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首页> 外文期刊>Protein Expression and Purification >Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp strain B11-1
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Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp strain B11-1

机译：嗜冷假单胞菌属菌株B11-1的具有独特一级结构的冷适应酯酶的克隆，异源表达，复性和表征

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A Gene coding for an esterase (PsEst1, 1911 bp in length) of the psychrotrophic bacterium Pseudomonas sp. B11-1 isolated from Alaskan soil was cloned and sequenced. The deduced amino acid sequence revealed a protein of 637 amino acid residues with a molecular mass of 69 kDa. Although the expression product, PsEst1, showed no appreciable sequence similarity (less than 15% identity) to any known proteins with the established biochemical functions, it is expected to be related to the alpha/beta hydrolase superfamily because it shared sequence motifs that have been identified with this superfamily. For example, a unique "nucleophilic 6 40 38 elbow" motif. -Gly(36)-Asp-Ser-Leu-Asn(40)-, was identified, and Ser(38) was predicted to constitute a catalytic triad with Asp(162) and His(303). PsEst1 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3) cells as an inclusion body. A Soluble denatured form of the enzyme was purified to homogeneity in the presence of 8 M urea, and the catalytically active form of the enzyme could be obtained by subsequent removal of urea by dialysis, where the addition of 0.1% Triton X-100 was essential for the efficient renaturation of the enzyme. To our knowledge, this was the first example of the successful renaturation of the recombinant cold-adapted enzyme. The enzyme efficiently hydrolyzed vinyl and aryl esters with the C-4-C-6 acyl chain. The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10 degreesC) was significantly lower than the value (79.9 kcal/mol) of the mesophilic lipase. It was observed that the K-m values for p-nitrophenyl butyrate in the growth temperature range of strain B11-1 (5-15 degreesC) were lower than those at higher temperatures. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 27]
机译：编码精神营养细菌Pseudomonas sp。的酯酶（PsEst1，长1911 bp）的基因。克隆和测序从阿拉斯加土壤中分离的B11-1。推导的氨基酸序列揭示了具有637个氨基酸残基的蛋白质，分子量为69kDa。尽管表达产物PsEst1与具有确定的生化功能的任何已知蛋白质均未显示明显的序列相似性（小于15％相同性），但由于它共享已被序列化的序列基序，因此有望与α/β水解酶超家族有关。被这个超家族所认同。例如，独特的“亲核6 40 38肘”图案。鉴定了-Gly（36）-Asp-Ser-Leu-Asn（40）-，并预测Ser（38）与Asp（162）和His（303）构成催化三联体。使用T7 RNA聚合酶转录（pET21a）系统在大肠杆菌BL21（DE3）细胞中将PsEst1过表达为包涵体。在8 M尿素存在下，将酶的可溶性变性形式纯化至均质，然后通过透析除去尿素即可获得酶的催化活性形式，其中必须添加0.1％Triton X-100使酶有效地复性。据我们所知，这是重组冷适应酶成功复性的第一个例子。该酶有效地水解了具有C-4-C-6酰基链的乙烯基酯和芳基酯。酶促对硝基苯基丁酸酯水解的活化能（在10摄氏度下为20.1 kcal / mol）显着低于中温脂肪酶的值（79.9 kcal / mol）。观察到，在菌株B11-1的生长温度范围（5-15℃）中，对硝基苯基丁酸酯的K-m值低于较高温度下的K-m值。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：27]

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