Cloning and characterization of an acidic lipase from a lipolytic bacterium in tempeh.

IF 3.6 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Naswandi Nur, Antonius Suwanto, Anja Meryandini, Maggy Thenawidjaja Suhartono, Esti Puspitasari, Hyung Kwoun Kim
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Abstract

Background: Lipases have emerged as essential biocatalysts, having the ability to contribute to a wide range of industrial applications. Microbial lipases have garnered significant industrial attention due to their stability, selectivity, and broad substrate specificity. In the previous study, a unique lipolytic bacterium (Micrococcus luteus EMP48-D) was isolated from tempeh. It turns out the bacteria produce an acidic lipase, which is important in biodiesel production. Our main objectives were to clone the acidic lipase and investigate its potential in biodiesel production.

Result: In this study, the gene encoding a lipase from M. luteus EMP48-D was cloned and expressed heterologously in Escherichia coli. To our knowledge, this is the first attempt at the cloning and expression of the lipase gene from Micrococcus luteus. The amino acid sequence was deduced from the nucleotide sequence (1356 bp) corresponded to a protein of 451 amino acid residues with a molecular weight of about 40 kDa. The presence of a signal peptide suggested that the protein was extracellular. A sequence analysis revealed that the protein had a lipase-specific Gly-X-Ser-X-Gly motif. The enzyme was identified as an acidic lipase with a pH preference of 5.0. Fatty acid preferences for enzyme activities were C8 and C12 (p-nitrophenyl esters), with optimum temperatures at 30-40 °C and still remaining active at 80°C. The enzyme was also shown to convert up to 70% of the substrate into fatty acid methyl ester.

Conclusion: The enzyme was a novel acidic lipase that demonstrated both hydrolytic and transesterification reactions. It appeared particularly promising for the synthesis of biodiesel as this enzyme's catalytic reaction was optimum at low temperatures and was still active at high temperatures.

豆豉溶脂菌酸性脂肪酶的克隆与特性研究。
背景:脂肪酶是一种重要的生物催化剂,具有广泛的工业应用前景。微生物脂肪酶由于其稳定性、选择性和广泛的底物特异性而引起了工业界的广泛关注。在先前的研究中,从豆豉中分离到一种独特的溶脂细菌(黄体微球菌EMP48-D)。事实证明,这种细菌会产生一种酸性脂肪酶,这在生物柴油的生产中很重要。我们的主要目标是克隆酸性脂肪酶并研究其在生物柴油生产中的潜力。结果:本研究克隆了黄体分枝杆菌脂肪酶基因EMP48-D,并在大肠杆菌中异种表达。据我们所知,这是首次从黄体微球菌中克隆和表达脂肪酶基因。该氨基酸序列与一个分子量约为40 kDa的451个氨基酸残基的蛋白核苷酸序列(1356 bp)相对应。信号肽的存在表明该蛋白是细胞外的。序列分析显示该蛋白具有脂肪酶特异性Gly-X-Ser-X-Gly基序。该酶被鉴定为酸性脂肪酶,pH值为5.0。脂肪酸对酶活性的偏好是C8和C12(对硝基苯基酯),温度在30-40°C时最适宜,在80°C时仍保持活性。该酶还被证明可以将高达70%的底物转化为脂肪酸甲酯。结论:该酶是一种具有水解和酯交换反应的新型酸性脂肪酶。由于该酶在低温条件下催化反应最佳,且在高温条件下仍具有活性,因此在生物柴油的合成中具有特别的应用前景。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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