Amino acids, neurotransmitters, purines, and pyrimidines are bioactive molecules that play fundamental roles in maintaining various physiological functions. Their metabolism is closely related to the health, growth, development, reproduction, and homeostasis of organisms. Most recently, comprehensive measurements of these metabolites have shown their potential as innovative approaches in disease surveillance or drug intervention. However, simultaneous measurement of these metabolites presents great difficulties. Here, we report a novel quantitative method that uses hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS), which is highly selective, high throughput, and exhibits better chromatographic behavior than existing methods. The developed method enabled the rapid quantification of 37 metabolites, spanning amino acids, neurotransmitters, purines, and pyrimidines pathways, within 6.5 min. The compounds were separated on an ACQUITY UPLC® BEH Amide column. Serum and brain homogenate were extracted by protein precipitation. The intra- and interday precision of all of the analytes was less than 11.34 %, and the accuracy was between -11.74 and 11.51 % for all quality control (QC) levels. The extraction recoveries of serum ranged from 84.58 % to 116.43 % and those of brain samples from 80.80 % to 119.39 %, while the RSD was 14.61 % or less for all recoveries. This method was used to successfully characterize alterations in the rat brain and, in particular, their dynamics in serum. The following study was performed to simultaneously test global changes of these metabolites in a serotonin antagonist p-chlorophenylalanine (PCPA)-induced anxiety and insomnia rat model to understand the effect and mechanism of PCPA. Taken together, these results show that the method is able to simultaneously monitor a large panel of metabolites and that this protocol may represent a metabolomic method to diagnose toxicological and pathophysiological states.

Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that propeptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

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