Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides

Abstract

Humans have exploited natural resources for a variety of applications. Chitin and its derivative chitin oligosaccharides (CHOS) have potential biomedical and agricultural applications. Availability of CHOS with the desired length has been a major limitation in the optimum use of such natural resources. Here, we report a single domain hyper-transglycosylating chitinase, which generates longer CHOS, from Enterobacter cloacae subsp. cloacae 13047 (EcChi1). EcChi1 was optimally active at pH 5.0 and 40 °C with a K_m of 15.2 mg ml^-1, and k _cat/K_m of 0.011× 10² mg^-1 ml min^-1 on colloidal chitin. The profile of the hydrolytic products, major product being chitobiose, released from CHOS indicated that EcChi1 was an endo-acting enzyme. Transglycosylation (TG) by EcChi1 on trimeric to hexameric CHOS resulted in the formation of longer CHOS for a prolonged duration. EcChi1 showed both chitobiase and TG activities, in addition to hydrolytic activity. The TG by EcChi1 was dependent, to some extent, on the length of the CHOS substrate and concentration of the enzyme. Homology modeling and docking with CHOS suggested that EcChi1 has a deep substrate-binding groove lined with aromatic amino acids, which is a characteristic feature of a processive enzyme.

Figure 1

pH, temperature optima, kinetic parameters and activity on different insoluble substrates of EcChi1. The pH and temperature optima were determined by incubating 30 mg/ml colloidal chitin and 30 μg of EcChi1 for 1 h at 40 °C in various buffers at different pH values, as follows: (A) 50 mM glycine-HCl (pH 2.0, ▼), 50 mM sodium citrate (pH 3.0–6.0, ■), 50 mM sodium acetate (pH 4.0–6.0, ▲), 50 mM sodium phosphate (pH 6.0–8.00, ●), 50 mM Tris-HCl (pH 8.0–9.0, ♦) and 50 mM glycine-NaOH (pH 9.0–10.0, ○) and at 10 °C to 80 °C (B), Different concentrations of colloidal chitin (2.5–60 mg/ml) and 30 μg of EcChi1 in 50 mM sodium citrate buffer, pH 5.0. The average of the triplicate data was fitted to the Michaelis-Menten equation by a nonlinear regression function of graphpad prism version 6.0 (C), The reaction mixture containing 30 μg EcChi1 and 1 mg ml⁻¹ of one of the polymeric substrates, was incubated at pH 5.0 and 40 °C for 1 h. The reaction mixture was centrifuged, and products were quantified using a reducing group assay (D). The error bars represent the standard deviations from three individual experiments.

Figure 2

Binding of EcChi1 with the soluble polymeric substrates. Affinity non-denaturating electrophoresis was performed in 8% native PAGE gels by preparing with or without substrate. (A) Proteins (EcChi1 or non-interacting BSA) without substrate, while (B) with 0.1% (wt/vol) substrate glycol chitin, (C) CM-cellulose and (D) laminarin incorporated in gels. Proteins were visualized by Coomassie blue G-250 staining after electrophoresis. Lane 1. BSA, Lane 2. EcChi1.

Figure 3

Hydrolysis of colloidal chitin by EcChi1. The EcChi1 (0.8 μM) was incubated with 30 mg/ml of the substrate in 900 μL reaction mixture for different time periods from 0 to 720 min at 40 °C. The reaction products were analyzed by binary gradient HPLC. (A) The topmost profile shows a standard mixture of CHOS ranging from DP1 to DP6. The remaining profiles are the reactions at different incubation times. Control represents the substrate without EcChi1 while standard represents CHOS ranging from DP1 to DP6. (B) Overview of the quantifiable CHOS products generated during hydrolysis. Products were quantified from respective peak areas using standard calibration curves of CHOS ranging from DP1 to DP4.

Figure 4

Time course hydrolysis and TG catalyzed by EcChi1 with DP3, DP4. The reaction mixture was incubated with 200 nM of EcChi1 and 1 mM of DP3/DP4 for different time periods from 0 to 720 min at 40 °C. Products were analyzed by binary gradient HPLC. (A and B) HPLC profiles of reaction products from DP3 (A) and DP4 (B) substrates. The topmost profile shows a standard mixture of CHOS ranging from DP1 to DP6 (A and B). The other profiles show the reaction products from DP3 and DP4 substrates at the indicated incubation times. The inset shows a magnified view of the low-peak-area products. Control represents the substrate without EcChi1. Overview of concentrations of CHOS products generated during reaction time courses with DP3 (C) and DP4 (D) substrates. Products were quantified from respective peak areas using standard calibration curves of CHOS ranging from DP1 to DP6.

Figure 5

Time course hydrolysis and TG catalyzed by EcChi1 with DP5 and DP6. The reaction mixture products were analyzed by binary gradient HPLC. (A and B) HPLC profiles of reaction products from DP5 (A) and DP6 (B) substrates. The topmost profile shows a standard mixture of CHOS ranging from DP1 to DP6 (A and B). The other profiles show the reaction products from DP5 and DP6 substrates at the indicated incubation times. The inset shows a magnified view of the low-peak-area products. Control represents the substrate without EcChi1. (C and D) Overview of concentrations of CHOS products generated during reaction time courses with DP5 (C) and DP6 (D) substrates. Products were quantified from respective peak areas using standard calibration curves of CHOS ranging from DP1 to DP6.

Figure 6

MALDI-TOF MS analysis of CHOS/TG products with DP5 and DP6 substrates catalyzed by EcChi1. The each 40 μL of reaction sample was concentrated under reduced pressure at 25 °C and dissolved in 10 μL of MilliQ H₂O. The 2 μL of the reaction mixture was mixed with equal volume of 2,5-dihydroxybenzoic acid (2,5-DHB), and the resultant solution was subjected to mass measurements using an Ultraflex MALDI – TOF/TOF instrument. The masses shown here are with Na adducts.

Figure 7

Substrate binding cleft of modelled EcChi1. (A) Aromatic residues lining along the substrate binding cleft (protein in surface representation and aminoacids in green and red sticks) and aminoacids interacting with chitin tetramer labeled in red colour. (B) Docked conformation of chitin tetramer (white sticks) and its possible interactions with residues (green lines) along with catalytic residue, Glu153 (yellow line) of EcChi1. (C) Deep active site cleft of EcChi1 showing catalytic (yellow colour) and aromatic (red colour) amino acids.