Title of Invention

A PROCESS FOR THE PERPARATION OF ALKALIP-HILIC AND THERMOPHILIC XYLANASE

Abstract

A process for the preparation of alkaliphilic and thermophilic xylanase which comprises cultivating Pseudomonas stutzeri such as herein described, in a nutrient medium such as herein described, containing assimilable nitrogen source, supplemented by conventional trace elements such as herein described, and a source of xylan in a known manner at a pH ≥ 7, separating cells from supernatant by conventional methods and recovering alkaliphilic and thermophilic xylanase from cell free supernatant by conventional precipitation method followed by conventional partial purification using dialysis technique.

Full Text

This invention relates to a process for the preparation of alkaliphilic and thermophilic xylanase. More particularly this invention relates to a microbial process wherein a extracellular microbial enzyme xylanase possessing activity at alkaline pH and high temperature of 80 to 120° C can be produced by means of xylanase producing strain of a mesophilic microorganism. Xylan is the major component of plant hemicellulose present in angiosperm cell walls ( Timell T E, 1967. Recent progress in the Chemistry of wood hemicellulose. Wood Sci. Technol, 1 : 45-70). It is probably the second most abundant carbohydrate polymer of plants. Xylans are heterogeneous polysaccharides consisting of a backbone of p-1,4- linked D- xylopyranosyl residues that often have O-acetyl, arabinosyl, and methylglucuronosyl substituents (Whistler R L & Richards E L,1970. Hemicelluloses, In : Pigman W & Horton D ( ed), The carbohydrates-chemistry and biochemistry, 2nd ed. Vol 2A. Academic Press, Inc., New York). A wide variety of microorganisms are known to produce xylanases, enzymes that are involved in the hydrolysis of xylan (Bastawade K B ,1992. Xylan structure, microbial xylanases and their mode of action. World Journal of Microbiology and Biotechnology, 8:353-368). In recent years increasing attention has been given to the study of xylan -degrading enzymes because of their potential application in different industrial processes. One area of application is in the pulp and paper industry where xylanase can be used for the bleaching of kraft pulps ( Viikari I, Kantelinen A, Sundquist J, Linko M.1994. Xylanases in bleaching : from and idea to industry. FEMS Microbiology Review, 13 : 335- 350). The use of xylanase prior to normal bleaching operation has been shown to significantly reduce the amount of chlorinated organic compounds formed during the bleaching process ( Senior D J, Hamilton J, Bernier R L, Manoir J, 1992. Reduction of chlorine use during bleaching of kraft pulp following xylanase treatment. Tappi Journal ,75 : 125-130). Since kraft process of pulp and paper making is carried out at alkaline pH and high temperature, the use of alkaline xylanases with higher temperature optima is considered to be advantageous. Alkaline xylanases will also find a number of other applications. For example because of high solubility of xylan at alkaline pH alkaline xylanases may have good potential for the hydrolysis of hemicellulosic waste to fermentable sugars. The prior art process normally employed for the production of xylanase is batch process using xylanase producing alkaliphilic or themophilic microbial strains. Reference may be made to Okazaki W, Akiba T, Horikoshi K, Akahoshi R, 1984. Production and properties of two types of xylanases from alkaliphilic thermophilic Bacillus sp. Applied Microbiology & Biotechnology, 19 : 335 - 340; Tsujibo H, Sakamoto T, Nishino N, Hasegawa T.lnamori Y, 1990. Purification and properties of three types of xylanases produced by an alkaliphilic actinomycete. Journal of Applied Bacteriology, 69 : 398-405; Dey D, Hinge J, Shendye A & Rao M, 1992. Purification and properties of extracellular endoxylanases from alkalophilic themophilic Bacillus sp. Canadian Journal of Microbiology, 38 : 436-442; Ratto M, Poutman K & Viikari I, 1992. Production of xylanolytic enzymes by an alkalitolerant Bacillus cirulans strain. Applied Microbiology and Biotechnology, 37 : 470-473. However xylanases produced from most of these alkaliphilic strains have their optimum pH around neutrality. Further most of these xylanases studied are active in slightly acidic conditions between pH 4 and 6 and temperature below 70° C. Due to high temperature and alkalinity of the pulp during pulp processing more thermophilic and alkalophilic xylanases are preferred over the currently used ones. We have discovered a xylanase enzyme which shows high activity at pH 5.5 to 9.0 and is -250 times more active at 80 to 100°C as compared to room temperature (28 to 30 °C). The enzymes also retains its ~42 % activity when sterilized at 120°C and 15 lb pressure for 10 min. These properties of the xylanase enzyme suggest commercial application especially in the paper and pulp industries. This xylanase enzyme can be isolated by cultivating a xylanase producing strain of Peudomonas stutzeri in a nutrient medium. The main object of the present invention is to provide a process for the preparation of alkaliphilic and thermophilic xylanase from a bacterium designated as Peudomonas stutzeri and deposited at NIO culture facility bearing culture number NIO-AB-001, which obviates the drawback as described above. Its morphology and physiology can be summarised as follows (all temperatures in degree centrigrades): Morphology on ZoBell Marine Agar at 28 + 2 °C Gram - negative, motile coccobacilli, colonies: 1 mm, offwhite, opaque, circular, entire, smooth, soft, not easily dispersed, butyrous.ln another embodiment a temperature of from 25 to 35°C is satisfactory, a temperature of about 28°C being optimal Physiology 28°C: (Table Removed) Accordingly the present invention provides a process for the preparation of alkaliphilic and thermophilic xylanase which comprises cultivating Pseudomonas stutzeri such as herein described, in a nutrient medium such as herein described, containing assimilable nitrogen source, supplemented by conventional trace elements such as herein described, and a source of xylan in a known manner at a pH ≥ 7, separating cells from supernatant by conventional methods and recovering alkaliphilic and thermophilic xylanase from cell free supernatant by conventional precipitation method followed by conventional partial purification using dialysis technique. In an embodiment of the present invention Pseudomonas stutzeri was isolated from the sediment biofilm developed on mild steel panels immersed in waters of a station in the Mandovi estuary. In another embodiment of the present invention the microorganism can be cultivated under aerobic conditions, in any convenient medium in which it will grow and produce extracellular xylanase. The typical medium include a chemically defined medium such as that described by Rodrogues and Bhosle (Biofouling, 4: 301 -308,1991) with supplementary carbon source of, for example, xylan. A supplement of about 0.1 % W7 V is desirable. In another embodiment of the present investigation the culturing may be effected in a batch-wise manner, according to conventional practice with a xylan- supplemented medium. In another embodiment of the present investigation xylanase enzyme production is enhanced during the stationary growth phase of the organism, preferably the pH of the medium may conveniently be about 7 to 10. In another embodiment of the present investigation the extracellular xylanase may be isolated from the culture supernatant (free from cells) by precipitation with 70% ammonium sulphate and deionized by using dialysis bags ( MW cut off of 8000 dalton). After dialysis, the isolated material can be used as a source of enzyme. In another embodiment the extracellular xylanase is partially purified by conventional dialysis technique. In another embodiment the preparation of alkaliphilic and thermophilic xylanase which comprises cultivating Pseudomonas stutzeri capable of producing xylanase in a nutrient medium containing assimilable nitrogen source, conventional trace elements and a source of xylan in a known manner at a pH more than 7, separating cells from supernatant by conventional methods and recovering alkaliphilic and thermophilic xylanase from cell free supernatant by conventional precipitation method followed by conventional partial purification using dialysis technique. In another embodiment the source of carbon used is selected from commercial xylan, xylose, galactose and sucrose. In yet another embodiment the pH of the growth medium is ranged between 7 to 10. In another embodiment the cultivation of Pseudomonas stutzeri is effected atleast 28 hours at temperature 28 - 30°C. In yet another embodiment the extracellular xylanase is precipitated with 70% ammonium sulphate, dissolved in glycine-NaOH buffer at a pH value of pH 9.5 and partially purified by conventional dialysis technique. . Enzyme production remained more or less same up to 22 days while biomass showed marginal decrease. A significant amount of xylanase was also produced when xylose was used as carbon source. However, compared to xylan lower xylanase activity was observed. The following examples illustrate the present invention and therefore should not be construed to limit the scope of the present invention. Example 1: Extracellular xylanase production by Pseudomonas stutzeri Extracellular xylanase production by Pseudomonas stuzeri was followed in a batch culture(Figure 1). The culture was grown in a 250 milliliter flask containing sixty milliliter litre of growth medium as defined above supplemented with 0.1 gram of xylan as carbon source for 48 hours. Aeration was provided by shaking the culture flask at 150 RPM using a Rotary shaker. The logarithmic growth continued for 18 hours during this stage xylanases production could be detected (Figure 1). Although xylanase production commence during the exponential phase of growth, the production was highest during the stationary growth phase. Example 2: Preipitation and recovery of xylanase The Pseudomonas stutzeri was grown in the growth medium as defined above supplemented with 0.1 grams of xylan as carbon source for 48 hour. Cells were removed by centrifugation at 8000 RPM and 4°C. Xylanase enzyme was isolated from the cell free supernatant by adding 70 grams of ammonium sulphate per 100 milliliters of the cell free supernatant. The precipitated enzyme was collected using a spatula and dissolved in 50 mM glycine-NaOH buffer of pH 9.5. The enzyme solution was dialysed overnight using the 50 mM glycine-NaOH buffer PH 9.5. The obtained dialysed solution of the enzyme was used to characterized the activities of the xylanase enzyme. Example 3: Effect of pH on xylanase activity and stability Effect of pH on the activity of the xylanase enzyme was studied using 50 mM sodium citrate(pH 4 to 6), 50 mM phosphate (pH 6 to 8) and 50 mM glycine-NaOH (pH 7.5 to 11) buffers. In order to assess the stability of the enzyme at various pH, one volume of the enzyme solution was mixed with one volume of above either of the buffer solution having different pH (5 to 11) and incubated for 30 minutes and the enzyme activity was assessed. The enzyme showed three maxima that is at pH 5.5 , 7, and 9.0 (Figure 2). Stability studies showed that the enzyme was more stable in the alkaline pH of 8 (Figure 3). Example 4: Effect of temperature on the xylanase activity and stability The effect of temperature was studied by assessing the activity of the enzyme at various temperatures ranging from 30 to 100° C.The enzyme activity increased with the increase in temperature (Figure 4). The enzyme showed highest activity at 80°C and then showed some decrease as the temperature was further increased to 100°C. The activity of the enzyme at 80°C was higher by a factor of about 250 as compared to that observed at 30°C. When the enzyme solution in a glycine-NaOH buffer of pH 9.5 was heated for 2 hour at 80°C (Figure 5) ~€3 % activity was retained Whereas, when the enzyme solution in glycine - NaOH buffer was heated for 80 minutes at 100°C ~45% enzyme activity was retained (Figure 6). Example 5: Effect of sterilisation on xylanase activity When the enzyme solution in glycine buffer of pH 9.5 was sterilized for 10 minutes at 120°C and 15 lb pressure the enzyme retained ~42 % of its activity (Table 1). Example 6: Effect of carbon sources on xylanase activity Effect of various carbon sources on the xylanase production was assessed by culturing Pseudomonas stutzeri in 50 milliliter of growth medium as defined above containing 0.1 grams of either xylan, xylose, arabionse, glucose, sucrose, galactose, cellobiose and carboxymethylcellulose as carbon source for 48 hours. Cells were removed by centifugation and supernatant solution was used to estimate the activity of the enzyme. Of these xylose produced higher amount of xylanase as compared to other sugars but relatively less than that obtained with xylan (Table 2). The other natural sources of xylan can also be utilised for the production of xylanase. Example 7: Effect of metal ions on xylanase activity Effect of metal ions like Ca, Mg, Fe, Hg, Cu and EDTA on the activity of xylanase was evaluated. Twenty microliter of xylanase enzyme was mixed 1mM of either Ca, Mg, Fe, Hg, Cu and EDTA for 30 min and the enzyme activity was measured. Ca, Mg, Fe, Cu, and EDTA did not inhibit the activity of the enzyme. Hg inhibited the enzyme activity (Table 3). The conclusion of the present invention is that a xylanase enzyme possessing activity at high temperature (80°C) and pH (9.0) is prepared by cultivation of Pseudomonas stutzeri. The xylanase possess useful properties which are similar and/or better than those currently available in the market. The main advantages of the present invention are: The enzyme shows good activity against xylan. The enzyme is active at pH 5.5 ,7 and 9.0 and shows better stability at pH 8. It shows highest activity at 80°C which is about 250 times higher than at 30°C. Enzyme retains -42% of its activity when sterilized at 120°C and 15 lb for 10 min. Xylose induced the production of the enzyme. Ca, Mg, Fe, Cu and EDTA did not inhibit the enzyme activity. Hg was found to inhibit the activity of the enzyme. A xylanase produced by Pseudomonas stutzeri was characterized by assessing the effect of various pH, temperature, temperature and pressure, substrate concentration, metal ions and sugars. The enzyme showed good activity against xylan and was active at high pH (9.0) and temperature (80°C) and was not inhibited by the presence of metal ions like Ca, Mg, Fe, Cu and EDTA and was induced by xylose. The said xylanase exhibits properties which are better than the presently available xylanase enzymes. Table 1 . Effect of sterlisation (Pressure 15lbs; Temperature 121 °C) for 10 mins on xylanase activity Table 2 . Effect of different sugars on xylanase activity Table 3. Effect of ions and chelator on xylanase activity (Table Removed) We Claim: 1. A process for the preparation of alkaliphilic and thermophilic xylanase which comprises cultivating Pseudomonas stutzeri such as herein described, in a nutrient medium such as herein described, containing assimilable nitrogen source, supplemented by conventional trace elements such as herein described, and a source of xylan in a known manner at a pH ≥ 7, separating cells from supernatant by conventional methods and recovering alkaliphilic and thermophilic xylanase from cell free supernatant by conventional precipitation method followed by conventional partial purification using dialysis technique. 2. A process as claimed in claims 1 wherein the nutrient medium used has following composition. NaCI 30.00 g KCI 0.75 g MgSO4 7.00 g NH4CI 1.00 g K2HPO4 (10%) 7.00 ml KH2PO4 (10%) 3.00 ml Trace metal solution 1.00 ml Distilled water 1000 ml pH 10.00 Sterilization at 121°C 15 min 3. A process as claimed in claims 1-2 wherein the nutrient medium was supplemented with trace metal solution which has the following composition Trace solution H3BO3 2.85 g MnCI2. 7 H2O 1.80 g FeSO4 7 H2O 2.49 g Na-Tartarate 1.77 g CuCI2 0.03 g ZnCI2 0.02 g COCI2 0.04 g Na2Mo04. 2H2O 0.02 g Distilled water 1000 ml 4. A process as claimed in claims 1 to 3 wherein the pH of the growth medium is 7 to 10. 5. A process as claimed in claims 1 to 4 wherein the cultivation of Pseudomonas stutzeri is effected for 28 hours at temperature 28 - 30°C. 6. A process as claimed in claims 1 to 5 wherein the extra cellular xylanase is precipitated with 70% ammonium sulphate, dissolved in glycine-NaOH buffer at a pH value of 9.5 7. A process for the preparation of alkaliphilic and thermophilic xylanase substantially as herein described with reference to the examples accompanying this specification.

Documents:

491-DEL-1999-Abstract-(31-12-2010).pdf

491-del-1999-abstract.pdf

491-DEL-1999-Claims-(31-12-2010).pdf

491-del-1999-claims.pdf

491-DEL-1999-Correspondence-Others-(31-12-2010).pdf

491-del-1999-correspondence-others.pdf

491-del-1999-correspondence-po.pdf

491-DEL-1999-Description (Complete)-(31-12-2010).pdf

491-del-1999-description (complete).pdf

491-del-1999-drawings.pdf

491-del-1999-form-1.pdf

491-del-1999-form-19.pdf

491-del-1999-form-2.pdf

491-del-1999-form-3.pdf

491-del-1999-petition-137.pdf

491-del-1999-petition-138.pdf