin、Cefadroxil、Cefatrizine、Cefaparole and Cefaperazon areβ-lactam antibiotics. The global market demand of β-lactam antibiotics reaches thousands of tons annually for medical treatment. Lipases (EC 3.1.1.3) catalyze the hydrolysis of esters formed from glycerol and long-chain fatty acids. The activity of lipases is dramatically increased upon binding to the lipid surface, due to a conformational change of the enzyme. Candida antarctica lipase B (CalB) is preferred in many applications, because of its versatility with respect to substrates, high resistance to organic solvents, high thermal stability, stereo specificity and high enanti-oselectivity. Therefore, CalB is one of the wildly used biocatalysts in industry, including ki-netic resolutions, aminolysis, esterification, and transesterification. Among the recently developed purification techniques, immobilized metal ion affinity membrane (IMAM) has been widely applied in the enzyme purification processes with ad-vantages such as no intra-particle diffusion, short diffusion path, low pressure drop and easier scale up, which limited in conventional packed-column systems. Besides, How to adopt effi-cient immobilization technique to overcome some of the problems of enzymes as industrial biocatalysts: enzyme recovery, enzyme stability, reusiblity and storage are a critical focal point. A suitable immobilized method is the key point that greatly increases the possibilities of success. In this paper, it contains four parts. From part 1 to 3, how to prepare hydrophilic im-mobilized metal ion membrane for DHTase purification and immobilization is our object. In part 4, hydrophobic immobilized metal ion membrane has been prepared and immobilized CalB. In part 1, the complex effects of metal ions on DHTase purification with an immobi-lized metal affinity membrane was explored. Batch DHTase adsorption experiments showed that the adsorption capacity varied remarkably for IMAMs with different metal ions. The maximum adsorption of DHTase (1.513 ± 0.12 mg) was reached when using Cu2+ as the che-lated ion, whereas the Co2+ showed the highest activity on membrane with only small amounts of protein adsorption. The Mn2+, Co2+, Ni2+, Fe2+ and Fe3+ additions showed a posi- tive effect on DHTase activity. The addition of Cu2+ showed a varied effect from the inhibition on original DHTase to the promotion on Ni-purified DHTase. The purification folds using IMAM chelated with Co2+, Ni2+, and Zn2+ were in the range of six to seven. SDS-PAGE anal-ysis showed that all of the IMAM-purified DHTase exhibited the same molecular weight, meaning DHTase adsorbed on IMAM was highly specific. The DHTase purified by different metal ions showed various levels of increased activity when adding the corresponding metal ions. The addition of Mn2+ or Co2+ displayed a dramatic increase (9- to 10-fold) in activity of DHTase purified by IMAM chelated with the same ion. In part 2, this study constructs the IMAM via using chemical reagents and nickel ion on the regenerated cellulose membrane (RC membrane) to immobilized DHTase. The immobili-zation conditions were studied and the optimal conditions are as follows. By employing an IMAM with nickel ion of 155.5±5 μmol/disc immersed in 0.1 M Tris-HCl buffer pH 8 (with 0.8 M sodium chloride) and immobilized time of 14 h, a DHTase activity of 4.2±0.3 U/disc was obtained. The immobilized DHTase membrane can achieve a larger pH and thermal tol-erant range than that of free enzyme. Meanwhile, the stability test showed that 99% of en-zyme activity could be retained after being repeated 15-times. The storage test also displayed 99% enzyme preservation after 7 weeks of storage (0.1M, pH8, Tris-HCl buffer at 4 ℃). In part 3, various IMAMs were prepared from the RC membrane and chelated with various metal ions such as Co2+, Ni2+, Cu2+ and Zn2+. The adsorption isotherm and the kinetic parameters Vmax, Km of DHTase on IMAMs were studied. The Co-IMAM was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4±4.7 μmol/disc with a DHTase activity of 4.1±0.1 U/disc. Only 0.08 mg/disc protein was coupled onto Co-IMAM, but exhibited a similar activity as that us-ing Ni as ligand. Owing to this characteristic, a remarkably high specific activity of 51.9 U/mg was obtained for Co-IMAM, which is 4.9-fold higher than that of Ni-IMAM. The phe-nomenon of pH and thermal tolerant is similar to Ni-IMAM. The 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C in 0.1M, pH8 Tris-HCl buffer. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobi-lization operation. In part 4, the hydrophobic immobilized metal affinity membranes were constructed by using 3-Glycidoxypropyltrimethoxysilane (GPTMS) to form the meshed structure support. HCl was the best reagent used to open the GPTMS epoxy ring. However, higher concentration of HCl would distort the RC membrane and weaken its structure for further use. According to the optimization approach, the optimal reaction conditions were found as follows: a RC membrane immersed in 24 ml, 18.75％ HCl and 1 ml GPTMS under 24 °C, 150 rpm for 12 h. IDA was used as the chelating agent to bind nickel ions. In this case, the chelated ions and coupling CalB activity were significantly increased to 71.5±1.5μmol/disc and 6.74 ± 0.2U/disc respectively. Meanwhile, the stability test showed that 95% of enzyme activity could be retained after 10-times repeated use. The storage test also displayed 97% enzyme could be preserved after 4 weeks of storage. 誌謝 . IX 摘要 . XI ABSTRACT . XIII CONTENTS . XVI LIST OF FIGURES . XIX LIST OF TABLES . XXI 1. INTRODUCTION . 1 1.1 FOREWORD . 1 1.2 HYDANTOINASE . 3 1.3 CANDIDA ANTARCTICA LIPASE B (CALB) . 7 1.4 IMMOBILIZED METAL ION AFFINITY CHROMATOGRAPHY (IMAC) . 9 1.4.1 Metal ions . 9 1.4.2 Chelating Ligands . 9 1.5 IMMOBILIZED METAL IONS AFFINITY MEMBRANES (IMAMS) . 13 1.5.1 Preparation of affinity membranes . 13 1.5.2 Active reagents . 14 2. MATERIALS AND METHODS . 18 2.1 MATERIALS AND CHEMICALS . 18 2.1.1 Hydrophilic immobilized metal ion membrane . 18 2.1.2 Hydrophobic immobilized metal ion membrane . 18 2.2 INSTRUMENT . 19 2.2.1 Taiwan lab. . 19 2.2.2 Germany lab. . 19 2.3 METHODS . 21 2.3.1 Preparation of DHTase enzyme . 21 2.3.2 IMAM preparation (via various metal ions as ligands) . 21 2.3.3 Batch DHTase purification with IMAM . 22 2.3.4 Metal ions addition test . 22 2.3.5 Protein assay (SDS-PAGE) . 22 2.3.6 DHTase activity assay . 22 2.3.7 Preparation of purified DHTases . 22 2.3.8 Preparation of IDM . 23 2.3.9 pH and thermal effects for DHTases . 23 2.3.10 pH and thermal stability of DHTases . 23 2.3.11 Reusability and storage stability for hydrophilic-IMAM . 24 2.3.12 Estimation of adsorption isotherm . 24 2.3.13 Determination of energy of activation (Ea) . 25 2.3.14 Nickel ions analysis . 25 2.3.15 Cobalt ions analysis . 25 2.3.16 Protein concentration assay . 25 2.3.17 Hydrophobic-IMAM preparation . 25 2.3.18 CalB immobilization . 26 XVI 2.3.19 CalB activity assay . 26 2.3.20 Water contents assay . 26 2.3.21 Reusability and storage stability for hydrophobic-IMAM . 26 2.3.22 Characterization . 26 3. EXPLORING THE COMPLEX EFFECTS OF METAL IONS ON D-HYDANTOINASE PURIFICATION WITH AN IMMOBILIZED METAL AFFINITY MEMBRANE 30 3.1 FOREWORD . 30 3.2 RESULTS AND DISCUSSION . 30 3.2.1 Ions effect on DHTase . 30 3.2.2 The adsorption capacity of IMAM with different metal ions . 31 3.2.3 DHTase purification using different metal ions . 31 3.2.4 Metal ions effects on purified DHTase . 32 3.3 CONCLUSION . 33 4. SIMULTANEOUS PURIFICATION AND IMMOBILIZATION OF D-HYDANTOINASE ON THE IMMOBILIZED METAL AFFINITY MEMBRANE VIA COORDINATION BONDS . 40 4.1 FOREWORD . 40 4.2 RESULTS AND DISCUSSION . 40 4.2.1 DHTase immobilized on the IMAM . 40 4.2.2 Selection of loading buffer on IDM . 41 4.2.3 Nickel ion adsorption and the amount of nickel ion on the IMAM . 41 4.2.4 pH and thermal stabilities of free DHTase and IDM . 43 4.2.5 Repeated use and storage test of the IDM . 43 4.3 CONCLUSION . 44 5. ENHANCED D-HYDANTOINASE ACTIVITY PERFORMANCE VIA IMMOBILIZED COBALT ION AFFINITY MEMBRANE AND ITS KINETIC STUDY . 51 5.1 FOREWORD . 51 5.2 RESULTS AND DISCUSSION . 51 5.2.1 Selection of chelated metal ions . 51 5.2.2 Adsorption isotherm . 52 5.2.3 Kinetic parameters . 52 5.2.4 Specific affinity for DHTase . 53 5.2.5 Kinetics of hydrolysis on free DHTase and Co-IDM . 54 5.3 CONCLUSION . 55 6. IMMOBILIZATION OF CANDIDA ANTARTICA LIPASE B ON HYDROPHOBIC IMMOBILIZED METAL AFFINITY MEMBRANES . 59 6.1 FOREWORD . 59 6.2 RESULTS AND DISCUSSION . 59 6.2.1 Selection of epoxy opening reagent . 59 6.2.2 Selection of optimal reaction time . 60 6.2.3 Repeated test and storage test . 60 6.3 CONCLUSION . 60 7. FURTHER . 64 REFERENCES .65
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