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.

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