INHIBITION OF CHITIN DEACETYLASE BY ACETIC ACID

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extract separated (centrifugation, 6000 r.p.m., 20 min, 4 °C) and salted out with ammonium sulphate (80% saturation) overnight at 4 - 6 °C. The solution was ...
INHIBITION OF CHITIN DEACETYLASE BY ACETIC ACID PRELIMINARY INVESTIGATION Małgorzata M. Jaworska, Ewa Konieczna-Mordas Faculty of Chemical and Process Engineering, Warsaw University of Technology, ul. Warynskiego 1, 00-645 Warsaw, Poland E-mail: [email protected],edu.pl

Abstract The current paper reports on a preliminary investigation of the mechanism of inhibition of chitin deacetylase isolated from Absidia orchidis vel coerulea by the acetic acid released during the deacetylation process. It is suggested that the process follows the competitive inhibition with respect to acetic acid. The reaction rate was correlated with the concentration of accessible GlcNAc mers in the polymer. Key words: chitosan, chitin deacetylase, competitive inhibition.

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M. M. Jaworska, E. Konieczna-Mordas

1. Introduction Chitin deacetylase EC 3.5.1.41 is an enzyme that catalyse the reaction of hydrolisis the linkage between the acetyl and amine groups in the mer of N-acetylglucosamine (GlcNAc) transforming it into the glucosamine mer (GlcN):

- GlcNHAc

chitin deacetylase --------------------------->

- GlcNH2 + AcOH

This transformation can be used for enzymatic modification of chitosan to obtain polymer with a lower degree of acetylation. The acetic acid formed during the deacetylation process was reported to be an inhibitor for the chitin deacetylase [1, 2]. It was shown that for the purified enzyme from Mucor rouxii ATCC 24905 [1] most organic acids acts as inhibitors: acetic, formic and propionic acids at a concentration of 250 mM decreased the activity to 10%, 50% and 15% of the initial value respectively. The influence of acetic acid on chitin deacetylase from Colletotrichum lindemutianum ATCC 56676 [2] was less significant, with the enzyme losing only 4% of its initial activity with a concentration of sodium acetate of 100 mM and only 30% with a concentration of 800 mM. The mechanism of this inhibition process has not been investigated or reported on to date. The aim of the current work was to investigate the mechanism of inhibition of chitin deacetylase by acetic acid. The presented paper reports on a preliminary investigation of the mechanism of inhibition the chitin deacetylase isolated from Absidia orchidis vel coerulea by the acetic acid released during the deacetylation process.

2. Materials and methods 2.1. Chitin deacetylase Chitin deacetylase was isolated from Absidia orchidis vel coerulea NCAIM F 00642 (National Collection of Agricultural and Industrial Microorganisms, Budapest Hungary). The fungi were cultivated in a 7.0-L batch culture (26 °C, pH 5.5, YPG nutrient medium [3]) and separated from the nutrient medium by centrifugation (6000 r.p.m., 20 min, 4 °C). Next the biomass was frozen and than slowly thawed and homogenised, and the crude cell extract separated (centrifugation, 6000 r.p.m., 20 min, 4 °C) and salted out with ammonium sulphate (80% saturation) overnight at 4 - 6 °C. The solution was diafiltrated with HCl (pH 4.0) to remove ammonium sulphate (using a membrane module with a 10 kDa cut-off) and than concentrated by utrafitration. This enzyme solution, adjusted to pH 4.0 with HCl, was used in the experiments.

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Inhibition of Chitin Deacetylase by Acetic Acid Preliminary Investigation

2.2. Chemicals The chitosan used in all the experiments was kindly donated by Gillet-MahtaniChitosan (France/India). It was of medium molecular weight (viscosity of 1% solution in 1% acetic acid solution at 25 °C μ = 85 Pa.s, according to the producer data), and acetylation degree of 39.8% (determined from the IR spectrum using the procedure of Domszy and Roberts [4]). Chitosan (5g) was mixed with 500 mL of HCl solution (pH 4.0). Next 0.1 M HCl was added in small portions (1 - 2 mL) to complete polymer dissolution (the pH was controlled during the dissolution and kept constant at pH 4.0) and it was adjusted to a final volume of 1.0 L with a HCl solution (pH 4.0). All other chemicals were analytical grade and purchased by POCH (Poland). 2.3. Inhibition experiments All experiments were carried out in a 1.5 L thermostated batch reactor at 50 °C and stirred at 250 r.p.m. Chitosan solution (1.0 L) with a concentration of 5.0 g/L prepared as described above was preincubated at 50 °C in a batch reactor for 30 min. At the same time native chitin deacetylase prepared as described earlier was preheated separately at 50 °C for 15 min. The reaction was initied by adding the enzyme to the chitosan solution. The reaction mixture was sampled (2.0 mL) every 10 min during the first hour of the experiment and every 20 min after this, and the reaction was stopped by addition of 0.1 mL of 1.0 M NaOH to the aliquot. The precipitated chitosan was centrifuged and the acetic acid concentration in the clear supernatant solution was determined. 2.4. Analytical methods Protein concentration was determined according to the Bradford method using a ready made reagent from Biorad (USA, cat. No. 500-0006) and bovine serum albumin as a standard. Acetic acid concentration in the clear solution was analyzed using the HPLC method: isocratic system (Varian ProStar 210) with HyperREZ XP Organic acid column (60 °C) and HyperREZ XO Carbohydrate H+ Guard Column, 0.0025 M H2SO4 as eluent (0.5 mL/min), and refractometer detector (Varian ProStar 350). The quantification limit was evaluated at 5 nmol/mL with a standard deviation of 8% of the mean value. The method was validated for acetic acid determination in chitosan-HCl (pH 4.0) solutions.

3. Results and Discussion Experiments were performed in a 1.5-L batch reactor containing 1.0 L of the chitosan solution with concentration of chitosan of 5 g/L. Experiments were carried out for 8 hours and the influence of the inhibitor, acetic acid, could be clearly observed. Data were compared with the assumed mechanism of inhibition (competitive, non-competitive, mix-type, substrate).

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The changes in acetic acid concentration in the reaction mixture (Figure 1.A) were transformed into changes in N-acetylglucosamine mers concentration (Fig.1.B.) – the reaction is equimolar. A.

4

10.5

3.5

10

[ mmol/ L ]

3

2.5

2

CGlcNAc

CAcOH [ mmol/ L ]

B.

11

1.5

9.5

9

8.5

1

8

0.5

7.5

7

0 0

100

200

300

Time [ min ]

400

500

0

100

200

300

Time [ min ]

400

500

Figure 1. The changes in acetic amid concentration (A.) and concentrations of GlcNAc mers (B.) during the deacetylation process in a batch reactor; concentration of enzyme was CE0 = 1,146 mg/L.

The mechanism of inhibition and corresponding kinetic equation were assumed. The equation was next integrated and transformed into the relationship t vs CGlcNAc and was compared with the original experimental data. In the equation the parameters of Michaelis-Menten equation determined earlier (data not published) are used: Rmax = k3×CGlcNAc = 0.4697 (µmol/mL)/min KM = 2.45 (mmol GlcNAc)/L It was also assumed that CGlcNAc = CGlcNAc, 0 - CAcOH and that the initial conditions for integration are: t = 0 and CGlcNAc = CGlcNAc, 0

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Inhibition of Chitin Deacetylase by Acetic Acid Preliminary Investigation

3.1. Non-competitive inhibition This kind of inhibition is described by the following equation:

that after integration can be converted into the relationship:

The original experimental data (Figure 1.B) were transformed into the relationship of t = f(CGlcNAc) and compared with the proposed model (Figure 2). The model of noncompetitive inhibition gave a rather poor approximation to the experimental data and this hypothesis was rejected.

500 450

competitive inhibition, R2 = 0.950

400

non-competitive inhibition, R2 = 0.742

Time [ min ]

350 300 250 200 150 100 50 0

7.0

7.5

8.0

8.5

9.0

9.5

CGlcNAc [µmol/ mL]

10.0

10.5

11.0

Figure 2. Comparison of the experimental data with assumed model of inhibition: competitive and non-competitive.

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3.2. Competitive inhibition In the next step the hypothesis on the competitive inhibition was examined. This kind of inhibition is described by the equation:

that after integration and rearrangement gives the following relationship:

Comparison of the experimental data with the proposed model gives good agreement (Figure 2) indicating that the mechanism of the inhibition of chitin deacetylase by acetic acid formed during the deacetylation process can be assumed as competitive. It can therefore be stated that the hypothesis on competitive inhibition has been confirmed. All other hypothesis on the mechanism of inhibition of chitin deacetylase by acetic acid (mixed-type, substrate) gave much worse approximations and were rejected as incorrect.

4. Conclusions The investigations that were focused on the mechanism of inhibition show that it can be assumed to be competitive with respect to the acetic acid. This mechanism is logical since for hydrolysis of the linkage between the amine and acetyl groups in the N-acetylglucosamine mers, the enzyme must posses the binding sites for both molecules. Due to this, the binding site of the acetyl group may be occupied either by the acetyl group of GlcNAc or by acetic acid. In such a case the competitive inhibition is quite possible.

5. Reference 1. Kafetzopoulos D., Martinou A., Bouriotis V.; (1993) ���������������������������������������� Bioconversion of chitin to chitosan: Purification and characterization of chitin deacetylase from Mucor rouxii Proc Nat Acad Sci USA, 1993, 90, pp. 2564-2568. 2. Tokuyasu K., Ohnishi-Kameyama M., Hayashi K.; (1996) Biosci Biotech Biochem, 30, pp. 239-242. 3. Jaworska M. M., Konieczna E.; (2001) The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis Appl. Microbiol.Biotechnol., 56, pp. 220-224. 4. Domszy J. G., Roberts G. A. F.; (1985) Evaluation of infrared spectroscopic techniques for analyzing chitosan. Makromol Chem, 186, pp. 1671-1673.

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