Enzymatic Production of Ascorbic Acid-2-phosphate by Recombinant Acid Phosphatase

Abstract

In this study, an environmentally friendly and efficient enzymatic method for the synthesis of l-ascorbic acid-2-phosphate (AsA-2P) from l-ascorbic acid (AsA) was developed. The Pseudomonas aeruginosa acid phosphatase (PaAPase) was expressed in Escherichia coli BL21. The optimal temperature, optimal pH, K, k, and catalytic efficiency of recombinant PaAPase were 50 °C, 5.0, 93 mM, 4.2 s, and 2.7 mM min, respectively. The maximal dry cell weight and PaAPase phosphorylating activity reached 8.5 g/L and 1127.7 U/L, respectively. The highest AsA-2P concentration (50.0 g/L) and the maximal conversion (39.2%) were obtained by incubating 75 g/L intact cells with 88 g/L AsA and 160 g/L sodium pyrophosphate under optimal conditions (0.1 mM Ca, pH 4.0, 30 °C) for 10 h; the average AsA-2P production rate was 5.0 g/L/h, and the AsA-2P production system was successfully scaled up to a 7.5 L fermenter. Therefore, the enzymatic process showed great potential for production of AsA-2P in industry.

Ascorbate Inhibits Proliferation and Promotes Myeloid Differentiation in TP53-Mutant Leukemia

“…For this reason, we included controls that mitigated any H 2 O 2 -induced toxicity by: (i) adding catalase to the medium, or (ii) using the redox-stable molecule phosphoascorbate. Phosphoascorbate is an ascorbate analogue that does not undergo oxidation to DHA and thus circumvents the generation of H 2 O 2 in cell culture media ( 22 , 38 , 39 ). When phosphoascorbate was added to the medium instead of ascorbate, SKM-1 intracellular ascorbate levels also increased, but at a slower rate.…”

Enzymatic Production of Ascorbic Acid-2-Phosphate by Engineered Pseudomonas aeruginosa Acid Phosphatase

“…Culture conditions, fed-batch fermentation, PaAPase purification, and transformation experiments were described in our previous study. 21 2.4. Protein Purification.…”

“…The total amounts of AsA and AsA-2P in the reaction mixture were determined using an Agilent 1260 HPLC system with an UV detector at 254 nm. 21…”

“…To evaluate the potential for increased production of AsA-2P, we performed fermentation experiments using WT and Var 5 according to the method described in our previous study. 21 As shown in Figure 6A, Var 5 reached the highest enzyme activity of 2080.4 U/L after 10 h of fermentation, and WT reached its highest activity of 1127.7 U/L after 12 h. Figure 6B shows that the dry cell weights (DCWs) of both the WT-and Var 5 -producing cells increased over the course of 18 h, reaching 8.5 g/L and 7.2 g/L, respectively. Notably, we harvest Var 5 and WT cells when their enzyme activity reaches the maximum (10 h and 12 h, respectively).…”

“…19 Additionally, Pseudomonas azotocolligans selected from 715 strains of microorganisms demonstrated the production of 31.6 g/L AsA-2P with a conversion rate of 27.6%. 20 To improve AsA-2P conversion, an enzymatic method for AsA-2P synthesis from AsA using recombinant Pseudomonas aeruginosa APase (PaAPase) was described in our previous study, reporting the highest AsA-2P titer (50.0 g/L) obtained by incubating 75 g/L intact cells with 88 g/L AsA and 160 g/L sodium pyrophosphate (PPi) for 10 h. 21 However, similar to previous methods, we obtained a low conversion rate of only 39.2% under optimal conditions. Nevertheless, the practical application of such an enzymatic synthesis method for the industrialscale production of AsA-2P remains a major challenge.…”