The chitosanase from Streptomyces sp. N174
Biochemical properties and recombinant production system

The chitosanase was first purified from supernatants of N174 strain cultures growing on chitosan as the main carbon source (31). The purification procedure involved precipitation with polyacrylic acid followed by two chromatographic steps on CM-Sepharose and Bio-Gel A 0.5 M. The purified enzyme was specific for chitosan degradation. No hydrolysis of colloidal chitin, purified chitin, CM-cellulose, Avicell, laminarin, N-N-diacetylchitobiose, p-nitrophenyl-b-D-N-acetyl-glucosaminide or p-nitrophenyl-b-D-glucosaminide was observed.

Maximal rates of hydrolysis was observed for chitosans with low degrees of acetylation (1-21%) but the N174 chitosanase was able to hydrolyse efficiently chitosans in the range of d.a. from 34 to 61%, still exhibiting half of the maximal hydrolysis rate against the most acetylated substrate. Glycol chitosan was hydrolysed at 35% of the maximal rate.

HPLC analysis of products obtained from the early stages of reaction indicated that the enzyme hydrolyses chitosan in an endowise manner.

The pH range for activity extended from 4.0 to 6.0 with a maximum at 5.5. The optimum temperature for a 10 min reaction at pH 5.5 was about 65 degrees C. The apparent Km for chitosan substrate (Sigma) was 0.029 mg/ml while kcat was 727.5/min.

Final products obtained from chitosan hydrolysis are dimers and trimers, accompanied by a very limited quantity of monomers (31).

Recently, Fukamizo and Brzezinski have extensively reviewed the properties of the chitosanase from Streptomyces sp. N174 (39).

In order to intensify enzyme production, a recombinant system was developed. The host strain, Streptomyces lividans TK-24, does not produce a typical chitosanase as shown in our earlier work (33).

S. lividans colonies on chitosanase detection medium (34): a minimal medium with microcrystaline chitosan as carbon source. Hydrolytic activity is visualized by clearing zones around the colonies. The topmost colony in the picture on the left is S. lividans TK-24 without plasmid; the other colonies are strains transformed with vectors harboring chitosanase genes. Click on the picture to see a larger more detailed version.

An efficient chitosanase production system was obtained by cloning the CSN gene into the pFD 666 vector constructed in our laboratory (Denis and Brzezinski, 1992) (35).

Click the image on the left to see a detailed restriction map of the vector.

Click here to retrieve the nucleotide sequence of pFD666

The neomycin/kanamycin resistance gene included in the vector pFD666 was subcloned from the plasmid pFDNeo-S. This plasmid contains a modified Neo-R gene originating from the transposon Tn5 of E. coli. The promoter of the neomycin/kanamycin resistance gene has been modified to increase the expression in streptomycetes. Furthermore, the resistance gene is flanked by restriction sites allowing an easy subcloning in a variety of plasmids, making a true cassette. Details can be found in Denis and Brzezinski (1991) (reference 47).

Click the image on the left to see a detailed restriction map of the vector.

Click here to retrieve the nucleotide sequence of pFDNeo-S

For the purification of chitosanase produced by recombinant strains, the procedure was simplified as the precipitation step is now omitted (36). After optimization of the culture conditions (37), quantities of chitosanase in the range of 0.5 - 1g/L can be routinely obtained.

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This page was created by Ryszard Brzezinski and Andrzej Neugebauer.
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Last updated: 2000/05/01