Abstract：
Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of β-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.
摘要：
肌肽是一种具有重要生理功能的天然生物活性二肽，广泛应用于食品和医药领域。粘质沙雷氏菌的二肽酶（PepD）可以在Mn2存在下催化β-丙氨酸与1-组氨酸的反向水解反应以合成肌肽。然而,由于酶的低活性和高成本，实践肌肽生物合成仍然具有挑战性。因此,开发具有高活性和稳定性的生物催化剂对合成肌肽具有重要意义。这里,我们建议将Mn2+螯合到聚乙烯亚胺（PEI），诱导快速形成磷酸钙纳米晶体（CaP），Mn-PEI@CaP通过静电相互作用用于PepD固定。以Mn-PEI@CaP为载体增强了固定化酶的稳定性。此外，负载在载体中的Mn2充当固定化PepD的原位活化剂，以促进肌肽合成的生物催化过程。制备的固定化酶（PepD-Mn-PEI@CaP）与游离的PepD加Mn2保持相似的活性（活性恢复，102.5%)，同时表现出提高的热稳定性和pH耐受性。此外，它表现出比游离PepD系统快大约两倍的肌肽合成。PepD-Mn-PEI@CaP在八次分批催化循环后保留了86.8％的原始活性，而在多个循环中没有添加游离的Mn2离子。这项工作为PepD和Mn2+的共固定化提供了一种新的策略，这大大提高了生物催化的可操作性，并证明了固定化PepD系统用于高效肌肽合成的潜力。
