Thematic Division: Structural Investigations.

______________________________________________________________________ Full Paper

Subdivision: Biochemistry and Biotechnology. Registration Code of Publication: bch2
Note: for the previous communication of this series see Butlerov Communications. 2001. Vol.1. No.4.1. (code: bch1)
Received October 3, 2002.

Thematic course: Natural polysaccharides. Part III.

RESEARCH OF ACID HYDROLYSES OF CHITIN-GLUCAN
AND CHITOSAN-GLUCAN COMPLEXES.
© Nataliya V. Shabrukova,*+ Liliya M. Shestakova, Diana R. Zainetdinova, and Valentina S. Gamayurova
Chair of Industrial Biotechnology. Kazan State Technological University.
K. Marx St., 68. Kazan 420015. Russia. Phone: +7 (8432) 389-423. E-mail: arsin98@cnit.ksu.ras.ru
____________________________________________
*Leader of the thematic course; +Corresponding author
Keywords: chitin-glucan complex, chitosan-glucan complex, hydrolysis, bridge peptide chains, D-glucosamine hydrochloride, N-acetyl-
D-glucosamine, chitooligosaccharide, ammonia, glucose, fructose.

Abstract
The use of the biomass of Aspergillus niger fungus - a producer of a citric acid as a raw source of chitin, demands carrying out a thorough
investigation of the structure of chitin-glucan complex (ChGC), in the form of which chitin is present in the cellular wall of this fungus.
The objective of the present work is to reveal the characteristic features of hydrolysis of ChGC by the concentrated acids.
By hydrolysis of chitin-glucan and chitosan-glucan complexes in concentrated hydrochloric and 55% sulfuric acids it has been shown that
degradation of these polymers results in isolation of ammonia and formation of D-glucosamine, glucose, fructose, acetic acid, as well as
chitooligosacharides and aminoacid fragments.
Detection of D-glucosamine, glucose, and fructose in hydrolysates was carried out with the use thin-layer chromatography (TLC) on silica plates
"Silufol" in various systems of solvents. As a result it has been revealed that:
• in the products of ChGC hydrolysis the intensity of fructose stain is insignificant as compared to the intensity of glucose stain;
• in the products of ChsGC the stains of glucose and fructose are comparable in intensity.
The explanation of these results is given below.

Introduction
Chitin and chitosan, its N-deacetylated derivative are biopolymers, which have recently found wide application in food,
pharmaceutical and biotechnological industries owing to such qualities, as non-toxicity, biodegradability, and biocompatibility. Chitin
and chitosan oligosacharides produced by acidic or enzymic hydrolysis of these biopolymers, have various biological properties. Being
immunostimulative [1], they serve as substrata for chitinous enzymes, such as lysozyme, chitinose, chitosanose and glucosamineose [2],
inhibit the growth of fungus and phytopathogens, and start the self-defense mechanism in plants [3]. At present, crab chitin is used as a
raw material for the production of these oligosacharides, while some micellar fungus are rather rich in chitin which is contained in them
mostly as covalent-bonded chitin-glucan complexes. Isolation of chitin-glucan complexes from the microbiological production waste of
fungus biomass, including that of mycelium fungus Aspergillus niger - a producer of a citric acid, and the partial hydrolysis of these
complexes with the purpose of obtaining biologically active oligosacharides for plant cultivation would be the most rational way for
utilizing these waste products.
As it is well known, the exhaustive hydrolysis of chitin in the concentrated acids proceeds with the formation of acetic acid and
D-glucosamine salt, and the hydrolyses of chitosan, its deacetylated derivative, results in the formation of D-glucosamine salt:
conc. НСI
[C6H9O5(NH-COCH3)]n n CH3COOH + n C6H9O5(NH2·HCl)
D-glucosamine hydrochloride
conc. НСl
[C6H9O5(NH2)]n n C6H9O5(NH2·HCl)

Nataliya V. Shabrukova - senior staff scientist of the chair of industrial biotechnology (PBT) of the Kazan state
technological university (KSTU), Cand. Chem. Sci.

Scope of scientific interests: studying chitin polysacharide, working out the technology of its isolation from the biomass
of fungus Aspergillbs niger and modification of the process.

Valentina S. Gamayurova - Head of the chair of industrial biotechnology (PBT) of the Kazan state technological
university (KSTU), professor, Doctor of Chemistry.

Scope of scientific interests: chemistry and ecology of arsenic compounds. In recent years her scientific interests have
been concentrated around the problems of biotechnology – the study and chemical modification of chitin biopolymer
("Biotechnology". 1997. No.6. P.30-33) as well as the study of enzyme activity in nonaqueous media ("Biotechnology".
1997. No.6. P.47-50. 1998. No.2. P.64-67.).

© Chemistry and Computational Simulation. Butlerov Communications. 2002. Vol.2 No.8. ________ K. Marx St., 68. 420015 Kazan. Tatarstan. Russia. ________ 57
Full Paper __________________________________ N.V. Shabrukova, L.M. Shestakova, D.R. Zainetdinova, and V.S. Gamajurova

In weak acids chitin is hydrolized with the formation of N-acetyl-D-glucosamine and salts of D-glucosamine:

HCl, H2O
[C6H9O5(NH-COCH3)]n xС6Н9О5(NHCOCH3) + N-acetyl-D-glucosamine
+ yС6Н9О5(NH2*HCl) + yCH3COOH

However, the low-temperature acidic hydrolysis of chitin results in degradation of chitin and its derivatives up to biologically
active fragments - water-soluble chitoligosacharides [4, 5].
Hydrolysis of ChGC, isolated from fungus Aspergillus niger biomass, as against ChGC hydrolyses from chitin and chitosan, is
not studied. We have just supposed, that the process of its hydrolysis is influenced by polypeptide bond bridges connecting, as well as in
the case of some other fungus [6, 7], the chains of chitin and glucan in this complex [8, 9].
The purpose of our work is revealing the features of hydrolysis of ChGC by concentrated acids.

Results and discussion

Earlier it was noticed, that the hydrolysis of ChGC at room temperature in concentrated acids (sulfuric, hydrochloric) is
accompanied by evolving ammonia bubbles from the hydrolysate. Thus, the titrimetric method of analysis used for studying the
hydrolysis of ChGC by 55% sulfuric acid, has shown, that the acidity of hydrolysate changes with time. Since the process of ChGC
hydrolysis and, accordingly, the changes in its hydrolysate acidity are aggravated by the removal of acetyl groups, with a view of
simplification of the further research in this experiment we used chitosane-glucan complex (ChsGC) obtained by deacetylation of
ChGC in 50% solution of sodium hydroxide in argon:

Simultaneously with deacetylation in the given reaction there take place the processes of degradation of the polymeric complex,
i.e. the removal of oligosacharides of glucan and chitosane chains and their dissolution in the reactionary mixture which results in the
reduction of molecular weight and ChsGC yield.
In the case of ChsGC the process of hydrolysis is also accompanied by evolving ammonia bubbles and reduction of acidity of
the hydrolysate, the least acidity of the latter being noted on the ninth day and then not practically changing. Collecting the evolved gas
in a flask with distilled water and definition of its alkalinity has shown the change of рН from 6 to 9, and the qualitative reaction on
NH4+-ions has revealed their presence in water, which is the evidence of ChsGC desamination in the course of hydrolysis. Presence of
NH4+-ions in the form of ammonium chloride is also detected in the products of hydrolysis of ChGC and ChsGC by concentrated
hydrochloric acid.
The study of the hydrolysis products of chitin-glucan and chitosane-glucan complexes by concentrated hydrochloric acid with the
thin-layer chromatography method on silica plates "Silufol" in different solvent systems: n-butanol: acetic acid: water (9:6:1), n-butanol:
acetic acid: water (4:5:1), propanol: pyridin: water (10:3:3), n-propanol: ethyl acetate: water (7:2:2) has shown that in the products of
hydrolysis there are, besides ammonium chloride, three more compounds. Comparison of Rf factors as well as the colors of spots of these
compounds with those of witnesses: D-glucosamine hydrochloride, glucose and fructose has shown their identity. Selivanov’s qualitative
reaction on ketohexose has also confirmed the presence of fructose in hydrolysates. Intensity of fructose spots in the products of ChGC
hydrolysis is insignificant as compared with the intensity of glucose spots. In some cases, after applying the mark with a small amount of
hydrolyses products on the plate, the fructose stain did not even appear. In the products of ChsGC hydrolysis the intensity of fructose
and glucose spots is approximately identical.
This can be explained only by the fact that fructose is the product of hydrolysis of those parts of the chitinous chain of chitin-
glucan and chitosane-glucan complexes which are connected with peptide bridges, or the fructose link is a part of these lateral bridge
chains. The content of bridge chains in ChsGC is richer than in ChGC, since at deacetylation of ChGC in 50% solution of sodium
hydroxide there also take place the processes of breakage of chitosane and glucan chains in the form of oligosacharides, soluble in the
reaction mixture, and insolubility of peptidoglycan fragments results, naturally, in the fact that ChsGC isolated from the reaction
mixture, as shown in works [6, 7], basically consists of peptidoglycan, insoluble in alkaline media:

1
Н NMR spectra of hydrolysates do not contradict the conclusion about the presence of hydrochloride D-glucosamine, glucose
and fructose in the products of ChGC 12N hydrolysis by hydrochloric acid. So, the signals of hydrogen protons - СН2ОН and -СН(ОН)
- groups of glucose and fructose should appear in the range of 3.3-3.6 and 3.4-3.85 ppm, accordingly, but owing to imposing they appear
as unsoluble multiplet in the range of 3.3-3.75 ppm, and signals of hydrogen protons at the first carbon atom of glucose appears as
doublets in the range of 4.52 ppm and 5.09 ppm, that corresponds to the presence of tautomeric forms of glucose, both aldehydic and
58 ________________ http://chem.kstu.ru _______________ © Химия и компьютерное моделирование. Бутлеровские сообщения. 2002. T.2. №8. 57.
RESEARCH OF ACID HYDROLYSES OF CHITIN-GLUCAN AND CHITOSAN-GLUCAN COMPLEXES._________________________________________________ 57-60
semiacetal. In spectrum there are also signals of hydrogen protons of acetyl groups of water-soluble chitoligosaccharides in the range of
2.2 ppm in the form of insoluble multiplet which in due course disappears. In 1Н NMR spectrum the signals of small intensity are
revealed also in the range of 5.38 and 5.60 ppm, corresponding, probably, to the fragments of the peptide chain.
Thus, the hydrolysis of chitin-glucan and chitosan-glucan complexes in concentrated sulfuric and hydrochloric acids results not
only in deacetylation of chitinous links and breakage of glycoside bonds, but also in desamination as evidenced by the detection of N-
acetyl-D-glucosamine and D-glucosamine, as well as ammonia, glucose and fructose in the hydrolysis products. The amount of fructose
formed in the process of ChsGC hydrolysis is much greater than in ChGC hydrolysis, which is, probably, due to the presence in ChsGC
of greater number of fragments of the chitinous links bound to lateral bridge chains.

Experimental
In work we used biomass of mycelial fungus A. niger, commercial strains P1 and P2, produced by the surface growth technique given in work [8]. ChGC isolated
by the technique given in work [10].
Chitosan-glucan complex (ChsGC) was obtained by deacetylation of CHGC in 50% solution of sodium hydroxide in argon at temperature 90-95° C. Yield - 22.2%.
Hydrolysis of ChGC and ChsGC by concentrated hydrochloric acid was carried out with the ratio of ChGC (or ChsGC): 12N НСI = 1 : 20 and temperature 80±1°
С for 1 hour with subsequent removal of hydrochloric acid in the air stream or under vacuum.
Hydrolysis of ChsGC by 55% sulfuric acid was carried out with the ratio of ChsGC: 55% H2SO4 = 1 : 20 at room temperature for 21 day, after 1, 2, 3, 7, 9 and 21
days passed, acidity of the hydrolysate was defined by titration 1N NaOH.
Presence of NH4+ ions was defined by the standard technique of qualitative analysis on NH4+-ions.
The analysis of products of hydrolysis of ChGC and ChsGC by 12N hydrochloric acid was carried out with the help of TLC method on silica plates "Silufol"
in the following systems of solvents:
• butanol-acetic acid - water (9:6:1) Rf of ChGC hydrolysis products: 0.44, 0.57. Rf of ChsGC hydrolysis products: 0.44, 0.57, 0.71. Rf of witnesses: 0.45
(D- glucosamine hydrochloride), 0.58 (glucose), 0.71 (fructose);
• butanol-acetic acid - water (4:5:1.) Rf of ChGC hydrolysis products: 0.33, 0.94, 0.95. Rf of ChsGC hydrolysis products: 0.32, 0.94, 0.95. Rf of witnesses: 0.34
(D-glucosamine hydrochloride), 0.92 (glucose), 0.95 (fructose);
• propanol - ethyl acetate - water (7:2:2.) Rf of ChGC hydrolysis products: 0.63, 0.70, 0.83. Rf of ChsGC hydrolysis products: 0.62, 0.67, 0.85. Rf of witnesses:
0.65 (D-glucosamine hydrochloride), 0.78 (glucose), 0.84 (fructose);
• propanol – pyridin - water (10:3:3.) Rf of ChGC hydrolysis products: 0.59, 0.78. Rf of ChsGC hydrolysis products: 0.61, 0.77. Rf of witnesses: 0.61 (glucose),
0.77 (fructose).
Development of the obtained chromatograms was performed with anilinphthalate developer.
Presence of fructose in hydrolysates of ChGC and ChsGC is also confirmed with Selivanov’s qualitative reaction on ketohexose [11].
NMR 1Н spectra were taken on NMR spectrometer Tesla 567 A of high resolution with the Fourier-converter and with the working frequency 100 MHz (solvents: a
hydrochloric acid + deuterowater and hydrochloric acid + deuteroacetone).

Conclusions
Hydrolysis of chitin-glucan and chitosan-glucan complexes isolated from the biomass of fungus Aspergillus niger by 12N
hydrochloric and 55% sulphuric acids results in the formation of N-acetyl-D-glucosamine, D-glucosamine, ammonia, glucose and
fructose. The amount of the fructose formed at the hydrolysis of chitosane-glucan complex is much greater, than at the hydrolysis of
chitin-glucan complex, which is probably conditioned by the presence in chitosane-glucan complex of a great number of chitinous
fragments connected by peptide bond bridges.

References
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2001. P.208-212. (Russian)
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[3] A.I. Usov. Uspekhi khimii. 1993. Vol.62. Iss.1. P.1119-1144. (Russian)
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2001. P.333-335. (Russian)
[9] N.V. Shabrukova, T.A. Zjablikova, D.R. Zajnetdinova, F.G. Halitov, V.S. Gamajurova. Chemistry and Computational Simulation. Butlerov Communications. 2001. Vol.1.
No.4. P.1. (Russian)
[10] V.S. Gamajurova, M.N. Kotlyar, N.V. Shabrukova, F.G. Halitov. Biotechnolog. 1997. No.6. P.30-33. (Russian)
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© Chemistry and Computational Simulation. Butlerov Communications. 2002. Vol.2. No.8. _____________ E-mail: info@kstu.ru __________________ 59