Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16

Abstract

Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

Keywords: Bacillus aryabhattai TCI-16; characterization; chitin deacetylase; genome sequencing; isolation.

FIGURE 1

Morphological andfeatures of strain TCI-16. (A) After incubation at 30°C for 48 h, TCI-16 hydrolyzed colloidal chitin and formed transparent circles on the selective medium; (B) after incubation at 30°C for 48 h, CDA producing colonies reacted chromogenically and exhibited yellow circles in the differential medium (right), but the control was unchanged (left); (C) after incubation at 30°C for 48 h, colony morphology of the TCI-16 strain in LB medium; (D) gram staining; (E) SEM image (×30.0k).

FIGURE 2

Phylogenetic tree of the strain TCI-16 based on 16S rDNA sequence.

FIGURE 3

Fermentation characteristics curve of Bacillus aryabhattai TCI-16.

FIGURE 4

Genome map of B. aryabhattai TCI-16 (outside to inside): first circle shows the identification of genome information; the second and third circles are CDS distribution on positive and negative strands, and different colors indicate the different functional classification in COG categories; the fourth circle represents rRNA and tRNA; the fifth circle means the GC content, the outward part (red) indicates that the GC content of the region is higher than the average value and the inward part (blue) indicates that is lower, with higher peaks denoting a greater difference from the average GC content; the innermost circle is GC-Skew value.

FIGURE 5

Carbohydrate-active enzyme family classification of B. aryabhattai TCI-16. Abscissa represents CAZy family classification and ordinate represents the number of genes.

FIGURE 6

Amino acid sequence analysis of the predicted CDA. (A) Hydrophilicity profile of amino acid sequence of the CDA in B. aryabhattai TCI-16, The minimum hydrophobicity of BaCDA was −2.033, which was located at 113th amino acid (Gln); the maximum value was 3.689, which was located at 11th (Val). Overall, the protein encoded by this gene is hydrophilic. Relative weight for window edges was 100% and the weight variation model was linear. (B) Signal P prediction of amino acid sequence of the CDA in B. aryabhattai TCI-16. The prediction of the N-terminal signal peptide from the amino acid sequence shows that the protein has no signal peptide. (C) Transmembrane helices prediction of the CDA in B. aryabhattai TCI-16. (D) Secondary structure of the CDA in B. aryabhattai TCI-16. The letter h denoted the alpha-helical structure, the irregular coil structure was denoted by c, t was the beta-turn structure, and e was the extended strand.

FIGURE 7

Comparison of the tertiary structures of BaCDA and ArCE4A proteins. (A) Tertiary structure of ArCE4A protein of Arthrobacter sp. (5lfz.1. A). (B) Tertiary structure of BaCDA protein, using the crystal structure of ArCE4A protein from Arthrobacter sp. (5lfz.1. A) as template sequence. (C) Structure comparison between BaCDA and ArCE4A. The solid part was BaCDA tertiary structure, while the translucent was ArCE4A. Arrows indicated amino acid positions with structural differences between the two.

FIGURE 8

Amino alignment of CDA from several microorganisms. Amino acid sequences of CDAs from Penicillium sp. RFL-2021a (KAF7717591.1), Salmonella enterica (AVB04285.1), Amylomyces rouxii (CAA79525.1), Colletotrichum lindemuthianum (AAT68493.1), Aspergillus niger (GAQ42027.1), Bacillus cereus (AHW57573.1), Aspergillus nidulans (ACF22100.1), Bacillus licheniformis (AOP14170.1), Mortierlla sp. GBA30(KAG020643.1), Aspergillus flavus (RMZ39112.1), Bacillus velezensis (ASB52338.1), Saccharomyces cerevisiae YJM789 (EDN59219.1), Bacillus sp. FJAT-27238 (KMZ42832.1), Mucor ambiguus (GAN03916.1), and Rhizopus microspores ATCC52813 (XP_023466809.1) were aligned. Identical or highly conserved residues are shaded in red, while similar residues are shaded in yellow or green. The catalytic domains with five conserved motifs 1∼5 were framed by the blue box.

FIGURE 9

Phylogenetic tree of B. aryabhattai TCI-16 CDA (BaCDA) with other CDA proteins from different microorganisms based on the amino acid sequence comparisons. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 9.99177349 is shown.