Enzymes are commonly used as biocatalysts for various biological and chemical processes. However, some major drawbacks of free enzymes (e.g. poor reusability and instability) significantly restrict their industrial practices. How to overcome these weaknesses remain considerable challenges. Enzyme immobilization is one of the most effective ways to improve the reusability and stability of enzymes. Cross-linked enzyme aggregates (CLEAs) has been known as a novel and versatile carrier-free immobilization method. CLEAs is attractive due to its simplicity and robustness, without purification. It generally shows: high catalytic specificity and selectivity, good operational and storage stabilities, and good reusability. Moreover, co-immobilization of different kinds of enzymes can be acquired. These CLEAs advantages provide opportunities for further industrial applications. Herein, the preparation parameters of CLEAs were first summarized. Next, characterization of structural and catalytic properties, stability and reusability are also proposed. Finally, some important applications of this technique in: environmental protection, industrial chemistry, food industry, and pharmaceutical synthesis and delivery are introduced. Potential challenges and future research directions, such as improving cross-linking efficiency and internal mass transfer efficiency, are also presented. This implies that CLEAs provide an efficient and feasible technique to improve the properties of enzymes for use in the industry.

Signal peptides are short peptides directing newly synthesized proteins toward the secretory pathway. These N-terminal signal sequences are ubiquitous to all prokaryotes and eukaryotes. Signal peptides play a significant role in recombinant protein production. Previous studies have demonstrated that the secretion amount of a given target protein varies significantly depending on the signal peptide that is fused to the protein. Signal peptide selection and signal peptide modification are the two main methods for the optimization of a recombinant protein secretion. However, the highly efficient signal peptide for a target protein with a specific bacterial expression host is not predictable so far. In this article, we collect several signal peptides that have previously performed well for recombinant protein secretion in gram-positive bacteria. We also discuss several factors influencing recombinant protein secretion efficiency in gram-positive bacteria. Signal peptides with a higher charge/length ratio in n-region, more consensus residues at the−3 and−1positions in c-region and a much higher proportion of coils are more likely to perform well in the secretion of recombinant proteins. These summaries can be utilized to the selection and directed modification of signal peptides for a given recombinant protein.