深海细菌Shewanella piezotolerans WP3中嗜冷脂肪酶的表达鉴定及体外诱变研究
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摘要
低温酶在理论研究和工业应用上的双重意义,使其成为近年来研究的热点。深海所拥有低温高压的极端环境,蕴藏有大量的低温微生物资源,它的开发对低温酶的研究具有重要意义。在生物催化剂的研究和应用中,微生物脂肪酶一直占据着非常重要的地位。而微生物低温脂肪酶由于具有反应温度低和对热敏感等特点,在食品、化妆品、皮革、饲料、废物处理等工业上有着其它脂肪酶无法比拟的优越性。
2003年我们在西太平洋暖池区1914米的沉积物中分离到一株具有脂肪分解能力的耐压低温细菌Shewanella piezotolerans WP3,其生长温度范围是0-28℃,最适生长温度是20℃。希瓦氏菌WP3脂肪酶基因的开放阅读框由759个核苷酸组成,编码由252个氨基酸组成,分子量约为30kDa的蛋白质。希瓦氏菌WP3脂肪酶属于第V家族的细菌脂肪酶,与Haemophilus influenzae Rd KW20酯酶的氨基酸序列有最大的同源性,它的催化三联体分别是Ser82、Asp202和His231,同时存在第V家族细菌脂肪酶未知功能保守序列同源的结构。
酶学性质分析表明,希瓦氏菌WP3脂肪酶是一个低温酶,最适反应温度是20℃,在4℃它还能保持80%的最大活性。它对热敏感,最适pH为7.0-8.0,能够分解中短链PNP底物。他的活性易受金属离子、抑制剂和去污剂及有机溶剂的影响,但是EDTA能够稍微增加其活性,说明希瓦氏菌WP3脂肪酶是一个非金属离子依赖的脂肪酶。氢键取代二硫键,成为维持脂肪酶三级结构稳定性最重要的相互作用。
我们在希瓦氏菌WP3脂肪酶中发现了一些适应低温的结构特征。代表蛋白柔性的Gly在脂肪酶活性位点附近出现,而代表蛋白刚性的Pro数量明显减少。希瓦氏菌WP3脂肪酶和其它低温脂肪酶的Arg与Lys+Arg的比值减少,而疏水区及疏水内核的氨基酸残基数也较少。同时,它的蛋白表面含有大量带电荷的残基,特别是Asp的比例增加,缺少在中温和高温脂肪酶中保守的二硫键。
为了进一步研究希瓦氏菌WP3脂肪酶结构与其低温活性和热稳定性的关系,我们构建了一系列氨基酸的点突变。结果表明,Leu14和Lys246是希瓦氏菌WP3脂肪酶低温活性的关键氨基酸,而Asp23和Gly97是其热稳定性的关键氨基酸。脂肪酶催化活性中心的蛋白质构象改变,一方面能够影响催化能力和底物结合能力而改变脂肪酶的低温活性,同时也能改变脂肪酶的热稳定性;另一方面也可能带来无活性产物的大量积累。此外,我们还初步研究了第V家族细菌脂肪酶的未知功能保守序列。我们推测,第V家族细菌脂肪酶未知功能保守序列与底物结合无关,其位于蛋白内部疏水区的部分与脂肪酶低温活性密切相关,而位于蛋白表面负电荷区的部分与脂肪酶的热稳定性密切相关。
最后,我们构建了一个包含约10000个突变子的希瓦氏菌WP3脂肪酶随机突变文库。通过筛选验证,我们获得了4个脂肪酶低温活性发生显著变化的突变子,其中2个突变子的脂肪酶低温催化效率(k_(cat)/K_m)分别提高到野生型的1.8倍和1.6倍。这就为今后我们进一步改造希瓦氏菌WP3脂肪酶,并使之具有工业应用价值打下了一定的基础。
希瓦氏菌WP3脂肪酶是第一个从希瓦氏菌中克隆表达并进行了酶学性质鉴定的脂肪酶,为进一步研究嗜冷脂肪酶的低温适应机制和稳定性提供了很好的材料。我们利用定点突变技术研究了对这个嗜冷脂肪酶低温活性和热稳定性起关键作用的氨基酸,并通过定向进化筛选到一些低温活性发生改变的突变脂肪酶,为利用蛋白质工程技术改造天然酶提供了理论依据。
Lipases are versatile tools for biotechnological applications not only their potential to catalyze the hydrolysis but also the synthesis of many useful ester compounds. Numerous lipases have already been isolated and characterized from animals, plants, and microorganisms. Most of the lipases used in industry are microbial enzymes, which have been extensively characterized biochemically. Psychrophilic lipases derived from extremophiles of deep-sea have lately attracted attention as a result of their increasing use due to their high activity at low temperatures and thermoliability, which are favorable properties for the production of fatty acids and interesterification of fats in the food, pharmaceutical, and fine-chemical industries.
A piezotolenrant and psychrotolenrant bacteria Shewanella piezotolerans WP3, showing lipolytic activity, was previously isolated from deep-sea sediments of 1914m depth in west Pacific Warm Pool. It could grow at a temperature range of 0-28°C and the best growth temperature was around 20°C. The open reading frame of Shewanella piezotolerans WP3 lipases consisted of 759 nucleotides, encoding a protein of 252 amino acids with a predicted molecular mass of approximate 30kDa. This lipase is a member of family V lipases, and had the highest homology with the esterases of Haemophilus influenzae Rd KW20. The catalytic triad of Shewanella piezotolerans WP3 lipases is composed of Ser82, Asp202 and His231. Additionally, the unknown conserved sequence of family V lipases could be found in N-terminal part of this lipase.
Shewanella piezotolerans WP3 lipase is a psychrophilic enzyme with a low optimum temperature of 20°C, and retains 80% activity even at 4°C. It is a thermoliable and pH neutral lipase, which is able to hydrolyze short to medium chain PNP esters. Metal ions, various reagents and organic solvents could inhibit the hydrolytic activity of Shewanella piezotolerans WP3 lipase except for EDTA, which indicate the independence of metal ions. H-bonds, rather than disulfide bridges, are the most important interactions in the stability of protein structure.
Structural modifications involved in cold-adaptation had been detected from structure analysis. A large amount of charged residues exposed at the protein surface, and a relative smaller hydrophobic core region. The lack of disulfide bridges, which are conserved in mesophilic and thermophilic lipases. A low proportion of arginine residues as compared to lysines, a low content in proline residues and a high content in glycine residues were also observed. All these properties should confer on the lipase a more flexible structure, in accordance with its low thermostability.
For further study of the relationship between structure and activity or stability, a series of single amino acid mutants had been constructed by site-directed mutagenesis. For Shewanella piezotolerans WP3 lipase, Leu14 and Lys246 may be the key residues of cold activity, while Asp23 and Gly97 may be the key residues of thermostability. Conformation changes of lipase active center, may affect cold activity by changes of catalytic ability and substrates affinity, but also the thermostability, and even lead to accumulation of non-active products. It is also predicted that hydrophobic region of unknown conserved sequence and cold activity are closely relative, and protein surface region of that and thermostability are closely relative, while it play no role in substrates affinity.
A high quality random mutagenesis of Shewanella piezotolerans WP3 lipase, containing around 10000 mutants, was constructed by error-prone PCR method. After screening, 2 of them were found to be favorable mutants with 1.8-fold and 1.6-fold catalytic efficiency(k_(cat)/K_m) of wild type lipase at low temperature.
Shewanella piezotolerans WP3 lipase is the first lipase expressed and characterized from Shewanella strains. This study on the cold-adaption of lipase may facilitate the research of relationship between enzyme structure and activity(stability), and the protein engineering of biocatalysts.
2003年我们在西太平洋暖池区1914米的沉积物中分离到一株具有脂肪分解能力的耐压低温细菌Shewanella piezotolerans WP3,其生长温度范围是0-28℃,最适生长温度是20℃。希瓦氏菌WP3脂肪酶基因的开放阅读框由759个核苷酸组成,编码由252个氨基酸组成,分子量约为30kDa的蛋白质。希瓦氏菌WP3脂肪酶属于第V家族的细菌脂肪酶,与Haemophilus influenzae Rd KW20酯酶的氨基酸序列有最大的同源性,它的催化三联体分别是Ser82、Asp202和His231,同时存在第V家族细菌脂肪酶未知功能保守序列同源的结构。
酶学性质分析表明,希瓦氏菌WP3脂肪酶是一个低温酶,最适反应温度是20℃,在4℃它还能保持80%的最大活性。它对热敏感,最适pH为7.0-8.0,能够分解中短链PNP底物。他的活性易受金属离子、抑制剂和去污剂及有机溶剂的影响,但是EDTA能够稍微增加其活性,说明希瓦氏菌WP3脂肪酶是一个非金属离子依赖的脂肪酶。氢键取代二硫键,成为维持脂肪酶三级结构稳定性最重要的相互作用。
我们在希瓦氏菌WP3脂肪酶中发现了一些适应低温的结构特征。代表蛋白柔性的Gly在脂肪酶活性位点附近出现,而代表蛋白刚性的Pro数量明显减少。希瓦氏菌WP3脂肪酶和其它低温脂肪酶的Arg与Lys+Arg的比值减少,而疏水区及疏水内核的氨基酸残基数也较少。同时,它的蛋白表面含有大量带电荷的残基,特别是Asp的比例增加,缺少在中温和高温脂肪酶中保守的二硫键。
为了进一步研究希瓦氏菌WP3脂肪酶结构与其低温活性和热稳定性的关系,我们构建了一系列氨基酸的点突变。结果表明,Leu14和Lys246是希瓦氏菌WP3脂肪酶低温活性的关键氨基酸,而Asp23和Gly97是其热稳定性的关键氨基酸。脂肪酶催化活性中心的蛋白质构象改变,一方面能够影响催化能力和底物结合能力而改变脂肪酶的低温活性,同时也能改变脂肪酶的热稳定性;另一方面也可能带来无活性产物的大量积累。此外,我们还初步研究了第V家族细菌脂肪酶的未知功能保守序列。我们推测,第V家族细菌脂肪酶未知功能保守序列与底物结合无关,其位于蛋白内部疏水区的部分与脂肪酶低温活性密切相关,而位于蛋白表面负电荷区的部分与脂肪酶的热稳定性密切相关。
最后,我们构建了一个包含约10000个突变子的希瓦氏菌WP3脂肪酶随机突变文库。通过筛选验证,我们获得了4个脂肪酶低温活性发生显著变化的突变子,其中2个突变子的脂肪酶低温催化效率(k_(cat)/K_m)分别提高到野生型的1.8倍和1.6倍。这就为今后我们进一步改造希瓦氏菌WP3脂肪酶,并使之具有工业应用价值打下了一定的基础。
希瓦氏菌WP3脂肪酶是第一个从希瓦氏菌中克隆表达并进行了酶学性质鉴定的脂肪酶,为进一步研究嗜冷脂肪酶的低温适应机制和稳定性提供了很好的材料。我们利用定点突变技术研究了对这个嗜冷脂肪酶低温活性和热稳定性起关键作用的氨基酸,并通过定向进化筛选到一些低温活性发生改变的突变脂肪酶,为利用蛋白质工程技术改造天然酶提供了理论依据。
Lipases are versatile tools for biotechnological applications not only their potential to catalyze the hydrolysis but also the synthesis of many useful ester compounds. Numerous lipases have already been isolated and characterized from animals, plants, and microorganisms. Most of the lipases used in industry are microbial enzymes, which have been extensively characterized biochemically. Psychrophilic lipases derived from extremophiles of deep-sea have lately attracted attention as a result of their increasing use due to their high activity at low temperatures and thermoliability, which are favorable properties for the production of fatty acids and interesterification of fats in the food, pharmaceutical, and fine-chemical industries.
A piezotolenrant and psychrotolenrant bacteria Shewanella piezotolerans WP3, showing lipolytic activity, was previously isolated from deep-sea sediments of 1914m depth in west Pacific Warm Pool. It could grow at a temperature range of 0-28°C and the best growth temperature was around 20°C. The open reading frame of Shewanella piezotolerans WP3 lipases consisted of 759 nucleotides, encoding a protein of 252 amino acids with a predicted molecular mass of approximate 30kDa. This lipase is a member of family V lipases, and had the highest homology with the esterases of Haemophilus influenzae Rd KW20. The catalytic triad of Shewanella piezotolerans WP3 lipases is composed of Ser82, Asp202 and His231. Additionally, the unknown conserved sequence of family V lipases could be found in N-terminal part of this lipase.
Shewanella piezotolerans WP3 lipase is a psychrophilic enzyme with a low optimum temperature of 20°C, and retains 80% activity even at 4°C. It is a thermoliable and pH neutral lipase, which is able to hydrolyze short to medium chain PNP esters. Metal ions, various reagents and organic solvents could inhibit the hydrolytic activity of Shewanella piezotolerans WP3 lipase except for EDTA, which indicate the independence of metal ions. H-bonds, rather than disulfide bridges, are the most important interactions in the stability of protein structure.
Structural modifications involved in cold-adaptation had been detected from structure analysis. A large amount of charged residues exposed at the protein surface, and a relative smaller hydrophobic core region. The lack of disulfide bridges, which are conserved in mesophilic and thermophilic lipases. A low proportion of arginine residues as compared to lysines, a low content in proline residues and a high content in glycine residues were also observed. All these properties should confer on the lipase a more flexible structure, in accordance with its low thermostability.
For further study of the relationship between structure and activity or stability, a series of single amino acid mutants had been constructed by site-directed mutagenesis. For Shewanella piezotolerans WP3 lipase, Leu14 and Lys246 may be the key residues of cold activity, while Asp23 and Gly97 may be the key residues of thermostability. Conformation changes of lipase active center, may affect cold activity by changes of catalytic ability and substrates affinity, but also the thermostability, and even lead to accumulation of non-active products. It is also predicted that hydrophobic region of unknown conserved sequence and cold activity are closely relative, and protein surface region of that and thermostability are closely relative, while it play no role in substrates affinity.
A high quality random mutagenesis of Shewanella piezotolerans WP3 lipase, containing around 10000 mutants, was constructed by error-prone PCR method. After screening, 2 of them were found to be favorable mutants with 1.8-fold and 1.6-fold catalytic efficiency(k_(cat)/K_m) of wild type lipase at low temperature.
Shewanella piezotolerans WP3 lipase is the first lipase expressed and characterized from Shewanella strains. This study on the cold-adaption of lipase may facilitate the research of relationship between enzyme structure and activity(stability), and the protein engineering of biocatalysts.
引文
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