Production, characterization and purification of chitosanase from Bacillus cereus

Production, characterization and purification of chitosanase from Bacillus cereus

F. A. T. PIZA, A. P. SILOTO, C. V. CARVALHO and T.T. FRANCO

Biochemical Engineering Laboratory, Chemical Engineering, State University of Campinas (UNICAMP), P.O. Box 6066, 13081-970, fax: [phone], Campinas- SP, Brazil, E-mail: [e-mail]

(Received: January 19, 1999; Accepted: April 20, 1999)

Abstract - A culture medium for a wild strain of Bacillus cereus was developed for chitosanase production by using an experimental design. The factors having the strongest influence on chitosanase production were ammonium sulfate concentration, aeration, pH and the interaction between the first two parameters. Optimal conditions for chitosan hydrolysis were pH 5.8 and 54 ^oC; however, hydrolysis activity drastically decreased at pH 7.0. The enzyme was purified (single-electrophoretic band) by partitioning in an aqueous two-phase system (ATPS), followed by cation-exchange chromatography with a 66% yield. Chitosanase was mainly collected in the top phase (K = 129) of a 22% PEG 1,500, 13% phosphate (pH = 5.8) and 12% NaCl (w/w) solution, and the main protein contaminants were evenly distributed between the phases (K = 1.07). The apparent molecular weight and the isoelectric point of the chitosanase, determined by SDS-PAGE electrophoresis and by isoelectric focalization, were 47 kDa and 8.8, respectively.

Keywords: Chitosanase, Bacillus sp., factorial experimental design, aqueous two-phase systems.

INTRODUCTION

Chitosan is a linear polysaccharide composed of b-1,4 linked d-glucosamine residues extracted from shellfish exoskeletons. In nature the polymer is partially acetylated, and the name chitosan describes a wide range of polymers corresponding to various proportions of d-glucosamine and n-acetyl-d-glucosamine (Figure ¹). It is currently obtained by deacetylation of chitin, and this transformation can be catalyzed by different enzymes, including proteases, some lipases and chitosanases. Chitosanase represents a class of hydrolytic enzymes found in bacteria, fungi and plants (Somashekar and Joseph, 1996). Oligosaccharides from chitosanase action have industrial, medical and agricultural applications. Transformation of chitosan into chitooligosaccharides could be potentially commercialized, as long as the hydrolysis processes were well controlled and reproducible (Muzarelli, 1993). Strains of microorganisms able to produce oligosaccharides by enzymatic processes have been isolated by different researchers. The chitosanases have been purified by conventional operations in several steps such as ammonium sulfate fractionation, gel filtration and ion exchange chromatography and by preparative isoelectric focusing, but most of the reported recovery processes include several chromatographic steps (Somashekar and Joseph 1996; Pelletier and Sygush 1990; Ushida and Ohtakara 1988). However, the improvement and simplification of the procedure for purification of chitosanase from its microbial sources would be desirable to reduce the cost of enzyme preparation, since extraction, purification and concentration steps are commonly responsible for up to 40% of the total cost of production.

Figure 1: Chemical structure of chitin and chitosan

Protein extraction in an aqueous two-phase system (ATPS) is a rapid procedure which avoids most of the problems of denaturing fragile molecules in conventional downstream processing. ATPS provides a gentle environment for biologically active proteins and may be employed on a large scale. In order to have a high yield, recovery and also a good purification factor for a target protein, a composition has to be selected for the ATPS to quantitatively extract the desirable protein from one of the phases with minimal concentration of contaminant molecules. It has been found that the debris are usually removed in one of the phases and that whole cells and large particles such as chromosomes partition at the interface. A single extraction step is usually sufficient for removal of some contaminants, but others can be eliminated by countercurrent extraction using fresh phase from identical systems without the sample or by a chromatographic step. It is desirable to find the highest difference values between their partition coefficients, and the volume ratio of the two phases has to be considered as it also affects the yield of enzyme purification (Franco and Kaul, 1998).

This article reports on the production of chitosanase by Bacillus cereus in shaken flasks, a study of optima conditions for the enzymatic hydrolysis of chitosan, the purification of the chitosanase produced from the main protein contaminants by partitioning in PEG-phosphate systems and a partial characterization of this enzyme.

MATERIALS AND METHODS

Microorganism

An isolated wild strain identified as Bacillus cereus was maintained on CDA (Chitosan Detector Agar, as described by Boucher et al. (1992).

Chitosanase Production

B. cereus was first grown on Tripticase Soytone (TSB) media (Pelletier and Sygush, 1990) for 14 hours at 30ºC and 200 rpm. Fractional optimization of the culture media was carried out in 500 mL Erlenmeyer flasks containing medium with fixed amounts of KH₂PO₄ (0.31g l^-1), MgSO₄.7H₂O (0.5 g l^-1) and Na₂HPO₄.12H₂O (1.7 g l^-1); trace element solutions of FeSO₄.7H₂O (0.001 g l^-1), ZnSO₄.7H₂O (0.0005 g l^-1), MnSO₄.H₂O (0.001 g l^-1) and CuSO₄.5H₂O (0.0001 g l^-1); and variable concentrations of (NH₄)₂SO₄ and chitosan (Sigma, St. Louis, MO, USA), as shown in Table ¹. The inoculated flasks were shaken continuously at 200 rpm and 30 °C for 16 and 32 hours. The fermented broth was then centrifuged and a clear supernatant was obtained.

Enzyme Activity Assay

Chitosanase activity was assayed by using a 0.2% chitosan solution (w/w) in 0.05 M of acetate buffer at pH = 5.6 as the substrate. The release of reducing sugars in 40 min. at 30 °C was measured as in Somogyi and Nelson (Spiro, 1966). One unit of chitosanase activity (U) is defined as the amount of enzyme that produces 1mmol of reducing sugars measured as glucosamine equivalent per min. at given assay conditions.

Culture Media

The culture media composition was investigated by fractional experimental design "type 2^5-1," design n⁰ 1 (Haaland). The design investigated a fraction of the combination of a two-level design with the following five factors: ammonium sulfate concentration, aeration, time of fermentation, pH and chitosan concentration (Table ¹). Statgraphics version 5.0 (Statistical Graphics Corp., Rockville, MD, USA) was used for plotting the graphs.

Chitosanase Optimal Conditions

Initially the conditions for chitosanase activity were investigated for pH, temperature and time. Then a complete two-level experimental design with three factors type 2³ (design n⁰ 2) was employed to study the effects of time of hydrolysis and temperature at a constant pH of 5.8 (Table ²). A constant volume ratio of 28 of substrate solution to enzyme solution was used for all conditions studied. Hydrolysis proceeded in a 25.0 mL stirred, jacketed reactor and was terminated by the removal of aliquots, followed by the addition of 1 mL of Somogyi reagent and subsequent heating to 100 °C for 6 min. The samples were left in an ice bath and the soluble-reducing sugars were measured by the Somogyi and Nelson assay (Spiro, 1966).

Chitosanase Purification

Purification of chitosanase by partitioning in aqueous two-phase systems (ATPS) was studied in five systems, as described in Table ³. Ten grams of ATPS were prepared in beakers by weighting solid PEG 1500, 40% phosphate stock solution at pH = 5.8 (w/w) and solid sodium chloride, and the remainder was the clear fermented broth. The systems were mixed and centrifuged for two minutes at 1,000 g to achieve phase separation. The partition coefficient (K) of the chitosanase was calculated as the ratio of the enzyme concentration in the top phase to that in the bottom phase at room temperature. In order to improve the separation of chitosanase from the protein contaminants, sixteen mililiterts of the diluted isolated top phase of the 22% PEG 1,500 + 13% phosphate + 12% NaCl system was applied to a HR 5/5 column packed with S-Sepharose (Amershan-Pharmacia), equilibrated with 50 mM sodium acetate buffer, pH 5.8 Elution was then achieved with a gradient of NaCl (0 to 1.5 M), and the fractions collected were analyzed for chitosanase activity and total proteins (Sedmark and Grossberg, 1977).

* in the predominant phase

RESULTS AND DISCUSSION

Chitosanase Production

The composition of the culture medium for Bacillus cereus was studied for the production of chitosanase by a fractional factorial design type 2^5-1 (design 1). The five two-level factors studied were ammonium sulfate concentration, chitosan concentration, aeration, pH and fermentation time (Table ¹). Aeration was defined in this work as the ratio of the volume of the Erlenmeyer flasks to the volume of the fermented media. Three central points were introduced in order to find the experimental error. Table ¹ summarizes the conditions investigated by this design and the levels of chitosanase produced, suggesting that an increase in aeration and (NH₄)₂SO₄ concentration would improve the enzyme yield. It was observed that chitosanase production is greatly affected by the composition of the culture medium, and the most important factors are the concentration of ammonium sulfate, aeration, pH and the interaction between the first two factors. The Pareto chart (Figure ²) represents the estimated effects of each factor and the interactions upon the production of chitosanase. The best culture medium composition for the highest level of chitosanase was obtained with 2% chitosan, 4% ammonium sulfate, an aeration of 10 (Erlenmeyer volume/culture media volume) at pH = 5.0 and 16 hours of fermentation (assay 13). It was observed that the enzyme level decreased after 32 hours of fermentation (assay 16), indicating that denaturation or proteolysis may have occurred during the last hours of fermentation, since the pH remained approximately constant. An important decrease in the production of chitosanase was obtained in the medium with the lowest aeration, despite the fact that it had the highest ammonium concentration (4%) added to the culture media (assays 9). Pelletier and Sygush (1990) produced 1U/mL of chitosanase from Bacillus megaterium in a culture medium, and a small concentration of chitosan flakes (1%, w/w) was found to be a powerful inducer of the enzyme. However, a 24 hour fermentation of Bacillus sp yielded 2.5 U/mL of chitosanase in a culture medium enriched with 0.8 % yeast extract and 0.4 % polypeptone at pH = 7.0 and 30 °C (Uchida and Ohtakara, 1988).

Figure 2: Pareto chart for experimental design n°1

Characterization of Chitosanase Activity

Initially the conditions for chitosanase activity were studied for pH, temperature and hydrolysis time. The preliminary results showed that a maximum of chitosanase activity was reached at pH = 5.8 for temperatures above 50 °C. A complete experimental design type 2³ (design 2) was then employed to optimize chitosanase activity for time of hydrolysis and temperature, at a constant pH of 5.8 (Table ²). The experimental results were fitted to a second-order polynomial function and response surface was analyzed. The model equation is described by (1).

Y=0.3497 - (0.05034T) + (0.11638t) - (0.14252T²) - - (0.0650t²) -(0.06534Tt) ₍₁₎

where

Y=chitosanase activity (U/mL)

T=temperature (°C) and

t = time (min).

The analysis of variance applied showed that this model is significant at a confidence level of 95%, indicating that this enzyme responded to hydrolysis time and temperature. The fit of the model was also expressed by the coefficient of determination R², which was 0.96 (Figure ³).

Figure 3: Surface response plot of experimental design n°2 for the optimization of chitosanase conditions

Table ² shows that the highest chitosanase activity was obtained at 54 °C and pH = 5.8 after 40 min. of hydrolysis and that it decreased drastically above 54 °C and its stability was also lowered. A possible denaturation of the enzyme may have occurred at pH values above 7.0, as its activity was almost lost after a ten-minute incubation at this pH. Pelletier and Sigush (1990) observed a sharp decrease in chitosanase activity produced by Bacillus megaterium at pH values above 7.0 and that the best pH for enzyme activity was between 4.5 and 6.5. They also found that the enzyme was most active around 50 °C, but thermal stability studies indicated a complete loss of activity at this temperature. A different enzyme, which was produced by Bacillus sp (Uchida and Ohtakara, 1988), was stable at pH values between 5.0 and 11.0 for 3 h. at 37°C and was only inactivated at pH values below 2.0 and above 12.0.

Chitosanase Purification

In order to separate the chitosanase from the main contaminant proteins, five different compositions of ATPS, which are usually employed (Franco et al., 1996; Johansson et al., 1998) for the purification of biomaterial, were investigated. Two of the aqueous systems tested were unable to increase the concentration of enzyme in one of the phases (systems 2 and 4). However, systems 1, 3 and 5 separated chitosanase from the contaminant proteins. A 1,800-fold increase in the partition coefficient of chitosanase was observed (from 0.04 to 72) with the addition of 12% NaCl (systems 2 and 3) but the K of the main protein contaminants was not strongly affected (from 0.92 to 1.72). The addition of 12% NaCl to a 22% PEG 1,500 + 13% phosphate increased the K_chitosanase almost 645-fold (from 0.2 to 129), with the main protein contaminants distributed between the two phases (K_proteins =1.07), but the purification factor remained the same (Table ³). Sixteen mililiters of isolated material from the top phase of system 5 were diluted 200-fold and injected into to the ion exchanger chromatographic media S-Sepharose. Chitosanase was strongly adsorbed by the cationic chromatographic media at pH 4.0, 5.0 and 5.8 and was eluted with 1.2 M, 0.7 M and 0.5 M of NaCl, respectively, suggesting that the isoelectric point (pI) of the chitosanase is located above 5.8, which was confirmed by isoelectric focalization. The pI and molecular weight were observed to be 8.8 and 47 kDa, respectively. A photograph of a SDS-PAGE gel (Figure ⁴) shows the protein profile of the crude fermentation broth, the isolated material from the top and bottom phases of an ATPS and the two step purified chitosanase (partitioning + chromatography). Chitosanase yield and the purification factor were 66% and 20 after just two simple purification procedures (partitioning + ion exchange adsorption).

Figure 4: Coomassie-stained SDS-PAGE of standard markers: phosphorilase B, bovine serum albumin, ovalbumin, carbonic anhydrase and trypsin inhibitor (lane 1); crude fermented broth (lane 2); top phase of system 4 (lane 3), bottom phase of system 4 (lane 4) and purified material collected after partitioning followed by IEC (lane 5)

Pelletier and Sigush (1990) purified three chitosanases in three steps: precipitation, ion exchange chromatography and gel permeation. The MW of the isolated enzymes were 23, 13 and 5.5 kDa. Uchida and Ohtakara (1988) identified a chitosanase produced by Bacillus sp and the molecular weight determined by SDS-PAGE was 41 kDa. However, a different chitosanase was produced by Bacillus circulans, with a MW of 31 kDa measured by SDS-PAGE.

CONCLUSIONS

A culture medium was developed for the production of chitosanase from Bacillus cereus. The factors having the strongest influence on chitosanase production were ammonium sulfate concentration, aeration, pH and the interaction between the first two parameters. The best composition was 2% chitosan and 4% ammonium sulfate, with an aeration of 10 (Erlenmeyer volume/culture medium volume) at pH = 5.0 for 16 hours of fermentation. An important decrease in the production of chitosanase was obtained in the medium with the lowest aeration, despite its having the highest ammonium concentration (4%) added to the culture media.

The best conditions for chitosan hydrolysis were pH = 5.8 and 54 °C, and enzymatic activity drastically decreased at pH = 7.0. Denaturation of the enzyme was observed at pH = 7.0 after 10 min.

The enzyme was partially purified by partitioning in 22% PEG 1,500 + 13% phosphate + 12% NaCl at pH 5.8. A high yield of recovery of chitosanase was obtained in the top phase (102%), and the main protein contaminants were evenly distributed between the phases. Cation exchange chromatography was employed in a second purification step to produce a pure enzyme, confirmed by a single electrophoretic band in SDS-PAGE, achieving a 66% yield of chitosanase.

ACKNOWLEDGMENTS

We thank Edilberto Princi Portugal at CPQBA/UNICAMP for the isolated microorganism. The financial assistance received from FAPESP in the form of scholarships for F.A.T.P., A.M.P.S. and C.V.C. are gratefully acknowledged. T.T. Franco thanks CNPq and FAPESP for financial support in the form of grants.

REFERENCES

Boucher, I.; Dupuy, A.; Vidal, P.; Negebauer, W.A. and Brzezinski, R., Purification and characterization of a chitosanase from Streptomyces N174, Applied Microbiology Biotechnology, 38, 188 (1992).

Franco, T.T.; Andrews A. T. and Asenjo, J. A., Use of chemical modified proteins to study the effect of a single protein property on partitioning in aqueous two - phase systems, Biotechnology and Bioengineering, 49, 300 (1996).

Franco, T.T. and Kaul, R., Single step partitioning in aqueous two-phase systems, cap.6. In Methods in Biotechnology - Aqueous Two-Phase Systems: Methods And Protocols. Ed. Dr. Rajini Hatti-Kaul, Humana Press, New Jersey, USA Editor Series: Prof. John M. Walker - University Of Hertforshire, UK, 1998 (In press).

Haaland, P.D., Experimental Design in Biotechnology. Marcel Dekker, Inc., New York (1989).

Johansson, H.O., Karlstrom, T. F. and Haynes, C., J.Chromatogr.B 711: 3-17 (1998).

Muzarelli. R.A.A., Biochemical significance of exogenous chitins and chitosans in animals and patients. Carbohyd. Polym., 20: 7-16 (1993).

Pelletier, A. and Sigush, J., Purification and characterization of three chitosanase activities from Bacillus megaterium P1, Applied Environmental Microbiology, 5, 844 (1990).

Sedmark, J. J. and Grossberg, S. E., A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250, Analytical Biochemistry, 79, 544 (1977).

Somashekar, D. and Joseph, J., Chitosanases - Properties and Applications: A Review. In: "Bioresource Technology" Elsevier Science (1996).

Spiro, R.G., Analysis of sugars found in glycoproteins, Methods in Enzymology, 8, 7 (1966).

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Table 1: Fractional Experimental Design (2^5-1) for the Production of Chitosanase

Table 2: Experimental Design Type 2³ (no 2) for the Optimization of Chitosanase Activity at pH 5.8

Table 3: Purification of Chitosanase by Partitioning in ATPS

Publication Dates

• Publication in this collection
  15 Sept 1999
• Date of issue
  June 1999

History

• Accepted
  20 Apr 1999
• Received
  19 Jan 1999