Purification and surface functionalization of Cyclotella meneghiniana frustules as supports for immobilizing xylose isomerase.

The hierarchically nanoporous frustules ofCyclotella meneghinianaprovide high mechanical strength and large surface area, making them an ideal support for enzyme immobilization. d-Xylulose, a rare sugar with broad applications in medicine and chemistry, is typically produced via the enzymatic conversion of xylose; however, this process is hindered by enzyme deactivation, poor reusability, and high costs. To address these limitations, several xylose isomerases were screened, and PirE2_XI, which exhibited the highest maximum reaction rate (Vmax), was selected for immobilization studies. To prepare the immobilization support, C. meneghiniana frustules (CF) were intensively purified. Methanol treatment removed most of the organic matter, but residual organics and pore‑blocking pigments remained; subsequent purification with K2Cr2O7 eliminated these residues and increased the protein loading and enzyme activity of immobilized PirE2_XI by 93.7% and 137.5%, respectively, compared with untreated frustules. After PirE2_XI adsorption onto K2Cr2O7‑purified frustules (PF), the resulting PirE2_XI@PF was surface-functionalized with polyethyleneimine (PEI) and crosslinked with glutaraldehyde (GA), forming a protective biopolymer coating that minimized enzyme leaching and enhanced operational stability. After six consecutive catalytic cycles, the immobilized PirE2_XI (PEI@GA@PirE2_XI@PF) produced an average of 6.9 g/L of d-xylulose per reaction. Collectively, frustule-based immobilization platform offers a robust and reusable system for enzymatic d-xylulose production.