水生栖热菌海藻糖合酶体外定向进化及重组菌BL35细胞透性化技术研究
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摘要
海藻糖是一种广泛分布于细菌、真菌和动植物体内的双糖。在生物体内,它不仅作为结构成分和能量物质存在,而且在热击和脱水等协迫条件下,对生物体和生物大分子起着良好的非特异性保护作用。由于其独特的生物学功能,使它广泛用于食品、医药、化妆品、农业、冷藏等方面,具有广阔的市场应用前景。
海藻糖(Trehalose),α-D-(?)比喃葡糖基(1→1)α-D-吡喃葡糖苷,是由两个葡萄糖残基通过一个α,α(1→1)糖苷键连接而成的非还原双糖。海藻糖合酶通过分子内转糖基作用将α,α-1,4糖苷键连接的麦芽糖,转化为α,α-1,1糖苷键连接的海藻糖,是酶法生产海藻糖的关键酶。现有多数海藻糖合成酶转化效率不高、热稳定性较差、反应时间较长等。因此,对其的研究显得尤为重要。
本研究首先克隆表达了海藻糖合酶基因,并从理性和非理性角度对海藻糖合酶进行了分子改造,以海藻糖合酶(trehalose synthase from Pyrococcus horikoshii)晶体结构为模板,在网站S WIS S-MODEL上对来自水生栖热菌FL-03(thermus aquaticus FL-03)海藻糖合成酶进行同源建模,结合分析不同来源的海藻糖合成酶基因的保守序列,选择了保守区的位点A197和H100进行定点(饱和)突变,并在保守区外的S547和H305,进行定点突变,获得高酶活突变体重组菌,并在此基础上进行细胞透性化技术的研究,促使重组菌细胞形成高效的固定化酶系统,大幅提高产率,同时简化工艺,降低生产成本,为工业化生产提供可行性依据。
主要取得的结果表明：
(1)采用巢式PCR法成功克隆并高效表达了水生栖热菌的海藻糖合酶基因TresC01。并通过同源建模获得了该重组酶的三维结构信息,预测了该酶的活性中心。
(2)通过同源建模获得了该酶的三维结构信息,活性中心的与底物结合的4个氨基酸残基中的A197、H100、H305和S547与该酶的催化活性有关,通过三维建模后,初步分析4个氨基酸残基变化可能影响海藻糖合酶的酶学性质。
(3)以克隆得到的海藻糖合酶基因T-TersC01为模板,通过PCR介导方法对海藻糖合酶基因进行饱和突变研究,结果显示A197和H100分别饱和突变为其它的19个氨基酸后,共获得了13个突变子,只有突变体pEA197H具有较高的活性其他均无活性；突变体pEA197H比酶活提高了1.52倍。
(3)结合ProSa理性设计,对H305和S547进行了定点突变,结果显示对H305和S547定点突变氨基酸His→Cys和Ser→Cys后酶活下降,降低为野生型(WT)的66%.S547变体没有明显变化,pEH305C比活力与野生型(WT)相比提高1.40倍。
(4)通过对野生型和突变型海藻糖合酶进行三维结构分析及将其与三维结构已知的海藻糖合酶(Pyrococcus horikoshii)进行比较,该海藻糖合酶同样具有(β/α)_8的桶状结构,符合大多数α/β桶酶的活性中心的定位；并探讨了它们酶学性质差异。推测酶活性中心处的催化氨基酸为A197、H305,同时具有α-淀粉酶家族的同源性。
(5)海藻糖合酶高通量筛选体系
采用高拷贝的克隆载体XL10-gold成功获得41个克隆载体,根据利用α互补法(蓝/白菌落筛选法)的直观反应,建立了一套海藻糖合酶高通量克隆载体筛选体系；同时采用DNS-ELISAM酶标法(糖化酶显色法)筛选在的高酶活突变酶,该方法可以大大降低背景的影响,简捷、高效。属于本实验室首创。
(6)透性化细胞可省略酶的纯化和胞内酶的固定化。研究了不同表面活性剂曲拉通X-100(TRITON X-100)、二甲基亚砜(DMOS)和十六烷基三甲基溴化胺(CTAB)对海藻糖合酶重组菌LB35细胞壁通透性的影响,低分子量物质的渗透障碍被部分或完全解除。结果表明：CTAB、TRITON-X100、DMOS对透性化细胞合成海藻糖活性都有提高作用,高浓度时CTAB和TRITON-X100对海藻糖合酶重组菌LB35具有明显抑制作用,其中DMOS对海藻糖合酶重组菌LB35在1.0%浓度范围内菌体生长抑制作用不是很明显,CTAB抑制较明显。而在细胞培养20h加入0.8%浓度TRITON-X100处理的透性化细胞,海藻糖酶活力提高了2.25倍。TRITON X-100对提高细胞通透性有较好的影响,能较大程度的提高海藻糖合酶重组菌LB35的细胞膜的通透性,促使麦芽糖进入细胞内部更充分的与胞内分泌的海藻糖合酶接触反应,进一步提高海藻糖产量。
通过对这些突变酶的性质变化分析和比较,进一步为研究海藻糖合成酶结构和功能的关系提供理论数据和信息,从而对海藻糖合成酶的分子改造提供理论依据。在国内外有关海藻糖合酶理性和非理性酶分子改性的系统研究还未见文献报道,本研究是首次开辟了这一领域的研究,为深入了解海藻糖合酶性质与结构的关系以及生物法高效生产海藻糖奠定了良好的基础。
Trehalose(α,α-trehalose) is a disaccharide formed byα,α-1,1 linkage of two D-glucose molecules. It occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant against the effects of freezing of dehydration. It also possesses physical and or chemical properties that are different than other sugars,which may make trehalose an attract ive ingredient in food, health and beauty and pharmaceutical products. This paper briefly reviews recent advances in the manufacture of trehalose, according to its biosynthetic pathways, and provides an overview of the maior applications of trehalose in food industry, biology, cosmetics,medical industry and agriculture.
Trehalose synthase play the key role in enzymatic process produced trehalose. Maltose can be transfered from anα,α-1,4-glycosidic linkage into a trehalose withα,α-1,4-glycosidic linkage. There were some disadvantages in the existing trehalose synthase which the transform effciency is too low;,such as the thermostability is too low and the reaction time is long. Therefore, there is much interest in researches on the biological catalyse method. It is necessary for the yeild to perform mutagenesis on trehalose synthase so that the high levels of total trehalose synthase activity to improve the industrial capability.
First of all,thermus aquaticus FL-03 chromosome genome DNA as a template, the trehalose synthase gene were cloned and high-expressed in E.coli by nested PCR amplification, was done with by PCR mediate genomic library screening. And more,trehalose synthase was evolved by PCR mediate and half-rational design. The three-dimensional model of TreS was from Pyrococcus horikoshii. It was constructed basis on the crystal structure of trehalose synthase from on the SWISS-MODEL Internet. United into analyzing the conserved region of trehalose synthase amio sequence from several different origin code and Site-directed Mutagenesis completely.In addtion, to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose. The productivity was improved, the cost decreased and the technological process which offer the basis of commercial production.The experimenta 1 results indicate that:
(1) the trehalose synthase gene TresC01 was cloned and high-expressed, and the 3D structure of the trehalose synthase was successful constructed by homology modeling,furthermore, the probable active site was forecasted.
(2)The relationship between the function groups and catalytic activity of enzyme revealed that acquired the 3D structure information of the homology modeling, A197, H100, H305 and S547 in 4 amino acids remnants radicle of combining with the domains related to the catalyst activity of S547 and passing the 3D model, the initial analyzes 4 amino acidses remnants the radicle change may effect the TresS catalize activity.
(3) T-TersC01 for mold plate, lie to lead through PCR the method matches TresC01 progress saturation to mutate a search to seaweed sugar, result display the A197 and H100 distinguish a saturated mutation for other of 19 amino acidses after, totally acquired 13 mutation, only wild-type pEA 197 H to have higher of activity other all have no activity; wild-type pEA197H lived to raise than TresC01 was 1.52 times.
(4) High throughput screening method of expressing.The results pointed out hat the enzyme activity was lost after A197and H100 which were converted to other 19 kinds of amio acids,and that the mutants A197X and H100Q were also indicated no activities expect of A197H. The reason of activity lost probably was that these stucture positions were important to hold on the structure stability of the protein domains, and they possibly were crucial residues or active sites of the enzyme. The specific activity of the mutants H305 and S547 (His→Cys and Ser→Cys) were decreased largely and reduced to 66%,99%, the wild-type pEH305C respectively was shown that the activity was increased Compared with the wild-type,four degree approxately,It can not only give aid for useful data providing and information in studying the relation of the sturcture and function by Studying and analysis, these changes about the properties of the mutants but also can afford the theoretic basis to improve the molecule evolution on trehalose synthase in the future.
(5) Directed evolution of trehalose synthase.The cloned that adoption higher-copies carries the body XL10-gold to successfully acquire 41 cloned to carry a body, according to the keeping of blue white spot view reaction, created the colon vector screening system; Adopt DNS-ELISA (diastatic enzyme) sieving at high enzyme activity, the method can consumedly lower the impact of background quickly and efficiently. Belong to this laboratory to found..
(6) Use of permeabilized cells allows the conventional enzyme purification and immobilization steps to be omitted and simplifies the synthesis procedure. The study on recombinant escherichia coli cell wall permeability was carried out by different surfactants (Triton-X100, DMSO,cetyltrimethy-lammonium bromide (CTAB)) of water, the results show that of Triton-X-100 to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose.
The article is the first systematic study to improve the property of trehalose synthase successfully by rational and irrational design evolution. This studies beneficial to understanding the relationship between 3D structures and properties of trehalose synthase,as well as the researches of producing threhalose highly by the gene engineering strain.
海藻糖(Trehalose),α-D-(?)比喃葡糖基(1→1)α-D-吡喃葡糖苷,是由两个葡萄糖残基通过一个α,α(1→1)糖苷键连接而成的非还原双糖。海藻糖合酶通过分子内转糖基作用将α,α-1,4糖苷键连接的麦芽糖,转化为α,α-1,1糖苷键连接的海藻糖,是酶法生产海藻糖的关键酶。现有多数海藻糖合成酶转化效率不高、热稳定性较差、反应时间较长等。因此,对其的研究显得尤为重要。
本研究首先克隆表达了海藻糖合酶基因,并从理性和非理性角度对海藻糖合酶进行了分子改造,以海藻糖合酶(trehalose synthase from Pyrococcus horikoshii)晶体结构为模板,在网站S WIS S-MODEL上对来自水生栖热菌FL-03(thermus aquaticus FL-03)海藻糖合成酶进行同源建模,结合分析不同来源的海藻糖合成酶基因的保守序列,选择了保守区的位点A197和H100进行定点(饱和)突变,并在保守区外的S547和H305,进行定点突变,获得高酶活突变体重组菌,并在此基础上进行细胞透性化技术的研究,促使重组菌细胞形成高效的固定化酶系统,大幅提高产率,同时简化工艺,降低生产成本,为工业化生产提供可行性依据。
主要取得的结果表明：
(1)采用巢式PCR法成功克隆并高效表达了水生栖热菌的海藻糖合酶基因TresC01。并通过同源建模获得了该重组酶的三维结构信息,预测了该酶的活性中心。
(2)通过同源建模获得了该酶的三维结构信息,活性中心的与底物结合的4个氨基酸残基中的A197、H100、H305和S547与该酶的催化活性有关,通过三维建模后,初步分析4个氨基酸残基变化可能影响海藻糖合酶的酶学性质。
(3)以克隆得到的海藻糖合酶基因T-TersC01为模板,通过PCR介导方法对海藻糖合酶基因进行饱和突变研究,结果显示A197和H100分别饱和突变为其它的19个氨基酸后,共获得了13个突变子,只有突变体pEA197H具有较高的活性其他均无活性；突变体pEA197H比酶活提高了1.52倍。
(3)结合ProSa理性设计,对H305和S547进行了定点突变,结果显示对H305和S547定点突变氨基酸His→Cys和Ser→Cys后酶活下降,降低为野生型(WT)的66%.S547变体没有明显变化,pEH305C比活力与野生型(WT)相比提高1.40倍。
(4)通过对野生型和突变型海藻糖合酶进行三维结构分析及将其与三维结构已知的海藻糖合酶(Pyrococcus horikoshii)进行比较,该海藻糖合酶同样具有(β/α)_8的桶状结构,符合大多数α/β桶酶的活性中心的定位；并探讨了它们酶学性质差异。推测酶活性中心处的催化氨基酸为A197、H305,同时具有α-淀粉酶家族的同源性。
(5)海藻糖合酶高通量筛选体系
采用高拷贝的克隆载体XL10-gold成功获得41个克隆载体,根据利用α互补法(蓝/白菌落筛选法)的直观反应,建立了一套海藻糖合酶高通量克隆载体筛选体系；同时采用DNS-ELISAM酶标法(糖化酶显色法)筛选在的高酶活突变酶,该方法可以大大降低背景的影响,简捷、高效。属于本实验室首创。
(6)透性化细胞可省略酶的纯化和胞内酶的固定化。研究了不同表面活性剂曲拉通X-100(TRITON X-100)、二甲基亚砜(DMOS)和十六烷基三甲基溴化胺(CTAB)对海藻糖合酶重组菌LB35细胞壁通透性的影响,低分子量物质的渗透障碍被部分或完全解除。结果表明：CTAB、TRITON-X100、DMOS对透性化细胞合成海藻糖活性都有提高作用,高浓度时CTAB和TRITON-X100对海藻糖合酶重组菌LB35具有明显抑制作用,其中DMOS对海藻糖合酶重组菌LB35在1.0%浓度范围内菌体生长抑制作用不是很明显,CTAB抑制较明显。而在细胞培养20h加入0.8%浓度TRITON-X100处理的透性化细胞,海藻糖酶活力提高了2.25倍。TRITON X-100对提高细胞通透性有较好的影响,能较大程度的提高海藻糖合酶重组菌LB35的细胞膜的通透性,促使麦芽糖进入细胞内部更充分的与胞内分泌的海藻糖合酶接触反应,进一步提高海藻糖产量。
通过对这些突变酶的性质变化分析和比较,进一步为研究海藻糖合成酶结构和功能的关系提供理论数据和信息,从而对海藻糖合成酶的分子改造提供理论依据。在国内外有关海藻糖合酶理性和非理性酶分子改性的系统研究还未见文献报道,本研究是首次开辟了这一领域的研究,为深入了解海藻糖合酶性质与结构的关系以及生物法高效生产海藻糖奠定了良好的基础。
Trehalose(α,α-trehalose) is a disaccharide formed byα,α-1,1 linkage of two D-glucose molecules. It occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant against the effects of freezing of dehydration. It also possesses physical and or chemical properties that are different than other sugars,which may make trehalose an attract ive ingredient in food, health and beauty and pharmaceutical products. This paper briefly reviews recent advances in the manufacture of trehalose, according to its biosynthetic pathways, and provides an overview of the maior applications of trehalose in food industry, biology, cosmetics,medical industry and agriculture.
Trehalose synthase play the key role in enzymatic process produced trehalose. Maltose can be transfered from anα,α-1,4-glycosidic linkage into a trehalose withα,α-1,4-glycosidic linkage. There were some disadvantages in the existing trehalose synthase which the transform effciency is too low;,such as the thermostability is too low and the reaction time is long. Therefore, there is much interest in researches on the biological catalyse method. It is necessary for the yeild to perform mutagenesis on trehalose synthase so that the high levels of total trehalose synthase activity to improve the industrial capability.
First of all,thermus aquaticus FL-03 chromosome genome DNA as a template, the trehalose synthase gene were cloned and high-expressed in E.coli by nested PCR amplification, was done with by PCR mediate genomic library screening. And more,trehalose synthase was evolved by PCR mediate and half-rational design. The three-dimensional model of TreS was from Pyrococcus horikoshii. It was constructed basis on the crystal structure of trehalose synthase from on the SWISS-MODEL Internet. United into analyzing the conserved region of trehalose synthase amio sequence from several different origin code and Site-directed Mutagenesis completely.In addtion, to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose. The productivity was improved, the cost decreased and the technological process which offer the basis of commercial production.The experimenta 1 results indicate that:
(1) the trehalose synthase gene TresC01 was cloned and high-expressed, and the 3D structure of the trehalose synthase was successful constructed by homology modeling,furthermore, the probable active site was forecasted.
(2)The relationship between the function groups and catalytic activity of enzyme revealed that acquired the 3D structure information of the homology modeling, A197, H100, H305 and S547 in 4 amino acids remnants radicle of combining with the domains related to the catalyst activity of S547 and passing the 3D model, the initial analyzes 4 amino acidses remnants the radicle change may effect the TresS catalize activity.
(3) T-TersC01 for mold plate, lie to lead through PCR the method matches TresC01 progress saturation to mutate a search to seaweed sugar, result display the A197 and H100 distinguish a saturated mutation for other of 19 amino acidses after, totally acquired 13 mutation, only wild-type pEA 197 H to have higher of activity other all have no activity; wild-type pEA197H lived to raise than TresC01 was 1.52 times.
(4) High throughput screening method of expressing.The results pointed out hat the enzyme activity was lost after A197and H100 which were converted to other 19 kinds of amio acids,and that the mutants A197X and H100Q were also indicated no activities expect of A197H. The reason of activity lost probably was that these stucture positions were important to hold on the structure stability of the protein domains, and they possibly were crucial residues or active sites of the enzyme. The specific activity of the mutants H305 and S547 (His→Cys and Ser→Cys) were decreased largely and reduced to 66%,99%, the wild-type pEH305C respectively was shown that the activity was increased Compared with the wild-type,four degree approxately,It can not only give aid for useful data providing and information in studying the relation of the sturcture and function by Studying and analysis, these changes about the properties of the mutants but also can afford the theoretic basis to improve the molecule evolution on trehalose synthase in the future.
(5) Directed evolution of trehalose synthase.The cloned that adoption higher-copies carries the body XL10-gold to successfully acquire 41 cloned to carry a body, according to the keeping of blue white spot view reaction, created the colon vector screening system; Adopt DNS-ELISA (diastatic enzyme) sieving at high enzyme activity, the method can consumedly lower the impact of background quickly and efficiently. Belong to this laboratory to found..
(6) Use of permeabilized cells allows the conventional enzyme purification and immobilization steps to be omitted and simplifies the synthesis procedure. The study on recombinant escherichia coli cell wall permeability was carried out by different surfactants (Triton-X100, DMSO,cetyltrimethy-lammonium bromide (CTAB)) of water, the results show that of Triton-X-100 to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose.
The article is the first systematic study to improve the property of trehalose synthase successfully by rational and irrational design evolution. This studies beneficial to understanding the relationship between 3D structures and properties of trehalose synthase,as well as the researches of producing threhalose highly by the gene engineering strain.
引文
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