Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Abstract

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.

FIG. 1

Expression and purification of ChBD_ChiA1. ChBD_ChiA1 produced in E. coli BL21 cells was purified by chitin affinity column chromatography from the soluble protein fraction of disrupted cells. Lane 1, soluble protein fraction of E. coli BL21(DE3) cells carrying pET3a (control); lane 2, soluble protein fraction of induced E. coli BL21(DE3) cells carrying pChBD; lane 3, proteins obtained by ammonium sulfate precipitation from the soluble protein fraction; lane 4, flowthrough fraction of chitin affinity column chromatography; lane 5, proteins eluted from the chitin affinity column with 20 mM sodium acetate buffer (pH 5.5); lane 6, purified ChBD_ChiA1 obtained by elution with 20 mM acetic acid; lane MW, molecular mass standards. The arrowhead indicates the position of the ChBD_ChiA1 protein band.

FIG. 2

Double-reciprocal plots of binding data for chitinase A1 (a), A1ΔChBD (b), and ChBD_ChiA1 (c). The binding assay mixture contained 1 mg (dry weight) of regenerated chitin and from 1 to 70 μg of each protein (14 to 1,000 pmol of chitinase A1, 15 to 1,050 pmol of A1ΔChBD, and 154 to 10,770 pmol of ChBD_ChiA1) in 1 ml of 20 mM sodium phosphate buffer (pH 6.0). The binding assay was performed by keeping the binding assay mixtures on ice for 1 h for chitinase A1 and A1ΔChBD and 3 h for ChBD_ChiA1. [B], bound protein concentration; [F], free protein concentration.

FIG. 2

Double-reciprocal plots of binding data for chitinase A1 (a), A1ΔChBD (b), and ChBD_ChiA1 (c). The binding assay mixture contained 1 mg (dry weight) of regenerated chitin and from 1 to 70 μg of each protein (14 to 1,000 pmol of chitinase A1, 15 to 1,050 pmol of A1ΔChBD, and 154 to 10,770 pmol of ChBD_ChiA1) in 1 ml of 20 mM sodium phosphate buffer (pH 6.0). The binding assay was performed by keeping the binding assay mixtures on ice for 1 h for chitinase A1 and A1ΔChBD and 3 h for ChBD_ChiA1. [B], bound protein concentration; [F], free protein concentration.

FIG. 2

Double-reciprocal plots of binding data for chitinase A1 (a), A1ΔChBD (b), and ChBD_ChiA1 (c). The binding assay mixture contained 1 mg (dry weight) of regenerated chitin and from 1 to 70 μg of each protein (14 to 1,000 pmol of chitinase A1, 15 to 1,050 pmol of A1ΔChBD, and 154 to 10,770 pmol of ChBD_ChiA1) in 1 ml of 20 mM sodium phosphate buffer (pH 6.0). The binding assay was performed by keeping the binding assay mixtures on ice for 1 h for chitinase A1 and A1ΔChBD and 3 h for ChBD_ChiA1. [B], bound protein concentration; [F], free protein concentration.

FIG. 3

Effect of pH on the binding of chitinase A1 and ChBD_ChiA1 to regenerated chitin. Assay mixtures contained 1 mg (dry weight) of regenerated chitin and 1 nmol of either chitinase A1 (70 μg) or ChBD_ChiA1 (6.5 μg) in 1 ml of buffers with various pHs. The buffers used in this experiment were 20 mM sodium citrate (pH 2.0 to 6.0), sodium phosphate (pH 7.0), Tris-HCl (pH 8.0), glycine-NaOH (pH 9.0 and 10.0), and disodium hydrogen phosphate-NaOH (pH 11 and 12). Assay mixtures were incubated on ice for 1 h for chitinase A1 and 3 h for ChBD_ChiA1. ●, chitinase A1; ×, ChBD_ChiA1.

FIG. 4

Time courses of binding of ChBD_ChiA1 at pH 9.0 and in the presence of NaCl. Assay mixtures contained 6 mg (dry weight) of regenerated chitin and 2.3 nmol (15 μg) of ChBD_ChiA1 in 1 ml of 20 mM sodium phosphate buffer (pH 6.0) (□), 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl (■), or 20 mM Tris-HCl buffer (pH 9.0) (+).

FIG. 5

Binding specificities of chitinase A1, A1ΔChBD, and ChBD_ChiA1. Binding assay mixtures contained 1 mg (dry weight) of various insoluble polysaccharides and 25 μg of protein in 1 ml of 20 mM Tris-HCl (pH 9.0). Assay mixtures were kept on ice for 24 h for binding.

FIG. 6

Hydrolysis of β-chitin microfibrils by intact chitinase A1 and A1ΔChBD. Reaction mixtures contained 200 μg (dry weight) of microcrystalline β-chitin from L. satsuma and 71 pmol of each chitinase. Reactions were performed at 37°C, and the amount of reducing sugar generated was monitored by the modified Schales procedure. ●, chitinase A1; ×, A1ΔChBD.

FIG. 7

Bright-field diffraction contrast micrographs of L. satsuma β-chitin microfibrils. (a) no enzyme treatment (control); (b) after treatment with chitinase A1; (c) after treatment with A1ΔChBD.

FIG. 8

Alignment of amino acid sequences that have similarity to ChBD of chitinase A1. Rows: 1, ChBD of B. circulans WL-12 chitinase A1; 2, N-terminal domain (ChBD) of B. circulans WL-12 chitinase D1; 3, C-terminal region of S. griseus proteinase C; 4, C-terminal region of Serratia marcescens 2170 chitinase CI; 5, C-terminal region of Aeromonas sp. strain 10S chitinase II; 6, C-terminal region of Janthinobacterium lividum chitinase 69a; 7 to 9, Bacillus sp. (strain N-4) cellulase A, cellulase B1, and cellulase B, respectively; 10, Alteromonas sp. (strain O-7) chitinase 85; 11-12, Aeromonas caviae chitinase A; 13, Vibrio harveyi chitinase A; 14 to 18, Aeromonas sp. strain 10S ORF1, chitinase II, ORF3, ORF4, and ORF2, respectively; 19, J. lividum chitinase 69b; 20 to 24, repeating units found in E. coli hypothetical 97.1-kDa protein YheB; 25, S. griseus chitinase C; 26, S. marcescens 2170 chitinase B; 27, E. chrysanthemi endglucasase Z. Highly conserved amino acid sequences in the upper and lower groups are indicated by a black background. The starting and ending amino acid positions, based on the translational start methionine being residue 1, are shown on both sides of each protein segment. The CLUSTAL X program was used to make the alignment.