L-Carnosine (L-Car) is a biologically active dipeptide with antioxidant and anti-aging effects, formed by condensation of β-alanine (β-Ala) with L-histidine (L-His), which is used widely in the pharmaceutical and cosmetic industries. Here we report a dipeptide hydrolase originating from Bacillus megaterium , designated as Bm PepD, whose synthetic activity reached 6.5 times that of Sm PepD, which is the highest active dipeptidase reported so far for L-Car biosynthesis in vitro . By site-directed saturation mutagenesis, we obtained a significantly improved variant (T171N) of Bm PepD. It showed a specific activity of 22.9 U mg −1 protein , reaching 1.4 times that of wild-type Bm PepD or 9.1 times that of Sm PepD, which is the highest ever reported among this enzyme class. After optimizing the reaction conditions of variant Bm PepD T171N , we performed the synthetic reaction of L-Car simply from the two free amino acids (0.2 M L-His and 6.5 M β-Ala), achieving a titer of 31.3 mM L-Car with a specific productivity of 283 g Car · g catalyst −1 . This work provides a highly active and substrate (β-alanine)-tolerant dipeptide hydrolase for the facile synthesis of dipeptide L-Car via reversed hydrolysis.
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.
Cui Jian Dong,Zhang Ya Nan,Zhao Gui Xia,Zhao Guo Qun,Zhang Si,Lu Jun,Jia Shi Ru
To develop a new method for producing aspartame (APM), a simple and efficient strategy for the isolation of certain microorganisms producing APM from soil samples was designed. A newly strain with secreting certain specific dipeptidase to directly synthesize APM from the substrates of l -aspartic acid ( l -Asp) and l -phenylalanine methyl ester ( l -PM) without protection for amino acid side chains was screened from soil samples. APM concentration in reaction mixture was quantified by high performance liquid chromatography (HPLC), yield reached 0.015 g/L. Examination of the general morphological characteristics and data from the sequence analysis of the rDNA-ITS gene led to identification of the isolate as a strain of fungal endophyte spp. The newly isolated strain had a high potential for application in industrial processes for APM production. In particular, this new method was low cost for synthesis of APM during the reaction due to avoiding protection for amino acid side chains and optical resolution of the mixtures. As we known it, this is first report that a newly strain with a high potential for selective synthesis of the APM was isolated from soil.
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