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Gene mining, recombinant expression and enzymatic characterization of N-acetylglucosamine deacetylase

  • Min Qiu ORCID logo , Xiaohui Dai ORCID logo , Jiliang Hu ORCID logo , Jianlong Zhang ORCID logo , Qiang Liu ORCID logo , Jiabao Luan ORCID logo , Qingmei Zhou ORCID logo , Yu Xia ORCID logo , Kunxiao Zhang ORCID logo EMAIL logo and Weiwei Liu ORCID logo EMAIL logo
Published/Copyright: January 28, 2025
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 50 Issue 1

Abstract

Objectives

Glucosamine (GlcN) is an important bioactive substance that is widely used in medicine, dietary supplements, cosmetics, and other fields. The traditional method of producing GlcN is mainly through chitosan hydrolysis catalyzed by strong acid, but this process is usually accompanied by environmental pollution and high energy consumption. Therefore, the development of green and efficient production methods of glucosamine has become the focus of current research.

Methods

In this study, N-acetylglucosamine (GlcNAc) was used as the substrate to facilitate the enzymatic synthesis of GlcN by deacetylase. Four deacetylases (TkDAc, PkDAc, PpDAc and AbDAc) were selected from marine thermophilic microorganisms, and Escherichia coli (E. coli) was used as the host for recombinant expression.

Results

The soluble expression of PpDAc was poor, so several groups of solubilizing labels were tried, and the results showed that the soluble expression of recombinant plasmid ArsC-PpDAc carrying pro-solubilization labels was greatly improved.

Conclusions

The effects of temperature and pH on enzyme activity were investigated by single factor analysis. Kinetic parameters further revealed that ArsC-PpDAc exhibited the highest catalytic activity, with a Kcat/Km value of 7.29, and achieved a conversion rate of over 95 %. The condition of ArsC-PpDAc was optimized, and the results showed that ArsC-PpDAc showed good tolerance to organic solvents, and its catalytic activity was not significantly affected.

Keywords: glucosamine; N-acetylglucosamine; deacetylase; enzyme catalysis; enzymatic properties

Introduction

Carbohydrates exist in various forms in the body, such as monosaccharides, oligosaccharides, polysaccharides and glycoproteins, and participate in various life activities of the human body. In recent years, carbohydrates have been widely used in the research and development of new drugs for anti-tumor, anti-virus and other diseases. Earlier in 2007, it was found that UDP-3-O- (R-3-hydroxymyristoyl) -N-acetylglucosamine deacetylase (LpxC) (EC 2.3.1.7) has been developed as an antibiotic target for the treatment of Gram-negative bacteria [1]. Glucosamine (GlcN) is the only basic monosaccharide in nature, which can combine with proteins and lipids to form glycoproteins, mucopolysaccharides and proteoglycans [2], and it is found in bacteria, yeast, filamentous fungi, plants and animals, and is one of the important components of the hexosamine pathway. As an important branch of the glycolytic pathway, the hexosamine pathway is essential for energy acquisition within organisms as well as multiple biosynthetic reactions [3]. GlcN is usually obtained by hydrolysis of chitin with strong acid. The structural change of this process is reflected in the substitution of the 2′-hydroxyl group of glucose by an amino group. Due to its unique functional groups, including hydroxyl, amide, and acetamide, on its structure, GlcN exhibits rich biological activity [4]. GlcN is considered a must supplement for patients with osteoarthritis [5]. The study showed that the combination of GlcN and chondroitin sulfate was significantly effective in a subgroup of patients with moderate to severe knee pain [6], 7].

There are three main forms of GlcN supplements commonly available on the market: glucosamine hydrochloride, glucosamine sulfate, and N-acetylglucosamine (GlcNAc) [8]. Crystalline glucosamine sulfate has been shown to provide temporary control of pain and have persistent effects on disease progression, providing analgesic effects equivalent to non-steroidal anti-inflammatory drugs but at moderate intensity [9]. At present, glucosamine is mainly produced by acid hydrolysis, microbial fermentation and enzymatic method [10], [11], [12]. Among them, the acid hydrolysis method involves depolymerizing chitin into GlcNAc through the action of strong acids, and subsequently hydrolyzing it with strong deacetylation reagents (such as hydrochloric acid and sodium hydroxide). During the hydrolysis process, GlcNAc can be converted into the main product GlcN, which is accompanied by the formation of acetic acid as a by-product [13]. Although the cost is relatively low, the environmental pollution and low product purity resulting from this method cannot be ignored. Microbial fermentation is a process in which glucose is converted into GlcN through microbial metabolism. Although GlcN produced through this method has been used in industry, its main substrate remains glucose rather than chitin, leading to higher industrial costs [14]. The reaction condition of this method is mild, and the purity of the product is high, but the operation process is relatively complex, and the technical difficulty is great. The enzymatic method primarily decomposes the exoskeletons of shrimp, crab, and other marine organisms through a deacetylation process, in order to prepare GlcN [15], usually using chitinase, chitosanase, lysozyme, etc. In contrast to the other methods, the enzyme is able to specifically cleave the β-1,4-glycosidic bond of the substrate under relatively mild conditions [16], enabling effective control of the degradation product, the conversion efficiency is high, and the enzyme can be reused, which can further reduce the production cost. Moreover, enzymatic digestion avoids the use of numerous organic solvents in the degradation process, and therefore causes less pollution to the environment.

This study first mined genome data from the National Center for Biotechnology Information (NCBI) database and screened out four kinds of deacetylases (EC 2.3.1.3) from marine thermophilic microorganisms, which were named TkDAc, PkDAc, ArsC-PpDAc, and AbDAc respectively. Furthermore, Escherichia coli (E. coli) was used as a host for the engineered expression of these enzymes, and a systematic study was conducted on their catalytic properties. Additionally, the organic solvent tolerance and metal ion preference of ArsC-PpDAc, which exhibited the optimal catalytic activity, were also investigated.

Materials and methods

Bacterial strains

Recombinant E. coli BL21(DE3)/TkDAc/HisA, BL21(DE3)/PkDAc/HisA, BL21(DE3)/PpDAc/HisA, BL21(DE3)/AbDAc/HisA carry the corresponding recombinant plasmids, respectively (Supplementary Figure 1). The synthesis and sequencing of the target gene were all synthesized by General Biologics (Anhui) Co., Ltd.

Main reagents and instruments

Nickel agarose chelate chromatography medium (Ni-NTA) was purchased from Jiangsu Qianchun Biotechnology Co., Ltd. Yeast powder and peptone were purchased from Thermo Fisher Oxoid. Other reagents were purchased from Shanghai trial analysis pure. The high-performance liquid chromatography (HPLC) detector is Japan (DGU-20A3R).

Screening of enzymes

We conducted targeted search in the NCBI database for the sequences of genes associated with deacetylase. Based on the published information about the deacetylase TkDAc [17] and combined with the alignment results, we selected a previously unreported deacetylase from marine thermophilic bacteria in the database.

Subsequently, we utilized MAGE 7 software to conduct in-depth analysis on the selected sequences and constructed a gene evolutionary tree, revealing the evolutionary relationships and potential phylogenetic patterns among these sequences (Figure 1). After analyzing the gene evolutionary tree and consolidating the database information, the initially reported TkDAc and unreported PkDAc, PpDAc, and AbDAc were selected for further research (Supplementary Table 1).

Figure 1: Results of the evolutionary tree analysis.
Figure 1:

Results of the evolutionary tree analysis.

Plasmid construction

In this experiment, we chose the pET28a plasmid with a His tag at its N-terminus. The TkDAc, PkDAc, PpDAc and AbDAc genes were cloned into the Nde I and Xho I polycloning sites of the pET28a vector and their expression was controlled by the T7 promoter and terminator. The final recombinant plasmids are pET28a-HisA-TkDAc, pET28a-HisA-PkDAc, pET28a-HisA-PpDAc and pET28a-HisA-AbDAc (Supplementary Figure 1).

Protein expression

Firstly, the recombinant plasmids pET28a-HisA-TkDAc, pET28a-HisA-PkDAc, pET28a-HisA-PpDAc, and pET28a-HisA-AbDAc were individually transformed into BL21(DE3) cells, plated onto agar dishes, and then incubated overnight in a 37 °C incubator. A single clonal colony was picked from the plate and inoculated into 5 mL of LB medium (consisting of 5 g/L yeast extract, 10 g/L NaCl, and 10 g/L peptone) containing 50 μg/mL kanamycin, and then cultured overnight in a 37 °C shaker. The activated E. coli culture was used as the seed liquid and was inoculated into 500 mL of LB medium at an inoculation rate of 1 % for expanded cultivation. When the bacterial strain is cultivated to reach an bacterial density (OD600) value of 0.6–0.8, isopropyl β-D-thiogalactoside (IPTG) is added to achieve a final concentration of 0.1 mM, and the culture is then incubated at 25 °C for 12 h. The bacterial cells were collected by centrifugation at 5,000 rpm for 10 min, resuspended in 20 mM PBS (phosphate-buffered saline) at pH 8.0, and then subjected to pressure disruption. Centrifugation at 10,000 rpm for 1 h yielded the supernatant. The results showed that except for PpDAc, which was not well expressed in solubleness, the other three proteins could be expressed normally, and the soluble expression of PkDAc and AbDAc was extremely high (Supplementary Figure 2).

Recombination of solubilizing labels

TkDAc, PkDAc, and AbDAc were successfully expressed by adjusting various parameters. However, the soluble expression of PpDAc was less effective. Therefore, in order to obtain a well-soluble PpDAc, a soluble tag was added to improve protein solubility. Arsenate reductase (ArsC) (EC 1.20.2.1) can enhance the soluble expression of aggregation-prone foreign proteins in E. coli. Firstly, primers were designed at both ends of the solubilization labels using pET28a-HisA-PpDAc as the carrier (Supplementary Table 2), which were synthesized by General Biology Company. Using PpDAc as a template, the plasmid was extracted for polymerase chain reaction (PCR) amplification of the target fragment. Then, the vector was digested with Fast Digest Xho I and Fast Digest Nde I. The target fragment was recombined with the label that facilitates the solution-based process, resulting in the recombinant plasmid, which was transformed into BL21 (DE3) cells. The monoclonal colony was grown in the incubator, and the soluble target protein was obtained after expression, followed by purification and verification (Figure 2).

Figure 2: Construction and expression of ArsC-PpDAc. (A) Construction of ArsC-PpDAc; (B) SDS-PAGE analysis of the ArsC-PpDAc (1: protein marker; 2: ArsC-PpDAc supernatant; 3: ArsC-PpDAc precipitate; 4: the purified ArsC-PpDAc).
Figure 2:

Construction and expression of ArsC-PpDAc. (A) Construction of ArsC-PpDAc; (B) SDS-PAGE analysis of the ArsC-PpDAc (1: protein marker; 2: ArsC-PpDAc supernatant; 3: ArsC-PpDAc precipitate; 4: the purified ArsC-PpDAc).

Protein purification

Ni2+ that has chelated with the agarose microsphere medium can bind to the imidazole ring on histidine in the supernatant [18]. The crude enzyme solution was kept at 4 °C for 1 h to fully bind to the nickel-chelated affinity agarose chromatography medium. Subsequently, a low-concentration imidazole solution of 50 mM was used to elute the impurity proteins. Finally, a high-concentration imidazole solution of 500 mM was employed to elute the target protein, resulting in the purified target protein. The supernatant, precipitate, and purified protein obtained after disruption were collected, and the purity of the target protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results and discussion

Phylogenetic analysis of N-acetylglucosamine deacetylase

To obtain GlcNAc deacetylase with good thermal stability from marine thermophilic microorganisms, an initial search was conducted in the NCBI database, resulting in the screening of 70 deacetylase sequences, and then build a phylogenetic tree (Figure 1). The results indicate that deacetylases are mainly classified into three branches, and all four deacetylases studied in this research belong to the same branch. Among them, the genes exhibiting N-acetylglucosaminidase deacetylation activity are underlined.

Multiple sequence alignment

Performing multiple sequence alignments on different sequences plays a crucial role in understanding protein functions and inferring their homologous relationships. Sequence alignments between the three screened, unreported protein sequences and TkDAc revealed an average identity of 17.33 % and an average similarity of 72.56 % (Figure 3). The four deacetylases from different sources exhibit certain conserved regions, indicating that they share similarities in protein function.

Figure 3: Multiple sequence alignment.
Figure 3:

Multiple sequence alignment.

Construction and expression of PpDAc after solubilizing label recombination

Soluble fusion tags play a crucial role in improving the soluble expression of proteins. Using the synthesized pET28a-HisA-PpDAc as the vector, seamless cloning primers were designed at both ends of the solubilizing tag sequence. The target gene and solubilizing tag, obtained after the PCR reaction, were connected through seamless cloning to obtain the recombinant plasmid pET28a-HisA-ArsC-PpDAc (Figure 2A). Expression and purification verification were conducted on the obtained recombinant plasmid that carried the solubilizing tag. Through SDS-PAGE analysis, it was found that the soluble expression of PpDAc after recombination with the solubilizing tag was greatly improved (Figure 2B). This indicates the significant role of the ArsC solubilizing tag in promoting the soluble expression of proteins. In subsequent experiments, we will utilize the soluble form of ArsC-PpDAc to conduct a comprehensive study of its enzymatic properties, aiming to elucidate its catalytic mechanisms and potential applications.

Effect of temperature on the enzyme activity

Since the stability of enzymes is often greatly influenced by temperature, TkDAc, PkDAc, ArsC-PpDAc, and AbDAc all belong to marine thermophilic bacteria, which exhibit good temperature tolerance and catalytic activity below 70 °C. Therefore, the experimental temperature range was selected to be 30–70 °C. With 50 mM GlcNAc chosen as the initial substrate concentration and at a pH of 8.0, reactions were carried out in 20 mM PBS buffer solution at temperatures of 30–70 °C for 10 min each. The analysis results indicate that the optimal temperatures for TkDAc, PkDAc, ArsC-PpDAc, and AbDAc are 50 °C, 40 °C, 70 °C, and 30 °C, respectively (Figure 4). The enzyme activities of PkDAc and AbDAc are significantly influenced by temperature; outside their optimal temperature ranges, any change in temperature results in a notable decrease in enzyme activity (Figure 4B–D). This phenomenon may be attributed to the inhibition of enzyme activity at excessively low temperatures. As the temperature rises, the kinetic energy of enzyme molecules increases, thereby enhancing the formation rate of enzyme-substrate complexes. However, at excessively high temperatures, the three-dimensional structure of proteins is disrupted, causing denaturation of the enzyme protein and subsequently leading to the loss of its catalytic activity. In contrast, temperature has minimal effect on the enzyme activities of TkDAc and ArsC-PpDAc within the range of 30–70 °C. Notably, ArsC-PpDAc exhibits higher activity than TkDAc and demonstrates the best catalytic efficiency, showing strong application potential (Figure 4A–C).

Figure 4: Effect of temperature (A, B, C, D) and pH on enzyme activity (E, F, G, H). ▲Citrate sodium citrate buffer (CPBS); ● phosphate buffer (PBS); ■ glycine-sodium hydroxide buffer (Glycine-NaOH buffer).
Figure 4:

Effect of temperature (A, B, C, D) and pH on enzyme activity (E, F, G, H). ▲Citrate sodium citrate buffer (CPBS); ● phosphate buffer (PBS); ■ glycine-sodium hydroxide buffer (Glycine-NaOH buffer).

Effect of pH on enzyme activity

Subsequently, using 50 mM GlcNAc as the substrate, the changes in enzyme activity of the four enzymes within the pH range of 5.0–9.0 were tested at their optimal temperatures, respectively. The results showed that TkDAc exhibited high activity within the pH range of 7–9, with the highest activity at pH 8.0 (Figure 4E). ArsC-PpDAc almost completely lost its activity at pH 5.0. However, when the pH was increased to 6.0, the activity increased significantly, and it remained high within the pH range of 6.0–9.0, with the optimum pH being 8.0 (Figure 4G). This may be due to the fact that changes in pH can affect the degree of dissociation of essential groups on the active center of the enzyme, as well as the degree of dissociation of substrates and coenzymes, thereby influencing the binding and catalysis of substrate molecules by the enzyme molecule. The optimal pH values of TkDAc and the three novel deacetylases generally align with the ambient growth temperatures of the strains in the database. Among them, TkDAc and ArsC-PpDAc exhibit a broader pH range, making them more suitable for practical production applications.

Using GlcNAc as the substrate and reacting at the optimal pH and temperature for each enzyme, the specific activities of the enzymes were determined by HPLC. The results indicated that TkDAc exhibited a specific activity of 78.25 U/mg, PkDAc had a specific activity of 1.68 U/mg, ArsC-PpDAc showed a significantly higher specific activity of 173.60 U/mg, and AbDAc displayed a specific activity of 7.77 U/mg. Among these enzymes, ArsC-PpDAc’s specific activity was notably superior, approximately twice that of TkDAc, 103-fold higher than PkDAc, and 22-fold greater than AbDAc (Table 1).

Table 1:

Enzyme activity of deacetylase.

Temperature pH Specific activity (U/mg)
TkDAc [19] 50 °C 8.0 78.25
PkDAc 40 °C 7.0 1.68
ArsC-PpDAc 70 °C 8.0 173.60
AbDAc 30 °C 7.0 7.77

Determination of the kinetic parameters

Through experiments examining the effects of temperature and pH on enzyme activity, the optimal reaction conditions for four deacetylases were determined. Since TkDAc has been reported, the enzyme kinetic parameters were measured as follows: PkDAc (40 °C, pH 7.0), ArsC-PpDAc (70 °C, pH 8.0), and AbDAc (30 °C, pH 7.0). Under the optimal temperature and pH conditions for each enzyme activity, specific enzyme activity assays were conducted on reaction solutions containing GlcNAc substrates at various concentrations (2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM). The kinetic analysis revealed that within 100 mM, the reaction rate positively correlated with the substrate concentration. However, as the substrate concentration further increased, the growth rate of the reaction velocity decelerated (Figure 5).

Figure 5: Kinetic curves.
Figure 5:

Kinetic curves.

Table 2 summarizes the kinetic parameters of the reported TkDAc, as well as the previously unreported PkDAc, ArsC-PpDAc, and AbDAc. The Kcat of the recombinant protein ArsC-PpDAc is 1.40 × 103 S−1, reflecting its catalytic capacity for the substrate GlcNAc. With a Kcat/Km value of 7.29 S−1mM−1, a comprehensive analysis of the affinity and catalytic ability of ArsC-PpDAc towards its substrate reveals that ArsC-PpDAc is significantly superior to the other three enzymes. This indicates that the catalytic efficiency of ArsC-PpDAc is much greater than that of enzymes such as PkDAc. In contrast, TkDAc and PkDAc had Kcat/Km values of 0.53 S-1 and 0.73 S−1, respectively, suggesting that their catalytic efficiencies are comparable to each other, but lower than that of ArsC-PpDAc.

Table 2:

Kinetic results.

Vmax, U/mg Km, mM Kcat, S−1 Kcat/Km, S−1 mM−1
TkDAc [20] 133 127 67.32 0.53
PkDAc 36.86 194.8 142.87 0.73
ArsC-PpDAc 1,048 191.8 1.40 × 103 7.29
AbDAc 101.3 92.18 119.18 1.29

Optimization of the conditions for the ArsC-PpDAc

The substrate GlcNAc was subjected to catalysis by ArsC-PpDAc for a period of 4 h. The conversion rate in the blank control group (without metal ions) was 96.86 %. When dichloromethane (DCM), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), ethyl acetate (EtOAc), acetonitrile (MeCN), and tert-butanol (t-BuOH) were present at a concentration of 10 %, their respective conversion rates were 95.57 , 95.81, 95.90, 95.78, 95.58, 95.88, and 95.45 %. These values remained at similar levels compared with the blank control group (Figure 6A). This result suggests that these organic solvents have a minor impact on the activity of the ArsC-PpDAc enzyme, indicating that the enzyme exhibits relatively high tolerance to these solvents. In contrast, the conversion rate in the presence of dimethyl sulfoxide (DMSO) was 77.41 %, indicating that DMSO inhibited the activity of ArsC-PpDAc (Figure 6A). In this experiment, DMSO may inhibit the catalytic activity of the ArsC-PpDAc enzyme by interacting with the enzyme or substrate, or by disrupting hydrogen bonds, hydrophobic bonds, and other interactions that maintain the active conformation of the enzyme protein.

Figure 6: Optimization of reaction conditions. (A) Effect of organic solvents; (B) effect of metal ions.
Figure 6:

Optimization of reaction conditions. (A) Effect of organic solvents; (B) effect of metal ions.

Studies have shown that the addition of appropriate divalent metal ions during enzymatic catalysis can significantly improve the catalytic efficiency of the enzyme, but metal ions have different effects on different enzymes. During the testing of metal ion preference, the substrate GlcNAc underwent catalysis by ArsC-PpDAc for 4 h. The conversion rate of the blank control group, which did not contain any metal ions, was 91.22 %. When various metal ions were added to the reaction system, significant differences were observed in their effects on the catalytic activity of ArsC-PpDAc towards GlcNAc. Specifically, the addition of 10 mM Mg2⁺, Ni2⁺, and Co2⁺ resulted in conversion rates of 91.26 , 91.57, and 90.97 % (Figure 6B), respectively, indicating that these ions had almost no significant impact on the enzymatic catalytic activity. This suggests that Mg2⁺, Ni2⁺, and Co2⁺ may not play a crucial role as key metal ions in the enzymatic catalytic process. However, when Ca2+ and Zn2+ are added, the conversion rate drops significantly, this may be due to the interaction between the metal ions and the enzyme, which alters the conformation of the enzyme and affects the binding of the substrate to the enzyme, thereby influencing its catalytic activity. However, the addition of Ca2⁺, Cu2⁺, and Zn2⁺ led to a sharp decline in catalytic activity, with Cu2⁺ almost completely inhibiting the activity of the enzyme.

The combined results suggest that ArsC-PpDAc may not necessarily require metal ions as a cofactor for its catalytic process. However, certain metal ions can indirectly affect the enzyme activity by altering the conformation and charge state of ArsC-PpDAc. By delving deeper into the metal ion preference results and understanding how different metal ions impact the catalytic activity of ArsC-PpDAc enzymes, we can provide valuable insights for the subsequent optimization of GlcNAc deacetylase reaction conditions.

Conclusions

Based on the demand for GlcN, we targeted its production and screened four deacetylases: TkDAc, PkDAc, PpDAc (specifically ArsC-PpDAc), and AbDAc. Using genetic engineering, we constructed plasmids and introduced them into E. coli to express four soluble recombinant deacetylases: TkDAc, PkDAc, ArsC-PpDAc, and AbDAc. These enzymes were then characterized to investigate the effects of temperature, pH, and kinetic parameters on their activity.

The results revealed that the optimal temperatures for TkDAc, PkDAc, ArsC-PpDAc, and AbDAc are 50 °C, 40 °C, 70 °C, and 30 °C, respectively. Notably, ArsC-PpDAc exhibited a broad temperature range of 30–70 °C with minimal temperature effect on its activity and maintained high enzyme activity. All enzymes displayed optimal catalytic activity under neutral or nearly neutral conditions, with optimal pH values of 8.0, 7.0, 8.0, and 7.0 for TkDAc, PkDAc, ArsC-PpDAc, and AbDAc, respectively.

Using GlcNAc as the substrate, we studied the kinetic parameters of the enzymes and found that, based on the Kcat/Km ratio, the catalytic efficiency was highest for ArsC-PpDAc, followed by AbDAc, TkDAc, and PkDAc. Due to its high enzyme activity and catalytic efficiency, ArsC-PpDAc was selected for further study to explore the effects of solvents and metal ions on its catalytic activity. The results showed that the enzyme was tolerant to most organic solvents, with DMSO having a significant impact on its activity. Additionally, Ca2⁺, Zn2⁺, and Cu2⁺ inhibited enzyme activity, with Cu2⁺ almost completely inhibiting it. This study provides a reference for the subsequent industrial application of N-acetylglucosamine and lays a theoretical foundation for the industrial production of GlcN.

Corresponding authors: Kunxiao Zhang, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, College of Pharmacy, Jiangsu Ocean University, Lianyungang 222005, China, E-mail: ; and Weiwei Liu, Jiangsu Key Laboratory of Marine Bioresources and Environment, College of Pharmacy, Jiangsu Ocean University, Lianyungang 222005, China, E-mail:

Min Qiu and Xiaohui Dai contributed equally to this work.

  1. Research ethics: The local Institutional Review Board deemed the study exempt from review.

  2. Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.

  3. Author contributions: Min Qiu: Research method design and experimental implementation. Xiaohui Dai: Data collection and paper writing. Jiliang Hu, Jianlong Zhang, Qiang Liu: Thesis writing and revision. Jiabao Luan, Qingmei Zhou, Yu Xia: Literature research and data analysis.

  4. Use of Large Language Models, AI and Machine Learning Tools: T improve language.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: None declared.

  7. Data availability: The raw data can be obtained on request from the corresponding author.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/tjb-2024-0191).

Received: 2024-08-15
Accepted: 2024-11-19
Published Online: 2025-01-28

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0191
Keywords for this article
glucosamine; N-acetylglucosamine; deacetylase; enzyme catalysis; enzymatic properties
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Targeting oxidative stress, iron overload and ferroptosis in bone-degenerative conditions
  4. Research Articles
  5. Assessing medical biochemistry professionals’ knowledge, attitudes, and behaviors regarding green and sustainable medical laboratory practices in Türkiye
  6. The efficacy of high pressure liquid chromatography (HPLC) in detecting congenital glycosylation disorders (CDG)
  7. Atypical cells parameter in sysmex UN automated urine analyzer: a single center study
  8. The frequency of single specific immunoglobulin E and allergen mixes with a MAST (multiple-antigen simultaneous test) technique
  9. Differences in second trimester risk estimates for trisomy 21 between Maglumi X3/Preaccu and Immulite/Prisca systems
  10. Comparison of classical and flowcytometric osmotic fragility and flowcytometric eosin-5-maleimide binding tests in diagnosis of hereditary spherocytosis
  11. Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells
  12. Trimethylamine N-oxide, S-equol, and indoxyl sulfate inflammatory microbiota players in ocular Behçet’s disease
  13. Genomic profiling of interferon signaling pathway gene mutations in type 2 diabetic individuals with COVID-19
  14. CDR1as/miR-7-5p/IGF1R axis contributes to the suppression of cell viability in prostate cancer
  15. Role of interferon regulatory factors in predicting the prognosis of Crimean-Congo hemorrhagic fever
  16. The significance of taurine for patients with Crimean-Congo hemorrhagic fever and COVID-19 diseases: a cross-sectional study
  17. Gene mining, recombinant expression and enzymatic characterization of N-acetylglucosamine deacetylase
  18. Ethanol inhibited growth hormone receptor-mediated endocytosis in primary mouse hepatocytes
  19. Gypsophila eriocalyx roots inhibit proliferation, migration, and TGF-β signaling in melanoma cells
  20. The role of kynurenine and kynurenine metabolites in psoriasis
  21. Tobacco induces abnormal metabolism of tryptophan via the kynurenine pathway
  22. Effect of vitamin D and omega-3 on the expression of endoplasmic reticulum-associated protein degradation and autophagic proteins in rat brain
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