Production, Characterization and Application of a Novel Chitosanase from Marine Bacterium Bacillus paramycoides BP-N07.

Chitooligosaccharides (COS), a high-value chitosan derivative, have many applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitosanase, which catalyzes the hydrolysis of chitosan, can cleave ß-1,4 linkages to produce COS. In this study, a chitosanase-producing Bacillus paramycoides BP-N07 was isolated from marine mud samples. The chitosanase enzyme (BpCSN) activity was 2648.66 ± 20.45 U/mL at 52 h and was able to effectively degrade chitosan. The molecular weight of purified BpCSN was approximately 37 kDa. The yield and enzyme activity of BpCSN were 0.41 mg/mL and 8133.17 ± 47.83 U/mg, respectively. The optimum temperature and pH of BpCSN were 50 °C and 6.0, respectively. The results of the high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of chitosan treated with BpCSN for 3 h showed that it is an endo-chitosanase, and the main degradation products were chitobiose, chitotriose and chitotetraose. BpCSN was used for the preparation of oligosaccharides: 1.0 mg enzyme converted 10.0 g chitosan with 2% acetic acid into oligosaccharides in 3 h at 50 °C. In summary, this paper reports that BpCSN has wide adaptability to temperature and pH and high activity for hydrolyzing chitosan substrates. Thus, BpCSN is a chitosan decomposer that can be used for producing chitooligosaccharides industrially.

Engineering chitin deacetylase AsCDA for improving the catalytic efficiency towards crystalline chitin.

Chitin deacetylase (CDA) catalyzing the deacetylation of crystal chitin is a crucial step in the biosynthesis of chitosan, and also a scientific problem to be solved, which restricts the high-value utilization of chitin resources. This study aims to improve the catalytic efficiency of AsCDA from Acinetobacter schindleri MCDA01 by a semi-rational design using alanine scanning mutagenesis and saturation mutagenesis. The quadruple mutant M11 displayed a 2.31 and 1.73-fold improvement in kcat/Km and specific activity over AsCDA, which can remove 68 % of the acetyl groups from α-chitin. Furthermore, structural analysis suggested that additional hydrogen bonds, contributing the flexibility of amino acids and increasing the negative charge in M11 increased the catalytic efficiency. The microstructure changes of α-chitin pretreated by the mutant M11 were observed and evaluated using 13C CP/MAS NMR spectroscopy, FT-IR spectroscopy, XRD and SEM, and the results showed that M11 more efficiently catalyzed the release of acetyl groups from α-chitin. This study would provide a theoretical basis for the molecular modification of CDAs and accelerate the process of industrial production of chitosan by CDAs.

Transcription Factors in Aureobasidium spp.: Classification, Regulation and a Newly Built Database.

Transcription factors (TFs) can regulate the synthesis of secondary metabolites through different metabolic pathways in Aureobasidium spp. In this study, a set of 16 superfamilies, 45 PFAM families of TFs with the DNA-binding domains, seven zinc finger families and eight categories of the C2H2 TFs have been identified in Aureobasidium spp. Among all the identified TFs, four superfamilies and six PFAM families are the fungal-specific types in this lineage. The Zn2Cys6 and fungal-specific domain regulators are found to be overwhelmingly predominated, while the C2H2 zinc finger class comprises a smaller regulator class. Since there are currently no databases that allow for easy exploration of the TFs in Aureobasidium spp., based on over 50 references and 2405 homologous TFs, the first TFs pipeline-the Aureobasidium Transcription Factor Database (ATFDB)-has been developed to accelerate the identification of metabolic regulation in various Aureobasidium species. It would be useful to investigate the mechanisms behind the wide adaptability and metabolite diversity of Aureobasidium spp.