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Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1. angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP. In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V. anguillarum serotype O1 strain MVM425. A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant. The angE-encoded protein was overexpressed in E. coli and purified by a three-step procedure. Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5'-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture. Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V. anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.

The morphological evolution of a single char particle with a low ash fusion temperature (T f) was investigated during the whole gasification process using a high-temperature stage microscope. The experimental results showed that the final shrinkage ratio of char particles at the temperature above T f (1300 °C) was higher than that below the deformation temperature (1000 °C). The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis results showed that the ash distribution in coal char was uniformly dispersed. Dispersed molten slag gradually aggregated to the molten slag layer during the gasification process at the temperature above T f, and the slag layer evidently hindered the diffusion of the gasifying agent. Therefore, there was a critical shrinkage ratio during the gasification process when the temperature was above T f and the critical shrinkage ratio of Xiaolongtan lignite (XLT) char particles was 0.7–0.8. The SEM–energy-dispersive X-ray spectroscopy (EDS) analysis results indicated that the critical points in the curve of the shrinkage ratio with time were related to the molten slag on the char surface. In addition, the reaction mechanism in this experiment was analyzed. The calculating results indicated that the critical thickness of the molten slag layer of XLT and Shenfu bituminous (SF) coal chars with different ash contents showed good agreement and the critical thickness of the slag layer was 6–18 μm. Finally, the sensitive analysis was performed to analyze the factors affecting the calculating results.

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