Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2

Abstract

A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extracellular chitinase was purified to >90% homogeneity from the culture filtrate. The purification involved hydrophobic-interaction and gel-filtration chromatographic separations with a yield of 58%. The purified enzyme (ChiNCTU2) is a monomeric protein with an estimated molecular mass of 36.5 kDa and a pI of 6.3. It is thermally stable at 60 degrees C and pH 6-8 for more than 3 h. The optimal activity is in the range of 50-60 degrees C at pH 7.0. Chitobiose is the predominant product throughout the enzymic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. Chito-oligosaccharides [with degree of polymerization (DP) values of 4-6] are good substrates of the purified enzyme, whereas a DP3 oligomer was slowly hydrolysed to form DP1 and DP2 sugars. The first 15 N-terminal amino acids of the enzyme were determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. A PCR cloning technique was employed to obtain the corresponding gene from Bacillus NCTU2. The gene sequence was determined to be 1080 bp, encoding a polypeptide of 360 amino acids with the first 27 amino acids as the signal peptide.

Purification and Characterization of Thermostable Chitinase from Bacillus licheniformis SK-1

“…Only few reports have shown that bacterial chitinases is stable at broad range of pH. The molecular basis for retaining of activity and stability at high pH of the enzyme remains to be elucidated [6][7][8][9]. The optimum temperature for the enzyme activity was found to be 55°C at pH 6.0 (Fig.…”

“…is regarded as a group of bacteria particularly efficient in the breaking down of chitin [5]. However, only a few thermostable chitinolytic enzymes from bacteria have been reported [6][7][8][9]. Previously, thermostable chitinases have been isolated from Bacillus licheniformis MB-2 [8] and Streptomyces thermoviolaceus OPC-520 [9].…”

“…Thus, chitinolytic enzymes have been studied and purified from many microorganisms, and their enzymatic properties have been investigated [2][3][4][5][6][7][8][9]. Bacillus spp.…”

Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1

“…Bacterial species such as Bacillus , Pseudomonas , Serratia and Arthrobacter have been proved in controlling the fungal diseases (Joseph et al 2007) Earlier reports showed that microorganisms capable of lysing chitin, which is a major constituent of the fungal cell wall, play an important role in biological control of fungal pathogens (Yu et al 2002; Zhang and Fernando 2004; Abdullah et al 2008). Fungi such as Trichoderma and bacteria such as Bacillus , Serratia and Alteromonas were reported to have chitinolytic activity (Mabuchi et al 2000; Someya et al 2001; Wen et al 2002; Huang et al 2005; Viterbo et al 2001). Non-pathogenic soil Bacillus species offer several advantages over other organisms as they form endospores and hence can tolerate extreme pH, temperature and osmotic conditions.…”

Purification of Chitinase enzymes from Bacillus subtilis bacteria TV-125, investigation of kinetic properties and antifungal activity against Fusarium culmorum

“…And, plants produce chitinase enzyme to defend themselves against fungal pathogens [9]. The baculoviruses used in biological control of insect pests produce chitinase for pathogenicity [10]. Chitooligosaccharides are produced via chitinase enzyme.…”