Diacetylchitobiose Deacetylase Research Articles

Functional analyses of bacterial genomes found in Symbiodiniaceae genome assemblies.

Bacterial-algal interactions strongly influence marine ecosystems. Bacterial communities in cultured dinoflagellates of the family Symbiodiniaceae have been characterized by metagenomics. However, little is known about whole-genome analysis of marine bacteria associated with these dinoflagellates. We performed in silico analysis of four bacterial genomes from cultures of four dinoflagellates of the genera Symbiodinium, Breviolum, Cladocopium and Durusdinium. Comparative analysis showed that the former three contain the alphaproteobacterial family Parvibaculaceae and that the Durusdinium culture includes the family Sphingomonadaceae. There were no large genomic reductions in the alphaproteobacteria with genome sizes of 2.9-3.9 Mb, implying they are not obligate intracellular bacteria. Genomic annotations of three Parvibaculaceae detected the gene for diacetylchitobiose deacetylase (Dac), which may be involved in the degradation of dinoflagellate cell surfaces. They also had metabolic genes for dissimilatory nitrate reduction to ammonium (DNRA) in the nitrogen (N) cycle and cobalamin (vitamin B12 ) biosynthetic genes in the salvage pathway. Those three characters were not found in the Sphingomonadaceae genome. Predicted biosynthetic gene clusters for secondary metabolites indicated that the Parvibaculaceae likely produce the same secondary metabolites. Our study suggests that the Parvibaculaceae is a major resident of Symbiodiniaceae cultures with antibiotics.

Directed evolution of diacetylchitobiose deacetylase via high-throughput droplet sorting with a novel, bacteria-based biosensor

Numerous biological disciplines rely on high-throughput cell sorting. Flow cytometry, the current gold standard, is capable of ultrahigh–throughput cell sorting, but measurements are primarily limited to cell size and surface marker. Droplet sorting technology is gaining increasing attention with the ability to provide an individual environment for the analysis of single-cell secretion. Although various droplet detecting methods, such as fluorescence, absorbance, mass spectrum, imaging analysis, have been developed for droplet sorting, it remains challenging to establish high-throughput sorting methods for numerous analytes. We aim to develop a high-throughput sorting system based on the glucosamine (GlcN) measurement for the directed evolution of diacetylchitobiose deacetylase (Dac), the key enzyme for GlcN production. To overcome the limitation that no high-throughput sorting system existed for GlcN, we designed a novel bacteria-based biosensor capable of converting GlcN to a positively correlated fluorescence signal. Through characterization and optimization, it was possible to detect GlcN in droplets for high-throughput droplet sorting. We recovered the best Dac mutant S60I/R157T/F168S after sorting ∼0.2 million Dac mutants; its activity was 48.6 ± 1.5 U/mL, which was 1.8-times that of our previously discovered Dac mutant R157T (27.2 ± 1.8 U/mL). This result successfully demonstrated the combination of high-throughput droplet sorting technology and a bacteria-based biosensor, which could facilitate the industrial production of GlcN and serve as a model for similar droplet sorting applications.

A Two-Step Cross-Linked Hydrogel Immobilization Strategy for Diacetylchitobiose Deacetylase

Free enzymes often face economic problems due to their non-recyclability, which limits their applications for industrial manufacturing. Organic biopolymers are frequently used to fabricate hydrogel for enzyme immobilization due to their advantages of non-toxicity, biocompatibility, biodegradability, and flexibility. However, for highly thermostable enzymes, simple cross-linking causes either low immobilizing efficiency or low thermal stability. Herein, we developed a novel enzyme immobilization strategy with two-step cross-linked gelatin hydrogel for thermostable enzymes working at high temperature. The hydrogel was firstly “soft cross-linked” to immobilize most enzyme molecules and then “hard cross-linked” to gain strong thermal stability. We selected the enzyme diacetylchitobiose deacetylase (Dac), which was firstly derived from hyperthermophilic bacteria, to demonstrate the advantages of our method. With the optimized fabrication steps, our hydrogel showed ~87% Dac immobilization efficiency and excellent stability against heating, dehydrating, long-time storing, and massive recycling. Importantly, our hydrogel showed ~85.0% relative enzyme activity at 80 °C and retained ~65.8% activity after 10 rounds of catalysis. This strategy showed high immobilizing efficiency and strong thermal stability and we believe it could improve the industrial potential for those enzymes.

Efficient production of D-glucosamine by diacetylchitobiose deacetylase catalyzed deacetylation of N-acetyl-D-glucosamine.

D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production. We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate. TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.

High level production of diacetylchitobiose deacetylase by refactoring genetic elements and cellular metabolism

Diacetylchitobiose deacetylase (Dac) from Pyrococcus horikoshii can realize the one-step production of glucosamine (GlcN). The efficient expression and secretion of Dac play a central role in the green production of GlcN. In this study, Bacillus subtilis WB600 was used as the expression host. Firstly, we screened 12 signal peptides, among which signal peptide NprB had the strongest ability of guiding Dac secretion. Further optimization of the functional region showed that the extracellular Dac activity of NprB mutant was increased to 3682.2 U/mL. Next, the extracellular Dac activity was increased to 4807.6 U/mL by RBS sequence optimization. Then we got a new recombinant B. subtilis C6 for plasmid-free expression of Dac by integrating comK gene and silencing bpr, nprB, aprE, mpr and nprE genes. Finally, the extracellular Dac activity of genome-integrating strain reached 6357.38 U/mL, which was the highest level reported so far.

Engineering diacetylchitobiose deacetylase from Pyrococcus horikoshii towards an efficient glucosamine production

In this study, semi-rational design based on site-directed saturation mutagenesis and surface charge modification was used to improve the catalytic efficiency of the diacetylchitobiose deacetylase derived from Pyrococcus horikoshii (PhDac). PhDac mutant M14, which was screened by site-directed saturation mutagenesis, showed a ~ 2.21 -fold enhanced catalytic efficiency (kcat/Km) and the specific activity was improved by 70.02%. To keep the stability of glucosamine (GlcN), we reduced the optimal pH of M14 by modifying the surface charge from −35 to −59 to obtain mutant M20, whose specific activity reached 2 -fold of the wild-type. The conversion rate of N-acetylglucosamine (GlcNAc) to GlcN catalyzed by M20 reached 94.3%. Moreover, the decline of GlcN production was slowed down by the reduction of pH when temperature was higher than 50 ℃. Our results would accelerate the process of industrial production of GlcN by biocatalysis.

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis.

Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5' untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s-1 in the wild type to 7.56 μM s-1 in the mutant R157T.IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

Biocatalytic Production of Glucosamine from N-Acetylglucosamine by Diacetylchitobiose Deacetylase.

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dacph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production.

Thermococcus kodakarensis is a hyperthermophilic archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N'-diacetylchitobiose in the medium. To enhance N-,N'-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation.IMPORTANCE Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the hyperthermophilic archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.

Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.

Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes.

Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus).

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-β-D glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation.

Multiple crystal forms of N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus.

Native N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. The N-terminal cadmium complex was involved in crystal contacts between symmetry-related molecules through hydrogen bonding to the N-termini. While all six N-termini of Se-Pf-Dac were involved in cadmium-complex formation, only two of the Pf-Dac N-termini participated in complex formation in the Cd-containing crystal, resulting in different crystal forms. These differences are discussed in light of the higher stability of the Cd-Se bond than the Cd-S bond. This work provides an example of the contribution of cadmium towards determining protein crystal quality and packing depending on the use of the native protein or the selenomethionine derivative.

Novel intersubunit active site of arcchaeal N,N′-diacetylchitobiose deacetylase

N,N′-diacetylchitobiose deacetylase (Dac) is involved in the archaea-specific chitinolytic pathway. In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on Dacs from P. horikoshii (Ph-Dac) and P. furiosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were thermostable up to 950C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer comprised of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic zinc situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. These factors give the archaeal Dacs unprecedented active site architecture as a zinc-dependent deacetylase.

Expression, refolding, and purification of active diacetylchitobiose deacetylase from Pyrococcus horikoshii

A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GlcNAc)2] as the end product. To further investigate the degradation mechanism of (GlcNAc)2 in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40mg of soluble, thermostable (up to 80°C) Ph-Dac was obtained from 1L of culture. The apparent molecular mass of the refolded Ph-Dac was 180kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GlcNAc)2, and the deacetylation site was revealed to be specific to the nonreducing end residue of (GlcNAc)2. These expression and purification systems are useful for further characterization of Ph-Dac.