Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5528
標題: 菌株Mesorhizobium sp. F28腈水合酶之純化及特性與應用
The characteristics and application of nitrile hydratase from Mesorhizobium sp. F28
作者: 馮筠書
Feng, Yun-Shu
關鍵字: nitrile hydratase;腈水合酶醯胺酶丙烯腈丙烯醯胺;amidase;acrylonitrile;acrylamide;acetonitrile;乙腈
出版社: 環境工程學系所
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摘要: 
目前腈化物已作為各式工業產物的原料,且有機腈化物亦做為民生消耗品如肥皂等物質之香味添加物的組成物,但絕大部分腈化物皆會對人體與生物具有致癌與基因突變等危害性,現今已有利用腈水合酶轉換腈化物至醯胺化物的研究,故本實驗目的為自含腈水合酶(nitrile hydratase, NHase)與醯胺酶(amidase)兩酵素系統的菌株Mesorhizobium sp. F28純化出腈水合酶,探討酵素活性最適pH值與溫度、不同抑制物質、碳氮源對活性的影響等酵素特性以獲得最適轉換腈化物累積醯胺化物的條件。並試驗酵素除了能將丙烯腈作為基質外,是否亦能催化水解丙腈(propionitrile)、異丁腈(isobutyronitrile)與苯甲腈(benzonitrile)。瞭解酵素特性後,亦藉由超過濾薄膜生物反應槽、海藻酸鈣與商業產品固定酵素來探討腈水合酶應用於連續轉換丙烯腈累積丙烯醯胺的操作穩定性。另外,本研究還以批次實驗試驗菌株Mesorhizobium sp. F28完整細胞生物轉換乙腈的基本特性。本研究已純化出菌株Mesorhizobium sp. F28腈水合酶,利用含鈷離子之R2A作為培養基培養細胞,約24.8小時後收集菌體,經含鈷離子50 mM potassium phosphate buffer (pH 7.5)清洗懸浮後,利用French press於壓力2793 psi下將細胞打破一次,離心,所得上澄液即為細胞粗萃取液。細胞粗萃取液經50%-80%百分飽合濃度之硫酸銨沉澱分劃、陰離子交換管柱層析 (ANX Sepharose 4 Fast Flow)與膠體過濾管柱層析 (Sephacryl S-200 high Resolution)後,所得最終酵素純化倍率相較未純化前的細胞粗萃取液為5.0,產率則為16% (以酵素總活性計算)。菌株Mesorhizobium sp. F28的腈水合酶分子量大小約為110 kDa,酵素活性最適pH與溫度範圍分別為pH 7.0-7.5與37-45度間。硫酸銅、亞硫酸銀、過氧化氫與高濃度的螯合劑 (EDTA與NaN3)會使酵素相對活性下降,而過硫酸銨與還原劑只會輕微抑制酵素活性,碳氮源如酵母抽出物、葡萄糖與丙酮酸鈉對純化酵素活性則無影響。以酵素基質特異性而言,菌株F28的腈水合酶可以丙烯腈、丙腈、異丁腈與苯甲腈做為基質,其中丙烯腈為最好利用的基質,酵素對丙烯腈的Km值為0.53 mM。文獻中提及能誘導腈水合酶的尿素在氮源豐富的培養基中,並不能有效增加菌株F28腈水合酶活性表現;而當培養基僅含丙烯腈無輔助因子(Co2+)存在時,無法監測到菌株F28的腈水合酶活性。實驗結果發現鈷離子對菌株F28表現高活性的腈水合酶是重要的,當菌株預培養與培養過程中的R2A培養基皆含鈷離子時,所獲得的腈水合酶活性可達328.5 U/ml,相較完全未添加鈷離子時的活性 (70.5 U/ml)高約近5倍。自菌株F28純化的腈水合酶可以超過濾薄膜生物反應槽應用於連續轉換丙烯腈累積丙烯醯胺,當溫度控制在22度與30度、水力停留時間6.7 hr、所用緩衝液為含鈷離子的磷酸鹽緩衝液 (pH 7.5)時,酵素能以10 mM丙烯腈作為基質穩定累積定量的丙烯醯胺。當腈水合酶固定於CNBr-activatedSepharose4 FastFlow(Pharmacia)此種商業產品時,可提高酵素的溫度與pH值忍受範圍。將此固定化酵素填充至管柱,酵素可在流速1 ml/min時穩定連續以濃度14.6 mM丙烯腈作為基質累積丙烯醯胺,出流水丙烯醯胺濃度在反應120分鐘-450分鐘可維持1.7 mM-2.8 mM。腈水合酶不易固定在海藻酸鈣內,改變海藻酸鈉濃度、酵素比例、靜置時間與球體大小皆無法獲的具有活性的固定化酵素。菌株F28除能有效轉換丙烯腈外亦能完全轉換濃度2.32-19.5 mM乙腈產生中間產物乙醯胺以及終產物乙酸與氨氮,於4.1小時內濃度19.5 mM乙腈轉換率達99%,且菌株能以轉換乙腈所獲得的終產物乙酸作為生長所需基質。控制pH值在6.5-7.5時,濃度18.3 mM乙腈能在2小時內完全被轉換至乙醯胺與乙酸,於反應49.4小時候,乙醯胺殘存濃度不高,乙酸累積濃度約為14.2 mM-15.0 mM;在低pH值(pH 4.2)情況下,濃度18.3 mM乙腈雖能再2小時內完全轉換,但菌株的醯胺酶活性會受到影響,於反應終點乙醯胺濃度依然殘存約16.0 mM,乙酸與氨氮累積濃度並不高。額外添加乙醯胺與乙酸的實驗結果顯示,20.3 mM乙醯胺會影響菌株轉換乙腈的變化情形,但影響不大;添加12.8 mM乙酸則會使乙腈達完全轉換的時間延長,濃度15.4 mM乙腈需在反應9.5小時轉換率才達94%,且反應終點亦殘存高濃度的乙醯胺,乙酸累積濃度僅約0.25 mM,菌株生長情形亦不佳,故環境中乙醯胺與乙酸高濃度的累積會影響菌株轉換乙腈的能力。

Nitrile compounds are used extensively in the manufacturing of a variety of polymers and some chemicals. For example, benzonitrile can be used as the feedstock solvent of pesticides (dichlobenil, buctril, etc); acrylonitrile is a precursor used in the synthesis of plastics, rubber, and nylon-66 polymers. Moreover, organonitriles are widely applied as components of fragrances incorporated into a variety of consumer products including soaps, shampoos, bath and cosmetic items. Therefore, substances containing nitrile compounds such as agricultural chemicals or industrial wastewater are likely to be discharged into the natural environment. Since nitriles have been reported as neurotoxic, potent carcinogenic, and mutagenic in nature, issues on the treatment of contaminated environment or industrial wastewater have been raised. In this study, Mesorhizobium sp. F28 containing the NHase/amidase enzyme system was isolated from the nitrile-polluted wastewater. The nitrile hydratase (NHase) was purified from this strain and its enzymatic properties about the optimums of pH values and temperature, the effects of the inhibitors, carbon and nitrogen sources on the enzyme activity, and substrate specificity were identified. Furthermore, the application of NHase to continuously convert acrylonitrile into acrylamide was investigated by ultrafiltration stirred-cell membrane reactor, Ca-alginate-immobilized NHase, and sepharose beads-immobilized NHase. On the other hand, the acetonitrile biotransformation by the whole cells of Mesorhizobium sp. F28 was also examined using batch experiments in this research. The strain F28 was cultivated in the R2A medium which contained 0.01 g/l CoCl2 H2Oat 30OC under aeration. After 24.8 hrs, the cells were harvested by centrifugation (6000Xg for 12 min). Harvested cells were washed with 50 mM potassium phosphate buffer (pH 7.5) and recentrifugated under the same conditions. The pellet was resuspended in a minimal volume of buffer containing 1 mM PMSF (pH 7.5) and then disrupted with a French press (FRENCH pressure cell press) under 2793 psi. The cell debris was removed by centrifugation. The cell-free extract was then brought to 50%-80% ammonium sulfate saturation, applied to an ANX Sepharose 4 Fast Flow, and finally purified by gel filtration through a Sephacryl S-200 High Resolution. Complete separation of the NHase and amidase was achieved during ion exchange chromatography. The enzyme was purified 5-fold with a yield of 16% from the cell-free extract. The NHase from Mesorhizobium sp. F28 had a molecular mass of 110 kDa. The highest activity of the NHase was between 37-45OC at pH 7.5. Examination of various inhibitors showed that the enzyme was sensitive to copper sulfate, silver sulfite, hydrogen peroxide, and high concentrations of chelating agents such as EDTA and sodium azide. Ammonium persulfate and the reducing reagent exhibited weak inhibition on the enzyme activity. Yeast extract, glucose and sodium pyruvate had no effect on the enzyme activity. The NHase could hydrolyze acrylonitrile, propionitrile, isobutyronitrile, and benzonitrile. Acrylonitrile was an especially good substrate for NHase, and the Michaelis-Menten constant (Km) for acrylonitrile was 0.53 mM.
Previous researches conclude that most NHases are inducible by urea, nitriles and amides, and either cobalt ions or iron ions are essential for the formation of NHase. It was found that urea did not induce NHase of Mesorhizobium sp. F28 in the nitrogen-rich medium (R2A liquid medium). Acrylonitrile, which is a good substrate for the NHase of Mesorizobium sp. F28, affected the NHase production except that cobalt ions were in the culture medium. As the medium supplemented with cobalt ions, the NHase activity reached 328.5 U/ml (R2A/Co-R2A/Co) that is almost a five-fold enhancement compared to the results for the medium without addition (R2A-R2A, 70.5 U/ml).The NHase from Mesorhizobium sp. F28 was applied to continuously convert acrylonitrile into acrylamide by ultrafiltration stirred-cell membrane reactor and sepharose beads-immobilized NHase. As using ultrafiltration stirred-cell membrane reactor, the enzyme stably converted 10 mM acrylonitrile and accumulated acrylamide in a 50 mM potassium phosphate buffer (pH 7.5) containing 0.01 g/l CoCl2 H2O at 22OC or 30OC under the hydraulic retention time(HRT) of 6.7 hr. The sepharose beads-immobilized NHase provided a stable environment for the enzyme and prevented the loss of enzyme activity at pH 9.0 and also at temperature up to 55OC. The process for the production of acrylamide was conveniently carried out in a column packed with sepharose beads-immobilized NHase operated at a flow rate of 1 ml/min while the substrate was 14.6 mM acrylonitrile in a 50 mM potassium phosphate buffer containing 0.01 g/l CoCl2H2O. The acrylamide concentrations in the effluent were maintained between 1.7 mM-2.8 mM with the reaction time from 120 to 450 mins. Ca-alginate was not suitable to immobilize the NHase. The Ca-alginate-immobilized NHase exhibited no enzyme activity, though the factors about sodium alginate concentrations, the amount of enzyme, immobilization time and so on were examined.
Mesorhizobium sp. F28 has the NHase/amidase enzyme system to convert acetonitrile into acetic acid via acetamide as an intermediate. This strain efficiently converted 2.32 mM-19.5 mM acetonitrile into acetic acid within 4 hours, and used acetic acid as a substrate to grow. The pH values were adjusted between 6.5 and 7.5 to maintain the amidase activity for complete conversion of 18.3 mM acetamide within 2 hrs. The amidase activity of Mesorhizobium sp. F28 decreased at a low pH value (pH 4.2). The residual concentration of acetamide was 16.0 mM and the concentrations of acetic acid and ammonia were low at the end of the reaction. The addition of 20.3 mM acetamide slightly inhibited the conversion of acetonitrile into acetamide by the NHase or the conversion of acetamide into acetic acid by the amidase. However, the high concentration of acetic acid in the medium significantly affected the biodegradation of acetonitrile. As adding 12.8 mM acetic acid, the conversion rate of 15.4 mM acetonitrile was 94% at 9.5 hrs, and the residual concentrations of acetiamide and acetic acid were respectively 15.1 mM and 0.25 mM at the end of the reaction. Moreover, the cell growth was decreased compared to that without addition of acetic acid. Thus, the appropriate operation (flow rate, HRT, feed load, and so on) of the bioreactor must be conducted to avoid the accumulation of acetic acid in the reactor as using Mesorizobium sp. F28 to continuously degrade acetonitrile.
URI: http://hdl.handle.net/11455/5528
其他識別: U0005-2907200817485000
Appears in Collections:環境工程學系所

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