PRODUCTION THEREOF.

The invention relates to a process for the production of depolymerized partially acetylated chitosan and to the new products obtained by this process, to the use of microorganisms of the genus Thermoactinomyces in a process for the production of an enzyme which depolymerizes partially acetylated chitosan and to a new strain of Thermoactinomyces .

Chitosan is the product of partial or complete deacetylation of chitin. Both chitinase (EC 3.2.1.14) and chitosanase act on partially acetylated chitosan. Such depolymerisation has been described with an enzyme of microbial origin, especially of the genus Bacillus and it has been tried to digest chitosan with chitinase produced by Streptomyces antibioticus. All these processes have not allowed commercial production of chitosan hydrolysates by enzymatic hydrolysis of industrial chitosans. Such products are of interest in various fields, especially in the fields which require the use of concentrated chitosan solutions of low viscosity and/or the use of well characterized chitosan fractions (acetylation degree, molecular weight distribution, viscosity).

It has been found that various strains of the genus Thermoactinomyces produce substantial quantities of an enzyme which depolymerizes partially acetylated chitosan when cultivated under appropriate conditions and that a new strain of Thermoactinomyce s is of special interest in this respect. This enzyme has some activity on colloidal chitin (i.e. 100% acetylated chitosan) and, therefore, acts as as chitinase. The right substrate, however, is a partially acetylated chitosan having an acetylation degree between 10% and 70% (which is the case with most industrial grades of chitosan). In the context of this specification the enzyme which depolymerizes partially acetylated chitosan will be called chitosanase.

The invention, therefore, relates to a process for the production of such chitosanase which is

characterized by the cultivation of Thermoactinomyces sp„ to a process for the production of such chitosan hydrolysates which is characterized by the use of such chitosanase and to the new strain of Thermoactinomyces which is identified by its deposition no. 1-1052 at the Institut Pasteur, Paris. The invention further relates to the new products obtained by this process and which are characterized by their viscosity, molecular weight distribution and degree of acetylation. In general, the molecular weight distribution will be determined by capillary viscosimetry and can vary from 10,000 to 1,000,000. The degree of acetylation can vary between 10 and 70%.

The genus Thermoactinomyces is a well known member of the actinomycetes family and is described in Bergey's Manual of Systematic Bacteriology, vol. 4 (1989), Williams & Wilkins, pages 2573-85. One of its character istics is the fact that colonies grow at high temperatures (55°C) and that they are extremely heat resistant. Typical species are T. vulgaris, T. thalpophilus, T. sacchari, T. dichotomicus, T. intermedius and T. putidus. (See the list of characteristics on pages 2583 and 2584 of the above-mentioned Manual).

In order to produce chitosanase under optimal conditions, i.e. to achieve maximum production of the enzyme in standard fermentation apparatus, preferably the strains are cultivated at 55°C in a culture medium containing an inducer like N-acetyl-D-glucosamine (hereinafter N-AGA). Other useful inducers are colloidal chitin, chitosan oligosaccharides and chitosan. The best inducer is N-acetylglucosamine. A typical culture medium contains 2 g/1 of such inducer, 2 g/1 of yeast extract, 2 g/1 casein hydrolysate, 50 ml/1 of a solution of mineral salts, 10 ml/1 of vitamins, 10 ml 1 of trace minerals, 1 g/1 of ammonium chloride and 50 mM MOPS buffer.

In the so-called fed-batch culture the first step is a batch culture during which the strain is grown on the standard medium except that N-acetylglucosamine is substituted by glucose as a carbon source. Under such conditions, biomass is produced and no enzyme synthesis can be observed because no inducer is present in the culture medium.

In the second step, the chitosanase synthesis is induced by adding sterile N-AGA at a constant flow rate of 0.6 g N-AGA/h into the fermentor with a peristaltic pump.

After a period of 24 hours typical amounts of 0.1 to 2.0 U/ml of chitosanase can be obtained. For the depolymerization of chitosan the culture broth can be used as such or the chitosanase is first isolated and concentrated before incubating it with the substrate. In fact, it is possible to depolymerize chitosan using various forms of the enzyme:

- whole culture (including cells);

- culture broth (supernatant);

- culture broth after concentration by ultrafiltration;

- liquid enzyme or after freeze-drying of the crude preparation; or

- purified enzyme.

The chitosanase activity of the culture broth is assayed at 60°C and pH 5 with a chitosan (20% acetylation degree) concentration of 1 % (w/v) in that the reducing power of the solution is measured after 5 and 10 minutes according to Kidby and Davidson, Anal. Biochem. 55(1), 321-25 (1973). Standard solutions of N-acetyl-D-glucosamine (N-AGA) are used as reference. One chito¬ sanase unit (U) corresponds to the appearance of one micromole of N-AGA per minute.

The purification of the chitosanase can be performed with known techniques. Best results have been obtained with a process consisting of 3 steps:

- diafiltration of the supernatant (obtained by centrifugation of the culture broth) using a membrane of 10,000 molecular weight cut-off in the presence of 20 mM Tris-HCl buffer, pH 8 at 20°C;

- 1st anion exchange chromatography using Sepharose Fast Flow support (Pharmacia). The buffer is 20mM Tris-HCl pH 8.0. In these conditions, chitosanase activity was not adsorbed

onto the support;

- 2nd anion exchange chromatography using Protein Pak HR DEAE 5 PW support (Millipore- Waters). The buffer was 5mM Tris-HCl, pH 8.0. In this case, chitosanase was adsorbed onto the support and further eluted by 50mM NaCl.

Especially interesting results are obtained with a new strain of Thermoactinomyces which is identified by its deposition no. 1-1052 at the Institut Pasteur, Paris (deposition date March 1, 1991). This new strain was isolated from a marshland in Hondschoote (France) in March 1989 and has the typical characteristics of the genus Thermoactinomyces as follows: thermophilic nature (growth at 55 ^β C), formation of aerial mycelium, Gram-positive reaction, single spores on both the aerial and substrate mycelium, resistance of the spores at lOO'C for 10 minutes and much ramification of the mycelium. Biochemical characteristics of the new strain in comparison to other Thermoactinomyce s strains are summarised in Table 1. In its properties the new strain is most comparable to T. sacchari DSM 43356 and T. vulgaris DSM 43062 which are however, less productive in chitosanase.

Table 1

Biochemical Characteristics that Distinguish the Different Species of the Genus Thermoactinomvces

^τ = type strain

^* = Strains DSM 43047, 43050 and 43062, referenced as T. vulgaris in the DSM catalog, are quite different from the "type strain" DSM 43016 (T. vulgaris ^τ ).

The new chitosanase which is obtained from Thermoactinomvces 1-1052 has the following physico-chemical characteristics:

-molecular weight: 43,000 daltons

-monomeric protein

-isoelectric joint (pi): 6.9

-max. temperature activity: 70°C

-optimal pH activity: 4.5

Its kinetic parameters can be determined for a specific substrate and are the following:

-Affinity constant, Km : 3.5 g/1

-Maximal initial rate, Vm : 160 U/mg protein

when using an industrial grade of chitosan (PROFLOC 340) with a degree of acetylation of around 20% as the substrate (dissolved in acetic acid, temperature 60°C, pH 4.7).

The process for the production of chitosan hydrolysates is performed in accordance with conventional methods well known to the man skilled in the art. It has been found that the preferred conditions for the depolymerization of chitosan are as follows:

- swell chitosan powder in acetic acid (or any convenient organic acid e.g. formic or lactic acid);

- pre-heat the chitosan solution at 30-60°C;

- add the chitosanase and mix with the chitosan solution;

- incubate the reaction mixture;

- denaturate the chitosanase by heating the reaction mixture (lh at 90°C) or by precipitating chitosan at alkaline pH.

Partially deacetylated chitin polymers with a degree of acetylation above 25% are not industrially

available at present. In order to obtain such products as starting materials, an industrial grade of chitosan with a degree of acetylation of about 20% can be submitted to partial reacetylation according to the method of Hirano S., Tsuchida M. and Nagao N., Biomaterials (1989), Vol. 10 (October), 574- . More particularly, reacetylation can be performed under the following conditions:

- dissolution of 300 g of chitosan in 6 liters of 2% aqueous acetic acid,

- addition of 30 liters of methanol, followed by vigorous stirring,

- addition of 42ml of acetic anhydride followed by a 10 hour rest period at room temperature,

- polymer precipitation by adding sodium hydroxide until the pH reaches a value close to 8,

- filtration and extensive washing of the precipitate,

- drying.

Starting with a chitosan with a Mv of 500,000 and a D.A. of 20%, a product with a Mv of 250,000 and a D.A. of 35% is obtained.

For the characterization of chitosan depolymerized fractions the following methods are used:

Molecular weight determination

The intrinsic viscosity ([η]) of each chitosan sample is determined using a capillary viscometer (Ubbelohde type, 25°C) at various polymer concentrations. Intrinsic viscosity allows the determination of viscosimetric average molecular weight (Mv) by using the following relation:

[η] = 1.81 x 10 ³ Mv ⁰⁹³

in which the values of the constants are strongly dependent on the degree of acetylation, the buffer used and the ionic strength according to Roberts and Domszy (Int. J. Biol. Macromol. 4 (6), 374-377 (1982).

Acetylation degree determination

The degree of acetylation (D.A.) is assayed by *H N.M.R. spectroscopy using an AC 300 Bruker model, at 30°C. Samples are purified by alkaline precipitation, lyophilized and solubilized in D ₂ O.

D.A. is determined using the -CH ₃ signal (1.91 ppm) and compared to Hj signal (reference).

With the above described process also chitosan oligosaccharides with low molecular weight which do not precipitate at alkaline pH (degree of polymerization between 2 and 12) can be obtained.

The new products obtained according to the invention can be used in various industrial applications. They are useful additives for pharmaceutical compositions in which they act as an efficient promotor of wound healing and inhibitor of fibroplasia. They are also of interest for wound dressings and production of absorbable sutures.

In the cosmetic field, the new products can be used for hair, skin and mouth care, where presently cosmetic formulations are limited by the poor solubility of high molecular weight polymers.

For such cosmetic and pharmaceutical applications, the abovementioned oligosaccharides are especially useful.

SUBSTITUTE SHEET

They can also be used as auxiliaries in paper making in which they replace (in part or in total) synthetic polymers used as flocculating agents, fixing agents, drainage agents and retention agents.

In the following examples, all temperatures are expressed in centigrades and all references to parts are understood to be parts by weight, unless stated otherwise.

Example 1

Isolation of Thermoactinomyces sp. 1-1052

Twenty samples collected from various ecological environments were screened for the isolation of chitosanase producing bacteria. This step of screening was performed at 55 ^β C in order to obtain thermophilic microorganisms.

The first step consisted of an enrichment investigated in liquid medium in erlenmeyer flasks of 125 ml containing 10 ml of medium in which was dispersed each sample.

The mineral salt solution contains 6g KH ₂ PO ₄ , 12g NaCl, 2.4g MgSO ₄ and 1.6g CaCl _s per liter H ₂ O. The vitamin solution contains 2mg biotin, 2mg folic acid, lOmg pyridoxin-HCl, 5mg thiamin-HCl, 5mg riboflavin, 5mg nicotinic acid, 5mg Calcium pantothenate, O.lmg vitamins B ₁₂

TITUTE SHEE ⁱ

and 5mg lipoic acid per liter H ₂ 0. The oligoelement solution contains 0.3g FeS0 ₄ .7 H ₂ 0, O.lg MnCl ₂ , O.lg CoCl ₂ .2 H ₂ O, O.lg ZmCl ₂ , 0.02g CuCl ₂ , O.Olg H ₃ BO ₄ , 0.017g NA ₂ SeO ₃ , 0.026g NiS0 ₄ .6 H ₂ O and 12.8g nitrilotriacetic acid per liter H ₂ O.

After incubation for 24 hours at 55°C under continuous agitation on an orbital shaker water bath, each sample was assayed at 60°C for chitosanase activity with the procedure mentioned above.

The samples showing enzymatic activity towards chitosan were selected for isolation of microorganisms on agar plates containing collidal chitin.

The second step of the screening consisted of an isolation of the microorganisms on agar plates containing colloidal chitin. The test for enzymic activity was based on the appearance of clearing zones around the active colonies. Due to its insolubility at pH 7.0, chitosan could not be used for agar plates and colloidal chitin was substituted for chitosan in this second step.

The composition of the agar plates was: Nutritive Gelose supplemented with colloidal chitin (4 g/1) prepared by the method described in Methods in Enzymology (vol. 161 (1988)).

Active colonies making clearing zones on agar plates containing colloidal chitin were subjected for chitosanase assay on chitosan 1% at 60°C pH 5 and one showed good activity. This strain was then selected and was maintained on agar plates containing colloidal chitin or frozen at -80°C after addition of 50% glycerol (v/v).

The new strain of Thermoactinomvces sp. was isolated from a marshland in Hondschoote (10 km from Dunkerque/Nord (59) France) in March 1989.

Example 2

Cultivation of Thermoactinomvces strains in erlenmeyer flasks

Medium composition

- inorganic solution 50 ml/1

- oligoelement solution 10 ml/1

- vitamins 10 ml/1 - NH ₄ C1 l g/1

- yeast extract (BioMerieux, France) 2 g/l

- casein hydrolysate (Biotrypcase, BioMerieux) 2 g l

- N-acetyl-D-glucosamine (N-AGA) l g/1

Culture conditions

- orbital shaker

- agitation 150 rpm -temperature 55° -volume 100 ml (total volume of the erlenmeyer flasks 500ml)

The activity of different Thermoactinomvces strains is determined according to the method described above using the culture broth after 24 hours of fermentation in erlenmeyer flasks. The results are summarised in Table 2

TABLE 2

Example 3

Cultivation of Thermoactinomvces sp. 1-1052 in a 2 liter fermentor

-batch culture -fed-batch culture

Culture conditions

-volume 1.5 liters

-temperature 55°

-agitation 400 rpm

-aeration lwm

a) Batch culture

A batch culture using the medium described in Example 2 allows the production of 0.56 U/ml after 24 hours of fermentation.

b) Fed-batch culture

The first step is a batch type culture during which the strain 1-1052 is grown on the medium described in Example 2 except that the N-acetylglucosamine is substituted by glucose. Under such conditions biomass is produced and no enzyme synthesis can be observed because no inducer is present in the culture medium.

In the second step, the chitosanase synthesis is induced by adding sterile N-acetylglucosamine (N-AGA) at a constant flow rate of 0.48g N-AGA/h into the 2 liter fermentor with a peristaltic pump (fed-batch process).

This (fed-batch) process allows the obtention of 2 U/ml of chitosanase after 24 hours of fermentation with the strain 1-1052.

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Example 4

Isolation of "chitosanase"

The cells of Thermoactinomvces sp. are eliminated by continuous centrifugation at 4°C. The supernatant is filtered on paper Whatman GFF (0.7 micron filter) and concentrated by ultrafiltration using a Millipore- Waters tangential flow system (cut-off 10,000 daltons, PTGC cassette). During this procedure the temperature is maintained ≤ 25°. The enzyme concentrate can be further micro-filtered using an Amicon hollow fiber system (cut-off 0.1 micron) in order to eliminate cell debris and insolubles still present in the enzyme solution. Finally, the enzyme solution can be freeze-dried, optionally in the presence of 5 g 1 mannitol as a stabiliser, or frozen below -18°. The enzyme solution can also be kept for several weeks at 4°.

Example 5

An industrial grade of chitosan (PROFLOC 340 from Protan Laboratories, Norway) with a degree of acetylation of around 20% is dissolved in 0.4% acetic acid to obtain a 1% (w/w) solution with pH 4.7 and submitted to enzymatic depolymerization by the purified chitosanase enzyme from Thermoactinomvces sp. 1-1052 at 60°C and using an enzyme concentration of 0.025 U/ml (2.5 U/g chitosan). Reducing sugars are assayed concomittantly with the decrease of viscosity of the reaction mixture during the incubation time. The results are illustrated in Figure 1.

Example 6

4 chitosan samples are used, all provided by ABER TECHNOLOGIES, France. One of these samples (ref. 920629) was reacetylated starting from the crude industrial chitosan (ref. Chit 78). The degree of acetylation is measured using ^! H NMR spectroscopy. In each case 1% chitosan solutions are treated with a crude chitosanase enzyme preparation at a concentration of 0.1 U/ml at 60°C and pH 4.7.

^" < — on 5CC I

The results are illustrated in Figure 2.

Example 7

4 chitosan samples are used with the indicated degree of acetylation (D.A.)

- chitosan from ABER TECHNOLOGIES 9% DA

- PROFLOC 340 from PROTAN LABORATORIES 20% DA

- reacetylated PROFLOC 340 60% DA

- reacetylated PROFLOC 340 87% DA

In this experiment, the degree of acetylation is determined by the measurement of amino groups using the Ride and Drysdale method (Physiol. Plant Pathol. (1972) Vol. 2, 7-15).

1.25% chitosan solutions are treated with purified chitosanase in low concentration (0.003 U/ml). The results are illustrated in Figure 3.

Example 8

Two sets of similar experiments are performed under the following conditions:

- chitosan concentration : 5% (w/w)

- acetic acid: 2% (v/v)

- pH: 4.7

- temperature: 60°C

- enzyme concentration: 4 U/g chitosan

- incubation time: 72 hours (experiment 2)

120 hours (experiment 1)

^{■ ■} ITΓ O ___ ^{■ p} ~ *-"τ

After depolymerization, sodium hydroxide is added to the reaction mixture until the pH is 8.6. The precipitate is centrifuged and extensively washed before freeze-drying. The results are presented in Table 3

TABLE 3: Characterization of chitosan fractions

*Reacetylated CHIT 78

Example 9

A sample of reacetylated chitosan (obtained from crude industrial chitosan - ref. CHIT 78, ABER TECHNOLOGIES, France) with a degree of acetylation of around 55% is dissolved in 4% acetic acid to obtain a 10% solution with pH 4.7 and submitted to enzymatic depolymerization by the purified chitosanase from Example 1 at 40°C and using an enzyme concentration of 2U/g chitosan. After incubation during 64 hours a product is obtained with viscosity at 30°C (Brookfield LVT) of 40 cps and Mv 11,000. Upon freeze-drying the solution after enzyme denaturation (30 minutes at 90°C) a powder with water content of 6.5% is obtained.

Examples 10-23

According to the methods described before, further samples of depolymerized chitosan are prepared. The intrinsic viscosity [η] and viscosimetric molecular weight (Mv) of the starting materials (PROFLOC 340 and FLONAC, Japan) and the corresponding depolymerized chitosans are indicated in Table 4. Purification of chitosan was performed by alkaline precipitation (pH 7.5) using ammonium hydroxide, and further dissolving them in acetic acid 0.1 N/sodium chloride 0.2 N (for viscosimetric measurement).

TE SHEET

TABLE 4

The comparison of the molecular weights obtained by gel filtration and by capillary viscosimetry is illustrated in Table 5.

TABLE 5

Example 24

A 10% chitosan solution of PROFLOC 340 (degree of acetylation < 30%; Protan Laboratories, Norway) is prepared using acetic acid with a final concentration of 4%. The pH of this solution is 4.8. The enzyme of Example 1 is added and the solution incubated at 60°C. The enzyme concentration is 0.25 U/ml, corresponding to 2.5 U/g of chitosan

TITUTE SHEET