Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.Chitin, an insoluble homopolymer of -(1,4)-linked
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...
Aims: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. Methods and Results: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single-and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87AE6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathioneSepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. Conclusions: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. Significance and Impact of the Study: The findings of this study provide important information on chitin degradation by microbial chitinases.
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