Production Of N-acetylglucosamine Research Articles

Engineering a Novel Acetyl-CoA Pathway for Efficient Biosynthesis of Acetyl-CoA-Derived Compounds.

Acetyl-CoA is an essential central metabolite in living organisms and a key precursor for various value-added products as well. However, the intracellular availability of acetyl-CoA limits the efficient production of these target products due to complex and strict regulation. Here, we proposed a new acetyl-CoA pathway, relying on two enzymes, threonine aldolase and acetaldehyde dehydrogenase (acetylating), which can convert one l-threonine into one acetyl-CoA, one glycine, and generate one NADH, without carbon loss. Introducing the acetyl-CoA pathway could increase the intracellular concentration of acetyl-CoA by 8.6-fold compared with the wild-type strain. To develop a cost-competitive and genetically stable acetyl-CoA platform strain, the new acetyl-CoA pathway, driven by the constitutive strong promoter, was integrated into the chromosome of Escherichia coli. We demonstrated the practical application of this new acetyl-CoA pathway by high titer production of β-alanine, mevalonate, and N-acetylglucosamine. At the same time, this pathway achieved a high-yield production of glycine, a value-added commodity chemical for the synthesis of glyphosate and thiamphenicol. This work shows the potential of this new acetyl-CoA pathway for the industrial production of acetyl-CoA-derived compounds.

Degradation of marine crustacean shell wastes through single-stage co-fermentation using proteolytic and chitinolytic bacteria.

Management of crustacean shell waste (SW) through an eco-friendly technique is an environmental obligation to control pollution. The present study showed a novel approach through the simultaneous application of proteolytic and chitinolytic bacteria to effectively degrade unprocessed crustacean SW. For this, the bacteria with concurrent chitinolytic and proteolytic activity (Bacillus subtilis, Priestia megaterium, or Bacillus amyloliquefaciens) were applied either alone or in combination with one proteolytic strain (Paenibacillus alvei) in the unprocessed lobster, crab, and shrimp SW. The method degraded the shells with high deproteinization (> 90%) and demineralization efficiency (> 90%). The degradation was confirmed through scanning electron microscopy. The highest weight loss achieved with shrimp, crab, and lobster shells was 93.67%, 82.60%, and 83.33%, respectively. B. amyloliquefaciens + P. alvei combination produced the highest weight loss in crab and lobster SW, whereas all combinations produced statistically similar weight loss in shrimp SW. There was a concurrent production of N-acetyl glucosamine (up to 532.89, 627.87, and 498.95 mg/g of shrimp, lobster, and crab shell, respectively, with P. megaterium + P. alvei and B. amyloliquefaciens + P. alvei in all SW) and amino acids (4553.8, 648.89, 957.27 μg/g of shrimp, lobster, and crab shells, respectively with B. subtilis + P. alvei in shrimp and B. amyloliquefaciens + P. alvei in crab and lobster). Therefore, it is concluded that, for the first time, efficient degradation of crustacean shell waste was observed using chitinolytic and proteolytic bacterial fermentation with the obtention of byproducts, providing a basis for further application in SW management.

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.

Two-stage process production of microbial lipid by co-fermentation of glucose and N-acetylglucosamine from food wastes with Cryptococcus curvatus

Microbial lipids were produced through a two-stage process with Cryptococcus curvatus by co-fermenting rice and shrimp shells hydrolysates. In the first stage, biomass production of glucose and N-acetylglucosamine was optimized by response surface methodology with the maximum biomass yield (17.60 g/L) under optimum conditions (43.2 g/L mixed sugar concentration, pH 5.8, 200 rpm, and 28 °C). In the second stage, according to a single-factor optimization setting (43.2 g/L sugar mixture solutions, pH 5.5, and shift time of 36 h), lipid titer of 10.08 g/L with content of 55.30 % was achieved. Scaling up to a 5-L bioreactor increased lipid content to 60.07 % with 0.233 g/g yield. When Cryptococcus curvatus was cultured in the blends of rice hydrolysates and shrimp shells hydrolysate, lipid content and yield were 52.25 % and 0.204 g/g. The fatty acid compositions of lipid were similar to those of typical vegetable oils.

Isolation, biochemical characterization, and genome sequencing of two high-quality genomes of a novel chitinolytic Jeongeupia species.

Chitin is the second most abundant polysaccharide worldwide as part of arthropods' exoskeletons and fungal cell walls. Low concentrations in soils and sediments indicate rapid decomposition through chitinolytic organisms in terrestrial and aquatic ecosystems. The enacting enzymes, so-called chitinases, and their products, chitooligosaccharides, exhibit promising characteristics with applications ranging from crop protection to cosmetics, medical, textile, and wastewater industries. Exploring novel chitinolytic organisms is crucial to expand the enzymatical toolkit for biotechnological chitin utilization and to deepen our understanding of diverse catalytic mechanisms. In this study, we present two long-read sequencing-based genomes of highly similar Jeongeupia species, which have been screened, isolated, and biochemically characterized from chitin-amended soil samples. Through metabolic characterization, whole-genome alignments, and phylogenetic analysis, we could demonstrate how the investigated strains differ from the taxonomically closest strain Jeongeupia naejangsanensis BIO-TAS4-2T (DSM 24253). In silico analysis and sequence alignment revealed a multitude of highly conserved chitinolytic enzymes in the investigated Jeongeupia genomes. Based on these results, we suggest that the two strains represent a novel species within the genus of Jeongeupia, which may be useful for environmentally friendly N-acetylglucosamine production from crustacean shell or fungal biomass waste or as a crop protection agent.

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..

Production, purification and characterization of chitinase from Micromonospora sp. AR17

N‐acetylglucosamine (NAG) is the monomer product of chitin, which has been widely used as a bioactive com‐ pound in applications such as anti‐tumor, anti‐microbial, and antioxidant activities. In production, biological processes using enzymes are preferable to chemicals due to environmental issues. This study aims to determine the activity, purity level, and molecular weight of purified chitinase from Micromonospora sp. AR17 determines the concentration of NAG produced by purified chitinase that has been characterized. Chitinase was produced by fermentation in colloidal chitin broth at 40 °C, pH 7, for 7 days, while chitinase activity was checked every 24 h. The optimal fermentation time was used to produce chitinase for a further purification step. Enzyme purification was carried out by ultrafiltration, ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose Fast Flow), and gel filtration (Sephacryl S‐300). The purified enzyme was then char‐ acterized for optimum time, pH, and temperature to produce NAG. The results suggested that the fourth day was the optimal time for chitinase production, with chitinase activity of 0.0040 U/mL and a NAG concentration of 7.62 µg/mL. The purifica‐ tion step successfully increased the purity by 6.82 times with chitinase‐specific activity at 1.4648 U/mg. Production of NAG with purified chitinase produced a NAG concentration of 32.472 µg/mL with an incubation time of 30 min at 40 °C and pH 7.

Functional characterization of N-acetyl glucosaminidase from Myrothecium verrucaria for bio-control of plant pathogenic fungi and bio-production of N-acetyl glucosamine

As a common structural element of invertebrates and fungi, chitin offers an ideal target for bio-control of agricultural pests as well as production of N-acetyl glucosamine (NAG). Therefore, chitin degrading enzymes have several biotechnological applications. In the present investigation, the bio-control and NAG production potential of Myrothecium verrucaria N-acetyl glucosaminidase (MvNAGase) were studied. Cloning and biochemical characterization of MvNAGase (1743 bp) suggest that it belongs to the glycosyl hydrolase-20 family. Chitooligosaccharides hydrolysis revealed the exo-acting nature of the enzyme with NAG as an end product. rMvNAGase displayed significant antifungal potential towards plant pathogenic fungi: Ustilago maydis, Bipolaris sorokiniana and Fusarium oxysporum. The bioprocess was optimized using an enzymic cocktail of earlier identified and characterized endo-chitinase and rMvNAGase to maximize NAG production from the chitin. Under specific experimental conditions, the chitin conversion was achieved up to 87%. The study propose the possible application of rMvNAGase in bio-control of agricultural pests and biochemical production of NAG from the crude chitin.

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine

The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility.

Biochemical purification and characterization of a truncated acidic, thermostable chitinase from marine fungus for N-acetylglucosamine production.

N-acetylglucosamine (GlcNAc) is widely used in nutritional supplement and is generally produced from chitin using chitinases. While most GlcNAc is produced from colloidal chitin, it is essential that chitinases be acidic enzymes. Herein, we characterized an acidic, highly salinity tolerance and thermostable chitinase AfChiJ, identified from the marine fungus Aspergillus fumigatus df673. Using AlphaFold2 structural prediction, a truncated Δ30AfChiJ was heterologously expressed in E. coli and successfully purified. It was also found that it is active in colloidal chitin, with an optimal temperature of 45°C, an optimal pH of 4.0, and an optimal salt concentration of 3% NaCl. Below 45°C, it was sound over a wide pH range of 2.0–6.0 and maintained high activity (≥97.96%) in 1–7% NaCl. A notable increase in chitinase activity was observed of Δ30AfChiJ by the addition of Mg2+, Ba2+, urea, and chloroform. AfChiJ first decomposed colloidal chitin to generate mainly N-acetyl chitobioase, which was successively converted to its monomer GlcNAc. This indicated that AfChiJ is a bifunctional enzyme, composed of chitobiosidase and β-N-acetylglucosaminidase. Our result suggested that AfChiJ likely has the potential to convert chitin-containing biomass into high-value added GlcNAc.

Mechano-Enzymatic Degradation of the Chitin from Crustacea Shells for Efficient Production of N-acetylglucosamine (GlcNAc)

Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase from Ostrinia furnacalis (OfHex1), endo-chitinase from Trichoderma viride (TvChi1), and multifunctional chitinase from Chitinolyticbacter meiyuanensis (CmChi1), were expressed in the Pichia pastoris system, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.

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.

Immobilization of Providencia stuartii Cells in Pumice Stone and Its Application for N-Acetylglucosamine Production.

SUMMARYResearch backgroundShrimp shells contain chitin that can be further processed into N-acetylglucosamine, which has been extensively used to treat joint damage. Providencia stuartii has a strong chitinolytic activity and may be utilized in the form of immobilized cells in repeated fermentation. Pumice is a porous and rigid stone that offers superior mechanical strength, making it suitable for immobilization.Experimental approachIn the research submerged fermentation with different pumice stone sizes and pumice stone/growth medium ratios (m/V) was carried out for 4 days at 37 °C and pH=7.0. The optimum pumice stone size and pumice stone/growth medium ratio (m/V) were used to determine the optimum fermentation cycle for the production of N-acetylglucosamine using immobilized P. stuartii.Results and conclusionsPumice stones of 1.0 cm×1.0 cm×1.0 cm and pumice stone/growth medium ratio of 1:5 were found to be the optimum conditions for successful immobilization of (90.0±1.6) % cells and production of (331.4±7.3) g/L N-acetylglucosamine. The highest N-acetylglucosamine concentration of (323.0±2.5) g/L was obtained in the first fermentation cycle, which then decreased and remained stable throughout the last three cycles.Novelty and scientific contributionP. stuartii, a strong chitinolytic bacterium previously isolated from rotten shrimp shells, was used for the first time in immobilized form to produce N-acetylglucosamine. The findings in this research showed the potential use of P. stuartii cells immobilized in pumice stone for continuous production of N-acetylglucosamine in repeated fermentation.

Immobilization of Providencia stuartii Cells in Papaya Trunk Wood for N-acetylglucosamine Production from Pennaeus vannamei Shrimp Shells

Highlight Research AbstractChitin is a natural compound found abundantly in shrimp shells. Chitin can be degraded to produce N-acetylglucosamine, which has wide applications in the food and pharmaceutical fields. Fermentation using chitinolytic microorganisms can be used to produce N-acetylglucosamine from shrimp shells’ chitin. One of the strong chitinolytic bacteria that was isolated from previous research was Providencia stuartii. To provide better stability and efficiency in fermentation, P. stuartii cells were immobilized using entrapment method in papaya trunk wood. The aims of this research were to determine the optimum papaya trunk wood size, ratio of papaya trunk wood and growth medium, as well as the optimum fermentation cycle to produce N-acetylglucosamine from P. vannamei shrimp shells using submerged fermentation method. The research used experimental method with treatment of different sizes of papaya trunk wood (1 x 1 x 1 cm3, 1.5 x 1.5 x 1.5 cm3, and 2 x 2 x 2 cm3), different ratio of papaya trunk wood and growth medium (1:10, 1:15 and 1:20), and 4 fermentation cycles. Results showed that papaya trunk wood with size of 1 x 1 x 1 cm3 and ratio (w/v) of 1:10 could immobilize 87.08±2.05% of P. stuartii cells and produce the highest N-acetylglucosamine concentration, which was 238177.78±3153.48 ppm. The highest N-acetylglucosamine production was obtained from first fermentation cycle and decreased over the last three cycles, but still produced high concentration of N-acetylglucosamine. Therefore, it is possible to perform continuous N-acetylglucosamine production from shrimp shells using P. stuartii cells immobilized in papaya trunk wood.

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.

N-Acetylglucosamine Production by Repeated-Batch Fermentation Using Immobilized Semi-Purified Chitinase Enzyme on Agar

Chitinolytic mold, such as Mucor circinelloidesis can be utilized to produce chitinase enzyme for shrimp shell’s chitin hydrolysis into N-acetylglucosamine (NAG). For that purpose, entrapment of chitinase on agar as a carrier could be an alternative way to improve NAG production. This study aimed to investigate the stability of immobilized semi-purified chitinase on agar for multiple cycles fermentation to produce NAG. In this study, 0.6 mL of semi-purified chitinase enzyme was immobilized into 3% of agar matrices and tested for four fermentation cycles to obtain highest NAG concentration and good enzyme activity. The results indicate that the immobilized chitinase could be used for 6 hours fermentation or three fermentation cycles. The NAG concentration produced after three cycle were 1042.22 ± 16.20 ppm. Besides, the immobilized enzyme was considerably stable up to the third cycles with activity value of about 4.74 U/mL.Keywords: agar; immobilized;NAG; repeated fermentation

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.

The effect of ultrasonication on enzymatic hydrolysis of chitin to N-acetyl glucosamine via sequential and simultaneous strategies

In this study, sequential and simultaneous strategies of ultrasonication (ultrasonic bath) were investigated to enhance enzymatic production of N-acetyl glucosamine (GlcNAc) from chitin powder. For the sequential strategy, the ultrasonic caused chitin powder to a visible fleecy structure, and Scanning electron microscopy (SEM) showed the surface of the treated chitin had a fiber-like structure with a diameter of 50 − 200 nm. Moreover, Fourier transform infrared spectroscopy (FTIR), Element analysis (EA), and X-ray diffraction (XRD) revealed that the crystallinity of the chitin decreased with little deacetylation. The simultaneous strategy is a one-pot treatment and enzymatic hydrolysis of chitin. The concentration of GlcNAc was 2.65 g/L for the strategy, which was 1.18- and 5.0-fold higher than the sequential strategy (2.25 g/L) and untreated chitin (0.53 g/L), respectively. In conclusion, this approach provided an efficient and environmentally friendly method for reducing the crystallinity of chitin and enhancing its enzymatic hydrolysis.

Bioconversion of chitin and concomitant production of chitinase and N-acetylglucosamine by novel Achromobacter xylosoxidans isolated from shrimp waste disposal area

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket–Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition—arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.

Chemoenzymatic Production and Engineering of Chitooligosaccharides and N-acetyl Glucosamine for Refining Biological Activities.

Chitooligosaccharides (COS) and N-acetyl glucosamine (GlcNAc) are currently of enormous relevance to pharmaceutical, nutraceutical, cosmetics, food, and agriculture industries due to their wide range of biological activities, which include antimicrobial, antitumor, antioxidant, anticoagulant, wound healing, immunoregulatory, and hypocholesterolemic effects. A range of methods have been developed for the synthesis of COS with a specific degree of polymerization along with high production titres. In this respect, chemical, enzymatic, and microbial means, along with modern genetic manipulation techniques, have been extensively explored; however no method has been able to competently produce defined COS and GlcNAc in a mono-system approach. Henceforth, the chitin research has turned toward increased exploration of chemoenzymatic processes for COS and GlcNAc generation. Recent developments in the area of green chemicals, mainly ionic liquids, proved vital for the specified COS and GlcNAc synthesis with better yield and purity. Moreover, engineering of COS and GlcNAc to generate novel derivatives viz. carboxylated, sulfated, phenolic acid conjugated, amino derived COS, etc., further improved their biological activities. Consequently, chemoenzymatic synthesis and engineering of COS and GlcNAc emerged as a useful approach to lead the biologically-active compound-based biomedical research to an advanced prospect in the forthcoming era.