Enzymes are efficient biocatalysts providing an important tool in many industrial biocatalytic processes. Currently, the immobilized enzymes prepared by the cross-linked enzyme aggregates (CLEAs) have drawn much attention due to their simple preparation and high catalytic efficiency. Combined cross-linked enzyme aggregates (combi-CLEAs) including multiple enzymes have significant advantages for practical applications. In this review, the conditions or factors for the preparation of combi-CLEAs such as the proportion of enzymes, the type of cross-linker, and coupling temperature were discussed based on the reaction mechanism. The recent applications of combi-CLEAs were also reviewed.

A rapid immobilization method for cellulase was developed. Functional graphene oxide was synthesized and grafted with hydrophobic spacer P-β-sulfuric acid ester ethyl sulfone aniline (SESA) though etherification and diazotization. The functionalized graphene oxide was characterized by Fourier-transform infrared spectroscopy and was used as the carrier for the immobilization of cellulase via covalent binding. The immobilization of cellulase was finished in a very short time (10 min) and very high immobilization yield and efficiency of above 90% were achieved after optimization. When compared with the free cellulase, thermal and operational stabilities of the immobilized cellulase were improved significantly. At 50 °C, the half-life of the immobilized cellulase (533 min) was six-fold higher than that of the free cellulase (89 min). Additionally, the affinity between immobilized cellulase (Km = 2.19 g·L⁻¹) and substrate was more favorable than that of free cellulase (Km = 3.84 g·L⁻¹), suggesting the immobilized cellulase has higher catalytic efficiency. The possible immobilization mechanism was proposed. The results strongly indicate that the immobilization is highly efficient and has great potential for the immobilization of other enzymes.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed K_m and V_max of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD⁺. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD⁺ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

When the nascent chain is released from the ribosome, its packing into the apolar environment of the lipid bilayer in the endoplasmic reticulum is facilitated by the Sec61 translocon. In this process, coupling of the conformational change of the channel is essential to transport the nascent chain and meanwhile maintain the membrane permeability barrier. Two molecular dynamics simulations were performed in the current work to investigate the resilience of the lateral gate and the linkage mechanism of the lateral gate, pore ring and plug. The results affirmed that the lateral gate is able to recover its partially-closed state rapidly after the nascent chain segment enters the bilayer. This triggers subsequent motions of the pore ring and plug, which prevent the small molecules passing through the pore. The pore diameter in the partially-closed state is about 6–7 Å. The plug would move upward ∼2 Å if the lateral gate could not close. Two waters permeate through the channel when the lateral gate was open. Water molecules could go across the bilayer via the gap of the open lateral gate due to the occluding of the pore ring and plug.