Abstract：
Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.
摘要：
果胶裂解酶（PNL）可以通过降解高度酯化水果中的果胶来提高果汁的透明度和风味，但是它们的耐酸性不足会导致果汁的活性迅速丧失。本研究旨在通过表面电荷设计提高黑曲霉PNLpelA的耐酸性。通过将pelA与蛋白质标签融合并在大肠杆菌中表达融合酶来建立修饰平台。使用计算工具鉴定了四个单点突变体以增加表面电荷。此外，组合的突变体M6(S514D/S538E)在pH3.0表现出99.8%的残余活性。然后使用多基因整合系统将M6基因整合到黑曲霉基因组中以获得重组PNLAM6。值得注意的是,AM6将橙汁的透光率提高到45.3%，比pelA高8.39倍。总之,AM6表现出最佳的耐酸性，使其成为工业果汁澄清的有希望的候选人。
