Chitinase from thermotolerant bacteria : enzyme characterization and gene cloning

Abstract

Thermotolerant bacteria isolate, SK-1, which produced high extracellular chitinase activity, was isolated from soil in Angthong Province, Thailand, optimum for growth for SK-1 in LB medium was at pH 7.5 and at 50 ℃. SK-1 can also grow in medium containing NaCl from 0 to 10% (optimum, 1% NaCl). The properties of the isolate, such as the formation of endospore, having peritrichous figella, and the partial 16S ribosomal RNA sequence, indicated that it belongs to Bacillus licheniformis. The enzyme production from B. licheniformis SK-1, in 0.02% colloidal chitin minimum medium, was found during log phase. The optimum for enzyme production from B. licheniformis SK-1 was cultured in 0.08%CCMM, pH 7.5 at 50 ℃. Crude enzyme contains at least 2 activities, a high activity chitinase, and chitobiase. The optimum pH and temperature of crude enzyme was 6.0/60 ℃. B. licheniformis SK-1 chitinase can hydrolyze regenerated chitin the best followed by, colloidal chitin, powdered chitin, partially N-acetylated chitin, chitosan, and flaked chitin, respectively. Products from this enzyme, analyzed by HPLC, a mixture of Nacetylglucosamine (GlcNAc) and chitobiose (GlcNAc)2. SDS-PAGE analysis of crude enzyme from culture medium from B. licheniformis SK-1 following by activity staining, using glycol chitin as substrate, eight chitinolytic activity bands were observed with molecular weight ranging from 20 to 72 kDa. Chitinase was partially purified from the culture medium of B. licheniformis SK-1 by colloidal chitin affinity adsorption followed by DEAE-cellulose column chromatography. The partial purified enzyme showed a single protein band on native polyacrylamide gel electrophoresis. The isoelectric point of the major component in the partial purified chitinase was 4.62. The partial purified chitinase showed a major band with MW 72 kDa and 2 minor bands with MW 58, 70 kDa on SDS-PAGE, respectively. The partial purified chitinase revealed two activity optima at pH is 6 and 8 when colloidal chitin was used as substrate and was stable in pH 6-8. The partial purified enzyme exhibited a broad activity temperatures ranging between 40 to 70 ℃, with optimum at 55 ℃ and retained 94%, 83 and 22% activity after heat at 40, 50 and 60 ℃ for 12 hrs, respectively. The Km and Vmax of the partial purified chitinase was 0.23 mg colloidal chitin m f' and 7.03 U mg-1. Shotgun cloning of the PstI partially cut genomic DNA of B. licheniformis SK-1 was investigated. After screening 5,000 transformants, no positive clones were found. Then the full length chitinase gene was amplified using primers, specific for Bacillus chitinase gene, BP-F and BP-R. The positive clone was screened on CCMM. The positive clone contained a plasmid with 2-kb insert fragment, pSKChi66. The plasmid pSKChi66 had one open reading of 1,797 bp which encoded a polypeptide of 598 amino acid residues, corresponding a to 66 kDa protein with an isoelectric point 5.02. The amino acid comparison indicated Chi66 is 84% similar to chitinase from B. subtilis TP-1 and chitinase from B. licheniformis. Crude chitinase from pSKChi66 was characterized. The optimum pH and temperature was pH 5.0 and 60 ℃. Crude enzyme hydrolyzed colloidal chitin the best followed by powdered chitin, PNAC, flaked chitin, chitosan and regenerated chitin, respectively. Determination of hydrolytic products by HPLC, found a NN’-diacetylchitobiose from colloidal chitin. SDS-PAGE analysis of chitinolytic enzyme from culture medium from recombinant clone showed three bands of protein with chitinase activity. The molecular weights were approximately 70, 65 and 58 kDa, which is similar with crude and partial purified enzyme from B. licheniformis SK-1.