Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium, Alteromonas sp. Strain O-7

Abstract

The chitinase B (ChiB) secreted by Alteromonas sp. strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced. The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30°C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0°C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The k cat /K m value for the ChiB reaction at 10°C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.Chitin, an insoluble homopolymer of ␤-(1,4)-linked N-acetylglucosamine (GlcNAc), is an abundant organic compound in nature. This polysaccharide is found in the exoskeletons and endoskeletons of many marine organisms, including mollusks, coelenterates, protozoa, fungi, and crustaceans. Since carbon and nitrogen are generally limited in the marine environment, chitin is a particularly important nutrient source for marine organisms. Chitinolytic marine bacteria play a crucial role in the recycling of chitinous materials for maintenance of the ecosystem in the marine environment (13). The concerted action of two chitinolytic enzymes, chitinase (EC 3.2.1.14) and ␤-N-acetylglucosaminidase (EC 3.2.1.30), is considered to be essential for the complete hydrolysis of chitin to GlcNAc. Chitinases cleave glycosidic linkages of GlcNAc randomly to produce soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by ␤-N-acetylglucosaminidases. Finally, the degradation products, mainly GlcNAc, are then taken up by the cells as a carbon and nitrogen source.Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin and an efficient producer of chitinolytic enzymes (33). This strain produces at least three chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), three ␤-N-acetylglucosaminidases (GlcNAcases A, B, and C), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), and a chitin-binding protease (AprIV) in the presence of chitin. We have cloned and sequenced...

Roles of Four Chitinases (ChiA, ChiB, ChiC, and ChiD) in the Chitin Degradation System of Marine Bacterium Alteromonas sp. Strain O-7

“…strain O-7 includes four chitinases (12,15,18,21), three ␤-N-acetylglucosaminidases (16,17,20), a transglycosylative enzyme (19), a chitin-binding protein (21), and a chitin-binding protease (11). The present study was conducted to elucidate why the strain produces multiple chitinases in the presence of chitin.…”

“…The recombinant ChiA, ChiB, and ChiD proteins were constructed as described previously (12,21). The expression plasmid pET-ChiC, coding for ChiC, was constructed as follows.…”

“…Chitinase activities were measured as described previously (12,14), with powdered chitin, colloidal chitin, glycol chitin (Seikagaku Co., Tokyo, Japan), or p-nitrophenyl-N,NЈ-diacetylchitobiose [pNP-(GlcNAc) 2 ] (Seikagaku Co.) as the substrate. One unit of chitinase activity was defined as the amount of enzyme releasing 1 mol of reducing sugar or 1 mol of p-nitrophenol per min.…”

“…strain O-7 is a gram-negative, flagellated, motile, aerobic, rod-shaped bacterium of marine origin and is an efficient producer of chitinolytic enzymes (14). We have already shown that the chitinolytic system of the strain consists of four chitinases (ChiA, ChiB, ChiC, and ChiD), three ␤-Nacetylglucosaminidases (GlcNAcase A, GlcNAcase B, and GlcNAcase C), a transglycosylative enzyme (Hex99), a chitinbinding protein (Cbp1), and a chitin-binding protease (AprIV) (11,12,(15)(16)(17)(18)(19)(20)(21). ChiA, ChiB, ChiC, and ChiD have sequence similarities to family 18 bacterial chitinases (4,7,8).…”

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

“…1), indicating that Vlchit1 was a member of glycosyl hydrolase family18. The potential substrate binding site (VMLSIGG) where X of XXXSXGG represents hydrophobic domain was located at aa site124 and the catalytic active site (FDGIDIDWE) was located at aa 163 site where glutamic acid (Glu171) is the critical residue involved as the proton donor in the catalytic doubledisplacement mechanism during hydrolysis (12,18).…”