Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthesis mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments, and gene manipulation revealed that l-threonine is the starting point involving l-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of l-threonine. The product of this reaction is l-2-amino-acetoacetate, which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate coenzyme A (CoA) ligase (KBL) catalyzes the cleavage of l-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, TMP can also be generated by Bacillus subtilis 168 by using l-threonine and d-glucose as the substrates and TDH as the catalytic enzyme. IMPORTANCE Despite alkylpyrazines' contribution to flavor and their commercial value, the synthesis mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates, and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differ from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthesis mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine’s synthesis pathway involving l-threonine-3-dehydrogenase as the catalytic enzyme and l-threonine as the substrate.

Chitosan/glycerophosphate thermosensitive hydrogel crosslinked physically was a potential drug delivery carrier; however, long gelation time limits its application. Here, chitosan-amino acid (AA) thermosensitive hydrogels were prepared from chitosan (CS), αβ-glycerophosphate (GP), and l-lysine (Lys) or l-glutamic acid (Glu). The prepared CS-Lys/GP and CS-Glu/GP hydrogel showed good thermosensitivity and could form gels in a short time. The optimal parameters of CS-Lys/ GP hydrogel were that the concentration of CS-Lys was 2.5%, the ratio of CS/Lys was 3.5/1.0, the ratio of CS-Lys/GP was 4.5/1.0. The optimal parameters of CS-Glu/GP hydrogel were that the concentration of CS-Glu was 3.0%, the ratio of CS/ Glu was 2.0/1.0, and the ratio of CS-Glu/GP was 4.0/1.5. Chitosan-amino acid (CS-AA) thermosensitive hydrogel had a three-dimensional network structure. The addition of model drug tinidazole (TNZ) had no obvious effect on the structure of hydrogel. The results of infrared spectroscopy showed that there were hydrogen bonds between amino acids and chitosan. In vitro release results showed that CS-Lys/GP and CS-Glu/GP thermosensitive hydrogels had sustained release effects. Thus, the chitosan-amino acid thermosensitive hydrogels hold great potential as a sustained release drug delivery system.

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