Abstract：
Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L-1 with a specific activity of 96 ± 0.32 U mg-1, resulting in a total enzyme activity of 19137 ± 1131 U L-1, demonstrating significant potential for industrial applications.
摘要：
人羧肽酶B1（hCPB1）对于重组胰岛素生产至关重要，在制药行业持有可观的价值。目前的挑战包括hCPB1酶活性有限。在这项研究中,重组hCPB1在毕赤酵母中高效表达。为了增强hCPB1的分泌，我们进行了信号肽筛选并删除了Vps10sortilin结构域，减少液泡分类错误。Sec4p的过表达增加了分泌囊泡与质膜的融合，并将hCPB1的分泌提高了20％。合理的蛋白质工程产生了22个单突变突变体，并鉴定了A178L突变，导致hCPB1比活性增加了30％。然而,所有增加比活性的组合突变均降低了蛋白质表达水平.因此,使用PROSS进行计算机辅助的全局蛋白质设计旨在提高比活性并保持良好的蛋白质表达。在六个设计的突变体中，hCPB1-P6显示催化速率常数(kcat)显著增加114%，米氏常数(Km)下降137%，催化效率提高了490%。大多数突变发生在hCPB1-P6的表面，有8个位点突变为脯氨酸。在5升发酵罐中，分泌增强的毕赤酵母底盘生产hCPB1-P6至199.6±20mgL-1，比活性为96±0.32Umg-1，总酶活性为19137±1131UL-1，显示出巨大的工业应用潜力。
