A. Konieczna-Molenda et al. / Carbohydrate Research 343 (2008) 3117–3119
3119
(Chitosan N-acetylglucosaminohydrolase; EC 3.2.1.132) (C0794
1.3. Reducing sugar determination
Sigma–Aldrich, Poznan, Poland) or chitinase from Serratia marces-
cens, lyophilized powder, ꢀ10 units/g solid (chitodextrinase
3,5-Dinitrosalicylic acid (DNS) in alkaline sodium potassium
tartrate was used as the reagent for reducing sugars according to
Southgate.22 Absorbance at 540 nm was recorded. Calibration
poly(1,4-b-[2-acetamido-2-deoxy-D-glucoside]) glycanohydrolase;
EC 3.2.1.14) (C1650 Sigma–Aldrich, Poznan, Poland)] was dissolved
in acetate buffer 0.1 M pH 5.5 (10 mL). Enzyme solution (2 mL) was
placed in a glass cell and illuminated from the 30 cm distance with
a KB 502 slit illuminator (Kabid, Chorzów, Poland) equipped with
150 W xenon arc (XBO 150, Oriel, Maidston, UK). An HN 22 linear
polarizing filter (Polaroid, Waltham MA, USA) with a glass filter
cutting off wavelengths below 500 nm was mounted between
the slit illuminator and the sample. In the experiments involving
non-polarized light, the HN 22 filter was removed. The light source
emitted continuous radiation in the visible range. Its energy flux at
the position of the sample was 8 mW/cm2 as checked by YSI radi-
ometer (Yellow Spring OH, USA).
curve was prepared using
ma–Aldrich Poznan, Poland) and N-acetyl-
D
-(+Glucosamine hydrochloride) (Sig-
-glucosamine (Sigma–
D
Aldrich, Poznan, Poland) were used as standards of substrates.
References
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B.; Vriend, G.; Vårum, K. M.; Eijsink, V. G. H. Proc. Natl. Acad. Sci. U.S.A 2006, 103,
18089–18094.
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Samples were illuminated at 4 °C (chitosanase) and 20 °C (chiti-
nase) for both 30 and 60 min. Control non-illuminated samples
were stored in the dark under the same conditions as the illumi-
nated samples.
8. Nordtveit, R. J.; Vårum, K. M.; Smidsrod, O. Carbohydr. Polym. 1994, 23, 253–257.
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1.2. Enzymatic reaction
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Eijsink, V. G. H. FEBS J. 2006, 273, 491–503.
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Eijsink, V. G. H. Biocatal. Biotransform. 2006, 24, 39–53.
13. Fiedorowicz, M.; Khachatryan, G. J. Agric. Food Chem. 2003, 51, 7815–7819.
14. Konieczna-Molenda, A.; Fiedorowicz, M.; Lai, V. M. F.; Tomasik, P. Biotechnol.
Prog., in press.
15. Konieczna-Molenda, A., private information.
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19. Fiedorowicz, M.; Khachatryan, G. J. Sci. Food Agric. 2003, 84, 36–42.
20. Fiedorowicz, M.; Khachatryan, G.; Konieczna-Molenda, A.; Yuryev, V. P.;
Wassermann, L. A. Starch. In Achievements in Understanding of Structure and
Functionality; Yuryev, V. P., Tomasik, P., Bertoft, E., Eds.; Nova Science: New
York, 2007; pp 147–165.
Either chitin [from crab shells, suitable for analysis of chitinase,
purified powder (Sigma–Aldrich, Poznan, Poland)] or chitosan [high
molecular weight deacetylated in >80% (Sigma–Aldrich, Poznan,
Poland)] was suspended in acetate buffer 0.1M pH 5.5 (32.0 mL, chi-
tin and chitosan concentration was 1 mg/1 mL) and heated at 85–
90 °C for 15 min. Chitin and chitosan solutions were cooled to room
temperature, then placed in a 37 °C water bath. Enzyme solutions
(1.4 mL) were added to the chitin and chitosan solutions.
After enzyme addition, samples were incubated with mild
agitation at 37 °C. Aliquots of the reaction mixture (4.0 mL) were
taken after 0, 10, 20, 40, 60, 80, and 100 min for reducing sugar
determination.
For comparison, enzymatic reactions were also illuminated
with non-polarized light under identical conditions as those
described for experiments with WLPL. All reactions were run in
duplicate.
21. Konieczna-Molenda, A.; Molenda, M.; Fiedorowicz, M.; Tomasik, P. Macromol.
Symp., in press.
22. Southgate, D. A. T. Determination of Food Carbohydrate; Elsevier Science: New
York, 1991.