臺灣博碩士論文加值系統

苯腎上腺素[(R)-phenylephrine, (R)-PE]常作為控制血壓或減緩鼻塞的藥劑使用，目前生產苯腎上腺素的方式是以化學合成為主，但此方法的立體選擇性不佳且對於環境造成傷害，通常亦會導致生產成本提高。利用分離純化的酵素或全細胞轉換進行不對稱的酮基還原，生產高純度的 (R)-PE是目前較佳的方式。先前之研究已從Serratia marcescens BCRC 10948篩選到NADPH-dependent short-chain dehydrogenase/reductase (SM_SDR)，能夠生產大於99%光學純度 (R)-PE之酵素，但其產率太低 [0.57 (mmole/l．h)]，並不利於日後工業生產。本實驗目的是利用已解析完成之SM_SDR晶體結構設計定點突變，選擇預期可改變酵素活性或偏好NADH 作為cofactor的Y40，A42，F98，T193，M195，N196，F202，L206等8個胺基酸位點進行定點突變。經篩選定序後總共得到35種變異酵素並於BL21(DE3)進行全細胞轉換，其中變異酵素A42和F98轉換活性並未明顯提昇，變異酵素Y40D、F98L、T193N、T193D、M195A、N196S、L206Y和N185Q偵測不到轉換活性。而預期可增加受質親和性或擴大結合口袋的F202變異酵素F202A、F202K、F202R、F202D和F202E，經利用10 mM HPAME反應3小時，(R)-PE產量至少提升了約1.3倍的產量。另外，實驗中亦分析全細胞轉換時，不同碳源和HPMAE受質濃度的最適化，以2% glycerol作為碳源，在40 mM 的HPMAE受質濃度，可作為野生株生產 (R)-PE的最佳條件。然而在50 mM 的HPMAE受質濃度反應48小時，變異酵素F202A、F202K、F202R、F202D和F202E的 (R)-PE產量較野生株分別提升了約1.84、2.02、1.53、1.64和1.87倍。另外，本研究也將SM_SDR野生株及F202A突變基因轉型至S. marcescens BCRC10948中表現並進行全細胞轉換。以5% 的細胞濃度在50 mM 的HPMAE受質濃度進行反應，野生株和變異酵素F202A經反應12小時後，分別得到23.82±0.75 mM及38.52±1.84 mM之 (R)-PE。當提高到10%的細胞濃度，變異酵素F202A能夠生產43.79±2.57 mM之 (R)-PE，但野生株產量無法增加。在重複利用菌體進行生物轉換方面，變異酵素F202A可進行6次轉換，若與第一次的轉換率作比較，其轉換率都保持在95%以上。此外，藉由饋料批次式反應 （fed-batch progress）來生產 (R)-PE，其產量可顯著提高，可得到134.09±2.97 mM (R)-PE，轉換率達到89.3% 和2.97 mmol/l·h之產率。

(R)-phenylephrine, (R)-PE, is often used as a medical treatment for blood pressure control and nasal decongestant. Conventional chemical synthesis is the current method being used for (R)-PE production. However, poor enantioselectivity and environmentally unfriendly are the major drawbacks and increase the production costs. Asymmetric bioreduction of prochiral ketone using purified enzymes or whole-cell system is more attractive method for production of optical pure (R)-PE. In our previous report, a novel NADPH-dependent short chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC10948 was used for the reduction of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone (HPME) to (R)-PE with more than 99% enantiomeric excess. However, a low conversion yield (51.06%) and productivity [0.57 (mmole/l．h)] limited the application of this method in industrial processes. In this study, the crystallographic structure of SM_SDR was used as a structural basis for site-directed mutagenesis to identify SM_SDR mutants that either enhance catalytic activities or change their cofactor preference from NADPH to NADH. A total of 35 mutants were constructed and then transformed into BL21(DE3) for whole cell conversion. Results showed that no significant change in conversion efficiency was observed for most of the mutants. However, mutational changes at Y40D, F98L, T193N, T193D, M195A, N196S, L206Y and N185Q resulted in completely lost of their enzymatic activities. Mutations at position F202 were designed to enhance the binding affinity with HPMAE or expand the binding pocket. Results revealed that F202A, F202K, F202R, F202D, F202E displayed at least 1.3-fold increase in (R)-PE production when using 10 mM HPMAE as substrate. The effect of different carbon sources and HPMAE concentrations on whole-cell bioconversion were evaluated. Results showed that 2% glycerol and 40 mM HPMAE, respectively, are the best conditions for (R)-PE production. However, the variants F202A, F202K, F202R, F202D and F202E have 1.84, 2.02, 1.53, 1.64 and 1.87 fold increase in (R)-PE production than wild type SM_SDR at 50 mM HPMAE at 48 h. Homologous expression of the cloned wild type and F202A SM_SDR in S. marcescens BCRC10948 were also carried out to enhance the (R)-PE production. When 5% of the recombinant cells was used for bioconversion, the wild type and F202A produced 23.82±0.75 mM and 38.52±1.84 mM of (R)-PE, respectively, after 12 h reaction using 50 mM HPMAE as substrate. The (R)-PE production can increase to 43.79±2.57 mM when 10% of the F202A but not for wild type recombinant cells were used for reaction. Moreover, the F202A recombinant cells can be recycled for 6 times and remaining over 95% conversion efficiency when compared to the first cycle. The fed-batch culture was performed to further improve the (R)-PE production, results revealed that (R)-PE production was dramatically increased to 134.09±2.97 mM with conversion yield of 89.3% and productivity of 2.97 mmol/l．h.

目錄
中文摘要………………………………………………………………i
Abstract………………………………………………………………ii
縮寫字對照表………………………………………………………………iii
前言………………………………………………………………1
材料與方法
I. 材料
1. 菌種與質體………………………………………………………………7
2. 藥品………………………………………………………………7
3. 分析管柱………………………………………………………………7
4. 酵素………………………………………………………………7
5. 引子………………………………………………………………7
6. 培養基及緩衝液………………………………………………………………8
II. 實驗方法
1. 小量質體DNA抽取………………………………………………………………9
2. DNA片段回收………………………………………………………………9
3. 接合作用 (ligation)………………………………………………………………9
4. 製備E. coli之熱休克勝任細胞……………………………………10
5. 熱休克轉型作用………………………………………………………………10
6. 製備S. marcescens BCRC10948之電穿孔細胞…10
7. S. marcescens BCRC10948之電轉型條件……………10
8. 菌種保存………………………………………………………………10
9. 聚合酶鏈鎖反應……………………………………………………………11
10. S. marcescens BCRC10948 sdr基因之定點突變…11
11. 重組蛋白表現、純化……………………………………………………11
11.1. 於BL21(DE3)進行蛋白表現………………………………………11
11.2. 於S. marcescens BCRC10948進行同源蛋白表現…12
11.3 蛋白定量標準曲線(Bio- Rad protein assay)…12
12. SDS-PAGE膠體電泳分析……………………………………………………13
13. 全細胞轉換HPMAE生產PE…………………………………………………13
13.1. 在E.coli BL21 (DE3)進行全細胞轉換…13
13.1.1. 不同碳源分析對於全細胞轉換之影響…13
13.1.2. 不同受質濃度對於全細胞轉換之影響…13
13.2. 在S. marcescens BCRC10948進行全細胞轉換…14
13.2.1. 不同反應條件對於全細胞轉換之影響…14
13.2.2. 重複利用菌體 (cell recycle)對全細胞轉換的影響………………………………………………………………14
13.2.3. 饋料批次式反應 (Fed batch process)對全細胞轉換的影響………………………………………………………………14
14. 高效能液相層析 (High Performance Liquid Chromatography, HPLC)………………………………………………………………14
15. 酵素活性分析………………………………………………………………15

結果
一、 於BL21 (DE3) 進行SM_SDR之表現及酵素活性分析………………16
二、利用BL21 (DE3) (pET30a-sdr10)全細胞轉換生產(R)-PE之最適化條件………………………………………………………………16
1. 不同的碳源影響……………………………………16
2. 不同濃度的glycerol影響………………17
3. 不同受質濃度的影響…………………………17
三、 SM_SDR變異酵素之構築及蛋白表現………………………………………17
四、 於S. marcescens 10948表現SM_SDR及其變異酵素進行全細胞轉換………………………………………19
1. 構築pUC-T5-sdr10和變異酵素並轉型送入S.marcescens BCRC10948…………………………………………19
2.於S.marcescens BCRC10948表現SM_SDR……………………………20
3. 比較不同 HPMAE受質濃度對全細胞轉換的影響…………………20
4. 重複利用菌體進行全細胞轉換的影響………………………………………21
5. 持續添加HPMAE進行全細胞轉換的影響…………………………………22
五、 選殖具轉換HPMAE為 (R)-PE能力之基因…………………………22

討論
一、 找尋在 E. coli 全細胞最適的轉換條件藉以提升 (R)-PE產量…23
二、構築提高酵素活性或受質親合性之SM_SDR變異酵素……………………24
三、 在S. marcescens 10948表現SM_SDR及變異酵素SM_SDR F202A ………………………………………25
四、 選殖具轉換HPAME為 (R)-PE能力的基因………………………………26

參考文獻………………………………………………………………28
表、………………………………………………………………33-40
圖、………………………………………………………………41-56

附錄
附錄一、 培養基與緩衝液………………………………………………………………57
附錄二、 不同環境因子分析S. marcescens BCRC10948 (pUC57-T5-sdr10)對於全細胞轉
換之影響9小時 (R)-PE的產量………………………………………………………………62
附錄三、 SM_SDR與SQ_SDR2胺基酸序列比對分析…………………………………63
附錄四、 SM_SDR與SQ_SDR胺基酸序列比對分析……………………………………64
 
表目錄

表ㄧ、本實驗所使用之菌種…………………………………………33
表二、本實驗所使用與構築之質體………………………………………………34
表三、本實驗所使用的核甘酸引子………………………………………………………………36
表四、以BL21 (DE3)表現不同變異酵素SM_SDR進行細胞轉換HPMAE為 (R)-PE………………………………………………………………39
表五、不同細胞密度對於全細胞轉換之影響對於S. marcescens BCRC10948 表現 SM_SDR and F202A變異酵素SM_SDR進行細胞轉換HPMAE為 (R)-PE………………………………………………………………40
 
圖目錄

圖一、 常見類交感神經胺類及其結構……………………………………………………41
圖二、SDS-PAGE分析不同濃度IPTG對BL21 (DE3) (pET30a-sdr10)表現SM_SDR之情形………………………………………………………42
圖三、以SDS-PAGE分析BL21(DE3) (pET30a-sdr10)表現及純化SM_SDR之情形………………………………………………………………43
圖四、利用2%不同的碳源進行BL21 (DE3) pET30a-sdr10全細胞轉換反應12小時PE的產量…………………………………………………44
圖五、利用不同濃度的glycerol以50 mM HPMAE進行BL21 (DE3) pET30a-sdr10全細胞反應………………………………………45
圖六、SDS-PAGE分析BL21 (DE3) (pET30a-sdr10)與變異酵素表現之情形……………………………………………46
圖七、利用2% glycerol以10 mM HPMAE進行BL21 (DE3) pET30a-sdr10和變異酵素進行全細胞反應………………………………47
圖八、以BL21 (DE3) (pET30a-sdr10 ) 和變異酵素表現SM_SDR在不同HPMAE濃度進行細胞轉換為PE之影響……………………48
圖九、以HPLC分析BL21 (DE3) (pET30a-sdr10) (A)與變異酵素sdr10 F202A (B), sdr10 F202K (C), sdr10 F202R (D), sdr10 F202D (E)和sdr10 F202E (F)轉換HPMAE為PE之旋光性…………………49
圖十、利用BL21 (DE3) (pET30a-sdr10 ) 和變異酵素表現SM_SDR菌株進行重複菌體生產PE之全細胞轉換反應……………………………………50
圖十一、質體pUC57-T5-sdr10 (A)及變異酵素pUC57-T5-sdr10 F202A (B)之構築………………………………………………………………51
圖十二、SDS-PAGE分析S. marcescens (pUC57-T5-sdr10)與變異酵素表現之情形………………………………………………………………52
圖十三、以S. marcescens (pUC57-T5-sdr10) (A)和變異酵素(pUC57-T5-sdr10 F202A)(B)表現SM_SDR在不同HPMAE 濃度進行細胞轉換為PE之影響………………………………………………………………53
圖十四、利用S. marcescens (pUC57-T5-sdr10 和pUC57-T5-sdr10 F202A )菌株進行重複菌體生產PE之全細胞轉換反應………………………………………………………………54
圖十五、S. marcescens (pUC57-T5-sdr10 F202A)持續添加HPMAE進行全細胞轉換反應………………………………………………………………55
圖十六、以HPLC分析BL21 (DE3) (pET30a-SQSDR2)轉換HPMAE為PE之旋光性………………………………………………………………56

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