Preparation of swollen chitin
The preparation of swollen chitin followed the method of Monreal and Reese . Briefly, 10 g of flake chitin was added into 100 mL of 85% phosphoric acid at 4 °C for 48 h. Then, the swollen chitin was precipitated by adding the gelatinous mixture into an excess of cold water. The excess of water was then removed and the precipitate was washed with distilled water until the pH became neutral.
Microorganism and production conditions of chitinase
Acinetobacter parvus HANDI 309 was originally isolated and identified from the feces of Korean Native calves, and the culture media was developed by Kim et al. . The isolated culture was inoculated (50 mL) into culture medium (1 L) containing colloidal chitin agar medium with the following composition: 1% swollen chitin, 1% peptone and 1% NaCl (pH 5.0). Then, 1 mL of the spore suspension was added to 100 mL of colloidal broth, and incubated in a rotary incubator at 220 rpm and 30 °C for 120 h. After the incubation period, the culture broth was centrifuged at 10,000g and 4 °C for 20 min. The collected supernatant consisted of crude chitinase which was further used for analysis. The culture supernatant was used for chitinase assay after overnight dialysis against 10 μM sodium acetate buffer (pH 5.0). Cell growth was also measured at OD 660 nm.
Enzyme activity assay by calorimetric method
For the determination of chitinase activity, colloidal chitin was used as a substrate. An amount of 0.5 mL of 1% colloidal chitin was added to 0.5 mL of enzyme solution and then the solution was incubated at 45 °C for 1 h. Next, 3 mL of 3,5-dinitrosalicyclic acid was added to stop the reaction, followed by incubation at 100 °C for 5 min. After centrifugation, the level of reducing sugar in the supernatant was carried out by the methods of Miller  with some modifications. The absorbance was noted at 540 nm using a UV spectrometer for the sample and also a blank. For the measurement of enzyme action, serial dilutions of GlcNAc was prepared and used. One millimole of GlcNAc was used as a standard.
Chitinolytic activity by Petri dish method
The hydrolytic activity of the isolated HANDI 309 bacterial strain from the feces of calves was measured by the serial dilution method and was screened by its capability to produce hydrolytic enzymes using the plate method. The sterile culture medium was developed on nutrient agar plates supplemented with 0.1% colloidal chitin, and 1.5% agar medium was inoculated with the isolated organism at 30 °C for 24 h. After the incubation period, 0.1% Congo red solution was poured over the plate, and the clear zone around the isolates observed after the incubation period is an indication of chitinase enzyme production [12, 13].
Molecular identification of A. parvus HANDI 309 for the production of chitinolytic enzyme
The 16S ribosomal DNA gene sequence was performed by method of Thompson et al. . In brief, genomic DNA was isolated from A. parvus HANDI 309 and purified by a Wizard genomic DNA purification kit (Promega, USA). The subsequent genomic DNA amplifications were sequenced with Taq DNA polymerase using the universal primers of 27F (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 1429R (5′-GGA TAT TAC GAC TTC TTG-3′). The amplified PCR products were purified by using the Wizard SV Gel and cleanup system (Promega). The purified PCR products were sequenced using an ABI PRISM 3730 DNA analyzer. The results were compared using sequence homology using the ClustalX program of Mega 2 and with the DNA sequence of ribosomal GENBANK using the BLAST program. During the similarity comparison, the sequence required an initial threshold of 99% homology when compared with the raw sequence.
Analysis of the hydrolytic products of swollen chitin
Thin layer chromatography (TLC)
In a 150-μL reaction mixture containing 0.5 mL of 1% of the substrate and 0.5 mL of A. parvus HANDI 309 chitinases in 50 mM of phosphate buffer at pH 7.0, those reaction mixtures were incubated at 37 °C for 12 h. Next, 10 μL of the response mixture was transferred to an Eppendorf tube containing 10 μL of 0.1 N NaOH to stop the reaction, and samples were stored in −20 °C until further analysis. Then, 20-μL aliquots from the reaction mixture were added to the chromatograph on silica gel plates, with a solvent system containing n-propanol, methanol, and ammonia water [7:3:1 (v:v:v)], and the hydrolyzed products were analyzed by spraying the plate with aniline-diphenylamine reagent and baking at 180 °C using a hot air gun for 3 min.
High-performance liquid chromatography (HPLC)
Analysis of the hydrolytic products of swollen chitin by A. parvus HANDI 309 extracellular chitinases was carried out by incubating the recombinant enzymes with the swollen chitin. The reaction mixture was added to 50 mM sodium phosphate buffer at pH 7.0, and incubated at 40 °C and 1300 rpm for 12 h. After that, 75 μL of the response mixture was mixed with 75 μL of 70% acetonitrile to stop the reaction, and then the reaction mixture was centrifuged at 16,100g for 10 min at 4 °C to remove the undigested swollen chitin. The supernatant was further concentrated until the complete evaporation of the solvent without heating. The remains were dissolved in 20 μL of 35% acetonitrile and the reaction mixture was stored at −20 °C until further analysis. The reaction mixture was analyzed by isocratic HPLC at 25 °C using a Shimadzu 10ATvp UV/VIS HPLC system (Shimadzu, Tokyo, Japan) with a Shodex Asahipack NH2P-50 4E column (4.6 ID × 250 mm). A sample or 20 ml of the reaction mixture was injected into the HPLC using a Hamilton syringe (Hamilton, Bonadzu, Switzerland). The liquid phase consisted of 67% acetonitrile and 33% MilliQ water and the flow rate was set to 0.70 mL/min, with the eluted chitooligosaccharides monitored by reading the absorption at 210 nm.
Production optimization of chitinase produced by A. parvus HANDI 309
Effect of incubation time and cell growth on chitinase activity
The effect of incubation time and cell growth on the activity of chitinase by A. parvus HANDI 309 was determined by using an inoculated flask containing the isolated culture inoculated in colloidal chitin agar medium, with the following composition (% w/v): colloidal chitin (1), yeast extract (0.1), K2HPO4 (0.07), KH2PO4 (0.03), MgSO4·7H2O (0.01), and FeSO4·7H2O (0.01) at pH 7.0, which was incubated using a rotary shaker at 200 rpm and 30 °C for 120 h. After that, the culture was centrifuged at 10,000 rpm for 20 min and the supernatant used for the chitinase activity. For the specific incubation time, the bacterial cells were grown for 120 h. The culture filtrate was harvested every 24 h and the enzyme production measured.
Impact of temperature and pH on chitinase activity
To determine the effect of incubation temperature and time, the incubating culture tryptic soy broth medium was measured at various temperatures (25, 30 and 35 °C) for 24 h on a shaking incubator (VS-8480SR; Vision Scientific, Korea) at 200 rpm. The effect of initial pH values on the chitinase production was analyzed by the inoculating broth culture medium composed of pancreatic digest of casein, 0.3% enzymatic digest of soy meal, 0.25% dextrose, 0.5% NaCl, and 0.25% dipotassium phosphate which was adjusted with 0.1 N HCl and 0.1 N NaOH to various initial pH of 5–9 for 24 h on a shaking incubator (VS-8480SR; Vision Scientific) at 200 rpm. Chitinase activity was measured as per the standard protocol, and the unit of chitinase activity was U/mL.
Effect of carbon source on chitinase activity
Four different types of carbon sources, glucose, sucrose, soluble starch and corn flour, were used to determine the impact of carbon sources on extracellular chitinase activity. These results were measured by adding different carbon sources (1% w/v) of supplemented media wihch were inoculated with inoculums (20 mL) fermented in optimized conditions. The culture medium contained different levels of glucose, which was adjusted to 0.5–2% on the shaking incubator. Chitinase activity was measured as per the standard protocol, and the unit of chitinase activity was U/mL. At the same time, the medium without carbon sources was used as a control.
Effect of organic and inorganic nitrogen source on chitinase activity
To explore the effect of organic and inorganic nitrogen sources on chitinase activity, various organic nitrogen sources (0.5% w/v), such as yeast extract, peptone, soybean flour and dry yeast were used, while the inorganic nitrogen sources (0.1% w/v) were urea, (NH4Cl2)SO4, NH4Cl, KNO3 and NaNO3, respectively. The different sources of both organic and inorganic sources inoculated with inoculums were incubated at pH 7.0 and 30 °C for 24 h under a shaking condition of 1200 rpm. The culture filtrate was used to measure chitinase activity after the incubation time, and the unit of chitinase activity was U/mole. An appropriate control was used for the study.
Effect of inorganic salts source on chitinase activity
The influence of various inorganic salts on chitinase activity was determined by inoculating the culture medium which was adjusted with different inorganic salts (1% w/v) of NaCl, K2HPO4, MgSO4·7H2O, MnSO4·5H2O and CaCl, and then incubated at pH 7.0 and 30 °C for 24 h under a shaking condition of 200 rpm. The culture filtrate was used to measure chitinase activity after the incubation time, and the chitinase activity was expressed as U/mol. An appropriate control was maintained for the above study.