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首页> 外文期刊>Journal of Biotechnology >Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems
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Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

机译：使用大肠杆菌表达系统分泌重组芽孢杆菌水解酶

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摘要

Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.
机译：芽孢杆菌是革兰氏阳性细菌，分泌大量具有工业意义的细胞外蛋白。在该报告中，克隆了三种芽孢杆菌胞外水解酶，即α-淀粉酶，甘露聚糖酶和几丁质酶，并在革兰氏阴性大肠杆菌中过表达。我们发现天然信号肽和大肠杆菌外膜蛋白OmpA均可用于指导重组酶的分泌。在琼脂平板上或酶谱图中观察到表达的酶为清除区。对不同细胞室中酶活性的测定表明，酶分泌到培养基中的能力取决于诱导时间，信号肽的类型和酶的分子量。过夜诱导后，可从培养物上清液中收获大部分酶活性（85-96％）。我们的结果表明芽孢杆菌属的各种信号肽。可以被大肠杆菌分泌机器识别。具有相似信号肽的其他酶似乎可以在大肠杆菌表达系统中同样很好地分泌。因此，我们的发现应该能够应用于其他芽孢杆菌水解酶以及其他蛋白质的克隆和细胞外生产。

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