Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in Escherichia coli. Extracellular enzymes from Bacillus subtilis, including β-1,4-xylanase, β-1,4-glucanase, and β-mannanase constructed with and without their signal peptides and intracellular enzymes from Pseudomonas stutzeri ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in E. coli BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of β-1,4-xylanase (Xyn), β-1,4-glucanase (Cel), and β-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in E. coli BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, E. coli can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.

Huperzine A (HupA), a sesquiterpene alkaloid isolated from Huperzia serrata, is clinically applied in China for Alzheimer’s disease and in the United States as a dietary supplement. In Thailand, Huperzia carinata (H. carinata) is an alternative and the richest natural source of HupA. However, poor solubility and low oral bioavailability restrict its therapeutic application. This study aimed to develop a self-emulsifying drug delivery system (SEDDS) to improve the solubility, dissolution, and efficacy of HupA from H. carinata extract in a mouse model of Parkinson’s disease (PD). The optimized SEDDS consisted of oleic acid (20%), polyethylene glycol-40 hydrogenated castor oil (70%), and propylene glycol (10%), containing 10% H. carinata extract. The HupA-loaded SEDDS displayed efficient self-emulsification, producing droplet sizes of 175.2 ± 8.3 nm to 253.4 ± 2.8 nm upon dilution in water. The SEDDS promoted rapid dissolution, achieving approximately 70% HupA release within 5 min and near-complete dissolution (100%) by 120 min in both gastric and intestinal media. In vitro, the SEDDS displayed significantly superior antioxidant and anti-inflammatory activities compared with the unformulated extract (p < 0.001). In vivo, MPTP-induced Parkinsonian mice receiving SEDDS containing HupA (0.75 and 1.00 μmol/kg body weight) showed significant improvements in locomotor function and motor coordination compared with both vehicle- and blank-SEDDS-treated groups (p < 0.001) at days 3 and 7 after MPTP induction. Biochemical assessments revealed declined malondialdehyde and nitrite levels in cortical and striatal brain tissues compared with the vehicle- and blank-SEDDS-treated groups (p < 0.05). Molecular studies confirmed that HupA-loaded SEDDS increased tyrosine hydroxylase and decreased tumor necrosis factor-α expression compared with the vehicle-treated group (p < 0.001) while protecting dopaminergic neurons in immunohistochemistry, indicating neuroprotective and antineuroinflammatory effects. The SEDDS formulation may represent a promising drug delivery system for the clinical application of HupA in PD and potentially other neurodegenerative disorders. This study contributes to Sustainable Development Goal 3 (good health and well-being) by developing neuroprotective formulations for neurodegenerative diseases.