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A novel peptide containing antimicrobial sequence and gelatinase cleavage sites was designed for Staphylococcus aureus detection. Since Staphylococcus aureus could secrete gelatinase, the fluorescein labeled peptide GKRWWKWWRRPLGVRGC could be recognized and cleaved. The obtained products were able to be analyzed by capillary electrophoresis with fluorescence detection. To explore the effect of Staphylococcus aureus concentration on enzyme digestion ability of peptide, Staphylococcus aureus with different concentrations were incubated with the peptide. Results indicated that capillary electrophoretic method was efficient for determining Staphylococcus aureus content. Compared with traditional approaches for Staphylococcus aureus detection, capillary electrophoresis possessed higher efficiency, enhanced sensitivity, and low sample consumption. Moreover, the proposed peptide also presented desirable antimicrobial activity. It suggested that the novel antimicrobial peptide used in this research opens a new path of detecting Staphylococcus aureus by capillary electrophoretic method.

Chondroitin sulfate (CS) is widely applied in the medical, clinical, and nutraceutical fields. However, all commercialized CS is extracted from animal tissues, which is associated with many problems, such as heavy reliance on antibiotics, animal excrement waste and long breeding time. Here, we developed a promising new green route for the de novo biosynthesis of non-animal CS from methanol, a one-carbon compound, using the engineered Pichia pastoris cell factory. Firstly, three exogenous genes, namely kfoC and kfoA from Escherichia coli K4 and tuaD from Bacillus subtilis, were introduced into P. pastoris to construct the chondroitin synthesis pathway, which yielded 5.5 mg L−1 chondroitin. After optimizing the expression of committed enzymes by codon optimization, the production of chondroitin improved by approximately 35-fold to 189.8 mg L−1. Thereafter, 6 kinds of Kozak sequences and 10 different endogenous promoters were compared and applied to achieve the expression of active chondroitin-4-O-sulfotransferase (41.3 U L−1). After integrating the chondroitin biosynthesis and sulfonation modules, the engineered strain, Pp006, successfully produced 182.0 mg L−1 CSA [GlcA-GalNAc (4S)] with a sulfation degree of 1.1%. We further improved the sulfation degree to 2.8% by strengthening the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) supply with the overexpression of adenosine-5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase. In the fed-batch cultivation, the final titer of CSA reached 2.1 g L−1 and the sulfation degree increased to 4.0%. Moreover, the results suggest that the P. pastoris cell factory could be used as a green route to produce other sulfated glycosaminoglycans.

The detection of protease activity in the body plays a significant role in the early diagnosis of diseases. However, enzymes inevitably come into contact with various complex biological fluids in the body during the flow, which greatly increases the detection difficulty. Therefore, protease detection in vivo has great challenges. Herein, we report a new assay for detecting protease using capillary electrophoresis inside a capillary with semicircular bends. We first designed a peptide substrate, and then the peptide was self-assembled with quantum dots to form a QDs-peptide substrate. The capillary was bent to semicircular-shaped turns and served as a micro-reactor to allow protease and substrate react in it. Due to the different electrophoretic velocity, the protease and the substrate were mixed inside the bent capillary with sequential injections and the cleavage of the substrate can be detected using capillary electrophoresis combined with Forster resonance energy transfer technology. This novel assay will greatly expand the detection of enzyme activity in vivo.