Hemu Xinya, Zhang Xiaohong, Nguyen Giang K T, To Janet, Serra Aida, Loo Shining, Sze Siu Kwan, Liu Chuan-Fa, Tam James P
School of Biological Sciences, Nanyang Technological University 637551 Singapore
WIL@NUS Corporate Lab, MD6 Centre for Translational Medicine, Wilmar International Limited, National University of Singapore 117599 Singapore.
RSC Adv. 2021 Jun 30;11(37):23105-23112. doi: 10.1039/d1ra03763c. eCollection 2021 Jun 25.
Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes.
Butelase-1是一种天冬酰胺内肽酶或豆球蛋白，是已知的典型且最快的Asn/Asp特异性肽连接酶。它在肽和蛋白质的工程化及大环化方面非常有用。然而，天然和重组Butelase-1的某些生化特性及应用仍未得到探索。在此，我们报告了提高天然和细菌表达的重组Butelase-1产量的方法，以及如何通过端到端环化利用它们来提高两种重要工业酶（脂肪酶和植酸酶）的稳定性和活性。首先，通过确定其在嫩组织（如嫩枝）中最高的分布，将天然Butelase-1的产量提高了3倍，达到15 mg/kg。通过促进细胞质二硫键折叠、密码子改变和N端前结构域的截短，提高了重组产生的可溶性Butelase-1的产量。天然和重组Butelase-1表现出相似的连接酶活性、物理稳定性和耐盐性。此外，通过蛋白质组学分析确定了天然和重组Butelase-1的加工和糖基化位点。还优化了两种形式的Butelase-1（冷冻或冻干）的储存条件。由可溶性或固定化的Butelase-1介导的脂肪酶和植酸酶的环化高效且简单，并导致热稳定性增加和酶活性增强。总体而言，提高Butelase-1的产量可用于通过端到端环化提高脂肪酶和植酸酶的生物催化效率。反过来，连接酶改良的酶可能是生产更稳定、高效工业酶的一种通用且环保的策略。
