A Novel Potent Crystalline Chitin Decomposer: Chitin Deacetylase from Acinetobacter schindleri MCDA01

Chitosan is a functional ingredient that is widely used in food chemistry as an emulsifier, flocculant, antioxidant, or preservative. Chitin deacetylases (CDAs) can catalyze the hydrolysis of acetyl groups, making them useful in the clean production of chitosan. However, the high inactivity of crystalline chitin catalyzed by CDAs has been regarded as the technical bottleneck of crystalline chitin deacetylation. Here, we mined the AsCDA gene from the genome of Acinetobacter schindleri MCDA01 and identified a member of the uraD_N-term-dom superfamily, which was a novel chitin deacetylase with the highest deacetylation activity. The AsCDA gene was expressed in Escherichia coli BL21 by IPTG induction, whose activity to colloidal chitin, α-chitin, and β-chitin reached 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg, respectively. In 12 h, the enzymatic hydrolysis of AsCDA removed 63.05% of the acetyl groups from α-chitin to prepare industrial chitosan with a degree of deacetylation higher than 85%. AsCDA, as a potent chitin decomposer in the production of chitosan, plays a positive role in the upgrading of the chitosan industry and the value-added utilization of chitin biological resources.


Introduction
Chitin, a linear homopolymer of β-1,4-glycosidically linked N-acetylglucosamine (GlcNAc) residues, is the second largest natural polymer renewable resource after cellulose. Some 6 to 8 million tons of chitin solid waste are generated during the fishery processing of crab, shrimp, and lobster, which are value-added processing by-products of aquatic foods [1][2][3]. However, natural chitin has a dense crystalline structure, a highly ordered three-dimensional network, and a high degree of polymerization and insolubility in regular solvents, which severely limits the development and commercial application of chitin [4][5][6]. Therefore, natural chitin bioresource is often considered to be a burdensome waste product of the marine and biotechnology industries [7]. Chitosan, the deacetylated derivative of chitin, prepared from natural chitin sources with a degree of deacetylation higher than 55%, is widely used in the food industry, medicine, environmental protection, and agriculture due to its safety, non-toxic, biodegradable, biocompatibility, anti-oxidation, and bacteriostatic properties [8][9][10]. Significantly, chitosan is most widely used in the food industry, and these characteristics and the chemical structure of chitosan may contribute to its functionality and application as an emulsifier, flocculant, antioxidant, or preservative in and biosynthetic potential. Sequencing the whole genome of the A. schindleri MCDA01 strain showed that the total length of the genome was 3.33 Mb (including the plasmid genome 0.17 Mb) with 43% GC content (see Supplementary Figure S1). The whole genome of A. schindleri MCDA01 encoded 3107 putative predicted genes, which were 84.31% of the genome sequence including 21 ribosomal rRNAs, 88 tRNAs, and 9 sRNAs ( Table 1). The function annotation of putative CDs was successfully performed on 1902 GO genes (61.21% of the genome sequence, Figure 1A), 1881 KEGG genes (60.57% of the genome sequence, Figure 1B), 2267 COG genes (72.96% of the genome sequence, Figure 1C), and 35 CAZy genes (1.12% of the genome sequence, Figure 1D). To further analyze whether the A. schindleri MCDA01 strain was a potential chitin decomposer, chitin degrading enzymes involved in chitin deacetylation and hydrolysis were identified by the CAZy database. The 11 genes were annotated as deacetylases or chitinases including five gene-encoding chitinases and six gene-encoding deacetylases (including AsCDA) (see Supplementary  Table S2), which were annotated for carbohydrate metabolic processes and catalytic and hydrolase activity toward carbon-nitrogen. The genome size, GC content, and CD quantity of A. schindleri MCDA01 were larger than other reported strains of A. schindleri, which may mean that more genes of the A. schindleri MCDA01 genome involved in the microbial metabolism process could adapt to different environmental conditions (see Supplementary  Table S3) [28]. To investigate the evolutionary specificity and diversity of A. schindleri MCDA01, we carried out a comparative genomic analysis of different strains. Comparative genomics analysis revealed that A. Schindleri MCDA01 has few homologous genes with B. anthracis Ames and B. cereus ATCC14579, but more homologous genes with A. schindleri CIP107287 and A. schindleri ACE ( Figure 2A) and these results were largely consistent with the previous study of Ye et al. [11]. Particularly, 418 genes (13.45% of the genome sequence) were shared, and unique genes of A. schindleri MCDA01 strain ( Figure 2B) were involved in signaling and cellular processing, genetic information processing, carbohydrate metabolism (including AsCDA), lipid metabolism, environmental information processing, glycan biosynthesis and metabolism, amino acid metabolism, and so on ( Figure 2C), which displayed that A. Schindleri MCDA01 has higher carbohydrate metabolism activity and is a potent chitin decomposer [29,30].

Comparative Genome Analysis of Acinetobacter schindleri MCDA01
To investigate the evolutionary specificity and diversity of A. schindleri MCDA01, we carried out a comparative genomic analysis of different strains. Comparative genomics analysis revealed that A. Schindleri MCDA01 has few homologous genes with B. anthracis Ames and B. cereus ATCC14579, but more homologous genes with A. schindleri CIP107287 and A. schindleri ACE ( Figure 2A) and these results were largely consistent with the previous study of Ye et al. [11]. Particularly, 418 genes (13.45% of the genome sequence) were shared, and unique genes of A. schindleri MCDA01 strain ( Figure 2B) were involved in signaling and cellular processing, genetic information processing, carbohydrate metabolism (including AsCDA), lipid metabolism, environmental information processing, glycan biosynthesis and metabolism, amino acid metabolism, and so on ( Figure 2C), which displayed that A. Schindleri MCDA01 has higher carbohydrate metabolism activity and is a potent chitin decomposer [29,30].

Genome Mining and Bioinformatics Analysis of AsCDA
There are four genes annotated deacetylases belonging to the CE4 family in the A schindleri MCDA01 genome database, and AsCDA was mined uniquely according to the molecular mass 31 kDa of natural chitin deacetylase [11]. The sequences of AsCDA were

Genome Mining and Bioinformatics Analysis of AsCDA
There are four genes annotated deacetylases belonging to the CE4 family in the A. schindleri MCDA01 genome database, and AsCDA was mined uniquely according to the molecular mass 31 kDa of natural chitin deacetylase [11]. The sequences of AsCDA were uploaded to GenBank with Accession NO. MW295944.1, but not opened. The AsCDA gene is a 975 bp encoding 324 amino acids with a Polysacc_deac_1 domain. To further determine the phylogenetic status of AsCDA, we performed a multiple sequence alignment, phylogenetic analysis, and homologous modeling of the AsCDA amino acid sequence with other 14 members of the deacetylase superfamily. The phylogenetic analysis identified these deacetylases from three different clades representing the CE4 superfamily, spore_pdaA superfamily, and uraD_N-term-dom superfamily, respectively [31,32]. AsCDA is clustered together with other members of the uraD_N-term-dom superfamily and has high sequence similarity with Schizosaccharomyces pombe SpCDA, polysaccharide deacetylase from Pseudomonas aeruginosa, CDA from Rhodococcus equi F6, and PuuE protein from Pseudomonas fluorescens, some of which had enzymatic hydrolysis to chitooligosaccharides or chitin pretreated by [TBA][OH] ( Figure 3A) [31,32]. Interestingly, multiple sequence alignment of these enzymes revealed that AsCDA has a few notable differences from other CDAs of the CE4 superfamily. The conserved Asp-His-His metal-binding triad of the CE4 family is replaced by Glu-His-Trp of AsCDA (active sites: Glu 51 , Glu 52 , His 140 , Trp 144 , and His 276 ; Glu 51 : base catalytic site; His 276 : acid catalytic site, and the catalytic amino acid residues are different, with Glu replacing Asp for the acidic amino acid) ( Figure 3B). Two conserved inserted segments (IS1 and IS2) of the AsCDA sequence do not have a counterpart in the CE4 superfamily ( Figure 3B). In the next section, we will analyze the crystal structure of AsCDA to explore and elucidate the enzymatic reaction mechanism of this arrangement in the AsCDA sequence. Importantly, these results indicate that AsCDA is a new PuuE homolog of the uraD_N-term-dom superfamily and has a high-enzyme activity to catalyze crystalline chitin deacetylation.

Heterologous Expression of AsCDA in Escherichia coli
The recombinant plasmid pET-28a-AsCDA was a heterologous expression in E. coli BL21. SDS-PAGE analysis showed that the molecular weight of purified AsCDA was approximately 37.5 kDa with 2.11 mg/mL by nickel affinity chromatography ( Figure 4A), which was expected on the MW of natural chitin deacetylase produced by A. schindleri MCDA01. Some previous studies observed that the MW of chitin deacetylases ranged from 15 to 100 kDa and most chitin deacetylases were in the range of 35-40 kDa [8,14,22,33,34]. The specific activity of purified AsCDA treated colloidal chitin, α-chitin and β-chitin were 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg by acetic acid assay kit, respectively ( Figure 4B). Previous results have shown that most CDAs had a high activity on chitooligosaccharides, water-soluble chitosan, and colloidal chitin, but low or no activity toward crystalline chitin [2,30]. Thus, AsCDA has potential application in the effective deacetylation of crystalline chitin substrates, especially α-chitin.

Heterologous Expression of AsCDA in Escherichia coli
The recombinant plasmid pET-28a-AsCDA was a heterologous expression in E BL21. SDS-PAGE analysis showed that the molecular weight of purified AsCDA was proximately 37.5 kDa with 2.11 mg/mL by nickel affinity chromatography (Figure which was expected on the MW of natural chitin deacetylase produced by A. schin MCDA01. Some previous studies observed that the MW of chitin deacetylases ran from 15 to 100 kDa and most chitin deacetylases were in the range of 35-40 [8,14,22,33,34]. The specific activity of purified AsCDA treated colloidal chitin, α-ch and β-chitin were 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg by acetic acid assay respectively ( Figure 4B). Previous results have shown that most CDAs had a high act on chitooligosaccharides, water-soluble chitosan, and colloidal chitin, but low or no a ity toward crystalline chitin [2,30]. Thus, AsCDA has potential application in the effec

Deacetylation of Crystalline Chitin Catalyzed by Chitin Deacetylase AsCDA
To determine characterized microstructure changes, chitosan, α-chitin, and AsCD Ch were performed for Fourier transform infrared spectroscopy (FT-IR) and scanni electron microscope (SEM). The results of SEM, as in Figure 5, show that untreated chitin was large dense particles stacked in a layered structure whose surface compac arrange a crystalline microfibril structure, which was compact, rough, and texture. T bulk structure of AsCDA-Ch, however, was severely damaged. Holes and dense rifts we observed on the surface of AsCDA-Ch and its fibers became blurred and indistinct a exhibited a less well-defined interface with separated sheets. The particle state of AsCD Ch was similar to chitosan, and was obviously different from α-chitin. The microstructu images of α-chitin and chitosan by SEM were reported in previous studies, which furth verified that deacetylation of α-chitin by AsCDA was similar to chitosan [35][36][37]. The re son for microstructure changes in α-chitin treated by AsCDA was the reduction in ace group content, which caused the intramolecular and molecular structure to be destroy or weakened [38].

Deacetylation of Crystalline Chitin Catalyzed by Chitin Deacetylase AsCDA
To determine characterized microstructure changes, chitosan, α-chitin, and AsCDA-Ch were performed for Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). The results of SEM, as in Figure 5, show that untreated α-chitin was large dense particles stacked in a layered structure whose surface compactly arrange a crystalline microfibril structure, which was compact, rough, and texture. The bulk structure of AsCDA-Ch, however, was severely damaged. Holes and dense rifts were observed on the surface of AsCDA-Ch and its fibers became blurred and indistinct and exhibited a less well-defined interface with separated sheets. The particle state of AsCDA-Ch was similar to chitosan, and was obviously different from α-chitin. The microstructure images of α-chitin and chitosan by SEM were reported in previous studies, which further verified that deacetylation of α-chitin by AsCDA was similar to chitosan [35][36][37]. The reason for microstructure changes in α-chitin treated by AsCDA was the reduction in acetyl group content, which caused the intramolecular and molecular structure to be destroyed or weakened [38]. The comparative analysis results of chitosan, α-chitin, and AsCDA-Ch by FT-IR showed that the structure of the three samples was basically the same, according to the similarity of the peak position and absorbance. The amide I, II, and III bands of the characteristic peaks were observed at 1655, 1560, and 1314 cm −1 in the FT-IR spectrum, and the peak position of 1655 cm −1 indicated that the samples were deacetylated products of αtype chitin ( Figure 6A) [39]. From the absorption bands at 1655 cm −1 (amide I) and 3450cm −1 (-OH group) emerged the displacement of the absorption peak in different chitin samples. The A1655/A3450 ratio linear with the degree of deacetylation of chitin was used to calculate the degree of deacetylation (DD) of samples [40]. The DD of the samples was determined on the basis of the A1655/A3450 value from FT-IR results, the degree of deacetylation of α-chitin, AsCDA-Ch, and chitosan were 25.44%, 88.49%, and 97.54%, respectively ( Figure 6B). AsCDA removed 63.05% of the acetyl groups for α-chitin pretreated for 12 h, and the degree of deacetylation did not change with the increase in treatment time ( Figure  S3). The results indicated that the enzymatic hydrolysis of AsCDA resulted in the release of an acetyl group and increased the degree of deacetylation of α-chitin. Compared to the NaCDA from Nitratireductor aquimarinus MCDA3-3 studied by our group, the AsCDA showed a significant improvement in reaction time and substrate concentration of the catalytic preparation of chitosan [35]. Chitosan was the deacetylated derivative of chitin with a high rate of deacetylation higher than 55% [2]. AsCDA has the highest activity toward The comparative analysis results of chitosan, α-chitin, and AsCDA-Ch by FT-IR showed that the structure of the three samples was basically the same, according to the similarity of the peak position and absorbance. The amide I, II, and III bands of the characteristic peaks were observed at 1655, 1560, and 1314 cm −1 in the FT-IR spectrum, and the peak position of 1655 cm −1 indicated that the samples were deacetylated products of α-type chitin ( Figure 6A) [39]. From the absorption bands at 1655 cm −1 (amide I) and 3450 cm −1 (-OH group) emerged the displacement of the absorption peak in different chitin samples. The A 1655 /A 3450 ratio linear with the degree of deacetylation of chitin was used to calculate the degree of deacetylation (DD) of samples [40]. The DD of the samples was determined on the basis of the A 1655 /A 3450 value from FT-IR results, the degree of deacetylation of α-chitin, AsCDA-Ch, and chitosan were 25.44%, 88.49%, and 97.54%, respectively ( Figure 6B). AsCDA removed 63.05% of the acetyl groups for α-chitin pretreated for 12 h, and the degree of deacetylation did not change with the increase in treatment time ( Figure S3). The results indicated that the enzymatic hydrolysis of AsCDA resulted in the release of an acetyl group and increased the degree of deacetylation of α-chitin. Compared to the NaCDA from Nitratireductor aquimarinus MCDA3-3 studied by our group, the AsCDA showed a significant improvement in reaction time and substrate concentration of the catalytic preparation of chitosan [35]. Chitosan was the deacetylated derivative of chitin with a high rate of deacetylation higher than 55% [2]. AsCDA has the highest activity toward deacetylation of crystalline chitin and it removes the acetyl group content of crystalline chitin, which had not been pretreated through various physical and chemical methods until this current study (see Supplementary Figure S1) (Figure 7). The enzymatic method is a promising alternative to these traditional physical and chemical methods to promote the chitosan industrial technology upgrade and quality improvement [7]. AsCDA is a potent chitin decomposer to catalyze chitin deacetylation to prepare chitosan and realize the value-added utilization of chitin biological resources. ecules 2022, 27, x FOR PEER REVIEW 9 of 1 deacetylation of crystalline chitin and it removes the acetyl group content of crystallin chitin, which had not been pretreated through various physical and chemical method until this current study (see Supplementary Figure S1) (Figure 7). The enzymatic method is a promising alternative to these traditional physical and chemical methods to promot the chitosan industrial technology upgrade and quality improvement [7]. AsCDA is a po tent chitin decomposer to catalyze chitin deacetylation to prepare chitosan and realize th value-added utilization of chitin biological resources.

Materials
Acinetobacter schindleri MCDA01 (CGMCC NO.13539)-producing chitin deacetylas was isolated from marine mud collected from Yanwei Harbor (34°29′0″ N, 119°47′0″ E) Lianyungang City, China, which was laboratory preservation strains [11]. Operationally the medium for the cell growth of A. schindleri MCDA01 was a 2216E broth at 20 °C and 180 rpm for 48 h. The α-chitin was kindly provided by Professor Fan Yimin from Nanjing Forestry University, which was purified from swimming crab (Portunus trituberculatus shells collected from Nantong, a seaside city in Jiangsu Province, China. Chitosan was th degree of deacetylation greater than 95% and average molecular weight (MW) of approx imately 2 × 10 5 , which was purchased from Sangon Biotech (Shanghai, China). All othe reagents and solvents were analytical grade. deacetylation of crystalline chitin and it removes the acetyl group content of crystalline chitin, which had not been pretreated through various physical and chemical methods until this current study (see Supplementary Figure S1) (Figure 7). The enzymatic method is a promising alternative to these traditional physical and chemical methods to promote the chitosan industrial technology upgrade and quality improvement [7]. AsCDA is a potent chitin decomposer to catalyze chitin deacetylation to prepare chitosan and realize the value-added utilization of chitin biological resources.

Materials
Acinetobacter schindleri MCDA01 (CGMCC NO.13539)-producing chitin deacetylase was isolated from marine mud collected from Yanwei Harbor (34°29′0″ N, 119°47′0″ E), Lianyungang City, China, which was laboratory preservation strains [11]. Operationally, the medium for the cell growth of A. schindleri MCDA01 was a 2216E broth at 20 °C and 180 rpm for 48 h. The α-chitin was kindly provided by Professor Fan Yimin from Nanjing Forestry University, which was purified from swimming crab (Portunus trituberculatus) shells collected from Nantong, a seaside city in Jiangsu Province, China. Chitosan was the degree of deacetylation greater than 95% and average molecular weight (MW) of approximately 2 × 10 5 , which was purchased from Sangon Biotech (Shanghai, China). All other reagents and solvents were analytical grade.

Materials
Acinetobacter schindleri MCDA01 (CGMCC NO.13539)-producing chitin deacetylase was isolated from marine mud collected from Yanwei Harbor (34 • 29 0" N, 119 • 47 0" E), Lianyungang City, China, which was laboratory preservation strains [11]. Operationally, the medium for the cell growth of A. schindleri MCDA01 was a 2216E broth at 20 • C and 180 rpm for 48 h. The α-chitin was kindly provided by Professor Fan Yimin from Nanjing Forestry University, which was purified from swimming crab (Portunus trituberculatus) shells collected from Nantong, a seaside city in Jiangsu Province, China. Chitosan was the degree of deacetylation greater than 95% and average molecular weight (MW) of approximately 2 × 10 5 , which was purchased from Sangon Biotech (Shanghai, China). All other reagents and solvents were analytical grade.

Isolation, Sequencing, and De Novo Assembly of the Acinetobacter schindleri MCDA01 Genome
The genomic DNA was extracted by Bacteria Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer's recommended protocol. Sequencing libraries were generated using a Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs Ltd). The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Subsequently, the libraries with highquality reads were sequenced using a HiSeq 2500 sequence platform provided by the Illumina company and PE125 strategies. A preliminary assembly of the filtered valid data was performed using SOAPdenovo2 (v.2.04.4; SOAPdenovo2, RRID: SCR_014986) [41][42][43]. Whole-genome data of A. schindleri MCDA01 was finally submitted to GenBank under accession number CP076603, Bioproject PRJNA733816, and Biosample SAMN19459915.

Comparative Genome Analysis and Gene Mining of Chitin Deacetylase
To determine the diversity and strain-specific features in A. schindleri MCDA01, the pan-genome was analyzed and searched for orthologous genes in the genome of different bacterial strains (Acinetobacter schindleri MCDA01, Acinetobacter schindleri CIP107287, Acinetobacter schindleri ACE, Bacillus anthracis Ames, and Bacillus cereus ATCC14579, genome information see Supplementary Table S2) with the software package GET_HOMOLOGUES v3.2.1, which can cluster homologous gene families using the bidirectional best-hit, COG triangles, or OrthoMCL v2.0 clustering algorithms [50][51][52]. For Acinetobacter schindleri CIP107287 and A. schindleri ACE, the most closely related to strain A. schindleri MCDA01, there were no known reports on producing chitin deacetylase [53,54]. Bacillus anthracis Ames and B. cereus ATCC14579, producing chitin deacetylase, were complete draft genomes [55,56]. The whole genome synteny between the genome pairs was performed by using the MUMmer-4.0.0beta2 with the '-mum' parameter [57]. In our previous studies, the molecular mass of natural chitin deacetylase was approximately 31 kDa produced by A. schindleri MCDA01, which was an important basis for the gene mining of AsCDA [11]. The putative CDAs were identified using a dbCAN2 meta-server for annotation of carbohydrate-active enzymes (CAZymes) [45]. A DNAMAN v9.0.1.116 (https://www.lynnon.com/index.html) was used for amino acid sequence alignment and conservative domain analysis. Phylogenetic analysis indicating the relationship of AsCDA to other chitosanases was performed using MEGA 6 software (see Supplementary File).

Heterologous Expression and Purification of AsCDA in Escherichia coli BL21
The cDNA of the AsCDA gene was amplified by PCR using the AsCDA1-F (BamHI:GGATCCACAACTGCTTACTTACGCGGA) and AsCDA1-R (HindIII:AAGCCT GTCCTGGTGCGCAGGAAAATG), verified by DNA sequencing, cloned into the pET-28a expression vector and transformed into Escherichia coli BL21 (TransGen Biotech, Beijing, China). The expression of the recombinant AsCDA was induced by the addition of isopropyl b-D-thiogalactoside (IPTG, final concentration of 1mM) in Luria-Bertani medium (LB medium, containing 50 ng/mL kanamycin) [58]. After overnight induction at 16 • C, the bacterial cells were collected, resuspended in lysis solution, and sonicated for 30 min. The sediment and supernatant were collected after centrifugation for 20 min (4 • C, 12,000 rpm/min). Recombinant AsCDA was purified by the AKTA-purifier protein purification system and Histidine Ni-Sepharose™ Fast Flow, analyzed by SDS-PAGE on a 12% gel after Coomassie brilliant blue R-250 staining. Protein concentration was determined by the bicinchoninic acid method using BSA as a standard.

Enzyme Assay of AsCDA
The enzymatic activity assay of AsCDA was performed as Acetic Acid Assay Kit (Acetate kinase analyzer format, Megazyme, Republic of Ireland) described by Bergmeyer with modification [59]. The Standard curve of acetic acid with ∆A 340 was drawn for enzyme assay of AsCDA ( Figure S2). The reaction mixture of 2 mg α-chitin and 10 µL AsCDA (2.11 µg/µL) was added to 190 µL phosphate buffer (pH 7.0) and incubated at 30 • C for 30 min; the enzyme reaction was terminated by boiling at 100 • C for 10 min in a sealed tube prior. The supernatant was collected by centrifugation at 12,000 rpm for 5 min, and the amount of acetic acid in 10 µL supernatant was determined to calculate the enzyme activity through the A 340 according to the assay procedure of the Acetic Acid Assay Kit. The minimum differential absorbance of the Acetic Acid Assay Kit was 0.005 absorbance unit, which was equivalent to 0.063 mg/L acetic acid in the sample solution. One unit of AsCDA activity was defined as the amount of enzyme required to catalyze the release of 1µmol acetic acid per minute.

Scanning Electron Microscope Observation of Deacetylation Effect
Chitosan, α-chitin, and α-chitin treated by AsCDA (AsCDA-Ch) were characterized microstructure changes by Scanning electron microscopy (Alexandridis, Ghasemi, Furlani, & Tsianou). AsCDA-Ch was a final concentration of 30 g/L chitin pretreated by AsCDA enzyme solution with 50 mM phosphate buffer with shaking fermentation at 30 • C for 12 h (treatment times were set at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18h, 22 h, and 24 h. After 12 h, the degree of deacetylation did not change with the increase in treatment time). All samples were dried to a constant weight at 100 • C and were respectively adhered to the metal sample stage with a conductive adhesive, and a metal film was sprayed in a vacuum evaporator and then observed under a scanning electron microscope at 20 kV [19,37].

Deacetylation Was Measured by Infrared Spectroscopy
Chitosan, α-chitin, and AsCDA-Ch carried out an infrared spectrometer in a wavenumber region of 400 to 4000 cm −1 using FT-IR spectra (Nicolet-iS10), which was based on previously described work by Chai et al. [35]. In the FT-IR spectrum of the enzymatic chitin, the absorption bands at 1659 −1 , 1560 −1 , and 1314 cm −1 , respectively, were the amide I, II, and III bands of the characteristic peaks of acetamido groups, and the absorbance bands at 1655 cm −1 and 3450 cm −1 used to determinate of the degree of deacetylation [60,61]. The difference in DD before (α-chitin) and after (AsCDA-Ch) enzyme treatment of AsCDA toward α-chitin was the removed acetyl groups. Infrared determination of the degree of deacetylation calculation formula:

Statistical Analyses
All tests were performed in triplicate and data were presented as the mean ± SE. One-way ANOVA and Duncan's multiple range tests were used to test for variance and significant differences using the SPSS software (version 26.0).

Conclusions
Chitosan is a biodegradable, biocompatible, and nontoxic aminopolysaccharide, which is widely used in food chemistry as an emulsifier, flocculant, and functional food additive. Chitin deacetylases catalyzed the crystal chitin deacetylation to prepare chitosan, which was a safe, controllable, and environmentally friendly process to replace the traditional current chemical methods. The low catalytic activity of CDAs for the deacetylation of crystalline chitin, however, did not meet the demand for the industrial production of chitosan. Thus, appropriate CDA should be explored to improve the efficiency of enzymatic hydrolysis to crystalline chitin for chitosan production. In this study, AsCDA, a member of the uraD_Nterm-dom superfamily, was mined uniquely through the A. schindleri MCDA01 genome database of bioinformatics analysis. AsCDA was biochemically characterized in E. coli BL21, which has a high activity rate and deacetylation reaction for crystalline chitin. The results of FT-IR and SEM confirmed that the enzymatic hydrolysis of AsCDA released the acetyl group and increased the degree of deacetylation of crystalline chitin, which removed 63.05% of the acetyl groups for crystalline chitin and prepared industrial chitosan in 12 h. AsCDA is a potent chitin decomposer to catalyze chitin deacetylation to prepare chitosan, and these findings will hopefully realize the effective conversion of chitin bioresources into their valuable derivatives: chitin nanofibers, chitosan, and chitooligosaccharides. The study on crystal structures of AsCDA will be a suitable method to elucidate the molecular mechanism of the deacetylation of long-chain chitin catalyzed by CDAs, which, in the future, will be the next scientific research subject of our research group.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27165345/s1, Figure S1: Circular graph of the Acinetobacter schindleri MCDA01 complete genome; Figure S2: Standard curve of acetic acid for enzyme assay of AsCDA; Figure S3: The determination of the degree of deacetylation of α-chitin treated by AsCDA with different time points; Table S1: CDAs with characterized activity on crystalline chitin; Table  S2: Chitin degrading enzymes in Acinetobacter schindleri MCDA01 identified by the CAZy database; Table S3: General genome features of seventeen strains in this study.