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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(6): 690-696                                     CN 31-1300/Q

Purification and Characterization of a Novel Chitinase from Bacillus brevis

LI Sheng, ZHAO Zhi-An, LI Ming, GU Zhen-Rong1, BAI Chen, HUANG Wei-Da*

( Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China;

1Shanghai Academy of Agricultural Sciences, Shanghai 201106, China )

Abstract    An extracellular chitinase secreted by Bacillus brevis was purified to homogeneity by a combination of ammonium sulfate precipitation, Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography. On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a mass of 85 kD even in the presence of b-mercaptoethanol, but shifted to 48 kD when heated in boiling water or treated with 8 mol/L urea at 50 for 10 min. The depolymerization of subunits was accompanied with the loss of chitinase activity, and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity. The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60 , and was most active at pH 8.0. The enzymatic activity was stable at pH 6-10, and inhibited by Ag+. Ten N-terminal amino acids were determined to be AVSNSKIIGY, demonstrating that the purified enzyme was a novel one. The hydrolysis pattern of the purified enzyme indicated that the chitinase was an endochitinase. The extraordinary thermo-stability and high resistance to proteolysis provide the enzyme with a good prospect to be used as a new tool for biocontrol.

Key words    endochitinase; Bacillus brevis; purification; dimer; disulfide bonds

Chitin (linear poly b-1,4-N-acetyl-D-glucosamine) is the second most abundant biopolymer on the earth[1] and can be found mainly in the cuticles of insects, shells of crustaceans, and cell walls of most fungi[2]. However, a wider range of organisms has the ability of producing chitinases (EC3.2.1.14), including those non-chitin-bearers such as bacteria, plant and vertebrates[3]. In fungi[4], invertebrates[5] partly composed of chitin chitinases are involved in morphogenesis, whereas in high plants 6] and vertebrates[7] chitinases function as defensive weapons against invasion of pathogens. Lots of pathogenic and parasitic microbes and invertebrates synthesize chitinases in order to aggress upon chitin-containing organisms[8]. In bacteria, chitinases are used mainly for their nutrition[9] and parasitism purpose[10]. Numerous chitinases from various origins constitute a super family and form a complex chitinolytic enzyme system, which is parallel to the cellulolytic enzyme complex[11]. To completely degrade chitin into free N-acetylglucosamine (GlcNAc), a synergistic and consecutive action of different types of chitinases and other enzymes is needed[12]. According to the characteristics of hydrolyzing chitin, the chitinases are classified into two types, exochitinase and endochitinase[13]. Endochitinases cleave randomly inside the chains of chitin and cut them into shorter segments[13]. Exochitinases (exo-N,N-diacetylchitobiohydrolase) or chitobiosidase, hydrolyze chitin from the terminal end and release chitobiose[13]. Another enzyme named N-acetylglucosaminidases (EC hydrolyzes short oligomers, typically chitobiose dimer units, and releases N-acetylglucosamine[13].

Recently chitin and chitinases are receiving more and more attention from biologists. A wide variety of medical applications of chitin and chitin derivatives have been reported over the last three decades[14]. N, N-diacetylchitobiose has been widely used as starting material for synthesis of biological active compounds[15,16]. Chitinases promise to be safer pesticides (than chemical ones) and microbial biocontrol agents due to the importance of chitinolytic enzymes in insect, nematode, and fungal growth and development[17]. Chitinase activity in human serum has recently been detected, and it may play a role in defending the invasion of fungal pathogens[18].

Bacteria, fungi, plants and insects are four major objects of chitinase research. In bacteria, Bacillus[19] and Streptomyces[20] are intensively studied for their high productivity of chitinases. Wiwat et al.[19]reported that Bacillus circulans WL-12 secreted chitinases into the culture medium, among which chitinase A1 showed strong affinity to chitin and played a major role in the hydrolysis of chitin. Chitinases have also been found in other Bacillus species including B.cereus, B.licheniformis and B.subtilis[21].

Bacillus brevis No.G1 is a newly isolated strain from soil in Shanghai of China for its high chitinase activity secreted in culture medium[22]. In this study we purified the extracellular chitinase to homogeneity from the fermented broth of B.brevis No.G1 and investigated its physico-chemical properties. With the determination of partial N-terminal amino acid sequence and its characteristics, we demonstrated that the purified enzyme was a novel endochitinase.

1  Materials and Methods

1.1  Bacterial strain and culture condition

Bacillus brevis No.G1 was isolated from soil in Shanghai of China as previously reported[22]. Cultures were maintained on nutrient agar slants and incubated at 30 for 72 h. The bacterial cells were then inoculated into a 500-ml Erlenmeyer flask containing 50 ml liquid medium, cultured at 30 for 48-72 h on a shaker until most spores broke off. The liquid medium for bacterial growth contained 20 g/L soybean powder, 4 g/L starch, 3 g/L peptone, 2 g/L yeast extract, 0.3 g/L KH2PO4, 0.2 g/L MgSO4, and 1 g/L CaCO3.

1.2  Chemicals

Phenyl-Sepharose CL-4B, DEAE-Sepharose Fast Flow, Sephadex G-150 were purchased from Pharmacia LKB (Uppsala Sweden). Purified chitin, chitosan and thin-layer chromatography (TLC) plates were purchased from Sigma. Other chemicals were of analytical grade.

1.3  Preparation of colloidal chitin  

Colloidal chitin was prepared from purified chitin according to the method of Roberts et al.[23] with minor modification. Ten grams of chitin powder were added slowly into 180 ml of HCl (37%, W/V) at 25 under vigorous stirring for 2 h. The suspension was poured into 1 liter of ice-cold 95% alcohol under vigorous stirring for 30 min, and stored at -20 until use. When in need, 10 ml of the suspension was centrifuged. The precipitate was washed with 50 ml of 0.1 mol/L sodium phosphate buffer (pH 7.0) for 3 times. The derived precipitate was dissolved in 90 ml 0.1 mol/L sodium phosphate buffer (pH 6.0), which was about 10 g/L colloidal chitin solution.

1.4  Purification of chitinase  

The fermented broth of B. brevis No.G1 was collected by brief centrifugation and the proteins fractionated with 50% saturation (NH4)2SO4 were collected by centrifugation at 8 000 g for 20 min. The protein precipitate was dissolved in 0.8 mol/L (NH4)2SO4 solution and the insoluble materials were removed by centrifugation at 15 000 g for 30 min. The derived supernatant was applied onto a Phenyl-Sepharose CL-4B column (f1.2 cm×10 cm) pre-equilibrated with 1 mol/L (NH4)2SO4. The column was washed with 1.5 bed volumes of 1 mol/L (NH4)2SO4, 2 bed volumes of 0.1 mol/L (NH4)2SO4, and then eluted with distilled water. The flow rate was maintained at 0.5 ml/min. The fractions with chitinase activity were pooled and dialyzed overnight at 4 against 10 mmol/L Tris-HCl buffer, pH 8.2. The dialysate was collected and immediately applied on a DEAE-Sepharose Fast Flow column (f1.2 cm×4 cm) pre-equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.2. The column was further washed with 1.5 bed volumes of 10 mmol/L Tris-HCl buffer (pH 8.2) and then developed with linear 0.04-0.14 mol/L NaCl gradient in 10 mmol/L Tris-HCl buffer, pH 8.2. The flow rate was maintained at 0.25 ml/min. The fractions with chitinase activity were pooled, concentrated and kept at -20 until use.

For N-terminal amino acid sequencing, the enzyme fraction was further purified by reverse-phase HPLC. Chitinase fractions derived from DEAE-Sepharose Fast Flow were loaded on a Zorbax 300SB-CN column (du Pont, f250 mm×4.6 mm I.D.), and the column was developed with acetonitrile gradient supplemented with 0.1% trifluoroacetic acid (TFA). The elution pattern was monitored by absorbance at 220 nm and 280 nm. The major peak was collected and lyophilized for automatic amino acid sequencing.

1.5  Enzymatic activity assay  

Two methods for enzymatic activity assay were used in this work. For rapidly tracing chitinase activity during the chromatographic separation processes, 100 ml of each fraction was mixed with 2 ml 10 g/L chitosan in 50 mmol/L acetate buffer (pH 5.0) and incubated at 50 for 10 min. The viscositic changes of the chitosan solutions were checked with naked eye to determine those fractions with chitinase enzyme activity. Quantitative assay of chitinase activity was carried out by colorimetric method described by Imoto et al.[24]. In a typical reaction, 50 ml enzyme solution, 0.05 ml 10 g/L colloidal chitin and 0.35 ml of 0.1 mol/L Tris-HCl buffer (pH 8.0) were mixed and incubated at 60 for 15 min. Reaction was terminated by heating in boiling water for 15 min, and mixed with 2.0 ml of 1.5 mmol/L potassium ferricyanide solution. The mixture was then heated in boiling water for another 15 min and cooled to room temperature. The supernatant was subjected to spectrophotometry measurement at 420 nm. The enzyme activity was calculated from a standard curve obtained with known concentration of GlcNAc. One unit of chitinase activity was defined as the amount of enzyme that liberated 1 mmol GlcNAc per min at pH 8.0 and 60 . Negative control tubes contained all components except substrate, and blanks contained all components except the enzyme.

1.6  Electrophoresis

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method described by Sambrook et al.[25]. The proteins were stained with Coomassie brilliant blue R-250. For isoelectric focusing (IEF) experiment, about 5 mg of sample proteins in 20 ml solution were loaded to a precasted capillary gel with 0.75% Ampholine (pH range 3.5-10)[26], and run under 200 V for 5 h.  Amyloglucosidase (pI 3.6), trypsin inhibitor (pI 4.6), b-lactoglubin A (pI 5.1), conalbumin (pI 6.0), myoglobin (pI 6.8, 7.2), lentil lectin (pI 8.2, 8.6, 8.8), and trypsinogen (pI 9.3) were used as markers.

1.7  Zymogram

To identify the protein of chitinase, zymographic approach was applied on samples derived from DEAE Fast Flow chromatography. Samples were separated on 10% polyacrylamide gel electrophoresis at pH 8.3. As soon as the electrophoresis was finished, the gel was immediately placed on an agarose slab gel containing 10 g/L colloidal chitin. After incubation for 1.5 h at 37 , a transparent band could be seen on the agarose slab. The sections of the polyacrylamide gel overlapping with the transparent band were carefully cut out and pestled with sample buffer in an Eppendorf tube. The derived paste was analyzed on 10% SDS-PAGE.

1.8  Gel filtration chromatography  

Gel filtration chromatography was used for determination of molecular weight of chitinase. Sephadex G-150 was packaged in a 1.2 cm ×60 cm column and pre-equilibrated with 10 mmol/L Tris-HCl buffer (pH 8.2) at a flow rate of 0.15 ml/min. About 0.4 ml concentrated enzyme obtained from the DEAE Fast Flow column was applied to the column and eluted with the same buffer. Protein profile was monitored at 280 nm. The molecular weight was estimated from a standard curve obtained from the proteins with their molecular weights known.

1.9  Determination of protein concentration

Protein concentrations were determined by Folin phenol method[27], with bovine serum albumin as the reference.

1.10  Silica thin layer chromatography

Silica thin layer chromatography (TLC) was performed according to the method described by Xia et al.[28]. Five ml of chitosan samples treated with HCl or digested with purified chitinase, were applied on TLC plate, and the plate was developed by n-butanol acetic acid water (2 1 1). For chitinase digestion, 1 ml of 1% chitosan in 50 mmol/L acetate buffer (pH 5.0) was mixed with 6 mg purified enzyme and incubated at 60 for 1 h, or at 37 for 24 h. For HCl treatment, 10 g/L chitosan in 4 mol/L HCl was prepared and heated at 90 for 2 h. GlcNAc and chitotetraose were used as markers for TLC.

1.11  N-terminal amino acid sequencing

Samples obtained from reverse-phase HPLC were lyophilized and subjected to N-terminal amino acid sequencing on an automatic protein sequencer (Model 473A, Applied Biosystems Inc, USA)

2  Results and Discussion

2.1  Purification of chitinase

B.brevis No.G1 was originally isolated from soil of Shanghai urban for its high chitinase activity secreted into the culture media[22]. We purified a chitinase secreted by this new strain of B.brevis for further study. Proteins in the fermented broth was recovered with (NH4)2SO4 precipitation at 50% saturation. The protein precipitates were dissolved in 0.8 mol/L (NH4)2SO4 solution and separated by Phenyl-Sepharose CL-4B hydrophobic interaction chromatography. In a typical separation, 30 ml of the sample solution containing 68.4 mg of crude proteins was applied to a 1.2 cm×10 cm column, and developed as described in Materials and Methods. Four protein peaks were detected at 280 nm as shown in Fig.1 and Fig.2. Chitinase activity was only found in the last peak eluted with distilled H2O.

Fig.1  Elution profile of chitinase on Phenyl-Sepharose CL-4B column

The protein solution was eluted stepwise with 0.1 mol/L (NH4)2SO4 and distilled water. Absorbance at 280 nm () and relative enzymatic activity () were determined.

Fig.2  Elution profile of chitinase on DEAE-Sepharose Fast Flow column choromatography

The protein solution was eluted with a linear gradient from 0.04 mol/L NaCl to 0.14 mol/L NaCl in 10 mmol/L Tris-HCl (pH 8.2). Absorbance at 280 nm () and relative enzymatic activity () were measured.

The fractions with chitinase activity were pooled and dialyzed against 10 mmol/L Tris-HCl buffer (pH 8.2) overnight. The dialysate containing 23.46 mg proteins was then applied onto a DEAE-Sepharose Fast Flow column for anion-exchange chromatography described as Materials and Methods. The chitinase activity was detected in the third peak as shown in Fig.2. When this peak was analyzed on 10% SDS-PAGE, a protein band with molecular weight of 85 kD (Fig.3) was shown. Meanwhile, zymographic approach was applied to identify the protein with chitinase activity. The protein recovered from the gel slice overlapping the transparent band on the agarose gel containing 10 g/L colloidal chitin, showed a single band on 10% SDS-PAGE with a mass of 85 kD (Fig.3). Therefore, the chitinase secreted by B. brevis No.G1 was identified to be the protein in the peak 3 derived from DEAE anion-exchange chromatography. Table 1 is the summary of purification.

Fig.3  SDS-PAGE analysis of chitinase under various conditions

15, protein markers (I: BSA, bovine serum albumin, 68 kD; II: ovalbumin, 45 kD; III: carbonic anhydrase, 31 kD); 2, chitinase treated with 2-mercaptoethanol; 3, chitinase treated with 0.1% TFA; 4, chitinase heated at 55 in 8 mol/L urea for 30 min; 6, chitinase recovered from the PAGE gel slice overlapping the transparent band on zymogram; 7, chitinase derived from DEAE chromatography.

2.2  N-terminal amino acid sequence

For N-terminal sequencing, the enzyme fractions from DEAE-Sepharose Fast Flow were further purified by reverse-phase HPLC. As shown in Fig.4, only one protein peak was detected. The protein peak was collected, lyophilized and subjected to amino acid sequencing. The first 10 amino acids in the N-terminal sequence were determined to be AVSNSKIIGY. The sequence was blasted against GenBank, however, no chitinase known showed significant similarity with this sequence. The 10 N-terminal amino acids of chitinase A1 from Bacillus circulans No.4.1 is APWNSKGNYA[19], which was the most close to the N-terminal amino acid sequence of the B.brevis No.G1 chitinase.

Fig.4  Elution profile of chitinase on reverse-phase HPLC

The CN column was developed by a linear gradient of acetonitrile from 35 % to 50% at the presence of 0.085% TFA.

2.3  Characteristics of the purified chitinase

The protein in the HPLC peak showed a mass of 48 kD on SDS-PAGE and no enzyme activity. The difference between molecular weights of proteins in DEAE-Sepharose Fast Flow fractions and HPLC fractions implied a dimer structure of chitinase. We performed a series of experiments or further characterization of this protein. On gel filtration chromatography, the molecular weight of chitinase was determined to be around 81 kD (Fig.5). The molecular weight of chitinase protein on SDS-PAGE differed depending on conditions. If the chitinase in DEAE fractions was heated in boiling sample buffer before SDS-PAGE analysis, the protein band on SDS-PAGE was at the position of 48 kD; and if not heated, at the position of 85 kD. After treatment of  the enzyme with 4% 2-mercaptoethanol, the molecular weight of the purified enzyme was still 85 kD on SDS-PAGE, implying that disulfide bond is not involved in the formation of chitinase dimers. Thus it is a reasonable conclusion that the chitinase dimer is most likely formed by hydrophobic interaction. Incubating the chitinase with 8 mol/L urea at 50 for 30 min could cause a total loss of enzymatic activity and a shift of the protein band position from 85 kD to 48 kD on SDS-PAGE (Fig.3). After dialysis of the enzymes depolymerized by 8 mol/L urea, by heated at 100 or by 0.1% TFA treatment against 10 mmol/L Tris-HCl buffer (pH 8.2), the enzymatic activity recovered by 79%, 75% and 87%, respectively; and the dimer was found to be the major component revealed by SDS-PAGE (data not shown). These results strongly suggest that the chitinase has a homodimer structure based on hydrophobic interaction. After incubating the chitinase with 8 mol/L urea and then dialyzing it against 40% alcohol, we obtained free chitinase subunits, which utterly lost the activity (the chitinase in 40% alcohol still exhibited hydrolytic activity). It was thus concluded that the compact structure of the dimer is necessary for chitinase activity.

Fig.5  Determination of molecular weight by gel filtration

A Sephadex G-150 column (1.2 cm×60 cm) equilibrated with 10 mmol/L Tris-HCl buffer (pH 8.2) was used for determination of the molecular weight of the purified enzyme. Conalbumin (86 kD), bovine serum albumin (68 kD) and ovalbumin (45 kD) were used as molecular weight markers. The arrow indicated the position where purified chitinase was eluted.

Up to now, no chitinase with dimer structure from bacteria has been reported. In other species only an insect a-amylase inhibitor/endochitinase from plant origin was shown to have a structure of dimer. This fact, as well as the result of the N-terminal amino acid sequence, makes us conclude that the chitinase produced by Bacillus brevis No.G1 is a novel chitinase with an unusual structure.

The purified chitinase was subjected to isoelectric focusing analysis and the pI of the chitinase was found to be 5.5 (data not shown). The optimal conditions  for enzymatic reaction were studied systemically. 6 mg purified chitinase was used to determine its characteristics. The optimal pH of the chitinase was 8.0 (Fig.6), which was similar to that of the chitinases from B.circulans No.4.1[19] and Alteromanas sp.strain O-7[29]. The chitinase was stable and could hydrolyze colloidal chitin at a wide pH range (from pH 6.0 to pH 10.0). The chitinase exhibited the highest activity at 60 and retained high activity even over 80 (Fig.7), but in the absence of substrate the chitinase lost its activity markedly above 60 (Fig.8), inferring that the substrate could protect the active center of the chitinase from denaturation.

Fig.6  Effect of pH on enzymatic activity of the purified chitinase

The chitinase activity was determined at different pH at 50 . Buffers used were 0.1 mol/L sodium acetate buffer (pH 3.0, 4.0, 5.0), 0.1 mol/L sodium phosphate buffer (pH 6.0, 7.0), 0.1 mol/L Tris-HCl buffer (pH 8.0), 0.1 mol/L glycine-NaOH buffer (pH 9.0, 10.0), 0.1 mol/L Na2HPO4-NaOH buffer (pH 11.0, 12.0).

Fig.7  Effect of temperature on enzymatic activity of the purified chitinase

The enzymatic activity of chitinase was measured at different temperatures in 0.1 mol/L Tris-HCl buffer (pH 8.0).

Fig.8  Effect of temperature on stability of the purified chitinase

The remained enzymatic activities after incubation at different temperatures in 0.1 mol/L Tris-HCl buffer (pH 8.0) were measured.

The Bacillus brevis No.G1 chitinase could be inactivated by Ag+ ion. After incubation with 1 mmol/L Ag+ at pH 8.0 and 30 for 30 min, only 60% of the enzyme activity remained.

2.4  Hydrolysis pattern of the purified chitinase

To clarify the action mode of the chitinase,viscositic changes of 10 g/L chitosan solution (in 10mmol/L acetate buffer, pH 5.0) caused by enzymatic hydrolysis were studied. It was found that the viscosity was promptly reduced in 5 minutes due to cleavage of chitosan long chains by the chitinase at 60 (Fig.9). Thus it was concluded that the purified chitinase had endo-splitting activity.

Fig.9  Hydrolysis of chitosan by the purified chitinase

Purified chitinase (60 mg) was added to 20 ml 10 g/L chitosan in 50 mmol/L acetate buffer (pH 5.0) and the mixture was incubated at 60 for digestion. Aliquots of 2 ml solution were removed at intervals and subjected immediately to viscosity measurement on an Ostwald viscometer.

After incubation of the enzyme with 1% colloidal chitin solution at 60 for 1 h (sample 3), and at 37 for 24 h (sample 4), 5 ml of the hydrolytic products were analyzed with silica TLC. An acid hydrolytic product (10 g/L chitosan in 4 mol/L HCl, 90 , 2 h) was used as the reference (Fig.10). We found that the final products of enzymatic hydrolysis were mainly oligomors much larger than chitotetroase. Compared with the hydrolytic results of the exochitinase from Aspergillus fumigatus YJ-407[28], the final product of which was a mixture of chitobiose, chitotriose and GlcNAc, it was concluded that the chitinase from B.brevis No.G1 had no exo-splitting activity and was an endochitinase simply.

Fig.10  TLC analysis of chitosan hydrolysis

1, mixture of 4 mg GlcNAc and 4 mg chitotetraose; 2, 10 g/L chitosan in 4 mol/L HCl at 90 for 2 h; 3, 10 g/L chitosan hydrolyzed by 6 mg of purified chitinase at 60 for 1 h; 4, 1% chitosan hydrolyzed by 6 mg of purified chitinase at 37 for 24 h.

2.5  Prospect in the field of biocontrol

The B.brevis  No.G1 chitinase was highly stable (retaining higher than 80% activity) in a wide range of pH (pH 6.0 to 10.0) and temperature (from 35 to 72 ). In comparison, the chitinase from B.circulans No.4.1 (from 25 to 50 )[19] and Aeromonas sp. 10S-24 (from 30 to 50 )[30] exhibit their enzymatic activities in a more narrow temperature range and are less stable. Furthermore, the purified chitinase from B.brevis No.G1 was strongly resistant to the hydrolysis by trypsin. A common condition (4 mol/L urea at 25 ) was not sufficient for trypsin digestion of the chitinase. The chitinase in fermented broth could be kept at 4 for at least two months without loss of enzymatic activity. The crude fermented broth of the B.brevis No.G1 has already been applied to vegetables against mold diseases with remarkable efficacy (to be published). Therefore, we expect that the B.brevis No.G1 chitinase could be widely applied as a new tool of biocontrol.

Acknowledgements    The research work was accomplished in the laboratory of biochemistry and molecular biology of Fudan University, Shanghai, China. The authors are grateful to Dr.WANG Wei-Rong, Dr.JIANG Pei-Hong and Dr.QIAN Zhi-Kang for their generous help.


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ReceivedMay 17, 2002    AcceptedJune 12, 2002

This work supported by a grant from Shanghai Hengda Scien. & Tech. Dev. Co., Ltd.

*Corresponding author:Tel/Fax,86-21-55522773; e-mail, [email protected]