GlcNAc Production Research Articles

A novel bi-functional cold-adaptive chitinase from Chitinilyticum aquatile CSC-1 for efficient synthesis of N-acetyl-D-glucosaminidase

In order to better utilize chitinolytic enzymes to produce high-value N-acetyl-D-glucosamine (GlcNAc) from chitinous waste, there is an urgent need to explore bi-functional chitinases with exceptional properties of temperature, pH and metal tolerance. In this study, we cloned and characterized a novel bi-functional cold-adaptive chitinase called CaChi18A from a newly isolated strain, Chitinilyticum aquatile CSC-1, in Bama longevity village of Guangxi Province, China. The activity of CaChi18A at 50 °C was 4.07 U/mg. However, it exhibited significant catalytic activity even at 5 °C. Its truncated variant CaChi18A_ΔChBDs, containing only catalytic domain, demonstrated significant activity levels, exceeding 40 %, over a temperature range of 5–60 °C and a pH range of 3 to 10. It was noteworthy that it displayed tolerance towards most metal ions at a final concentration of 0.1 mM, including Fe3+ and Cu2+ ions, retaining 122.52 ± 0.17 % and 116.42 ± 1.52 % activity, respectively. Additionally, it exhibited favorable tolerance towards organic solvents with the exception of formic acid. Interestedly, CaChi18A and CaChi18A_ΔChBDs had a low Km value towards colloidal chitin (CC), 0.94 mg mL−1 and 2.13 mg mL−1, respectively. Both enzymes exhibited chitobiosidase and N-acetyl-D-glucosaminidase activities, producing GlcNAc as the primary product when hydrolyzing CC. The high activities across a broader temperature and pH range, strong environmental adaptability, and hydrolytic properties of CaChi18A_ΔChBDs suggested that it could be a promising candidate for GlcNAc production.

Engineering Corynebacterium glutamicum for the efficient production of N-acetylglucosamine

N-acetylglucosamine (GlcNAc) is significant functional monosaccharides with diverse applications in medicine, food, and cosmetics. In this study, the GlcNAc synthesis pathway was constructed in Corynebacterium glutamicum and its reverse byproduct pathways were blocked. Simultaneously the driving force of GlcNAc synthesis was enhanced by screening key gene sources and inhibiting the GlcNAc consumption pathway. To maximize carbon flux, some competitive pathways (Pentose phosphate pathway, Glycolysis pathway and Mannose pathway) were weakened and the titer of GlcNAc reached 23.30 g/L in shake flasks. Through transcriptome analysis, it was found that dissolved oxygen was an important limiting factor, which was optimized in a 5 L bioreactor. Employing optimal fermentation conditions and feeding strategy, the titer of GlcNAc reached 138.9 g/L, with the yeild of 0.44 g/g glucose. This study significantly increased the yield and titer of GlcNAc, which lay a solid foundation for the industrial production of GlcNAc in C. glutamicum.

Highly efficient production of N,N'-diacetylchitobiose according to substrate modification and changes in enzyme kinetics

The purpose of this study is to improve the method for characterizing the kinetics of enzymes on chitin and to improve the inconvenience of colloidal chitin production and use. Therefore, we prepared a low molecular weight (MW) substrate by partially hydrolyzing and acetylating high molecular weight chitosan (HMWC) with chitosanase. The activity of chitinase was confirmed under optimal reaction conditions using acetylated low molecular weight chitin (AcCOS). In the hydrolysates of AcCOS, N, N′-diacetylchitobiose (GlcNAc)2 was identified as the main product under optimal reaction conditions using TLC. Residual N-acetylglucosamine (GlcNAc), which could not be sufficiently identified by the sensitivity of TLC, was confirmed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. However, from a quantitative point of view, the TLC results suggest that the content of (GlcNAc)2 is much higher than that of GlcNAc. These results are significant for use as a specific substrate capable of evaluating the reaction rate of a chitinase substrate. Taken together, our results provide novel results for the highly efficient production of (GlcNAc)2 and the evaluation of its biological activity by chemical modification of the substrate.

Production of N-acetylglucosamine with Vibrio alginolyticus FA2, an emerging platform for economical unsterile open fermentation

Members of the Vibrionaceae family are predominantly fast-growing and halophilic microorganisms that have captured the attention of researchers owing to their potential applications in rapid biotechnology. Among them, Vibrio alginolyticus FA2 is a particularly noteworthy halophilic bacterium that exhibits superior growth capability. It has the potential to serve as a biotechnological platform for sustainable and eco-friendly open fermentation with seawater. To evaluate this hypothesis, we integrated the N-acetylglucosamine (GlcNAc) pathway into V. alginolyticus FA2. Seven nag genes were knocked out to obstruct the utilization of GlcNAc, and then 16 exogenous gna1s co-expressing with EcglmS were introduced to strengthen the flux of GlcNAc pathway, respectively. To further enhance GlcNAc production, we fine-tuned promoter strength of the two genes and inactivated two genes alsS and alsD to prevent the production of acetoin. Furthermore, unsterile open fermentation was carried out using simulated seawater and a chemically defined medium, resulting in the production of 9.2 g/L GlcNAc in 14 h. This is the first report for de-novo synthesizing GlcNAc with a Vibrio strain, facilitated by an unsterile open fermentation process employing seawater as a substitute for fresh water. This development establishes a basis for production of diverse valuable chemicals using Vibrio strains and provides insights into biomanufacture.

A novel microbial-derived family 19 endochitinase with exochitinase activity and its immobilization

A novel chitinase gene of 888bp from Streptomyces bacillaris was cloned and expressed in Escherichia coli BL21. The purified recombinant enzyme (SbChiAJ103) was identified as the first microbial-derived family 19 endochitinase that showed exochitinase activity. SbChiAJ103 exhibited the substrate preference for N-acetylchitooligosaccharides with even degrees of polymerization and the capability to specifically hydrolyze colloidal chitin into (GlcNAc)2. Mono-methyl adipate was employed as a novel linker for the efficient covalent immobilization of chitinase on magnetic nanoparticles (MNPs). The immobilized SbChiAJ103, SbChiAJ103@MNPs, exhibited superior pH tolerance, temperature stability, and storage stability than free SbChiAJ103. Even after incubation at 45°C for 24h, SbChiAJ103@MNPs could retain more than 60.0% initial activity. As a result, the enzymatic hydrolysis yield of SbChiAJ103@MNPs increased to 1.58 times that of free SbChiAJ103. Moreover, SbChiAJ103@MNPs could be reused by convenient magnetic separation. After 10 recycles, SbChiAJ103@MNPs could retain almost 80.0% of its initial activity. The immobilization of the novel chitinase SbChiAJ103 paves the way to the efficient and eco-friendly commercial production of (GlcNAc)2. KEY POINTS: • The first microbial GH19 endochitinase with exochitinase activity was reported. • Mono-methyl adipate was first employed to immobilize chitinase. • SbChiAJ103@MNPs showed excellent pH stability, thermal stability, and reusability.

Production of N-acetylglucosamine from carbon dioxide by engineering Cupriavidus necator H16

The conversion of CO2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner–Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO2 under normal conditions..

Optimization of chitinase production from bacillus cereus smg 1.1 using response surface methodology

Shrimp shell waste are potential to be processed further into value-added products, such as N-acetylglucosamine (GlcNAc). In the production of GlcNAc, biological approach is preferred and environmentally friendly to chemical treatment. Chitinase is an enzyme that plays a vital role in bioprocessing of shrimp shell waste into GlcNAc. Previously, Bacillus cereus SMG 1.1 was isolated from fermented shrimp paste (terasi) and showed the ability to produce chitinase. This study was designed to determine the optimum medium for the production of chitinase from B. cereus SMG 1.1 through the Response Surface Method (RSM) using a factorial design with 15 treatments. The optimization of the media was carried out by determining the factors that influence the production of chitinase through the Plackett-Burman design followed by optimization of the concentration of the media through the Box-Behnken design. The test was designed to assess the effect of the independent variables on chitinase activity. Placket Burman analysis shows that colloidal chitin, fructose, and MgSO4.5H2O were the significant components in the medium affecting the production of chitinase by B. cereus SMG 1.1. Box-Behnken analysis developed a linear model capable of predicting the response. The highest response value was achieved at a concentration of 0.75% fructose, 1.5% colloidal chitin, and 0.075% MgSO4.5H2O resulted in optimum chitinase activity of 0.0016 U/ml.

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

AbstractN‐acetylneuraminic acid (NeuAc), which has been widely used as a nutraceutical and pharmaceutical intermediate, plays an important role in improving brain development and cognition while enhancing immunity. Bacillus subtilis, generally regarded as a food‐safe microorganism, is suitable for developing as a chassis cell for efficient NeuAc synthesis. However, accumulated intermediates can lead to metabolic bottlenecks for NeuAc synthesis. To eliminate the accumulated byproduct N‐acetylglucosamine (GlcNAc), the UDP‐GlcNAc epimerase pathway without GlcNAc production was first reconstructed and optimized in B. subtilis, resulting in the NeuAc titer increase of 5.9 g/L with GlcNAc elimination. In addition, to reduce another accumulated byproduct N‐acetylmannosamine (ManNAc), the directed evolution of N‐acetylneuraminic acid synthase and the enhancement of phosphoenolpyruvate supply was implemented. Using this strategy, ManNAc decreased by 46.3%, and the NeuAc titer increased by 54.9%, reaching 7.9 g/L. Finally, the maximum titer of NeuAc in a 3‐L fermenter reached 21.8 g/L with a productivity of 0.34 g/L/h.

Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine.

In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, kcat, and kcat/Km of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min–1 l–1, 39.99 μmol l–1, 4667.07 s–1, and 116.71 ml μmol–1 s–1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.

Synergistic utilization of carbon sources for efficient biosynthesis of N-acetylglucosamine

N-acetylglucosamine (GlcNAc) is an amino monosaccharide that has a variety of bioactivities and is widely used in pharmaceutical and food industries. Production of GlcNAc by chitin hydrolysis is limited by the supply of raw materials and encounters the risk of shellfish protein contamination. For efficient biosynthesis of GlcNAc, one challenge is to balance the carbon distribution between growth and production. Here, we applied the strategy of synergistic carbon utilization, in which glycerol supports cell growth and provides the acetyl group of GlcNAc while glucose serves as the precursor to glucosamine. The efficiency of GlcNAc production was stepwise improved by blocking the product re-uptake and degradation, strengthening the biosynthetic pathway and synergistically utilizing two carbon sources. With these efforts, the final strain produced 41.5 g/L GlcNAc with a yield of 0.49 g/g of total carbon sources. In addition, we also explored the feasibility of using acetate as a cheap carbon source to partly replace glycerol. This study provides a promising alternative strategy for sustainable and efficient production of GlcNAc.

Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant.

Shellfish waste is a primary source for making N-acetyl-D-glucosamine. Thus, establishing a high-efficiency and low-cost bioconversion method to produce N-acetyl-D-glucosamine directly from shellfish waste was promising. A mutant C81 was obtained from Chitinolyticbacter meiyuanensis SYBC-H1 via 60Co-γ irradiation. This mutant C81 showed the highest chitinase activity of 9.8 U/mL that was 85% higher than the parent strain. The mutant C81 exhibted improved antioxidant activities, including total antioxidant capacity, superoxide radical ability, and hydroxyl radical scavenging ability, compared to that of the parent strain. Four out of nine organic solvents increased the chitinase activity by 1.9%, 6.8%, 11.7%, and 15.8%, corresponding to methylbenzene, n-heptane, petroleum ether, and n-hexane, respectively. The biphase system composed of aqueous and hexane presented a five-fold reduction of cell viability compared to the control. Using a continuous fermentation bioconversion process, 4.2g/L GlcNAc was produced from crayfish shell powder with a yield of 80% of the chitin content. This study demonstrated that the mutant C81 is suitable for converting crayfish shell powder into GlcNAc in an aqueous-organic system.

Property and Function of a Novel Chitinase Containing Dual Catalytic Domains Capable of Converting Chitin Into N-Acetyl-D-Glucosamine.

A novel multifunctional chitinase (CmChi3)-encoding gene was cloned from Chitinolyticbacter meiyuanensis and actively expressed in Escherichia coli. Sequence analysis showed that CmChi3 contains two glycoside hydrolase family 18 (GH18) catalytic domains and exhibited low identity with well-characterized chitinases. The optimum pH and temperature of purified recombinant CmChi3 were 6.0 and 50°C, respectively. CmChi3 exhibited strict substrate specificity of 4.1 U/mg toward colloidal chitin (CC) and hydrolyzed it to yield N-acetyl-D-glucosamine (GlcNAc) as the sole end product. An analysis of the hydrolysis products toward N-acetyl chitooligosaccharides (N-acetyl COSs) and CC substrates revealed that CmChi3 exhibits endochitinase, N-acetyl-β-d-glucosaminidase (NAGase), and transglycosylase (TGase) activities. Further studies revealed that the N-terminal catalytic domain of CmChi3 exhibited endo-acting and NAGase activities, while the C-terminal catalytic domain showed exo-acting and TGase activities. The hydrolytic properties and favorable environmental adaptations indicate that CmChi3 holds potential for commercial GlcNAc production from chitin.

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli.

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

Model-based dynamic engineering of Escherichia coli for N-acetylglucosamine overproduction

N-acetylglucosamine (GlcNAc), a glucosamine derivative, has a wide range of applications in pharmaceutical fields, and there is an increasing interest in the efficient production of GlcNAc genetic engineered bacteria. In this work, Escherichia coli ATCC 25947 (DE3) strain was engineered by a model-based dynamic regulation strategy achieving GlcNAc overproduction. First, the GlcNAc synthetic pathway was introduced into E. coli, and through flux balance analysis of the genome-scale metabolic network model, metabolic engineering strategies were generated to further increase GlcNAc yield. Knock-out of genes poxB and ldhA, encoding pyruvate oxidase and lactate dehydrogenase, increased GlcNAc titer by 5.1%. Furthermore, knocking out N-acetylmuramic acid 6-phosphate etherase encoded by murQ and enhancing glutamine synthetase encoded by glnA gene further increased GlcNAc titer to 130.8 g/L. Analysis of metabolic flux balance showed that GlcNAc production maximization requires the strict dynamic restriction of the reactions catalyzed by pfkA and zwf to balance cell growth and product synthesis. Hence, a dynamic regulatory system was constructed by combining the CRISPRi (clustered regularly interspaced short palindromic repeats interference) system with the lactose operon lacI and the transcription factor pdhR, allowing the cell to respond to the concentration of pyruvate and IPTG to dynamically repress pfkA and zwf transcription. Finally, the engineered bacteria with the dynamic regulatory system produced 143.8 g/L GlcNAc in a 30-L bioreactor in 55 h with a yield reaching 0.539 g/g glucose. Taken together, this work significantly enhanced the GlcNAc production of E. coli. Moreover, it provides a systematic, effective, and universal way to improve the synthetic ability of other engineered strains.

Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70 °C and 90 °C respectively, and exhibited good thermostability over a range of temperature below 70 °C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc.

N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

The Draft Genome Sequence and Analysis of an Efficiently Chitinolytic Bacterium Chitinibacter sp. Strain GC72.

A novel chitinolytic bacterium Chitinibacter sp. GC72, which produces an enzyme capable of efficiently converting chitin only into N-acetyl-D-glucosamine (GlcNAc), was successfully sequenced and analyzed. The assembled draft genome of strain GC72 is 3,455,373 bp, containing 3346 encoded protein sequences with G + C content of 53.90%. Among these annotated genes, 17 chitinolytic enzymes including 12 glycoside hydrolase family 18 chitinases, three family 19 chitinases, one family 20 β-hexosaminidase, and one auxiliary activity family 10 lytic polysaccharide monooxygenase, were found to be essential in the production of GlcNAc from chitin. The genomic information of strain GC72 provides a reference genome for Chitinibacter bacteria and abundant novel chitinolytic enzyme resources, and allows researchers to explore potential applications in GlcNAc enzymatic production.

A novel bacterial \u03b2-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

BackgroundN-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4–(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs.ResultsThe gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min−1 mg−1, 0.50 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3–(GlcNAc)7) from (GlcNAc)2–(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers.ConclusionsThe observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Biochemical Characterization and Structural Analysis of a β-N-Acetylglucosaminidase from Paenibacillus barengoltzii for Efficient Production of N-Acetyl-d-glucosamine.

Bioproduction of N-acetyl-d-glucosamine (GlcNAc) from chitin, the second most abundant natural renewable polymer on earth, is of great value in which chitinolytic enzymes play key roles. In this study, a novel glycoside hydrolase family-18 β-N-acetylglucosaminidase (PbNag39) from Paenibacillus barengoltzii suitable for GlcNAc production was identified and biochemically characterized. It possessed a unique shallow catalytic groove (5.8 Å) as well as a smaller C-terminal domain (solvent-accessible surface area, 5.1 × 103 Å2) and exhibited strict substrate specificity toward N-acetyl chitooligosaccharides (COS) with GlcNAc as the sole product, showing a typical manner of action of β-N-acetylglucosaminidases. Thus, an environmentally friendly bioprocess for GlcNAc production from ball-milled powdery chitin by an enzyme cocktail reaction was further developed. By using the new route, the powdery chitin conversion rate increased from 23.3% (v/v) to 75.3% with a final GlcNAc content of 22.6 mg mL-1. The efficient and environmentally friendly bioprocess may have great application potential in GlcNAc production.