Production Of N-acetylglucosamine Research Articles

Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine

Glucosamine and N-acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l −1. Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l −1. Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N-acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N-acetylglucosamine by over-expressing a heterologous glucosamine-6-P N-acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l −1 of N-acetylglucosamine was produced.

Production of chitinase by Fusarium species.

The presence of chitinase activity without inducers in the enzymic precipitates from the culture fluid of 25-day-old autolyzed cultures of 17 Fusarium species has been studied. In all cases endochitinase and beta-N-acetylglucosaminidase activities were found. The chitinase activity as a joint action of these two enzymes with production of N-acetylglucosamine was also determined. A correlation among endochitinase, beta-N-acetylglucosaminidase, and chitinase was always found. Fusarium oxysporum f.sp. lini, F. subglutinans, and F. moniliforme were the best producers of chitinase activity. Fusarium species could be a good source of chitinases for production by fungi.

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β-N-acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum. Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a Km of 0.80 mmol 1-1 for p-nitrophenyl-β-N-acetylglucosaminide and 1.03 mmol 1-1 for p-nitrophenyl-β-N-acetylgalactosaminide. The Ki with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate was 1 μmol 1-1. Hg2+, Ag+ and Fe3+ were effective inhibitors. β-N-acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum, hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N-acetylglucosamine.

Immobilization of chitinolytic enzymes and continuous production of N-acetylglucosamine with the immobilized enzymes

Chitinolytic enzymes containing chitinase and β- N-acetylhexosamimidase (NAHase) from Nocardia orientalis IFO 12806 were immobilized on various carriers by different methods for the continuous production of N-acetylglucosamine (GlcNAc). The immobilized enzyme containing both enzymes prepared by physical adsorption to tannin-chitosan was very useful for GlcNAc production. The optimum pHs of the immobilized chitinase and NAHase on tannin-chitosan were shifted lower than those of the free forms. The optimum temperature of the immobilized chitinase decreased from 60°C for the free form to 40–50°C, although the thermostability of the immobilized form increased in comparison with that of the free form. As for NAHase, little difference was observed between the immobilized and free forms in optimum temperature and thermo-stability. In GlcNAc production using a column reactor packed with the immobilized enzyme, GlcNAc was continuously obtained from chitin-oligosaccharides with a yield of above 90% over 130 d. After the operation, the activities of immobilized chitinase and NAHase remained at 47.2% and 32.5% of the initial activities respectively.

β-N-Acetylglucosaminidase fromAspergillus nidulanswhich degrades chitin oligomers during autolysis

A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.

Beta-N-acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis.

A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.

Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid

We have studied the kinetic mechanism for the production of product N-acetylglucosamine from the enzymatic hydrolysis of chitin oligosaccharides by the binary enzyme system, chitinase plus β-N- acetyl- d-glucosaminidase , isolated from pharate pupal moulting fluid of Manduca sexta (L.). A synergism occurs with the rate of monomer production by the enzyme mixture greater than the sum of the rates of the individual enzymes. For the production of monosaccharide from tetrasaccharide, pentasaccharide and hexasaccharide the time courses exhibited by a mixture of purified insect enzymes are essentially identical to those developed using crude moulting fluid. Satisfactory agreement with the experimental data is obtained when using theoretical calculations based on the following: (1) a random attack mechanism with no interaction between the two enzymes, (2) monosaccharide is not produced to a significant extent from the original substrate, (3) chitinase forms both productive and nonproductive complexes with substrate, (4) nonproductive substrate binding to chitinase is more favorable than productive binding, (5) chitinase forms multiple substrate nonproductive complexes, and (6) intermediate metabolite concentrations are low during the course of hydrolysis. β- N-Acetylglucosaminidase is dependent to a great extent on chitinase for production of small fragments from which monosaccharide can be released.

Purification and characterization of chitinase enzymes from healthy and Verticillium albo-atrum-infected tomato plants, and from V. albo-atrum

Constitutive chitinases were purified from healthy tomato stem and culture filtrates of a tomato isolate of Verticillium albo-atrum. Both chitinases hydrolyzed chitin, chitosan and 3,4-dinitrophenyl-tetra-N-acetyl-β-d-chitotetraoside, but the tomato enzyme showed enhanced production of N-acetylglucosamine from chitin in the presence of chitobiase. The tomato chitinase, purified to homogeneity, had a molecular weight of 27 000–31000 and functioned as an endo-enzyme. The partially purified chitinase from V. albo-atrum had a molecular weight of 63 000, appeared to be an exo-enzyme and was unaffected by the addition of chitobiase to the reaction mixture. It differed from the tomato enzyme in the hydrolysis of chitobiose and in its inhibition by 2-acetamido-2-deoxy-d-gluconolactone.The origin of increased chitinase activity in V. albo-atrum-infected tomato stem was investigated by comparing the response of chitinase in extracts from healthy and infected stem to pH and temperature and by examining the patterns of activity in healthy and infected stem extracts after gel isoelectric-focusing. The enzymes from healthy and infected stem and from V. albo-atrum were also compared during the various purification stages. The increased activity in infected tissue was due primarily to increased production of the constitutive endo-enzyme found in healthy tomato stems. An additional chitinase similar to the fungal enzyme was also present.

STUDIES ON CHITAN (β-(1 → 4)-LINKED 2-ACETAMIDO-2-DEOXY-D-GLUCAN) FIBERS OF THE DIATOM THALASSIOSIRA FLUVIATILIS HUSTEDT: II. PROTON MAGNETIC RESONANCE, INFRARED, AND X-RAY STUDIES

The extracellular fibers attached to the diatom Thalassiosirafluviatilis have been shown to be pure β-(1 → 4)-linked 2-acetamido-2-deoxy-D-glucan. This polysaccharide was given the systematic trivial name chitan to distinguish it from chitin, which is not a chemically distinct species and which has a radically different X-ray diffraction pattern and infrared spectrum. Proton magnetic resonance studies on the acid hydrolysis of chitan have established that the hydrolysis occurred in three distinct steps: (a) the degradation of the polysaccharide to smaller polymeric units, (b) the production of N-acetylglucosamine from the latter, and (c) the conversion of N-acetylglucosamine into glucosamine and acetic acid. X-ray and infrared studies have shown that chitan has a different macrostructure from that of arthropod chitin. Chitan is completely crystalline and can be converted irreversibly into a form indistinguishable from that of chitin by treatment with hot aqueous lithium thiocyanate. Boiling chitan in water converts it into a second crystalline modification, possibly a hydrate, which can be reconverted into the starting material by drying invacuo. The exceptionally sharp infrared spectrum of chitan allows a more unequivocal assignment of a number of bands common to chitan and chitin, and provides information about the nature of the bands at 1 626 and 1 656 cm−1 in the spectrum of chitin. The correlation of the bands in the region of 840 to 890 cm−1 with the configuration at the anomeric center of glycopyranose derivatives is discussed.