Collection of samples and chitinolytic bacteria isolation
A total of 10 samples were collected from different areas of Jiangsu China. Four soil samples were collected form Lihu Lake (Wuxi) and Xihu Lake (Hangzhou), and the remaining 6 samples were collected from maize rhizophore and the fish market. For screening purposes, agar media containing colloidal chitin and colonies with a clear zone were used and considered for further study for chitinase, as these colonies specify chitinase-producing bacteria.
Colloidal chitin preparation
For colloidal chitin, preparation powder chitin (case no.1398-61-4) was purchased from Sigma-Aldrich and prepared by slight modification in Hsu and with the Lockwood method . Ten grams of powdered chitin was prepared by dissolving 300 mL conc. HCL gradually, acid was added to chitin with stirring, and mixture was allowed to stand at room temperature with alternating stirring until the chitin dissolved (2 h). By using 8 layers of cheesecloth (to remove large chitin chunks), the chitin-HCL solution was poured into a 2-l ice-cold (4 °C) D2H2O beaker and kept in the fridge overnight at (4 °C). After 24 h, there will be white cake-like chitin in the solution. Next, we have to centrifuge the white cake-like chitin solution at 18,000 rpm (F0485 Fix-Angle-Rotor) for 15 min. We then proceeded to wash it with D2H2O, at least 4 times until pH 5 is reached. Lastly, we loosened the colloidal by using 100 mL distill water to make w\w solution and stored it at 4 °C with an aluminum covering.
Culture media preparation
For identification of bacteria, LB (Luria broth) is the most commonly used medium; for screening chitinase-producing bacteria, selective medium is used. Selective medium containing (g L−1) KH2PO4, 14; K2HPO4, 6; (NH4)2SO4, 2; Na3C6H5O7, 1; MgSO4, 0.12; Agar, 15; with 1% (w/v) colloidal chitin at pH 7, were incubated at 30 to 37 °C. The clearance zone was formed by chitin hydrolysis and was recorded up to 8 days. Based on the size of clearance zone, two of the best samples were chosen for further study.
Gram staining procedure
The standard gram staining procedure was performed for the characterization of isolate .
Different chemical tests were performed to test the motility and oxidase. The nitrate reduction test and urease test were used for characterization .
Colloidal chitin was used as the substrate for measuring the chitinase activity according to originally modified protocol . Reaction mixture consists of 0.5 mL enzyme which was added to 0.5 mL colloidal chitin (1% w/v); it was then incubated at 45 °C for 1 h. The reaction was terminated at 90 °C for 5 min after incubation. The reaction mixture was then centrifuged at 20,000g (F0485 Fix-Angle-Rotor) for 5 min, and the supernatant was collected. The supernatant was boiled with 0.8 M potassium tetraborate of 100 μL quantity and boiled for 3 min, followed by the addition of 1 mL of dimethylamino benzaldehyde for 30 min at room temperature, and then incubated until it appeared to be a pink color. Absorbance was measured at 560 nm by using a spectrophotometer. A blank solution was used as a negative control. One chitinase enzyme unit was defined as a change in the absorbance of 0.01 min–1 .
Molecular characterization of microorganism DNA sequencing
The gene coding 16S rRNA was sequenced to identify the genus of each strain. The genomic DNA of the selected strains were isolated by using the TIANamp Bacterial DNA kit, and amplification was performed by using the PCR master mix kit with initial heating step for 2 min at 95 °C followed by denaturation step 30 cycles of 95 °C for 30s, annealing for 60 s at 55 °C, and 2 min extension at 72 °C, and then followed by final extension at 72 °C for 7 min. Two universal primers FC 27 (forward) and RC1429 (reverse) were used for amplification. The amplified product was than purified by using DNA purification kit, the purified product was sent to Sangon Biotech Shanghai for sequencing, and 16S rRNA sequence was obtained. 16S rRNA sequence analysis showed that this strain belongs to genus Myxococcus and species Myxococcus fulvus.
The gene coding 16S rRNA partial sequence obtained from Sangon Biotech Shanghai (www.sangon.com) was compared to other bacterial sequences to search for their pairwise identities by using NCBI, BLAST (BLAST; http://www.ncbi.nlm.nih.gov/BLAST/), and online software. Multiples sequence alignments of highly similarity sequences were available at the data bank and were performed by using MEGAX (Version 10.1.5). By using the neighbor-joining method (NJ), phylogenetic analysis was carried out with the MEGAX (Version 10.1.5) . Partial gene coding 16S rRNA sequence was then submitted to NCBI Gene bank with the name UM01 under the accession number MN811202.1 .
Determination of different substrates and effect on enzyme production
To determine the best substrate for chitinase production, various substrates such as (1%) colloidal chitin, powdery chitin, crab shell, and chitosan were used. Final 20 mg/mL substrate concentration was used in the final reaction mixture and the increase reductively was determined .
Effect of temperature and pH on enzyme production
The effect of temperature for optimum enzyme production was determined at 20 to 55 °C by using colloidal chitin as a substrate, and the pH value effect on enzyme production was examined by varying pHs from pH 3.5 to pH 9 in culture medium at optimized temperatures and incubation period. Tris-HCL buffer (pH 6.5 to 9) and acetate buffer (pH 3.5 to 6.0) were used for this purpose.
Chitinase gene amplification
Five starter primers were used for amplification of chitinase gene from UM01 strain:
BBGA (F): ATGAGCACAAATAACATTATTAATGC, BBGA (R): TTAGGCGATGAGCTGCC
BBGB (F): ATGCAGCTTCCAGGATTCC, BBGB (R): TTACGGGTAGGTGTCCAGGT
BBGC (F): ATGCAGAGGTCCCTCGC, BBGC (R): TTAGCGCACG AACTCCC
BBGD (F): ATGTCTGGCAATTTTGTTTCAC, BBGD (R): TTAGCAGCCCAGGTTGC
BBGE (F): ATGGCCATGGCCGTG, BBGE (R): TTAGGGCTGGAAGGCTTCC
All these primers were designed by searching the database (NCBI) to find the closest relative sequence. By putting all these sequences in FASTA format in a text document, we were able to do multiple alignments using Clustal Omega. For amplification of the chitinase gene, genomic DNA from strain UM01 was used by using TIANamp bacterial DNA kit and amplification was performed by using PCR master mix kit by following standard PCR protocol. The obtained PCR product was analyzed by gel electrophoresis and an amplified purified product was sent to Sangon Biotech Shanghai for sequencing.
Cloning of (UMCda) chitinase gene
The coding region of the chitinase gene (UMCda) was amplified by PCR with the flanking restriction enzyme by using forward primer BBG71F: CCGGAATTCATGAACAAAACTTCCCGTAC and reverse primer BBG7R: CCCAAGCTTTTATTTGCTCAGGTTGACGT, and PCR products were purified by using PCR purification kit (Thermo fisher). By using the restriction enzyme EcoR1 and HIND111, the PCR product were digested and then ligated into pET-28 plasmid vector, which was digested with the same restriction enzyme. By using the heat shock method, the recombinant vector transformed into E. coli DH5a. The recombinant E. coli DH5a strain was grown in LB medium containing 100 mg/mL kanamycin at 37 °C in a rotatory shaker 180 rpm. When the optical density (OD = 600) of culture media has reached up to 0.5, expression of recombinant protein was induced by IPTG 0.5 mM and culture was grown at 37 °C at 12 h.
Purification of recombinant chitinase and chitinase assay
The reaction mixture containing 0.1 mL enzyme solution and 0.1 mL (1%) colloidal chitin which was incubated at 45 °C for 30 min, and the reducing sugar was determined . The one unit of enzyme activity is defined 1 μmol of GlcNAc obtained from enzyme hydrolysis during 1 min using 1 mL of enzyme.
For purification of recombinant chitinase, 1 mL of overnight culture was induced with IPTG; the cell was then harvested by using centrifugation at 4 °C at 10,000 rpm and re-suspended again in 1 mL PBS buffer then centrifuged again 12,000 rpm for 1 min, followed by a second wash with 55 μL PBS buffer. The cell was disrupted by using sonication and again centrifuged at 4 °C at 10,000 rpm for 30 min to obtain suspension. Purification of the enzyme was done by using NI-NTA affinity chromatography and imidazole used as an eluent. Column chromatography purified chitinase enzyme was loaded to DEAE-cellulose A52 (2.6 × 20 cm) column. A different salinity of tris 0.05 M HCL and pH 7 was applied. Elution step was preceded in a stepwise gradient of NaCl (0–1.0%) at the flow rate of 0.5 mL/min. The selected fraction was applied for Sephadex G-100 column (1.6 × 30). Purified column was eluted with tris 0.05 M HCL and at pH 7 with the rate of 0.4 mL/min. The fraction of each 3 mL was collected and chitinase activity with protein content of each fraction was determined accordingly . The purified beak was chosen for determining the properties of chitinase enzyme. SDS-PAGE using 12% gel in Tris-glycine buffer pH 8.3  was done to determine enzyme homogeneity. Chitinase activity was measured by using a modified method .
Antifungal activity of chitinase gene
To evaluate the antifungal activity of the recombinant chitinase gene, it was measured against Trichoderma reesei IFO 31329 using a chitinase plate assay by using a slightly modified method of the previous study . Recombinant strain was streaked at the center of the agar plates containing 1:1 (v/v) ratio of potato dextrose agar (PDA) and yeast extract mannitol) (YEM) with the addition of 0.5% colloidal chitin and was incubated at 37 °C for 24 h for bacterial growth. Next, we inoculated each bacterial (recombinant) colony and the mentioned strain in a straight line. After a 24-h incubation period at 37 °C, a mycelial lump of T. reesei was placed on the center of the plate. One plate with bacterial inoculation was used as a control. Both plates were incubated at 37 °C for 3 days, and the antifungal activity of recombinant was evaluated by visual examination.