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论文类型：期刊论文

发表时间：2019-02-02

发表刊物：MICROBIAL CELL FACTORIES

收录刊物：SCIE、PubMed、Scopus

卷号：18

期号：1

页面范围：27

ISSN号：1475-2859

关键字：l-Alanyl-l-glutamine; -Amino acid ester acyltransferase; Immobilization; Codon optimization; Recycle

摘要：Backgroundl-Alanyl-l-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln.ResultsIn this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of -amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3days at 26 degrees C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 degrees C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln=1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles.ConclusionsCharacterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.