Abstract

Pseudoenzymes are homologs of active enzymes that have amino acid substitutions in the active center and, therefore, usually do not possess enzymatic activity. In this work, a recombinant proprotein (proSerPH122) of the homolog of serine peptidases of the S1 family from the yellow mealworm (Tenebrio molitor) was obtained in the yeast producer strain Komagataella kurtzmanii. The target His₆-tagged protein was produced in a glycosylated form during secretion in yeast. The properties of both glycosylated and deglycosylated forms were studied. The proSerPH122 homolog with the replacement of active site Ser with Thr was pretreated with trypsin to study the enzymatic properties. The processed, mature homolog SerPH122 was shown to have low, but reliably detectable activity on the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA, and this activity did not depend on the glycosylation level.

中文翻译：

---

重组黄粉虫SerPH122——丝氨酸肽酶的蛋白水解活性同源物的制备及性质

摘要

假酶是活性酶的同源物，在活性中心有氨基酸取代，因此通常不具有酶活性。在这项工作中，在酵母生产菌株Komagataella kurtzmanii 中获得了来自黄粉虫（黄粉虫）的 S1 家族丝氨酸肽酶同源物的重组前蛋白 (proSerPH122) 。目标他的₆-标记蛋白在酵母中分泌期间以糖基化形式产生。研究了糖基化和去糖基化形式的特性。将活性位点 Ser 替换为 Thr 的 proSerPH122 同源物用胰蛋白酶预处理以研究酶学特性。经加工的成熟同源物 SerPH122 对显色底物 Suc-Ala-Ala-Pro-Phe-pNA 具有低但可可靠检测的活性，并且该活性不依赖于糖基化水平。