Bioeconomic production of high-quality chitobiose from chitin food wastes using an in-house chitinase from Vibrio campbellii – Bioresources and Bioprocessing

Preparation of chitin

Chitin used in this study was obtained from three different sources: shrimp shells (Marine Bio Resources Co., Ltd., Thailand), crab shells (Practical grade, Sigma-Aldrich, Germany), and squid pens (Marine Bio Resources Co., Ltd., Thailand). Colloidal chitin was prepared by acid treatment using the protocol described previously (Murthy and Bleakley 2012). In brief, 20 grams of chitin flakes in a 1000-mL glass beaker were added into 150 mL of 12 M HCl, added slowly with continuous stirring, and then stirred at 25 °C overnight. The supernatant containing HCl was then discarded after centrifugation at 3,924 xg for 30—60 min at 4 °C and the chitin pellet was washed thoroughly with ice-cold distilled water (DI) until the pH of the chitin suspension was close to 7.0. The acid-treated chitin (at this stage called colloidal chitin) was air-dried in an oven at 60 °C, ground in a mortar and used as substrate for the preparation of chitobiose by VhChiA.

Expression and purification of VhChiA

The DNA fragment encoding full-length VhChiA was cloned into the pQE60 expression vector and expressed in E. coli M15 cells at a high level as described previously (Suginta et al. 2004). For recombinant expression, the transformed cells were grown at 37 °C in LB medium containing a final concentration of 100 μg.mL⁻¹ ampicillin and 50 μg.mL⁻¹ kanamycin until the OD₆₀₀ of the culture reached 0.6–0.8. The cell culture was then cooled on ice before chitinase expression was induced by the addition of isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.2 mM. Cell growth was continued at 25 °C for an additional 18 h, and the cell pellet was collected by centrifugation at 3,924x g at 4 °C for 30 min. The pellet was resuspended in freshly prepared lysis buffer (20 mM Tris HCl, pH 8.0, 150 mM NaCl, 1 mM PMSF and 1 mg.mL⁻¹ egg white lysozyme) and further lysed on ice using an ultrasonic processor (Cole-Parmer, Vernon Hills, Illinois, USA) with a 1-cm diameter probe (Amp: 30%, Pulse ON: 20 s, OFF: 40 s, Timer: 40 min). Unbroken cells and cell debris were removed by centrifugation at 16,773x g for 45 min at 4 °C. The supernatant was immediately applied to a Ni-charged Resin affinity column (GenScript USA, Inc. Piscataway, NJ08854, USA) and chromatography was carried out under gravity at 4 °C; after loading, the column was equilibrated with equilibration buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl) containing 20 mM imidazole. The bound proteins were eluted with 150 mM imidazole in equilibration buffer. Eluted fractions (5 mL) were collected, and each fraction was analyzed by 12% SDS-PAGE to confirm purity. Fractions containing chitinase were pooled and then subjected to multiple rounds of dialysis (Snake Skin™ Dialysis Tubing, 3.5 K MWCO, 35-mm dry I.D, Thermoscientific, Meridian Rd, Rockford, U.S.A) in equilibration buffer for the complete removal of imidazole. The purified protein was concentrated using an Amicon Ultra-15 centrifugal filter unit, 30 K-cut off (Merck Millipore, Tullagreen, Cork, Ireland) to 19 mg.mL⁻¹, then aliquots were stored at -80 °C until use.

Chitinase activity assay

Chitinase activity was determined by a colorimetric assay using pNP-(GlcNAc)₂ (Megazyme, Neogen, Ireland) as substrate. The assay was carried out in a 96-well microtiter plate, with a 100-μL reaction mixture containing various concentrations of pNP-(GlcNAc)₂ (0, 6.25, 12.5, 25, 50, 100, 250, 500 μM), the protein sample (0.2 μg.μL⁻¹) and 0.1 M sodium acetate buffer, pH 5.5. The mixture was incubated at 30 °C for 10 min with constant agitation, and the reaction terminated by the addition of 100 μL of 3 M sodium carbonate (Na₂CO₃). The amount of liberated pNP was determined spectrophotometrically at 405 nm in a microtiter plate reader (ThermoFisher scientific, Ratastie 2, Finland) and a standard curve was obtained with pNP solution (0 to 50 nmol). One unit (U) of the chitinase activity is defined as the amount of enzyme required for the liberation of 1 nmol of pNP per min. Chitinase activity assays were usually carried out in triplicate, unless otherwise stated.

Enzyme stability study

VhChiA was tested for stability at different reaction times. The reaction mixture (100 μL) in a microplate reader (ThermoFisher scientific, Ratastie 2, Finland), containing pNP-(GlcNAc)₂ (500 μM), VhChiA (10 U) and 0.1 M sodium acetate buffer, pH 5.5, was incubated at 30 °C with constant agitation. At each time point the reaction mixture was withdrawn and flash-frozen in liquid N₂. The chitinase activity was determined as described above using pNP-(GlcNAc)₂ as the substrate. The effect of bovine serum albumin (BSA) on the enzyme stability was also tested. Reactions were carried out in the absence and presence of 4 μg per well of BSA (ACROS, New Jersey, USA) and the residual chitinase activities under the two different conditions were examined at different times of reaction from 0 to 24 h.

Substrate specificity

The specificity of VhChiA was studied using different polysaccharide substrates including mannan, partially deacetylated chitin (chitosan), Avicel® microcrystalline cellulose and squid pen chitin. The time-courses of degradation of these polysaccharides were determined and the reaction products analyzed by thin layer chromatography (TLC) (Tanaka et al. 1999). The reaction mixture, consisting of 2 mg of each polysaccharide, 40 U (8 μg) of VhChiA and BSA (16 μg or 1:2 ratio of chitinase to BSA) in 2 mL of 0.1 M sodium acetate buffer, pH 5.5 was incubated at 30 °C with continuous agitation. For product analysis, the TLC silica plate (TLC silica gel 60 F254, aluminum sheets, Merck, Germany) was pre-heated at 60 °C prior to sample application to remove any absorbed moisture. Aliquots of the reaction mixture withdrawn at different incubation times from 0 min to 24 h were applied six times (1 μL each) to the silica plate and chromatographed four times (1 h each), followed by spraying with aniline–diphenylamine reagent and the plate was heated until visible spots were observed. The mobile phase used for TLC contained butanol:methanol:28% ammonia solution:water (10:8:4:2 v/v). The substrate specificity of VhChiA was also tested with different chitooligosaccharides (GlcNAc)_2–6 (a final concentration of 1.5 mM of each CHO, prepared in distilled water) using the protocol described above (data not shown).

A small-scale production of chitobiose and product analysis by TLC

Chitobiose production was carried out in two steps: first, pretreatment of crystalline chitin with HCl and second, enzymic hydrolysis. For small-scale production (2 mL), chitin from squid pens or shrimp or crab shells (5 mg of each) was incubated with 100 U (20 μg) of enzyme and 40 μg of BSA as a stabilizer in 0.1 M sodium acetate buffer at 30 °C and hydrolysis was carried out for 24 h. Aliquots of 120 μL were withdrawn after different time intervals (0, 2.5, 5.0, 10.0, 30.0 min, and 1.0, 16, 24 h) and the reaction was terminated by heating in a block at 98 °C for 5 min followed by centrifugation at 13,817 xg at 4 °C for 20 min. The degradation products were then analyzed by TLC, following the method described earlier.

Quantitative analysis by HPLC

Chitooligosaccharide products generated from chitin hydrolysis by VhChiA were determined with a gel-filtration column connected to an HPLC system. Each aliquot (20 μL) obtained from the reaction mixture at various reaction times was injected into a TSK Gel G2000 PW column (7.5 mm ∅ × 30 cm L) connected to a high-performance liquid chromatography (HPLC) system (Shimadzu, High Performance Liquid Chromatography Prominence – I LC – 2030 series, Nexera -I LC 2040 series) (Shimadzu Bara Scientific Co., LTD, Bangkok Thailand). Deionized water was used as the mobile phase, and the column was operated isocratically at 150 psi with a flow rate of 0.1 mL.min⁻¹ and temperature 25 ± 1 °C. The products were monitored by absorption at 200 nm (A₂₀₀) using a photodiode array detector (PDA). The data were extracted and plotted in GraphPad Prism v.5.01 and the concentrations of the hydrolytic products were calculated from the standard curves obtained with the mixture of CHOs, (GlcNAc)_1–4.

Hundred-milligram scale production of chitobiose

Larger scale production of chitobiose was carried out in an Erlenmeyer flask containing 1 g of dried shrimp colloidal chitin, 20,000 U (4 mg) of VhChiA and 8 mg of BSA in 0.1 M sodium acetate buffer, pH 5.5 in total volume of 1L.After incubation for 24 h at 30 °C, the reaction mixture was centrifuged at 2359 g for 40 min at 4 °C to remove the remaining chitin substrate from the reaction mixture and concentrated using a centrifugal concentrator (Amicon Ultra-15 centrifugal filter unit, 30 K-cut off (Merck Millipore, Tullagreen, Cork, Ireland) at 4129 xg for 30 min at 4 °C. The solution was then dried in a rotavapor (IKA RV 10 digital Rotary Evaporator line, IKA® Works (Thailand) Co. Ltd., Bangkok, Thailand) supported with an IKA MVP10 basic compact vacuum pump and Model K-015 chiller Circulator (heating temperature 40 °C, cooling temperature 2 °C, pressure 0.29 psi, rotation at 40 rpm). The completely dried products were dissolved in 10 mL deionized water and passed through a gel filtration column (2.1 cm × 160 cm) packed with cellulose beads (Cellufine, Lot No. R2407, JNC Corporation, Japan), and the eluted fractions were collected by gravity using a fraction collector (Bio Rad Model 21,100, USA). Fractions with ultraviolet absorption at 200 nm were analyzed by TLC and the fractions corresponding to chitobiose were then pooled and concentrated in a rotavapor, then freeze-dried. Chitobiose in powder form was stored at room temperature (25 ± 2 °C) in vacuo.

Purification and salt elimination of chitobiose by preparative HPLC

In the final step, salt was eliminated from chitobiose to obtain highly purified chitobiose. One gram of the chitobiose/salt powder obtained from Cellufine gel filtration column was dissolved in 3 mL of deionized water. Then, aliquots of 45 µL of the solubilized sample were injected multiple times into an Asahipak NH₂P-50 10E preparative column (10.0 mm × 250 mm, Shodex China Co., Ltd) connected to a Shimadzu HPLC system. The HPLC separation was conducted at a temperature of 25 ± 1 °C under a pressure of 2000 psi with a flow rate of 1.0 mL.min⁻¹ and elution by a gradient of acetonitrile:water (70:30 v/v) as the mobile phase. The separated products were detected at 200 nm using a PDA detector. Eluted fractions (6.0 mL each) corresponding to the chitobiose peak were pooled and concentrated in a Rotavapor followed by freeze-drying and the purified chitobiose powder was stored in vacuo at room temperature.

Mass identification of chitobiose by QTOF-MS

Chitobiose obtained from quantitative HPLC was further analyzed by quadrupole-time‐of‐flight‐mass spectrometry (QTOF-MS) (Bruker Biospin AG, Bangkok, Thailand). Chitobiose dissolved in water (2 mg.mL⁻¹, 100 μL) was injected into the instrument. A mass range of 50–1000 was selected for data acquisition. Positive ionization mode was chosen using source type Electrospray Ionization (Bruker Apollo II, Thailand). The capillary and charging voltage were set at 4500 V and 2000 V, respectively.