Carbon as a Simple Support for Redox Biocatalysis in Continuous Flow

Abstract

A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also considered as optimization targets, such as low E factors between 2.5-11 and space-time yields of up to 22.9 g L^-1 h^-1. The long-term stability of the biocatalytic reactor was also demonstrated, with full conversion maintained over more than 30 h of continuous operation.

Scheme 1. (A) NADH-Dependent Biocatalytic Conversion of Pyruvate to Lactate; (B) Flow Reactor Setup

Legend for flow reactor setup: (i) reaction solution vessel; (ii) syringe pump; (iii) packed bed reactor; (iv) fraction collector; LDH = lactate dehydrogenase; FDH = formate dehydrogenase; ● = enzyme-modified carbon particle; ○ = glass bead.

Figure 1

Effect of varying (A) tRes, (B) the NAD⁺ mol %, (C) the number of equivalents of formate, and (D) the Tris-HCl buffer concentration on the conversion of pyruvate to lactate in the continuous packed bed reactor. Unless otherwise stated, all experiments were performed with 50 mM pyruvate, 5 equiv of formate, 1 mol % NAD⁺, 50 mM Tris-HCl buffer (pH 8.0), 3 mg of LDH, 30 mg of FDH, and 30 mg of carbon at room temperature. Symbols: green circles, tRes = 10 min; blue squares, tRes = 20 min; red triangles, tRes = 30 min.

Figure 2

Contour profiles showing the combination of predicted responses for conversion (red), STY (green), and E factor (blue) for the continuous biocatalytic conversion of pyruvate to lactate for various operating conditions: (A) tRes vs NAD⁺; (B) tRes vs formate; (C) NAD⁺ vs formate. Colored sections denote the operating conditions where the desired response limit is not achieved, and the white sections denote the optimum operating windows in which all of the desired response limits are achieved. The profiles were generated using the statistical design software JMP 14.

Figure 3

Long-term operation of the continuous biocatalytic packed bed reactor for the conversion of pyruvate to lactate. Conditions: 50 mM pyruvate, 5 equiv of formate, 1 mol % NAD⁺, 50 mM Tris-HCl buffer, pH 8.0, room temperature, tRes = 30 min, 16 μL min^–1, reactor volume = 0.48 mL.