Yaraguppi Deepak A, Bagewadi Zabin K, Mahanta Nilkamal, Singh Surya P, Khan T M Yunus, Deshpande Sanjay H, Soratur Chaitra, Das Simita, Saikia Dimple
Department of Biotechnology, KLE Technological University, Hubballi 580031, India.
Department of Chemistry, Indian Institute of Technology, Dharwad 580011, India.
Gels. 2022 Jun 25;8(7):403. doi: 10.3390/gels8070403.
Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from . To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.
生物表面活性剂是推荐用于强化采油技术的环保型表面活性分子。在本研究中，从……合成了一种编码伊枯草菌素A基因的潜在脂肽（生物表面活性剂）。为了提高脂肽在特定应用中的产量，当前的研究倾向于对重组肽进行工程改造和表达。对伊枯草菌素A基因序列进行密码子优化，用基因特异性引物扩增，并连接到pET - 32A表达载体中以实现高水平的蛋白质表达。将质粒构建体转化到BL21 DE3宿主中以评估表达情况。在镍 - 亚氨基三乙酸（Ni - NTA）琼脂糖柱上纯化高表达的重组伊枯草菌素A脂肽。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）显示伊枯草菌素A的纯度和分子量为41 kDa。发现重组伊枯草菌素A的产量为60 g/L，与我们之前发表的关于野生菌株的研究相比增加了6.7倍。克隆部分伊枯草菌素A功能片段的方法导致脂肽产量增加。当使用机油时，重组蛋白伊枯草菌素A显示出生物表面活性剂特性，乳化指数（E24）为74 ± 1.9%。通过质谱对纯化的重组蛋白伊枯草菌素A进行表征。胰蛋白酶消化的基质辅助激光解吸电离飞行时间（MALDI - TOF）光谱（蛋白质/胰蛋白酶比例为50:1和25:1）显示出该蛋白质所需的消化质量峰，进一步证实了伊枯草菌素A的身份。基于拉曼光谱中独特的光谱带阐明了伊枯草菌素A的结构，该光谱带揭示了肽主链和脂质的存在。通过填充砂柱使用重组伊枯草菌素A进行强化采油，额外产油量为61.18 ± 0.85%。因此，作为一种有前景的用于从……采油的生物表面活性剂，高水平表达伊枯草菌素A（脂肽）的新方法鲜有报道。因此，重组伊枯草菌素A展示了其在高效采油方面的潜力，在石油工业中具有特定应用。
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