The catalytic efficiency of carnitine CoA ligase from Escherichia coli ( EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiC F380M/N430D, which was then cloned into the plasmid pET- EcCaiC F380M/N430D- DegoPPK2. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Here, we obtained the best EcCaiC F380M/N430D double mutant with a k cat/ K m value 1.5 times higher than that of the wild type via mechanism-based protein engineering.
The carnitine-CoA ligase from Escherichia coli ( EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. The return of the products is accompanied by the return of the respective document.Pyrrolidone is a high value-added monomer and an important active drug intermediate. Returns of products are accepted within 14 days from the moment of receipt and if the products are in good condition and their packaging has not been unsealed. In any other case where the weight of the products of the order is greater than 4kg, there is an additional charge of 0.90€ for each additional kilo. We cooperate with: Alpha Bank, Eurobank, National Bank and Piraeus Bank.ĥ) Through Paypal secure transaction systemįor orders where the purchase cost and the weight of the products do not exceed 39€ and 4kg respectively, the shipping cost is 2€.įor orders where the purchase cost is greater than 39€ and the weight of the products remains less than 4kg, there is no shipping cost.
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