Characterization of thermostable chitinase from Bacillus licheniformis B2

Chitinases is an enzyme capable of degrading chitin into oligomers to produce chitin derivatives products which are more useful. Thermostable-chitinase is of important in the relevant industrial application, since the degradation process oftently requires prety high temperature. This research report a characterization of chitinase isolated from thermophilic microorganism. The chitinase was obtained from Bacillus licheniformis B2 isolated from Ijen hot spring, East Java. It has the best chitinolytic activity at pH 7 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 50 °C to 70 °C, optimally at 55 °C. It was stable at 50 °C up to 90 min, at 60 °C up to 60 min and at 70 °C up to 30 min. At neutral pH this enzyme has negative charge but further purification is needed to determine its pI. The Km and Vmax of this chitinase for colloidal chitin were 101.96 mg mL −1 and 2.72 μmol (min mL)−1, respectively. Addition of NaCl, KNO3 and MgSO4 decreased the activity of chitinase following mixed inhibitor mode. This enzyme should be a good candidate for applications in the recycling of chitin waste.


Introduction
Chitinase (EC 3.2.1.14) is a group of enzyme capable of degrade chitin to low-molecular-weight products. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shellfish wastes that not only solves environmental problems but also promotes the economic value of marine products [1]. Chitinase can be isolated from many kind of organism, such as fungi, bacteria [2], plant and animal. Thermostable-chitinase usually produce by thermophilic microorganism at geothermal environment [3] for instance volcano area and hot springs.
Enzyme work optimally in specific condition, such as temperature, pH, substrate concentration and the presence of inhibitor and activator [4]. Those aspect affect three dimension comformation and polypeptide folding of enzyme. Ion addition to enzymatic reaction can enhance enzyme activity and work as activator or inhibit enzyme and work as inhibitor [5]. In this experiment, NaCl, KNO 3 and MgSO 4 used because of its soluble property in phosphate buffer. Many study has been done to characterize thermostable chitinase produced by microorganism but the role of each ion in enzyme activity varies depending on the type chitinase produced and the producing bacteria species.
Previously, Bacillus licheniformis B2 that produced a particular high activity of thermostable chitinase was obtained when cultured in the medium containing colloidal chitin as the sole carbon

Production of chitinase
Colony of B. licheniformis B2 inoculated to thermus broth without chitin and incubated at 50 °C with 180 rpm shaking for 12 h or until maximum density. Afterwards, to make starter add 7.5 mL of culure to 67.5 mL of thermus broth media and incubated for 16 h at 50 °C with 180 rpm shaking (1 rpm = 1/60 Hz). Total of 75 mL starter then added to fermentor with 675 mL thermus broth media. The production of chitinase is carried out with following condition, temperature 50 °C, pH 5, 200 rpm agitation and 2 L min -1 aeration. After incubation, all media at fermentor centrifuged at 11 000 rpm for 5 min to separate chitin and cell debris (pellet) with chitinase crude extract (supernatant).

Enzyme assay
Chitinase activity was assayed in a 0.5 mL reaction mixture containing 0.1 g mL -1 colloidal chitin in 0.02 M phosphate buffer, pH 7, and 0.5 mL of enzyme solution. After incubation at 55 °C for 90 min, the reaction was stopped by centrifugation. For every 10 min, the tube was shaken to maximize enzyme-substrate contact. Reducing sugar produced was measured by Nelson-Somogyi method. Chitinase activity was defined using equation (1) Notes: GlcNAc released per mL = GlcNAc mass per mL (μg mL -1 ) Mr GlcNac = 221.21 (g mol -1 )

Optimum temperature and pH
The optimum temperature and pH were measured using colloidal chitin as a substrate. The enzyme activity was assayed for 90 min at temperatures 40 °C to 65 °C and pH 4.0 to 8.0 using 0.02 M phosphate buffer.

Thermal stability
The thermal stability was investigated by incubating the enzyme for 2 h at temperatures 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C in phosphate buffer pH 7.0. Aliquots were taken every 30 min, and residual activity of the enzyme was determined under standard assay condition.

pI determination
To determine enzyme's pI, buffer pH 4 to 9 was used with addition of five drops 0.1 % CTAB (cationic wetting agent) for each mL of buffer. Buffer with CTAB was then added to 0.2 mL of enzyme with different pH at separate tube then turbidity of each tube was observed visualy.

Ion exchange chromatography
Column resin was prepared corresponding to each protocol, i.e. bio-rex resin for cation exchange chromatography and DEAE cellulose resin for anion exchange chromatography. Resin was to column and flow rate was set to 1 mL min -1 . The amount of 2 mL enzyme was loaded then eluted using phosphate buffer pH 7 with gradient concentration of NaCl. Protein concentration was determined using Bradford method in every fraction obtained from chromatography.

Molecular weight determination
Separating gel (12.5 %) was prepared with the composition as follows: 3.125 mL acrylamide 30 % (29.2 g acrylamide and 0.8 g bis-acrylamide in 100 mL aquades); 2.75 mL Tris-HCl 1 M pH 8.8; 1.505 mL aquadest; 75 µL SDS 10 %; 75 µL APS 10 % and 6.25 µL TEMED. Stacking gel was prepared on top of separating gel with the composition as follows: 0.45 mL bis-acrylamide 30 %; 0.38 mL Tris-HCl 1 M pH 6.8; 2.11 mL aquabidest; 30 µL SDS 10 %; 5 µL TEMED and 30 µL APS 10 %. Sample was prepared by adding sample buffer and boiled for 5 min. Total of 10 µL to 20 µL of sample was then loaded into the well. After electrophoresis run for 20 min to 30 min with 110 mA, the gel was stained with staining solution (1 g Coomassie Brilliant Blue G-250, 450 mL methanol, 450 mL aquadest and 100 mL acetic acid) and then destained with destaining solution (100 mL methanol, 100 mL acetic acid and 800 mL aquadest). Protein bands shown from sample were then compared to protein marker to determine enzyme molecular weight.

Effect of salt addition
To determine effect of salt addition, following variation was added to enzyme assay: 0.47 mM and 1.3 mM NaCl, 0.47 mM and 1.3 mM KNO 3 , 0.47 mM and 0.238 mM MgSO 4 . Enzyme activity was tested in 7 different substrate concentration (2.5 mg mL -1 to 17.5 mg mL -1 colloidal chitin) and compared to the activity without any salt addition.

Temperature and pH characterization
The enzyme showed optimal activity at pH 7 (figure 1). At pH 7, the enzyme had the highest chitinolytic activity at 55 °C (figure 2). The half-life at 50 °C was about 90 min, whereas at 60 °C and 70 °C, no significant activity lost was observed within 60 min and 30 min, respectively (figure 3).

Protein charge determination
The result of pI determination using CTAB can be observed at figure 4. All solution gave almost same turbidity in pH 4 to pH 10 and enzyme pI can't be determined. As shown at the graph (figure 5 and figure 6), enzyme can't attach to column with negative charge and can be detect at early fraction beside at column with positive charge, high concentration of salt is needed to unattached enzyme from column so enzyme present in later fraction.

Molecular weight determination
Enzyme in crude extract visualized using SDS PAGE method with CBBG R250 staining. Visualization done to determine enzyme molecular weight. As shown in figure 7, in crude extract from B. licheniformis there were several protein bands shown. Proteins contained in crude extract sized between 50 kDa and 75 kDa, between 25 kDa and 35 kDa, between 15 kDa and 25 kDa, and also between 10 kDa and 15 kDa. It remains unclear which protein has chitinolytic activity.

Enzyme kinetic
The kinetic of the enzyme was studied on colloidal chitin. The kinetics of chitinase from B. licheniformis B2 followed the classical Michaelis-Menten kinetics. The Km and Vmax value calculated from Lineweaver-Burk plots was 101.96 mg mL -1 and 2.72 µmol (min mL) -1 , respectively (table 1)