绿豆环氧水解酶基因克隆、表达及酶学性质表征
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摘要
环氧水解酶(Epoxide hydrolase, EH)能催化合成光学纯环氧化合物和邻位二醇,在医药、绿色精细化工等领域具有重要应用前景。近年新发现的绿豆EH (mbEH)能对映归一性水解消旋环氧化物产生单一构型的R-邻位二醇,底物转化率与立体选择性达90%以上,是更一种实用的生物催化剂。本论文首次获得了一种mbEH的编码序列,实现了该基因在大肠杆菌中的克隆与高效表达,对重组绿豆EH进行了纯化和酶学性质表征。
首先,比较了UNIQ-10柱式法、Trizol法和改良Trizol法对绿豆种子、绿豆胚芽和绿豆芽总RNA抽提效果,选定改良Trizol法抽提目标RNA;RT-PCR仅从绿豆胚芽RNA中扩增到部分绿豆EH编码序列,说明该基因的表达存在时间差异性；利用cDNA末端快速扩增(RACE)法获得mbEH A全长序列,该序列含有960bp的开放阅读框、117bp和239bp的上下游非编码序列；通过三维建模方法获得衍生的mbEH A的三维模型。
其次,成功构建了mbEH A的重组质粒pET-28a-mbEH A,转化大肠杆菌BL21(DE3)获得重组表达菌；经IPTG诱导,重组mbEH A表达量约占菌体总蛋白的30%,分子量为41 kDa,等电点pI为6.02；金属螫合亲和层析法一步纯化了重组酶,纯度和回收率均在85%以上；以对硝基苯乙烯氧化物(pNSO)为底物,重组酶活力比天然酶提高了近500倍。
酶学性质分析表明,重组mbEH A能对映归一性水解pNSO,且主要产生R-二醇；相对顺式-pNSO而言,更倾向于优先水解反式-pNSO该酶的最适反应pH和最适反应温度分别为pH7.0和25℃,米氏常数Km为2.05 mM,最大反应速率Vmax为14.8μmol·min-1·mg-1;重组mbEH A基本不耐热性,在pH6-10较稳定；有较强的有机溶剂耐受性,10%苯、甲苯或二甲基亚砜中活力残留50%左右,甲醇、异丙醇对酶活有促进作用；聚乙二醇、吐温、曲拉通等也能明显促进重组酶活力。
最后,初步摸索了重组mbEH A真空冻干条件。低浓度的海藻糖、山梨醇、乳糖、蔗糖、甘露醇、甘氨酸均对重组酶的冻干有良好保护性,5%海藻糖和1%蔗糖的保护效果最好,分别为92.4%和92.1%；对大规模冻干而言,1%蔗糖作为保护剂更经济实用。
Epoxide hydrolases (EHs) have attracted significant interest as biocatalysts of industrial potential. Recently, two novel interesting EHs that catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols was found in Vigna radiate L. In this thesis, for the.first time, a novel gene, mbEH A, encoding one of the two EHs from Vigna radiate L, mbEH A, was obtained, cloned and highly expressed in Escherichia coli. Recombinant mbEH A was one-step purified and characterized.
First, total RNA was isolated from seed, germ and sprouts of Vigna radiata by UNIQ-10 column, Trizol and modified Trizol methods, respectively. Modified Trizol method was selected for further use because of high yield, high purity and integrity of RNA. After RT-PCR, partial mbEH A was only amplified from RNA of Vigna radiata germ. Whole length mbEH A was obtained by rapid amplification of cDNA ends (RACE), which composed of a 960 bp open reading frame,117 bp 5'-upstream and 239 bp 3'-downstream non-coding regions, respectively. The three-dimensional (3D) structure of the deduced mbEH A was predicted and compared to that of potato EH.
Next, recombinant expression plasmid pET-28a-mbEH A was successfully constructed and transformed into Escherichia coli BL21(DE3). Recombinant mbEH A was about 30% of total bacterial protein after IPTG induction, which had a molecular mass of 41 kDa and pI value of 6.02. Recombinant mbEH A was purified to 85% pure by one-step nickel chelating affinity chromatography. The recovery yield was over 85%. Its activity was increase by 500-fold compared to that of purified native enzyme using p-nitrostyrene oxide (pNSO) as substrate.
Then, enzymatic properties assay showed that recombinant mbEH A can catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols and display a enantiopreference to trans-pNSO; its pH and temperature optima were pH 7.0 and 25℃, the Km and Vmax were 2.05 mM and 14.8μmol·min-1·mg-1, respectively; it was stable in buffers within pH6-10 and showed high tolerance against methanol, Tween-40 and Triton X100, which can even strongly activate the enzyme.
At last, cryoprotectants for recombinant mbEH in freeze-drying process were preliminarily screened. All cryoprotectants tested (trehalose, lactose, sucrose, mannitol and glycine) showed better protect at low concentration. The optima was 5% trehalose(92.4%) and 1% sucrose (92.1%). For large scale freeze-drying process,1%sucrose is better and more economic.
首先,比较了UNIQ-10柱式法、Trizol法和改良Trizol法对绿豆种子、绿豆胚芽和绿豆芽总RNA抽提效果,选定改良Trizol法抽提目标RNA;RT-PCR仅从绿豆胚芽RNA中扩增到部分绿豆EH编码序列,说明该基因的表达存在时间差异性；利用cDNA末端快速扩增(RACE)法获得mbEH A全长序列,该序列含有960bp的开放阅读框、117bp和239bp的上下游非编码序列；通过三维建模方法获得衍生的mbEH A的三维模型。
其次,成功构建了mbEH A的重组质粒pET-28a-mbEH A,转化大肠杆菌BL21(DE3)获得重组表达菌；经IPTG诱导,重组mbEH A表达量约占菌体总蛋白的30%,分子量为41 kDa,等电点pI为6.02；金属螫合亲和层析法一步纯化了重组酶,纯度和回收率均在85%以上；以对硝基苯乙烯氧化物(pNSO)为底物,重组酶活力比天然酶提高了近500倍。
酶学性质分析表明,重组mbEH A能对映归一性水解pNSO,且主要产生R-二醇；相对顺式-pNSO而言,更倾向于优先水解反式-pNSO该酶的最适反应pH和最适反应温度分别为pH7.0和25℃,米氏常数Km为2.05 mM,最大反应速率Vmax为14.8μmol·min-1·mg-1;重组mbEH A基本不耐热性,在pH6-10较稳定；有较强的有机溶剂耐受性,10%苯、甲苯或二甲基亚砜中活力残留50%左右,甲醇、异丙醇对酶活有促进作用；聚乙二醇、吐温、曲拉通等也能明显促进重组酶活力。
最后,初步摸索了重组mbEH A真空冻干条件。低浓度的海藻糖、山梨醇、乳糖、蔗糖、甘露醇、甘氨酸均对重组酶的冻干有良好保护性,5%海藻糖和1%蔗糖的保护效果最好,分别为92.4%和92.1%；对大规模冻干而言,1%蔗糖作为保护剂更经济实用。
Epoxide hydrolases (EHs) have attracted significant interest as biocatalysts of industrial potential. Recently, two novel interesting EHs that catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols was found in Vigna radiate L. In this thesis, for the.first time, a novel gene, mbEH A, encoding one of the two EHs from Vigna radiate L, mbEH A, was obtained, cloned and highly expressed in Escherichia coli. Recombinant mbEH A was one-step purified and characterized.
First, total RNA was isolated from seed, germ and sprouts of Vigna radiata by UNIQ-10 column, Trizol and modified Trizol methods, respectively. Modified Trizol method was selected for further use because of high yield, high purity and integrity of RNA. After RT-PCR, partial mbEH A was only amplified from RNA of Vigna radiata germ. Whole length mbEH A was obtained by rapid amplification of cDNA ends (RACE), which composed of a 960 bp open reading frame,117 bp 5'-upstream and 239 bp 3'-downstream non-coding regions, respectively. The three-dimensional (3D) structure of the deduced mbEH A was predicted and compared to that of potato EH.
Next, recombinant expression plasmid pET-28a-mbEH A was successfully constructed and transformed into Escherichia coli BL21(DE3). Recombinant mbEH A was about 30% of total bacterial protein after IPTG induction, which had a molecular mass of 41 kDa and pI value of 6.02. Recombinant mbEH A was purified to 85% pure by one-step nickel chelating affinity chromatography. The recovery yield was over 85%. Its activity was increase by 500-fold compared to that of purified native enzyme using p-nitrostyrene oxide (pNSO) as substrate.
Then, enzymatic properties assay showed that recombinant mbEH A can catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols and display a enantiopreference to trans-pNSO; its pH and temperature optima were pH 7.0 and 25℃, the Km and Vmax were 2.05 mM and 14.8μmol·min-1·mg-1, respectively; it was stable in buffers within pH6-10 and showed high tolerance against methanol, Tween-40 and Triton X100, which can even strongly activate the enzyme.
At last, cryoprotectants for recombinant mbEH in freeze-drying process were preliminarily screened. All cryoprotectants tested (trehalose, lactose, sucrose, mannitol and glycine) showed better protect at low concentration. The optima was 5% trehalose(92.4%) and 1% sucrose (92.1%). For large scale freeze-drying process,1%sucrose is better and more economic.
引文
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