Journal of Advanced Research ( IF 11.4 ) Pub Date : 2022-12-23 , DOI: 10.1016/j.jare.2022.12.005 Riyuan Wang 1 , Yuancheng Wang 2 , Jianxin Song 1 , Chi Tian 1 , Xinyuan Jing 1 , Ping Zhao 2 , Qingyou Xia 2
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Introduction
Conventional hot-alkaline cocoon degumming techniques greatly weaken the physicochemical and mechanical properties of silk fibroin fiber, thus affecting the quality of silk fabric. Moreover, it causes massive energy waste and serious environmental pollution.
Objective
This study aims to establish a novel cocoon self-degumming method by genetic modification of silkworm varieties and silk fibers.
Methods
The self-degummed cocoon material was generated by specifically overexpressing trypsinogen protein in the sericin layer of silk thread; the effect of cocoon self-degumming method was evaluated by the degumming rate of sericin protein, the cleanliness and equivalent diameter of silk fibroin fiber; the basic characteristics of silk fibroin fiber degummed by cocoon self-degumming method and conventional hot-alkaline degumming technique were determined by electron microscopy, Fourier infrared spectroscopy, X-ray diffraction and tensile tests; the composition and biological activity of degummed sericin protein was respectively analyzed by liquid chromatograph-mass spectrometry and cytological experiments.
Results
The genetically engineered self-degumming cocoon containing trypsinogen protein was successfully created, and the content of trypsinogen protein in silk was 47.14 ± 0.90 mg/g. The sericin protein in the self-degumming cocoon was removed out in water or 1 mM Tris-HCl buffer (pH = 8.0). Compared to alkaline-degummed silk fibroin, self-degummed silk fibroin had better cleanliness, thicker equivalent diameter, more complete silk structure and better mechanical property. In addition, sericin protein degummed from self-degumming cocoons significantly promoted cell proliferation and caused no obvious cytotoxicity.
Conclusion
Compared to conventional hot-alkaline degumming technique, the cocoon self-degumming method by genetically overexpressing trypsinogen protein in sericin layer of silk thread can self-degummed in a mild degumming condition, and gain silk fiber with better quality and more biologically active sericin protein products. This strategy can not only reduce the environmental impact, but also generate greater economic value, which will accelerate its application in the silk and pharmaceutical industries.
中文翻译：
蚕茧自脱胶新方法及其对丝纤维的影响
介绍
常规热碱茧脱胶技术极大削弱了丝素纤维的理化性能和机械性能，从而影响了丝织物的质量。此外，还造成大量的能源浪费和严重的环境污染。
客观的
本研究旨在通过对蚕品种和蚕丝纤维进行基因改造，建立一种新型茧自脱胶方法。
方法
自脱胶茧材料是通过在丝线的丝胶层中特异性过表达胰蛋白酶原蛋白而产生的；通过丝胶蛋白的脱胶率、丝素纤维的清洁度和当量直径来评价茧自脱胶的效果；通过电镜、傅里叶红外光谱、X射线衍射和拉伸试验测定了茧自脱胶法和常规热碱脱胶工艺脱胶丝素纤维的基本特性；通过液相色谱-质谱联用法和细胞学实验分别分析了脱胶丝胶蛋白的组成和生物活性。
结果
成功制作出含胰蛋白酶原蛋白的基因工程自脱胶茧，蚕丝中胰蛋白酶原蛋白含量为47.14±0.90 mg/g。在水或 1 mM Tris-HCl 缓冲液（pH = 8.0）中去除自脱胶茧中的丝胶蛋白。与碱脱胶丝素相比，自脱胶丝素具有更好的清洁度、更粗的当量直径、更完整的丝结构和更好的机械性能。此外，自脱胶茧脱胶的丝胶蛋白可显着促进细胞增殖，且无明显细胞毒性。
结论
与传统的热碱脱胶技术相比，在丝胶层中基因过表达胰蛋白酶原蛋白的茧自脱胶方法可以在温和的脱胶条件下进行自脱胶，获得质量更好、生物活性更强的丝胶蛋白产品。 。这一战略不仅可以减少对环境的影响，还可以产生更大的经济价值，这将加速其在丝绸和制药行业的应用。
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