Co-immobilization of Cellulase and Glucose Oxidase Layer-by-Layer and Chain Catalytic Reaction

A dual-enzyme cascade catalytic system (Cu-rGO-Fe₃O₄-GA-CEL-PEI-GOD) was prepared by co-immobilizing cellulase (CEL) and glucose oxidase (GOD) on a nanocomposite (Cu-rGO-Fe₃O₄) to efficiently catalyze the conversion of carboxymethyl cellulose (CMC) to gluconic acid. A layer-by-layer strategy was used by adding polyethyleneimine (PEI) to allow the upper enzyme layer to attach to the lower neighboring layer, increasing the loading capacity of the support. The loading capability of CEL and GOD on Cu-rGO-Fe₃O₄-GA-CEL-PEI-GOD were 55.034 mg g⁻¹ and 12.4 mg g⁻¹, respectively. The specific activity of CEL on Cu-rGO-Fe₃O₄-GA-CEL was 74.3 U·g⁻¹, and that of immobilized CEL after cross-linking PEI was 25.45 U·g⁻¹, which could retain 34.253% of the enzyme activity. The specific activity of GOD on Cu-rGO-Fe₃O₄-GA-CEL-PEI-GOD was 30.9 U·g⁻¹. The carrier Cu-rGO-Fe₃O₄ has peroxidase-like activity, which can timely remove harmful H₂O₂ to the enzyme, thereby improving the yield of gluconic acid and the stability of biocatalysts. The yield of gluconic acid with Cu-rGO-Fe₃O₄-GA-CEL-PEI-GOD reached 96.04% within 2 h, higher than the control systems for comparison. In addition, the Cu-rGO-Fe₃O₄-GA-CEL-PEI-GOD maintained 82.61% of catalytic activity even after undergoing seven cycles of reaction. The dual-enzyme catalytic systems had shallow temperature and pH optima of 40 °C and 4.5. Such a chemoenzymatic cascade system provides a new strategy for the conversion from CMC to gluconic acid in one-step.

Graphical Abstract

Enzymatic conversion of sodium carboxymethyl cellulose (CMC-Na) to gluconic acid.