PRODUCTION AND PARTIAL PURIFICATION OF ANTIFUNGAL CHITINASE FROM Bacillus cereus VITSD3

: The current work was designed to isolate and characterize chitin degrading bacteria. Among the 55 bacterial colonies isolated from 7 different soil samples, 4 isolates were capable of degrading chitinase, among which one strain VITSD3 was found to be potent. Based on the morphological, biochemical and molecular characterization of VITSD3 the isolate was confirmed as Bacillus cereus (Genbank accession number: KC961638), designated as Bacillus cereus VITSD3. The crude enzyme had a total activity of 220 U, precipitated with 44.8 U and 22.5 U for dialysed sample. The hydrolysed product NAG (N-Acetyl Glucosamine) from chitin was analysed by high-pressure liquid chromatography (HPLC).The molecular weight of the chitinase was determined using SDS PAGE and found to be 55 kDa. The partially purified chitinase produced from Bacillus cereus VITSD3 showed antifungal activity against Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) and Aspergillus flavus (15 mm). Hence the investigation suggests a potential benefit of partially purified chitinase extracted from Bacillus cereus VITSD3 which will serve as an excellent antifungal potential with therapeutic use.


INTRODUCTION
Chitin is well-known as an insoluble structural polysaccharide that occurs in the exoskeleton and gut linings of many insects, invertebrates such as crustaceans, protozoa, fungi and diatoms which could be hydrolysed by chitin degrading enzymes such as chitinases. (KRAMER et al., 1986). It is the second most abundant polymer and it is a monomeric protein that forms a single strand of polypeptide with molecular weight range between 21 kDa to 70 kDa and also known as poly 1, 4 -(N-acetyl--D-glucosaminide) glycanohydrolase (JEUNIAUX et al., 1996). Recently, chitinases have receiving attention because of their possible applications for biological control also for the exploitation of natural chitinous materials (OHNO et al., 1996). Chitinases plays an important biological and physiological roles: lysis of the cell wall (separation of cells after division, hypha autolysis), nutritional requirements, morphogenetic formation (sporulation, spore germination, hyphal growth) and antagonistic actions against other microorganisms (SAHAI et al., 1993). Among their applications, chitinolytic enzymes have studied as potential antifungal agents against chitin bearing plant pathogens. The enzymes play a key role in the mechanism of parasitic entry into host cells (DAHIYA et al., 2006;MAVROMATIS et al., 2003). The fungal cell wall has a cross-linked complex structure composed of chitin, glucans, and other polymers (AGRIOS, 2005). Enzymes that hydrolyze these components play a significant role in cell wall lysis of the pathogens (ADAMS, 2005;CARSOLIO et al., 1994). The antifungal activity and highly biocompatible quality make the chitin and its derivatives particularly useful for biomedical applications such as wound healing, cartilage tissue engineering, drug delivery and nerve generation (NAGWA et al., 2004). Almost all industrial microbiology processes require the initial isolation of cultures from nature by a suitable method, followed by small-scale cultivation and optimizations (JEYANTHI et al., 2013). The discoveries of novel secondary metabolites are currently the thirst area of research (JEMIMAH NAINE et al., 2012). Chitinase is a potential antifungal agent that can be isolated from a wide range of organisms. (HORSCH et al.,1997;HARMAN et al.,2004;LI, 2006;MERZENDORFER et al.,2003;RAST et al.,2003). Further screening of microbes for enzymes, has received ever-increasing interest. Hence the main aim of the current study was to isolate and screen chitinase producing bacteria from soil samples. This study also emphasizes the antifungal activity of the chitinase from Bacillus cereus VITSD3

Isolation of chitin degrading bacteria
Seven different soil samples were collected from Bhramapuram, Vellore and CBMR nursery, VIT, Vellore, Tamil Nadu, India. One gram of soil sample was serially diluted on colloidal chitin agar. Isolated colonies were sub cultured on nutrient agar 961 Production and partial purification… DEVI, C. S. et al. plates. Pure cultures of the organisms were maintained and stored at 4 o C.

Colloidal chitin preparation
Colloidal chitin was prepared by grinding chitin flakes manually and ground on a mortar and pestle for 5 minutes to fine powder and 10 N HCl was added and kept overnight at 4°C with vigorous stirring. The suspension was added to 50% cold ethanol with rapid stirring and kept overnight at 25 °C. The precipitate was collected by centrifugation at 10,000 rpm for 20 min and washed with sterile distilled water until the colloidal chitin becomes neutral (pH 7.0). The supernatant was discarded and the pellet was stored at 4 o C till further use (PRIYA et al., 2001).

Screening
Primary screening was performed by streaking all the four bacterial isolates on colloidal chitin agar (CCA) and incubated at room temperature for 2 days. The zone of clearance due to chitin hydrolysis was recorded up to 3 days. The secondary screening was performed with the culture filtrates of selected bacterial isolates using well diffusion method. After 24 h, the development of clear zone around the well was observed (KAVI KARUNYA et al., 2011).

Morphological and biochemical characterisation
The potent producers of chitin degrading isolate VITSD3 was further studied for its morphological and biochemical properties. The strain was identified according to the Bergey's manual of determinative bacteriology (Holt, 1994).

Molecular characterisation 16S rRNA gene amplification and sequencing
16S rRNA gene sequencing of Bacillus sp was amplified using forward primer 27 F 5'AGAGTTTGATCMTGGCTCAG3' and reverse primer 1792 The sequences were subjected to homology search using BLAST programme (ALTSCHUL et al., 1990). The results were compared manually with the data in NCBI. The 16S rRNA gene sequences determined were aligned along with the sequences of the type strains using the CLUSTAL W program version 2.1 (CHENNA et al.,2003). The phylogenetic tree was constructed by neighbourhood joining method (SAITOU et al., 1987). Individual branches in phylogenetic tree were determined by bootstrap analysis based on 1000 samplings (FELSENSTEIN et al., 1985).

Chitinase production
Colloidal chitin broth (100 mL) in 250 mL capacity Erlenmeyer flasks was inoculated with 1 mL ( OD 600 =1.0) of potent strain Bacillus cereus VITSD3. It was incubated at 27 °C in a rotary incubator (150 rpm) for 72 h. Culture supernatant was collected from 2days cultures by centrifugation at 12,000g for 20 min. The supernatant was then concentrated by ammonium sulphate precipitation.

Partial purification of enzyme
Ammonium sulphate was added to the culture supernatant in small quantities with constant stirring in order to achieve saturation. Then the supernatant was concentrated by precipitation with ammonium sulphate to 60-70% levels. The precipitates were dissolved in 50 mM phosphate buffer (pH 7) and dialyzed over night against the same buffer. The resultant dialysates were regarded as partially purified chitinase and were used for further studies.

Total protein estimation
Total protein content of the crude, precipitated and the dialysed samples were estimated by standard Lowry's method (LOWRY et al., 1951).

Enzyme assay
The reaction mixture composed of crude enzyme solution, 1mL 0.5% colloidal chitin in 0.1M citrate buffer (pH 7.0) was incubated at 37 o C in a shaking water bath for 30min. The reaction was terminated by adding 2 mL DNS reagent. The colour was developed in the tubes after 5 min. The optical densities were measured at 575nm using UV-Vis spectrophotometer (Model-Hitachi U-2800) against a blank containing 1mL of 0.5 substratebuffer solution %, 1mL buffer and 2mL DNS (Miller et al.,1959). Then the optical densities were compared with the control. The amount of chitinase released was determined from N-acetyl-Dglucosamine calibration curve, where one unit of chitinase activity (U) is defined as the amount of enzyme that releases 1µmol of glucose /min under the above mentioned conditions.

SDS PAGE
The partially purified enzyme was subjected to sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) with lower separating gel (pH 8.8), upper stacking gel (pH 6.8) and 12% acrylamide concentration. A broad range protein molecular weight marker was used to compare the 962 Production and partial purification… DEVI, C. S. et al.
protein bands with the standards, for the confirmation of the enzyme.

HPLC analysis
The oligosaccharides obtained by enzymatic hydrolysis were analyzed by HPLC (LC-10 AT vp model). The mobile phase-acetonitrile: water (70:30 v/v) at the flow rate of 1 mL/min and detected at 210 nm with NH 2 P 50 -4E column. The retention times for the peaks detected in the crude samples of hydrolytic products were compared (CHANG et al., 2003).

Antifungal activity
The partially purified chitinase enzyme was checked for antifungal activity against Agar well diffusion assay was performed on potato dextrose agar. The plates were incubated for 5 days at 30 o C. After incubation the zone of inhibition was measured.

Isolation and screening
In the present study, a total of fifty five organisms were isolated from seven different soil samples. The preliminary screening revealed four isolates were capable of degrading chitinase, among which one strain VITSD3 was found to be potent. Chitinolytic bacteria isolated from soil were identified by the formation of distinct clearing zones on the substantially darker background of the medium within 2-3 days of incubation. All the four strains were subjected to preliminary screening which revealed the potential producers of chitinase (Figure1).

Figure1. Screening for chitinase production on colloidal chitin agar
Interestingly, the isolate obtained from the rhizosphere soil VITSD3 remarkably hydrolyzed the colloidal chitin and produced a prominent and maximum clear zone on CCA plate. The culture filtrate of the potent isolate produced a clear zone of 13mm. The morphological characteristics of the isolate VITSD3 was found to be whitish colored colonies with irregular margin and an opaque density, convex elevation with a lobate configuration on nutrient agar. Based on the gram staining results the VITSD3 strain was found to be gram positive rod shaped bacilli in singles and pairs ( Figure 2). Based on the isolate VITSD3 was identified as Bacillus sp. (Table 1) The adoption of colloidal chitin in the media has provided easier and more accurate screening of chitinolytic Bacillus cereus VITSD3. These finding coincides with previous studies that suggest employing colloidal chitin as sole carbon source for highest chitinase production (PRIYA et al., 2011).

Molecular characterisation
Sequence results of 16S rRNA was exported to the database and checked for the homologous alignment. Based on the alignment results, strain VITSD3 was found as Bacillus cereus which showed 99% similarity. The strain name was designated as Bacillus cereus VITSD3. The partial 16S rRNA sequence was deposited in Gen Bank under the accession number KC961637. The phylogenetic tree based on 16S rRNA of the strain Bacillus cereus VITSD3 is shown in Figure  3.

Figure 3. Phylogenetic tree of Bacillus cereus VITSD3
Total protein estimation and enzyme assay Total protein estimation of crude enzyme, precipitated and dialyzed sample showed differences in OD readings was due to its variable presence of total protein content. The concentration of N-Acetyl glucosamine in the sample was derived using different concentration of NAG. The specific enzyme activity of the crude enzyme was found to be 3.2 U/mg whereas the precipitated was determined as 12.3 U/mg and 13.2 U/mg for dialysed sample. The partially purified dialysed enzyme showed the yield of 24.2% with the purification fold of 9.8 (Table 2). Similar results were reported in the previous studies, the protein content extracted from sample was found to be 1010 mg with a specific activity of 0.063, 0.016 and 0.053 AU/mg (KHALID THABIT et al.,2013).

SDS-PAGE
The molecular weight of the partially purified chitinase was found to be 55 KDa and hence it confirms the chitinase produced by Bacillus cereus VITSD3 (Figure 4). 965 Production and partial purification… DEVI, C. S. et al.  (NAGWA et al.,2004) . Similar results were indicated by the other chitinases using SDS-PAGE analysis of enzyme from Bacillus sp around 25-80kDa (DHAR et al., 2010).

HPLC analysis
HPLC analysis revealed the presence of NAG (N-Acetyl Glucosamine) from chitin hydrolysates. The retention time of the principal peak was observed at 3 min ( Figure 5). The previous reports support the present findings (JEYANTHI et al.,2013). Similar results were obtained for the products. The partially purified chitinase from the enzyme precipitated sample showed the principal peak at 3.17 min which indicates the presence of chitinase.

Antifungal activity
Antifungal activity of the partially purified chitinase was determined. It showed inhibitory activity against Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) and Aspergillus flavus (15 mm). (Figure 6). The relevant data reported by the chitinase inhibition against Aspergillus niger (7 mm), Aspergillus flavus (5 mm) (MUHAMAD, 2009). The present result is supported by the previous literature as the chitinolytic bacteria are active against the fungal pathogens (SID AHMED et al., 2003;DE BOER et al.,1999). 966 Production and partial purification… DEVI, C. S. et al. Recently, chitinases have been receiving attention because of their possible application for biological control and gaining importance for their biotechnological applications as it is biodegradable with various applications in the environmental, agricultural, food technology and cosmetics. Despite the potential applications of chitinase, the most promising future applications in therapy of fungal diseases and potential as therapeutic of antifungal drugs. Although chitinase have been isolated and characterized from a wide variety of sources, it is still important to screen for new sources for production of chitinase with more economical values and enhanced properties to expand their usefulness. Hence the study is unique when compared to previously reported studies on chitinase producing on the related genera. Pilot scale production and enzyme kinetics of chitinase is yet to be adopted for maximum production.

ACKNOWLEDGEMENT
We are greatly indebted to Vellore Institute of Technology for the constant encouragement, help and support for extending necessary facilities.