Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51

This paper reports the isolation and identification of chitinase-producing Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125 kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer.


Introduction
Chitin, a linear β- (1,4)-linked N-acetylglucosamine (Glc-NAc) polysaccharide, is the main structure component of the fungal cell wall and the exoskeletons of invertebrates, such as insects and crustaceans. Chitinase plays a variety of important roles in these organisms ranging from nutrition to defense and control of ecdysis in insects. The importance of chitinases in many biological processes makes their inhibitors important targets for potential antifungal and insecticidal agents as well as antimalarial agents [1].
It is one of the most abundant naturally occurring polysaccharides and has attracted tremendous attention in the fields of agriculture, pharmacology, and biotechnology. Chitin is the second most abundant component of biomass on earth [2]. This linear polymer can be hydrolyzed by bases, acids or enzymes, such as lysozyme, some glucanases, and chitinase. Chitinases (EC 3.2.1.14), essential enzymes catalyzing the conversion of chitin to its monomeric or oligomeric components (low-molecular-weight products), have been found in a wide range of organisms, including bacteria, plants, fungi, insects, and crustaceans. Because chitin is not found in vertebrates, it has been suggested that inhibition of chitinases may be used for the treatment of fungal infections and human parasitosis [3]. Biological control, using microorganisms to repress plant disease, offers an alternative environmentally friendly strategy for controlling agricultural phytopathogens [4]. 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, so chitinases have been studied and purified from many microorganisms, and their enzymatic properties have been investigated [5]. Global annual recovery 2 Biotechnology Research International of chitin from the processing of marine crustacean waste is estimated to be around 37.300 metric tons [6].
2.1.1. Isolation of Bacteria. Crabs, campus soil, and compost were mixed and held to approximately 30 days outside (exposure sun, rain or etc.). After this period, sterile distilled water was added to material and homogenized. 10 mL supernatant wae transferred to glass tube and incubated at 80 • C for 10 min to eliminate vegetative forms of bacteria. 1 mL sample inoculated to 10 mL LB and incubated at 37 • C for 6 hours and then sample was diluted 10 −6 − 10 −7 fold with freshly prepared LB, then streaked on N1 agar plate. After incubation at 37 • C overnight, single colonies were selected (550 colonies in total) and stock cultures prepared [7].

Preparation of Colloidal
Chitin. Preparation of colloidal chitin was performed from commercial chitin (C9752, Sigma-Aldrich Co, USA) according to Wen et al. [3] with minimal modifications. Fivegrams of chitin powder were added into 60 mL of concentrated HCL and left in refrigerator (at 4 • C) overnight with vigorous stirring. The mixture was added to 2 litres of ice-cold Et-OH (95%) with rapid stirring and kept overnight at room temperature (at 22 • C). The precipitate was collected by centrifugation at 6, 000 ×g (4 • C) for 25 min. The precipitate was washed with sterile distilled water until the colloidal chitin became neutral (pH 7.0) and then stored and at 4 • C for further applications.

Screening Chitinase Producer Strains.
All strains (total 550) were selected from soil including chitin wastes, tested one by one for chitinase activity on the CHDA (chitinasedetection agar). Chitinase producer strains were determined after 3-5 days at 37 • C by visualizing the clear zone formed surrounding of the colonies on the CHDA plate. CHDA agar, used first detection of chitinase positive strains and preparation of stock culture, by adding 10% g of colloidal chitin and 20 g of agar in M9 medium containing (g/L); 0.65 NaHPO 4 , 1.5 KH 2 PO 4 , 0.5 NH 4 Cl, 0.12 MgSO 4 , 0.005 CaCl 2 , 0.25 NaCl, pH = 6.5 [3,8].

Enzyme
Production. HBK-51 strain (Bacillus thuringiensis subsp. kurstaki) was used as chitinase producer. 1 mL of HBK-51 strain fresh culture was inoculated into 10 mL of LB medium (containing 1% colloidal chitin as the sole source of C and N) and incubated overnight at 37 • C with shaking (150 rev min −1 ). The culture was transferred into a 2 litres glass bottle containing 500 mL M9-Medium which was supplemented with 1% colloidal chitin and incubated at 37 • C on a rotary shaker at 150 rev min −1 for 3 days [3]. The culture was centrifuged for 20 min at 9000 ×g, at 4 • C and supernatant was separated from cell debris. Then supernatant was filtered (with filter paper) and precipitated with Et-OH (ethyl-alcohol) [9]. All experiments were done with this enzyme preparation.

Purification of the Chitinase.
Proteins in the culturesupernatant fluid were precipitated with Et-OH (ethyl-alcohol) at −33 • C overnight. Et-OH (ethyl-alcohol) was added 70% of the original volume of supernatant. The precipitate was recovered by centrifugation (12, 000 ×g (4 • C) for 30 min.), dissolved in a small amount of 0.1 M Na-phosphate buffer (pH 7.0), and stored at 4 • C [10,11].
2.6. Measurement of Enzyme Activity. Chitinase activity was measured with chitin-azure as a substrate [12]. Enzyme solution (0.5 mL) was added to substrate solution (0.5 mL), which contained 1.0% chitin azure in a sodium acetate buffer (100 mmol/L, pH 7.0), and the mixture was incubated at 50 • C for 30 min. After centrifugation (12, 000 ×g, 15 min, at 4 • C), enzyme activity was measured at 595 nm absorbance using spectrophotometer. One unit of chitinase was the amount of enzyme that produced an increase of 0.01 in the absorbance [12,13].

Thermal
Stability. The effect of the temperature on the stability of chitinolytic enzyme was determined by exposure of the enzyme solution in 50 mM Tris-HCl buffer, pH: 9.0 (optimum pH) to different temperatures (30-110 • C) for 3 h. The residual activity was then assayed under standard conditions using chitin-azure as the substrate [14,15].

pH Stability.
For pH stability assay, the enzyme solution was preincubated for 3 h at room temperature in the buffers of various pH values (pH: 5.0-12.0) and then the residual activity was determined under standard conditions [14,15]   The amplified PCR product was sequenced by the Beckman Coulter-CQ 8800 model sequence analyzer with their methods. The 16S rDNA sequence was aligned with other 16S rDNA bacterial sequence obtained from GenBank by basic local alignment search tool (BLAST) program [20].

SDS-PAGE Analyses.
The enzyme molecular weight was determined with sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE) [22]. The gel was prepared Con: Concentration, Act: Activity. The enzyme was incubated with various ions and reagents at room temperature for 30 min., then chitinase activity was assayed under standard conditions. The enzyme activity without any additive was taken as 100%.
The HBK-51 strain was identified according to morphological and physiological characteristics (are presented in Table 1) [17]. According to Bergey's Manual of Systematic Bacteriology [7], strain HBK-51 was classified as a bacteria  belonging to the genus Bacillus. Further sequence analysis of the gene encoding 16S rRNA and fatty acid analyses (FAME, data not shown) confirmed the isolate as being Bacillus genus and according to BLAST confirmation, it was Bacillus thuringiensis subsp. kurstaki. Theamplified16S rRNA sequence was submitted to GenBank for possible identification. The result showed 99.00% identity with Bacillus thuringiensis subsp. kurstaki. The accession number is EU153549. Chitinolytic activity of HBK-51 on CHDA is shown in Figure 1.
According to the plasmid-curing tests, the gene of encoding chitinase of Bacillus thuringiensis subsp. kurstaki HBK-51 was located on chromosomal.

pH and Temperature
Optima. The pH effect on the chitinase activity was determined using three buffer systems at various pH values. The HBK-51 chitinase was active at broad range of pH (3.0-10.0) but had optimum activity at pH 9.0 (data shown Figure 5), when assayed with chitin azure as a substrate. On the other hand, enzyme was active from 30 to 120 • C and exhibited maximum activity at 110 • C ( Figure 3). The enzyme was stable (after 3 hours incubation period) at 30-120 • C and pH 9.0-12.0 and generally, protected original activity approximately 92.4% ( Figure 4) and 98% (Figure 6), respectively, at 90 • C chitinase was 100% active and at 100-110 • C had shown 96% retain activity. Therefore, HBK-51 chitinase was called as thermostable at high temperatures. Siwayaprahm et al. [23] reported that for Bacillus circulans No. 4.1. recombinant chitinase, the optima pH and temperature were 7.0 and 45 • C, respectively, and it was stable in the pH range of 5.0-9.0 and at temperatures up to 50 • C. Lee et al. [24] reported that Bacillus sp. DAU101 chitinase had optimum activity pH 7.5 and 60 • C, Wen et al. [3] reported chitinase activity of Bacillus sp. NCTU2 had in the range of 50-60 • C at pH 7.0. Kim et al. [14] reported Serratia sp. KCK chitinase had broad range of pH (5.0-10.0) with an optimum value 0f 8.0 and 40 • C, respectively. Li et al. [25] reported Bacillus cereus strain CH2 had optimum activity at pH 7.1 and temperature at 40 • C. According to these results, Bacillus thuringiensis subsp. kurstaki HBK-51 chitinase is a thermotolerant and alkaline enzyme. Guo et al. [26] reported that Thermomyces lanuginosus SY2 chitinase exhibited optimum catalytic activity at pH 4.5, 55 • C and enzyme was stable at 50 • C and its half-life time at 65 • C was 25 min. On the other hand, they reported that the thermostable chitinase had major advantages over industrial catalysis for its high activity at high temperature. Thermostable chitinase could be useful for the chitin industry and biotechnological applications.
Chitinases have a broad spectrum of different industrial applications such as bio-control agents against plant pathogens (fungi) and insects (bio-insecticide friend with environment) and bioconversion of chitin waste to single-cell protein, Et-OH and fertilizer [14].
The thermostable chitinase had major advantages over industrial catalysis for its high activity at high temperature [26]. The properties of thermostable chitinase suggest that it could be useful for the chitin industry and biotechnological applications.