Department of Food Science and Technology, Faculty of Agricultural Engineering, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran, Iran.
Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.
Mol Biol Rep. 2019 Jun;46(3):2599-2608. doi: 10.1007/s11033-018-4468-1. Epub 2019 Apr 24.
Bioactive peptides are short amino acid sequences with desirable health effects which are derived from animals, plants, and marine sources. In this study, recombinant production of a bioactive peptide (GIISHR) from spotless smooth-hound (Mustelus griseus) muscle and its antioxidant properties is discussed. A gene composed of 12 tandem copies of the peptide sequence was cloned in pET-28a and expressed as a His-tagged polypeptide in Escherichia coli. The recombinant polypeptide was then purified by Ni-NTA affinity chromatography, cleaved by Trypsin and purified by ultrafiltration. DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) and hydroxyl radical scavenging activity assays, ferric reducing antioxidant power (FRAP) assay and β-carotene bleaching test were used to characterize the antioxidant activity of the GIISHR. Liquid chromatography-mass spectrometry analysis revealed 60% purity for released bioactive peptide. Production yield was estimated as 60-80 mg GIISHR active peptide per 1 L bacterial culture. Antioxidant activity assays indicated that the antioxidant activity was increased with increase in peptide concentration. Though the DPPH radical scavenging activity, FRAP and β-carotene bleaching power of the peptide were lower than those of the synthetic antioxidant tert-butylhydroquinone (TBHQ), the ABTS and hydroxyl radical scavenging activities of the peptide (at a concentration of 20 mg/mL) were similar to those of TBHQ (at a concentration of 0.1 mg/mL). The findings of the present study may be helpful in development of a process for production of the bioactive antioxidant peptides and its application in food industry.
生物活性肽是具有理想健康效应的短氨基酸序列，来源于动物、植物和海洋资源。本研究讨论了来自皮光星鲨（Mustelus griseus）肌肉的生物活性肽（GIISHR）的重组生产及其抗氧化特性。由 12 个串联肽序列组成的基因被克隆到 pET-28a 中，并在大肠杆菌中表达为 His 标记的多肽。然后，通过 Ni-NTA 亲和层析纯化重组多肽，用胰蛋白酶切割并通过超滤纯化。使用 DPPH（1,1-二苯基-2-苦基肼）、ABTS（2,2'-联氮双-3-乙基苯并噻唑啉-6-磺酸）和羟基自由基清除活性测定、铁还原抗氧化能力（FRAP）测定和β-胡萝卜素漂白试验来表征 GIISHR 的抗氧化活性。液相色谱-质谱分析显示释放的生物活性肽纯度为 60%。估计每 1 L 细菌培养物可生产 60-80 mg GIISHR 活性肽。抗氧化活性测定表明，随着肽浓度的增加，抗氧化活性增加。尽管肽的 DPPH 自由基清除活性、FRAP 和β-胡萝卜素漂白能力低于合成抗氧化剂叔丁基对苯二酚（TBHQ），但肽的 ABTS 和羟基自由基清除活性（浓度为 20 mg/mL）与 TBHQ 相似（浓度为 0.1 mg/mL）。本研究的结果可能有助于开发生物活性抗氧化肽的生产工艺及其在食品工业中的应用。
