Chitin extraction of crab
Blue crab (Callinectes sapidus) was purchased from market and washed with water, and shells were separated. The shells were oven dried at 105 °C and crushed to powder. Chitin polymer was extracted according to the method of Rhazi et al. (2000). Finally, extracted chitin powder was dried and saved in bottles.
Processing for colloidal chitin
Chitin was processed according to the method of Jabeen and Qazi (2014). The extracted chitin in filter paper was washed several times by autoclaved distilled water till the spent wash water’s pH became 7. The colloidal chitin was removed from the filter paper, weighed, and stored in dark bottle at 4 °C.
Sampling and isolation of the chitinolytic bacterium
Termites were sampled from trees of Quaid-e-Azam campus area, University of the Punjab, Lahore, Pakistan. The samples were saved in tightly capped sterilized bottles and transported to laboratory for further processing. The dead termites were enriched in selective medium prepared after Furukawa et al. (1978). The medium contained 1% prepared colloidal chitin as sole carbon source; NH4SO4, 1.0; KH2PO4, 0.2; K2HPO4, 1.6; NaCl, 0.1; MgSO4 7H2O; FeSO4·7H2O, 0.01; CaCl2·2H2O, 0.02 (g/L) dissolved in distilled water. After 5 days of incubation at 30 °C temperature, dilution was made and spread on chitin agar plates, incubated at 30 °C for 3 days. Zones of clearance appeared around colonies and then pure cultured through nutrient agar.
Chitinase assay
Chitinolytic activity of the isolate was determined by the estimation of released reducing sugars from the chitin as described by Sadafi et al. (2001). The standard curve was plotted with N-acetylglucosamine (NAG) in the range of 100 to 600 μg/ml. One unit of chitinolytic activity was described as 1 μmol of liberation of NAG per milligram of protein per minute.
Protein test
The protein content was estimated by the method described by Bradford (1976). Bovine serum albumin (BSA) prepared from 2 to 10 μg/ml range with two class intervals was used as standard. Calibration curve was then plotted by performing regression analysis of A595 absorbance versus corresponding concentrations of the standards.
16S rRNA gene sequencing
Freshly grow bacterial colony was suspended in 5 ml sterilized nutrient broth and grown for overnight. The culture was centrifuged at 10,000 for 10 min, and pellet was processed for DNA extraction. Bacterial 16S rRNA gene was amplified by using the universal primers 27F(5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R(3′-TACGG{Y}TACCTTGTTACG-5′) (Oligo, USA). Extraction and amplification was performed according to the method described in Jabeen and Qazi (2014). Sequencing was done from Korea and matched with the nucleotide database available at Gene Bank, using BLAST tool in NCBI (http://www.ncbi.nlm.nih.gov) for recognition of the highest percentage similarity with the described species.
Phylogenetic analysis
BLAST sequences were imported into the clustalW program package. The sequence was aligned with the closest relative and phylogenetic tree was plotted by using the neighbor joining program in clustalW package (Fig. 1).
Optimization of chitinase production
Effects of temperature, pH, and nitrogen source on chitinase production were studied by growing the isolate in chitin broth at different temperature ranging from 20 to 60 °C, pH ranging from 4 to 11 for 5 days. Different nitrogen sources such as ammonium chloride, trypton, gelatin, peptone, ammonium oxalate, ammonium dihydrogen phosphate, yeast extract, and urea were supplemented to the chitin medium to study their influence on chitinase production.
Enzyme characterization
The enzyme was characterized by incubating enzyme to different pH levels from 4 to 11 by using different buffers acetate (4–5), phosphate (6–7), Tris HCl (8–9), and glycine NaOH buffers (10–11) similarly temperatures ranging from 20 to 60 °C for 30 min in shaking water bath. The crude enzyme was also incubated with different concentrations of chitin substrate at optimum pH and temperature for 30 min. The enzyme activity was estimated, as described before.
Amplification of chitinase gene
The extracted DNA was also used to amplify chitinase gene(s) employing the primer sequence used by Lee et al. (2007), i.e., F(5′-AATGGGGAATTCGCAAAAGCCAGTTCTGAC-3′), and R(5′CTCTCTCTTTATC-CTCGAGTATCAAACTGAT-3′) (Macrogen, Korea) using Promega Go-Taq® Flexi DNA Polymerase (MGW Biotech, Germany). Following Lee et al. (2007), PCR was prepared and amplified. The gel was electrophoresed then along with 1 Kb plus ladder of Invitrogen™ (catalog number: 10787018) and PCR product. The PCR products were purified by using QIA quick PCR purification kit protocol, and purified product was got sequenced commercially from Macrogen, Korea.
Partial purification of chitinase enzyme
The extracellular chitinases was purified through ammonium sulphate precipitation and dialysis. Five hundred milliliter supernatant of selected strain was precipitated with ammonium sulphate at different saturation levels (20–80%) by increasing the concentration of salt by 10% each time following the method described by Jabeen (2011). Chitinase activities and protein concentrations were measured every time during addition of salt fractions to calculate the specific activities of enzyme.
Experiments on termites
Collection of termites
Termites’ members of Heterotermes and Coptotermes were collected from the field and transported to the laboratory. Termites were allowed to remain within the piece of bark of tree for 24 h. Termites were recovered and placed in a cool place (28 ± 2 °C) in a glass small box covered with black cloth till further use.
Concentrating bacterial chitinases for termiticidal assessment
The bacterium was grown in 50 ml FJ medium at their corresponding enzyme optima. The growth was centrifuged in sterile centrifuge tubes, and three different concentrations were made as 1X, 5X, and 10X. Three test tubes were prepared with 10 ml of supernatant in each sterile capped test tube. The 10 ml supernatant in two of the test tubes (5X, 10X) was dried by exposing to room temperature at sterile conditions till it becomes 5 and 1 ml to attain 5X and 10X concentrations, respectively. A third tube was remained as it is as 1X.
Exposing termites to the bacterial isolates
Filter paper discs of 10 mm were made, sterilized by autoclaving, and loaded with prepared concentrations, i.e., 1X, 5X, and 10X, of the bacterial culture fluids. The discs loaded with 10 μl/disc of given preparation were placed in Petri plates and 10 termites free of extraneous dust etc. were exposed to them. A control was also run having filter paper discs moistened with simple sterilized distilled water. The Petri plates were covered and kept under darkness at 26 ± 2 °C and were observed for the termite mortality after every hour up to 8 h and then at 24 h post-exposure.
Preparation of bagasse made chipboard
One gram of Sugarcane Bagasse (SCB) was sterilized in each Petri plate and was mixed with 10 ml of a chitinolytic bacterial cell free cultural fluid. The chitinolytic chips of the SCB thus prepared were exposed to the termites. Mortality rate of the termite was observed and recorded as mentioned before. The experiments were conducted in triplicates.