Title of Invention	"AN ENZYMATIC PROCESS FOR THE PREPARATION OF LOW MOLECULAR WEIGHT CHITOSAN"
Abstract	The present invention relates to an enzymatic process for the preparation of low molecular weight chitosan. The process employs the depolymerization of chitosan in aqueous acidic medium by a commercially available partially purified enzyme pectinase from Aspergillus niger.

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The present invention relates to an enzymatic process for preparing low molecular weight chitosans. The invention, in particular describes the production of low molecular weight chitosans QLMWCVwhich having wide range of applications in various fields such as medicine, agriculture and biotechnology.) Chitin is the second most abundant natural biopolymer which is ectodermal in origin and found in the exoskeletons of crustaceans and cell walls of fungi, moulds and insects. Chemically it is a polymer of N-acetyl-D-glucosamine, joined by p 1,4-linkages. Chitosan is a collective name representing a family of de-N-acetylated chitins, deacetylated to different degrees. Because of the presence of amino groups, both chitin and chitosan find extensive applications in various fields. In India, the production of chitosan per annum is over 300 metric tonnes. / For Chemistry, biochemistry and functional aspects of chitin/chitosan, G.S. Braek, T. Anthonsen and P.A. Sand ford (Eds.), Chitin and Chitosan, Elsevier Applied Science, NY, 1989 may be referred. The wide application of these biomolecules is because of their biological activity, biocompatibility, biodegradability and their less toxic nature. However, the high molecular weight of chitosan that restricts the solubility in the aqueous medium and high viscosity even at low concentrations are the limiting factors of chitosan to be used for specific applications. The potentiality of chitosan mainly depends on its molecular weight and the consequent ease of its solubility in aqueous medium and hence, of late more importance is given to low molecular weight chitosans (LMWC), which are easily soluble in aqueous acidic or neutral medium. LMWC has various applications because of its antibacterial, antitumor, hypocholesterolemic, antihypertensive and immunopotentiating activities. Acidic and enzymatic degradations are the methods available for the depolymerization of chitosan to get LMWC. Enzymatic method is the preferred choice as the process of hydrolysis can be controlled precisely by varying the factors such as the reaction time, pH of the reaction medium, reaction temperature or enzyme concentration. Reference may be made to CFTRI patent No.433/DEL/01 entitled A process for preparation of low molecular weight chitosan, wherein chitosan is depolymerized by potassium persulphate induced free radical reaction for preparing LMWC. The drawbacks of this method are the possibility of random cleavage of chitosan, structural modification induced by persulphate free radicals and recovery of moderately high molecular weight products. Reference may be made to D. Somashekar and R. Joseph, Bioresource Technol., 55 (1996) 35-45, wherein chitosanases from different sources are used for the production of low molecular weight chitosans and chitooligomers. The drawback of the process is the unavailability of the reported enzymes in bulk quantities and also the high cost of the enzymes. Reference may be made to You-Jin-Jeon, Pyo-Jam Park, Se-Know Kim, Carbohydr. Polymer, 44 (2001) 71-76, wherein chitosan solution is passed through a column containing immobilized enzyme to get partially hydrolyzed chitosan (PHC) and again adding the latter to the ultrafiltration membrane reactor system for further enzymatic conversion of PHC to chitooligosaccharides. The drawbacks of the process are cost of the immobilized enzyme column and long duration of the reaction. Reference may be made to Japanese patent No. 02011601A2, entitled Production of low molecular weight chitosan, wherein a neutral protease such as 'Protein P' or lipase Qipase P) is used for chitosan depolymerization process. The drawbacks of the process are very lengthy reaction time (upto 100 h) required and production of very low molecular weight chitosans. The main object of the present invention is to provide an enzymatic process for preparing low molecular weight chitosans which obviates the drawbacks discussed above. Another object of the invention is to prepare a range of low molecular weight chitosans, which find application in various fields such as medicine, agriculture and biotechnology. The novelty embodying the present invention is the depolymerization of chitosan in aqueous acidic medium by a commercially available partially purified pectinase from Aspergillus niger, which is a non-specific enzyme, to low molecular weight chitosans and chitooligomers in fairly high yield and high purity. The probable reaction mechanism is as follows, Accordingly, the present invention provides an enzymatic process for the preparation of low molecular weight chitosan, which comprises: [a] dissolving chitosan in 0.5N acetic acid having pH of 3.0 to 4.0 by stirring; [b] adding 5 to 15 units of pectinase from Aspergillus niger having MTCC No. CFR 1047 to the dissolved chitosan as obtained in step [a] and incubating the reaction mixture at a temperature of 27 to 47 degree C for a period of 1 to 6 hours; [c] arresting the reaction mixture of step [b] by boiling at 100 degree C for a period of 5 to 10 minutes followed by adding 2 N sodium hydroxide solution to obtain a precipitate having low molecular weight chitosan; [d] separating the low molecular weight chitosan from the precipitate as obtained in step [c] by conventionally known methods; [e] removing excess of sodium hydroxide from the separated low molecular weight chitosan as obtained in step [d] by washing with distilled water to obtain the desited low molecular weight chitosan. In an embodiment of the present invention, the aqueous acidic medium may be 0.5N acetic acid. In an another embodiment of the present invention, the incubation of the reaction mixture may be carried out at specific temperature of 35 to 40 degree C for a period of 2 to 3 hours. The preparation of low molecular weight chitosan is accomplished as described in the Scheme I The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 3.0 using dilute hydrochloric acid. To this 10 units of A niger pectinase was added and incubated at room temperature (27°C) for 3 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 70%). The molecular weight as determined by viscometric method was about 20000 Da. Example 2 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 3.0 using dilute hydrochloric acid. To this 10 units of A niger pectinase was added and incubated at 37°C for 3 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 65%). The molecular weight as determined by viscometric method was about 11000 Da. Example 3 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 3.0 using dilute hydrochloric acid. To this 10 units of A niger pectinase was added and incubated at 37°C for 6 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 72%). The molecular weight as determined by viscometric method was about 6000 Da. Example 4 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 3.0 using dilute hydrochloric acid. To this 10 units of A niger pectinase was added and incubated at 47°C for 2 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 68%). The molecular weight as determined by viscometric method was about 11000 Da. Example 5 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 4.0 using dilute sodium hydroxide solution. To this 10 units of A niger pectinase was added and incubated at room temperature (27°C) for 3 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 72%). The molecular weight as determined by viscometric method was about 60000 Da. Example 6 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 4.0 using dilute sodium hydroxide solution. To this 10 units of A niger pectinase was added and incubated at 47°C for 2 h. The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by centrifugation (10 min at 4000 rpm), and washed with distilled water to get low molecular weight chitosan (yield 69%). The molecular weight as determined by viscometric method was about 60000 Da. Example 7 Chitosan (1.75 g) of molecular weight 95,000 Da was dissolved in 200 ml of 0.5N acetic acid by stirring and the solution pH was adjusted to 3.0 using dilute hydrochloric acid. To this 10 units of A niger pectinase was added and incubated for 1 h at room temperature (27°C). The reaction was arrested by boiling for 5 min and adjusting the pH to 8.0 with 2N NaOH. The precipitate was separated by milrifugation (10 min at 4000 rpin), and washed with distilled water to get low molecular weight chitosan (yield 72%). The molecular weight as determined by viscometric method was about 60000 Da. Charactrization of LMWC I he low molecular weight chilosans were characterized by measurements of their solution viscosity, molecular weight, and spectral data. The spectra showed extra peaks reflecting the presence of chitosan polymorphs. The main advantages of the present invention are, 1. Use of low cost and commercially available A.niger pectinase enzyme 2. Shorter incubation time and milder conditions compared to the reported processes 3. Tlu- molecular weight of chitosan can be controlled by adjusting the reaction conditions, and therefore leading to tailor made end products in fairly very good yields 4. The low molecular weight chitosans, obtained were of high purity (95%) and can be used as such without any additional purification steps. We claim: 1. An enzymatic process for the preparation of low molecular weight chitosan, which comprises: [a] dissolving chitosan in 0.5N acetic acid having pH of 3.0 to 4.0 by stirring; [b] adding 5 to 15 units of pectinase from Aspergillus niger having MTCC No. CFR 1047 to the dissolved chitosan as obtained in step [a] and incubating the reaction mixture at a temperature of 27 to 47 degree C for a period of 1 to 6 hours; [c] arresting the reaction mixture of step [b] by boiling at 100 degree C for a period of 5 to 10 minutes followed by adding 2 N sodium hydroxide solution to obtain a precipitate having low molecular weight chitosan; [d] separating the low molecular weight chitosan from the precipitate as obtained in step [c] by conventionally known methods; [e] removing excess of sodium hydroxide from the separated low molecular weight chitosan as obtained in step [d] by washing with distilled water to obtain the desited low molecular weight chitosan. 2. A process as claimed in claim 1, wherein incubation of the reaction mixture is carried out at a temperature of 35 to 40 degree C for a period of 2 to 3 hours. 3. An enzymatic process for the preparation of low molecular weight chitosan substantially as herein described with reference to the foregoing examples.

Documents:

232-DEL-2002-Abstract-(07-07-2008).pdf

232-del-2002-abstract.pdf

232-DEL-2002-Claims-(07-07-2008).pdf

232-del-2002-claims.pdf

232-del-2002-complete specifiction (granted ).pdf

232-DEL-2002-Correspondence-Others-(07-07-2008).pdf

232-del-2002-correspondence-others.pdf

232-del-2002-correspondence-po.pdf

232-del-2002-description (complete)-07-07-2008.pdf

232-del-2002-description (complete).pdf

232-del-2002-drawings.pdf

232-DEL-2002-Form-1-(07-07-2008).pdf

232-del-2002-form-1.pdf

232-del-2002-form-18.pdf

232-del-2002-form-2.pdf

232-del-2002-form-3.pdf