Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer’s disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.

Contributions of the evolutionarily conserved A16Leu and B17Leu to insulin foldability were characterized by evaluating folding properties of single-chain insulin analogs. The results showed A16Leu had much more significant effects on the foldability of insulin than B17Leu.

Relaxin-3 (also known as INSL7) is the most recently identified member of the insulin-like family. It is predominantly expressed in the nucleus incertus of the brain and involved in the control of stress response, food intake, and reproduction. In the present work, we have established a simple approach for the preparation of the mature human relaxin-3 peptide. We first designed and recombinantly expressed a single-chain relaxin-3 precursor in E. coli cells. After purification by immobilized metal ion affinity chromatography, refolding in vitro through disulfide reshuffling, and digestion by endoproteinase Asp-N, mature human relaxin-3 was obtained in high yield and at low cost. Peptide mapping and circular dichroism spectroscopy studies suggested that the recombinant relaxin-3 adopted an insulin-like fold with the expected disulfide linkages. The recombinant mature relaxin-3 was fully active in both RXFP3 binding and activation assays. The activity of the single-chain precursor was very low, suggesting that a free C-terminus of the B-chain is necessary for receptor-binding and activation of relaxin-3. Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.Graphical abstractDownload full-size imageResearch highlights▶ A single-chain precursor of relaxin-3 with a mini C-peptide and a 6×His tag was designed. ▶ The precursor was expressed well in E. coli cells. ▶ After purification, in vitro refolding, and enzymatic digestion, fully active human relaxin-3 was obtained in high yield and at low cost. ▶ Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.