The gene encoding an extracellular chitinase from marine Alteromonas sp. strain 0-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase ofAlteromonas sp. strain 0-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.Chitin is a major component of the exoskeletons of insects and crustaceans, and it is abundantly distributed throughout nature. This polysaccharide is also an important nutrient source of both carbon and nitrogen in the marine environment. Yu et al. (38) pointed out that the oceans would be completely depleted of carbon and nitrogen in a relatively short time if chitin could not be returned to the ecosystem in a biologically usable form. However, marine sediment contains relatively little chitin, despite the production of huge amounts of this insoluble polysaccharide by crustaceans. ZoBell and Rittenberg (39) reported that chitinolytic bacteria, which are abundant and widely distributed in the sea, play an important role in converting insoluble chitin into a biologically usable form. Chitinases (EC 3.2.1.14) are enzymes which degrade chitin, and they have been detected in various microorganisms (3,25,28,30), plants (1, 18), insects (5, 13), and crustaceans (19). Alteromonas sp. strain 0-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin. This strain excretes chitinase into the growth medium in the presence of chitin (32). We have already reported the purification, properties, and partial amino acid sequence of the enzyme from this strain (33). Recently, bacterial chitinase genes from terrestrial and marine bacteria such as Serratia marcescens (6, 10), Bacillus circulans (35, 36), Vibrio harveyi (28), and Vibno vulnificus (37) have been cloned and sequenced. However, the mechanism of hydrolysis, the relationship between structure and function, and the regulatory system involved in enzyme induction are still unclear. In this report, we describe the cloning of a gene coding for chitinase and the purification of the gene products. The nucleotide sequence of the gene was determined, and the deduced amino acid s...

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

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when ␣-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc) 2 from chitin. The optimum temperature and pH of ChiD were 50°C and 7.0, respectively.Chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant polymer in nature. This polysaccharide is particularly an important nutrient source for maintaining the ecosystem in the marine environment (7). Chitinolytic marine bacteria play a critical role in the process of recycling chitinous materials such as the exoskeletons of crustaceans and insects. If the insoluble form of chitin could not be returned to the ecosystem in a biologically usable form, the marine environment would be completely depleted of a carbon and nitrogen source in a relatively short time (39). To degrade chitin, chitinolytic microorganisms produce two classes of enzymes: chitinases (EC 3.2.1.14) and ␤-N-acetylglucosaminidases (GlcNAcases; EC 3.2.1.30). Chitinases hydrolyze chitin to soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by GlcNAcases. 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 (30). This strain was isolated from a sediment sample at Sagami Bay in Japan as an efficient producer of chitinolytic enzymes (30). We have been studying the chitin degradation system of the strain as a model for defining the various components involved in chitin utilization. It was made clear that the chitinolytic system of the strain consists of at least four chitinases (Chi85, ChiA, ChiB, and ChiC) and three ␤-Nacetylglucosaminidases (GlcNAcaseA, GlcNAcaseB, and GlcNAcaseC). The genes for these enzymes have been cloned and characterized previously to clarify the role of individual enzymes in the ...

No 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...