Title of Invention

A PROCESS FOR PRODUCTION OF XYLAN-DEGRADING ENZYMES

Abstract

A process for production of xylan-degrading enzymes: This invention provides a process for production of the xylan-degrading enzyme endo-1,4-ß -D-xylanase, using a marine strain of the fungus Aspergillus niger. The endo-xylanase produced by this culture does not act on cellulose and is thus cellulose-free. Besides, the culture is able to grow and produce xylanase in the presence of 0.1 - 0.2% black liquor and marine water added to nutrient medium. The said fungus also produces other xylan - degrading enzymes such as p -xylosidase and a -L-arabinofuranosidase in the culture medium.

Full Text

This invention relates to a process for production of xylan-degrading enzymes. This invention particularly relates to a process for production of the xylan-degrading enzyme endo-1,4-ß-D-xylanase, using a marine strain of the fungus Aspergillus niger, NIOCC isolate # 3. Further, this invention relates to production of xylanases, using conventional nutrient media. Furthermore, the endo-xylanase produced by the said fungus A.niger NIOCC isolate #3 is thermostable at 55°C for more than 4 h and has optimum activity at pH 3.5 and 8.5 and at temperature 50°C and 90°C. The endo-xylanase produced by this culture does not act on cellulose and is thus cellulase-free. Besides, the culture NIOCC #3 is able to grow and produce xylanase in the presence of 0.1-0.2% black liquor added to nutrient medium. The said fungus also produces other xylan-degrading enzymes such as ß-xylosidase and α-L-arabinofuranosidase in the culture medium. One of the major applications of xylanases is in the paper and pulp industry. In the process of making paper, lignin is removed from the pulp by cooking at a high temperature at alkaline pH. This process is termed Kraft process. The residual lignin that remains on the pulp is dark brown in color. In order to obtain high-quality white paper, all of the lignin must be removed. This is traditionally done with chlorine-based chemicals and is called bleaching. This bleaching process although highly effective in obtaining white paper, is very polluting, because it produces chlorinated lignins called chlorolignins. These are mutagenic and difficult to degrade. Therefore, the pulp and paper industries are actively looking for alternatives which would reduce or eliminate chlorine bleaching. Thus microbial enzyme system with high xylanase and very low to none cellulase activity (so that the cellulose part of the pulp is no degraded) is preferred in this process. Xylanases are hemicellulases that hydrolyze xylan which is a major constituent of hemicellulose complex in the plant cell wall. Xylan is composed of (3-1,4-linked xylopyranose units with branches containing L-arabinofuranosyl and glucopyranosyl residues. Attempts to use fungi and/or enzyme systems from fungi and bacteria to modify or decrease the lignin content in the pulp to obtain brightness are reported in literature. However, very few enzyme systems have been found which selectively act on hemicellulose and lignin and not adversely affect cellulose content of pulp. Further, hemicellulases, that are active at alkaline pH and high temperature are of great potential since they can be introduced at different stages of bleaching without much changes in temperature and pH. Moreover, the enzyme should be active in the presence of extensively modified and oxidized dark brown residual lignin in the pulp after the Kraft process. Moreover, complete degradation of a branched, acetylated xylan requires the concerted action of several different hydrolytic enzymes, including endo-1,4-ß-xylanase, ß-xylosidase, a-L-arabinofuranosidase, ot-glucuronidase and acetylesterase or acetylxylanesterase (Biely.P. 1985. Microbial xylanolytic systems. Trends in Biotechnology 3:286-290). Due to the importance attached to the prevention of environmental pollution, environmental agencies all over the world, including India are imposing strict regulations for mitigation of pollution from industries. The effluents from the paper manufacturing mills are a major source of concern for environmentalists, since such effluents contain toxic compounds such as chlorinated lignins (Springer, 1985. Industrial Environmental Control, pulp and paper industry. John Wiley & Sons, Inc., New York). Thus, the present invention particularly relates to production of xylanases for use in biobleaching of pulp in paper manufacturing mills by the fungus A. niger deposited at the National Institute of Oceanography, Dona Paula, Goa 403 004, India and having the accession number NIOCC 3. The said fungus can be grown in conventional media or in powdered sugarcane bagasse suspended in distilled water or half-strength sea water for large-scale production of the enzyme. The said fungus A.niger, NIOCC isolate # 3 produces xylan-degrading enzymes such as endo-1,4-ß-xylanase (EC 3.2.1.8), 4-ß-D-xylosidase (EC 3.2.1.37) and a-L-arabinofuranosidase (EC 3.2.1.55) when grown in conventional media prepared with distilled water or half-strength sea water supplemented with oat spelts xylan or birchwood xylan or larchwood xylan or sugarcane bagasse suspended in distilled water or natural or synthetic half-strength sea water. Xylanase produced by this fungus does not act on cellulose and is thus cellulase-free. Therefore, this enzyme would not affect the cellulose component of the pulp. Xylanases produced by this fungus would also be capable of reducing chlorine consumption to achieve desired brightness of the pulp, and thus help in reducing the toxic material from the effluent. Therefore, the present invention will be very useful for abating environmental pollution. Further, the enzyme endo-1,4,4-ß-D-xylanase produced by this fungus acts at pH 3.5 and 8.5. It shows temperature optimum at 50°C and 90°C. It is stable at 55°C and retains ca. 60% of its activity for more than 4 h at this temperature. Purified enzyme has high specific activity and its stability at 85°C increases with increasing concentration of protein in the enzyme solution. The said fungus produces xylanase, also when black liquor is added to the growth media. The optimum temperature and pH for ß-xylosidase activity are 70°C and 4.0 respectively. The optimum temperature and pH for α-L-arabinofuranosidase activity are 60°C and 4.0 respectively The filamentous fungus Aspergillus niger belonging to the class Deuteromycotina, is a ubiquitous terrestrial fungus. However, we isolated it from mangrove detritus using common fungal medium, namely com meal agar medium prepared with half-strength sea water. Normally, the enzyme xylanase having the international enzyme nomenclature code of EC 3.2.1.8, is prepared by growing a suitable microorganism in culture media containing a carbon source suitable for the production of enzyme, as well as nitrogen and mineral sources, using water as an aqueous medium. The above process resulting in production of the said enzyme has been particularly employed for bleaching of paper pulp. Thus, various organisms have been used for production of xylanases for use in paper and pulp industry. (i) A reference may be made to a US patent wherein, the fungus Thermomyces lanuginosus , strain # DSM 5826 is cultivated in a medium containing com cobs for the production of xylanase having optimum activity at pH 5.0-7.0 and temperature of 65*C. Besides xylanase, it also showed p-xylosidase, arabinosidase, acetylesterase, acetylxylan esterase and mannanase activity (Wizani et al., 1991. Preparation of xylanase by cultivating Thermomyces lanuginosus DSM 5826 in a medium containing corn cobs. Patent No. US 5183753). Our method of cultivating the fungus in sugarcane bagasse medium is advantageous, as it uses raw material readily available in a paper mill, (ii) A reference may be made to a US patent wherein, a bacterial strain, Thermomonospora fusca has been grown on wheat bran for the production of the said enzyme (Casimir-Schenkel et a/., 1991. Pulp bleaching with thermostable xylanase of Thermomonospora fusca. Patent No. US 5407827). Temperature and pH optima for this strain are reported to be 65°C and 6.0. The maximum activity of xylanase reported in this strain is only 2.1 nmol/min/ml, whereas our fungus strain produces 5 times more than this. (iii) A reference may be made to a US patent wherein, an actinomycete, Microtetraspora flexuosa produces thermostable xylanase with an optimum pH of 7-9 and an optimum temperature of 70-80°C and is claimed to be more efficient in delignifying and brightening pulp than prior xylanases (Bodie et al., 1997. Enhancing delignification and bleaching of wood pulp. Patent No. US 5683911). However, our strain produces other xy Ian-degrading enzymes such as beta-xylosidase and alpha-arabinofuranosidase in the same culture medium, (iv) A reference may be made to a patent wherein, xylanase produced by a bacterium, Acidothermus cellulolyticus (ATCC 43068) with a optimum pH of 3.6-4.2 and optimum temperature of about 70-80°C (Clarkson et al., 1997. Novel purified xylanase produced e.g. from Acidothermus sp. Patent No. WO 9720920). The drawback of this xylanase is that it is not active at alkaline pH. (v) A reference may be made to a patent wherein, a method for producing recombinant xylanase APX-II in the yeast Saccharomyces cerevisiae is claimed (Liang et a/., 1997. New isolated xylanase APX-II gene. Patent No. US 5591619). According to the claim, an isolated recombinant DNA molecule encoding Aureobasidium pullulans (Y-2311-1) endo-1, 4-ß -D-xylanase is cloned into the yeast S. cerevisiae , which successfully produces xylanase APX-II. It is claimed to degrade hemicellulose for potential use in pulp bleaching, (vi) A reference may be made to a patent wherein, a claim for removing color from wood pulp using endo-1,4-beta-D-xylanase, specifically xyl3, obtained from Streptomyces roseiscleroticus (NRRL B-11019) is made (Jefferies et a/., 1996. A method for removing color from Kraft wood pulps. Patent No. US 5498534). The enzyme works optimally at pH 7.0-8.5 but no claim regarding its thermostability is made. (vii) A reference may be made to a patent wherein, a novel bacterium which produces endo-1,4-beta-D-xylanase having activity at pH 4.5-9.0., having activity up to 80° C and stability up to 70°C (Dunlop et al., 1995. Novel bacterium which produces xylanase. Patent No. WO 9527779). However, xylanase produced by our fungus has these characteristics, and besides it also shows p-xylosidase and a-L-arabinofuranosidase activities and these three enzymes can have cumulative or synergistic effect on degradation of hemicellulose. (viii) A reference may be made to a publication wherein, Aspergillus niger isolated from terrestrial source is reported to produce two endo-xylanases, both having temperature optimum of 45°C and pH optimum of 5.5 and 6.0.(Frederick MM, Kiang CH, Frederick JR, Reilly PJ. 1985. Purification and characterization of endo-xylanases from Aspergillus niger. I Two isozymes active on xylan backbones near branch points. Biotechnology Bioengineering 27: 525-532). However, our isolate poduces endo-xylanases which show two peaks of activity, namely at 50°c and 90°C and two peaks of activity, namely at 3.5 and 8.5 in the crude culture filtrate. The main objective of the present invention is to provide a process for production of xylanases using the marine strain of the fungus Aspergillus niger NIOCC # 3 for possible use in bleaching of pulp and degradation of hemicellulose from plant cell wall in other processes which obviates drawbacks as detailed above. Another objective of the present invention is to provide a method for growing the said fungus on a large scale using inexpensive and readily available raw material like sugarcane bagasse suspended in distilled water or tap water or half-strength sea water supplemented with some nitrogen source like ammonium tartrate. Still another object of the present invention is to provide a culture having thermostable and alkaline xylanase which is cellulase-free. Still another object of the present invention is to provide a xylanase which is active in the presence of black liquor (untreated raw effluent of paper mill). Yet another object of the present invention is to provide a culture which besides having endo-1,4,-p-D-xylanase has other hemicellulose-degrading enzymes like 4-ß-D-xylosidase and α-L-arabinofuranosidase. The presence of these three xyIan-degrading enzymes could have cumulative or synergistic effect. In the drawings accompanying this specification Figure 1a represents production of endo-1,4,-p-D-xylanase by NIOCC isolate # 3 in the presence of various xylan-containing substrates in distilled water or half-strength sea water. Figure 1b represents relative production of xylanase in acidic as well as alkaline media. Figure 2 represents xylanase activity assayed at various pHs from 3.5-10.5. Figure 3 represents xylanase activity of the culture supernatant of NIOCC isolate # 3 grown in xylan containing medium assayed at various temperatures ranging from 30°C-100°C at pH 4.5 and 8.5. Figure 4 represents effect of pH on stability of xylanase at 0°C and at 30°C. Figure 5 represents thermostability of xylanase enzyme at 55°C, 60°C and 70°C from 1-4 hours. Figure 6 represents stability of xylanase at 85°C depending directly on protein concentration of the enzyme. Figure 7 represents effect of pH on ji-xylosidase activity of the culture supernatant from NIOCC isolate # 3. Figure 8 represents effect of temperature on p-xylosidase activity. Figure 9 represents effect of pH on a-L-arabinofuranosidase activity of the culture supernatant from NIOCC isolate # 3. Figure 10 represents effect of temperature on a-L-arabinofuranosidase activity. Accordingly the present invention provides a process for producing xylan-degrading enzymes which comprises, growing of a marine strain of the fungus Aspergillus niger, having characteristic such as herein described, in conventional media containing xylan-containing substrates as carbon source selected from 5% concentration of sugarcane bagasse and conventional nitrogen source preferably 2.4 mM ammonium tartrate, at pH ranging 3.5 and 8.5, in the presence or absence of black liquor obtained from paper mill at a concentration of 0.1 to 0.2%, for a period of 4 days to get xylan-degrading enzymes. In an embodiment of the present invention, the xylan-degrading enzymes include but not limited to 1,4-ß-D-xylanase, 4-ß-D-xylosidase and a-L-arabinofuranosidase enzymes. In another embodiment of the present invention the xylanase enzyme thus obtained is cellulose-free and has pH optimum at 3.5 and 8.5. In another embodiment of the present invention, the xylanase enzyme thus obtained has optimum activity at temperature 50°C and 90°C and the enzyme is thermostable at 55°C for at least 4 hours. In another embodiment of the present invention, 4-ß-D-xylosidase enzyme activity has optimum pH of 4 and temperature of 70°C. In yet another embodiment of the present invention, the enzyme a-L-arabinofuranosidase has optimum activity at pH 4 and at temperature of 60°C. In yet another embodiment of the present invention, the stability of the endo-xylanase at 85°C is dependent on protein concentration in the enzyme solution. In yet another embodiment of the present invention, the carbon source for growing the fungus used is selected from shredded or powdered sugarcane bagasse at 1 to 5% concentration and oat spelts xylan, birchwod xylan and breakfast grade cereal oats. In yet another embodiment of the present invention, the nitrogen source used for growing the fungus is ammonium tartrate at about 2.4mM concentration. In still another embodiment of the present invention, the culture medium can be prepared with distilled water or tap water or half-strength natural or synthetic sea water. In still another embodiment of the present invention, the culture medium for growing the fungus can have acidic or alkaline pH. In still another embodiment of the present invention, black liquor at 0.1 to 0.2% concentration can be added and at least 75% of the endo-xylanase activity is retained. The organism given in the present invention is a deuteromycete fungus isolated from decaying mangrove leaves from Mandovi estuary in Goa and identified as Aspergillus niger, having the accession number NIOCC # 3 and is deposited in National Institute of Oceanography, Dona Paula, Goa 403 004, India. The said fungus can be grown in malt extract broth containing malt extract and peptoneThe fungal mat grown this way may be macerated and used as starter inoculum for the experimental cultures containing sugarcane bagasse (shredded or powdered at 1 to 5% concentration) or commercially available xylan (1%) as carbon source. Ammonium tartrate at minimum concentration of 2.4mM may be used as nitrogen source. The pH of the medium can be adjusted to 4.5 or 8.5. This medium can be prepared with distilled water, tap water or half-strength sea water. Black liquor from paper mill at a concentration of 0.1 to 0.2% can be added to this culture medium. The cultures are grown as shallow static cultures at room temperature. The culture can be grown for 4-7 days. The cell-free clear supernatant is used as the source of endo-1,4-4-ß-D-D-xylanase, p-xylosidase and a-L-arabinofuranosidase enzymes. Endo-1,4-ß-D-xylanase activity is assayed by using 3,5-dinitrosalicylic acid to measure the amount of xylose-equivalent reducing sugars liberated from oat spelts xylan. p-D-xylosidase activity is assayed using p-nitrophenol-ß-D-xylopyranoside. a-L-arabinofuranosidase activity is measured using 1 mM p-nitrophenol arabinofuranoside. The said fungus Aspergillus niger NIOCC isolate # 3 is capable of growth and production of xylan-degrading enzymes in simple, inexpensive medium containing shredded or powdered sugarcane bagasse in distilled water, tap water or half-strength sea water, supplemented with ammonium tartrate as nitrogen source. The culture is fast-growing and by following our method of inoculation, maximum enzyme production can be obtained by day 4. Xylanase production is seen in the presence of black liquor and this novel feature has an added advantage because during bleaching of pulp in paper mills, the cooked pulp is covered with oxidized brown modified lignins which sometime are highly inhibitory to the action of bleaching enzymes during the process of biobleaching. The said fungus produces endoxylanase which has two pH and temperature optima in the crude culture filtrate itself. The same culture filtrate also shows activity of other xylan-degrading enzymes such as p-xylosidase and α-L-arabinofuranosidase. These three enzymes could have concerted effect on degradation of hemicellulose during bleaching process. These two enzymes also have optimum activity at 70°C and 60°C respectively. Thus, these enzymes can be used in bleaching of pulp without bringing down its temperature in the processing line in a paper mill. 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 The said fungus A.niger, NIOCC isolate # 3 is grown in malt extract broth containing 2% malt extract and 0.3% peptone in distilled water or half-strength sea water. The 7 day old fungal mat grown this way may be macerated and used as starter inoculum for the experimental cultures containing sugarcane bagasse (shredded or powdered at 1 to 5% concentration) or commercially available xylan (1%) as carbon source. The basal medium to which the above-mentioned xylan containing substrates are added independently, contains ammonium tartrate at minimum concentration of 2.4mM as nitrogen source. Besides it contains 2g KH2PO4, 1.45g MgSO4.7H2O, 0.132g CaCI2.2H2O, 1mg thiamine-HCI. The pH of the medium can be adjusted to 4.5 or 8.5 with sodium acetate or Tris-HCI buffer. This medium can be prepared with distilled water, tap water or half-strength sea water. The cultures are grown as shallow static cultures at room temperature. Black liquor at the concentration of 0.1 to 0.2% can be added to basal medium or to sugarcane bagasse medium. The culture can be grown for 4-7 days, filtered through cheese clothe or double-layered muslin clothe or filter paper or centrifuged at SOOOxg at 5°C for 10 min to obtain cell-free culture filtrate. The clear supernatant thus obtained is used as the source of endo-1,4-ß-D-xylanase. Endo-1,4-ß-D-xylanase activity is assayed by using 3,5-dinitrosalicylic acid to measure the amount of xylose-equivalent reducing sugars liberated from oat spelts xylan in 20mM sodium acetate buffer at pH 4.5 or Tris-HCI buffer at pH 8.5 and incubated at 50°C for 30 min in a final volume of 1ml. The reaction is terminated by adding 3ml of dinitrosalicylic acid reagent (Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry 31:426-428). The samples are cooled to room temperature, centrifuged at SOOOxg for 2 min and the absorbance read at 640nm. One unit of activity is defined as the amount of enzyme capable of releasing reducing sugars equivalent to one nmole of xylose per minute under the conditions described (Nakamura S, Wakabayashi K, Nakai R, Aono R, Horikoshi K. 1993. Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. Strain 41M-1. Applied Environmental Microbiology 59:2311-2316). Substrate and enzyme blanks were maintained for all the assays. All the assays were run in triplicates. Different concentrations of xylose were used to prepare a standard graph. Accordingly, Figure 1a of the drawings accompanying this specification shows comparative production of endo-xylanase in media containing sugarcane bagasse, breakfast oats, oat spelts xylan and birchwood xylan in media prepared with distilled water or half-strength sea water. Accordingly, Figure 1b of the drawings accompanying this specification shows relative production of endo-xylanase in acidic and alkaline media wherein the enzyme assay was carried out at acidic as well as alkaline pH. Although, maximum xylanase production was seen in alkaline medium, this was produced on day 13 whereas 60% of it was produced on day 4 itself in acidic medium. Table 1 shows relative production of xylanase in various concentrations of sugarcane bagasse added to basal medium or suspended in tap water with ammonium tartrate at a concentration of 2.4 mM. Table 1. Effect of concentration of sugarcane bagasse on the production of xylanase (Table Removed) Accordingly, to make the process cheaper, xylanase can be produced by •^ ; ' growing the fungus in 5% concentration of sugarcane bagasse/suspended in tap ,,v , water with 2.4 mM ammonium tartrate as nitrogen source. The crude culture filtrate showed no cellulase activity, both with crystalline cellulose as well as carboxymethyl-cellulose and is thus cellulase-free. Production of xylanase in bagasse medium in the presence of black liquor was tested by adding different concentrations of black liquor in tap water containing sugarcane bagasse and ammonium tartrate as mentioned in the above example. Accordingly, Table 2 shows production of xylanase in sugarcane bagasse medium in the presence of black liquor. Table 2. Effect of black liquor on the production of xylanase (Table Removed) EXAMPLE 2 The said fungus Aspergillus niger NIOCC isolate # 3 was grown as described in the Example 1 and the clear culture supernatant was used for assaying the optimum pH for endo-xylanase activity. The effect of pH on the reaction was assessed by incubating the reaction mixture containing 50mM sodium acetate buffer for pH range 3-6; 100rnM phosphate buffer for pH range 6-8 and sodium borate buffer for pH range 8-10. Endo-xylanase activity was measured as described in the Example 1. Accordingly, Figure 2 of the drawings accompanying this specification shows optimum activity at pH 3.5 and a second peak of activity at pH 8.5, where about 50% of the activity was measurable. EXAMPLE 3 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for assaying the optimum temperature for endo-xylanase activity. The effect of temperature was assessed by incubating the reaction mixtures adjusted to pH 4.5 or 8.5 at different temperatures in the range of 30° to 100°C. Endo-xylanase activity was measured as described in the Example 1. Accordingly, Figure 3 of the drawings accompanying this specification shows optimum activity at 50°C at all the pHs tested, followed by a second optimum peak of activity at 90°C. About 50% of the total activity measured at 50°C could be detected at 90°C. EXAMPLE 4 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for studying the pH stability of the enzyme preparation. This was done by assaying the endo-xylanase activity following a 30 min pre-incubation of the enzyme sample with different buffers, at 0°C or 30°C (room temperature). The buffers used were 50mM acetate buffer at the pH range 3 to 6, 100mM phosphate buffer at the pH range 6 to 8 and 25 mM borate buffer at the pH range 8 to 10. Endo-xylanase activity was measured as described in the Example 1. Accordingly, Figure 4 of the drawings accompanying this specification shows that when the enzyme was incubated at 0°C at various pHs, there was an almost 50% reduction in activity by pH 8.0, before showing a rise at pH 9.0. On the other hand, when the enzyme was incubated at room temperature 4-ß-D0°C), there was a steady decrease in activity up to pH 5.0, while 90% of the activity was detectable at pH 10. EXAMPLE 5 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for studying the thermostability of the enzyme preparation. This was monitored by incubating the enzyme for the specified period (1 to 4 h) at different temperatures (55, 60 and 70°C) before rapidly cooling in ice and carrying out the routine assay at 50°C as described in the Example 1. Accordingly, Figure 5 of the drawings accompanying this specification shows that the enzyme retained 60-65% of the activity after 1-4 h incubation at 55°C. At 60°C it retained only 28% of the activity at the end of 1 h. Incubation at 70°C totally inactivated the enzyme after 1h, while it appears that more than 50% of the activity may be retained even after 15 min of incubation at 60°C as well as at 70°C. EXAMPLES The said fungus A. niger NIOCC isolate # 3 was grown in oat spelts xylan medium at pH 4.5 as described in the Example 1 and the cell-free culture supernatant was used for studying the effect of protein concentration in the enzyme preparation on the stability of enzyme at higher temperature v/z. at 85°C. For this purpose endo-xylanase activity was measured after incubating enzyme solutions containing various concentrations of protein (0-42 µg/ml) at 85°C for 15 min after which the residual activity was measured in the presence of oat spelt xylan at 50°C. Accordingly, Figure 6 of the drawings accompanying this specification shows that with increasing protein concentration, the specific activity followed a hyperbolic curve. EXAMPLE 7 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for assaying the optimum pH for p-D-xylosidase activity. The effect of pH on the reaction was assessed by incubating the reaction mixture containing 50mM sodium acetate buffer for the pH range 3-6; 100mM phosphate buffer for the pH range 6-8 and Tris:NaOH buffer for pH range 8-10. The activity was assayed at 50°C using p-nitrophenol-ß-D-xylopyranoside dissolved in the buffers mentioned above. The reaction was terminated by addition of 0.4M glycine buffer (pH 10.8) and absorbance read at 430 nm. One unit of activity is the amount of enzyme liberating 1 jxmol p-nitrophenol min'1 (Smith DC, Wood TM. 1991. Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production extracellular xylanase and ß-xylosidase while maintaining low protease production. Biotechnology and Bioengineering 38:883-890). Standard graph was prepared using different concentrations of p-nitrophenol. Accordingly, Figure 7 of the drawings accompanying this specification shows that maximum activity of p-xylosidase was observed at pH 4.0. EXAMPLE 8 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for assaying the optimum temperature for p-D-xylosidase activity. The effect of temperature was assessed by incubating the reaction mixtures adjusted to pH 4.0 at different temperatures in the range of 30° to 90°C. p-D-xylosidase activity was measured as described in the Example 7. Accordingly, Figure 8 of the drawings accompanying this specification shows that maximum activity of 4-ß-D-xylosidase was observed at 70°C. EXAMPLE 9 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for assaying the optimum pH for a-L-arabinofuranosidase activity. The effect of pH on the reaction was assessed by incubating the reaction mixture containing 50mM sodium acetate buffer for the pH range 3-6; 100mM phosphate buffer for the pH range 6-8 and Tris:HCI buffer for pH range 8-10. The activity was assayed at 50°C using p-nitrophenol-a-L-arabinofuranoside dissolved in the various buffers mentioned above. The reaction was terminated by adding 1 ml of ice cold 0.5 M sodium carbonate solution and the color developed was read at 405 nm. (Saha BC, Bothast RJ. 1998. Purification and characterization of a novel thermostable α-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans. Applied and Environmental Microbiology 64:216-220). Standard graph was prepared using various concentrations of p-nitrophenol. Accordingly, Figure 9 of the drawings accompanying this specification shows that maximum activity of a-L-arabinofuranosidase was observed at pH 4.0. EXAMPLE 10 The said fungus A. niger NIOCC isolate # 3 was grown as described in the Example 1 and the cell-free culture supernatant was used for assaying the optimum temperature for a-L-arabinofuranosidase activity. The effect of temperature was assessed by incubating the reaction mixtures adjusted to pH 4.0 at different temperatures in the range of 30° to 90°C. The activity of α-L-arabinofuranosidase was measured as described in the Example 9. Accordingly, Figure 10 of the drawings accompanying this specification shows that optimum activity of α-L-arabinofuranosidase was observed at 60°C. The main advantages of the present invention are: 1. The marine isolate of the fungus Aspergillus n/grer NIOCC isolate # 3 can be grown on a large-scale using inexpensive, readily available raw material, namely sugarcane bagasse suspended in tap water, distilled water or half- strength sea water with ammonium tatrate as nitrogen source for production of xylan-degrading enzymes. 2. The said fungus produces maximum amount of endo-xylanase within 4-7 days. 3. The said fungus can be cultured in acidic or alkaline media to obtain endo- xylanase. 4. Endo-xylanase thus produced by the said fungus shows optimum activity at pH 3.5 and 50% of this activity at pH 8.5. 5. Endo-xylanase thus produced by the said fungus shows optimum activity at temperature of 50°C and has a second optimum at 90°C. 6. Endo-xylanase of A.niger, NIOCC isolate # 3 shows thermostability at 55°C for at least 4 h by retaining around 68% of the activity. 7. Endo-xylanase of the said fungus shows increasing stability at 85°C for 15 min with increasing concentration of protein in the enzyme solution. 8. The said fungus is also able to produce endo-xylanase in the presence of black liquor in culture medium. 9. The said fungus also produces other xylan-degrading enzymes such as 4-ß-D-D- xylosldase and a-L-arabinofuranosidase in the same culture medium. 10. The enzyme ß-D-xylosidase shows optimum activity at 70°C and pH 4.0 and . a-L-arabinofuranosidase at 60°C and pH of 4.0. Claim: 1. A process for producing xylan-degrading enzymes which comprises, growing of a marine strain of the fungus Aspergillus niger, having characteristic such as herein described, in conventional media containing xylan-containing substrates as carbon source selected from 5% concentration of sugarcane bagasse and conventional nitrogen source preferably 2.4 mM ammonium tartrate, at pH ranging 3.5 and 8.5, in the presence or absence of black liquor obtained from paper mill at a concentration of 0.1 to 0.2%, for a period of 4 days to get xylan-degrading enzymes. 2. A process as claimed in claim 1, wherein, the xylan-degrading enzymes include but not limited to 1,4-p-D-xylanase, p-xylosidase and a-L-arabinofuranosidase enzymes. 3. A process as claimed in claim 1-2, wherein, xylanase enzyme obtained is cellulose-free and has pH optimum at 3.5 and 8.5. 4. A process as claimed in claim 1-3, wherein, the xylanase enzyme activity is optimum at temperature 50° and 90°C and the enzyme is thermostable at 55°C for at least 4 hours. 5. A process as claimed in claim 1-4, wherein, (3-xylosidase enzyme activity has optimum pH of 4 and at temperature of 70°C. 6. A process as claimed in claims 1-5 wherein, a-L- arabinofuranosidase enzyme has optimum activity at pH 4 and at temperature of 60°C. 7. A process as claimed in claims 1 - 6, wherein, the stability of the endoxylanase at 85°C is dependent on increasing protein concentration in the enzyme solution. 8. A process as claimed inc claims 1-7, wherein, the carbon source for growing the fungus used is selected from shredded or powdered sugarcane bagasse at 1 to 5% concentration, oat spelts xylan, birchwood xylan and breakfast grade cereal oats. 9. A process as claimed in claims 1-8,wherein, the nitrogen source used for growing the fungus is ammonium tartrate at about 2.4 mM concentration. 10. A process as claimed in claims 1-9, wherein, the culture medium can be prepared with distilled water or tap water or half-strength natural or synthetic sea water. 11. A process as claimed in claims 1-10, wherein, the culture medium can have acidic or alkaline pH. 12. A process as claimed in claims 1-11, wherein, black liquor at 0.1 to 0.2% concentration can be added and at least 75% of endo-xylanase activity is retained. 13. A process for production of xylan-degrading enzymes , substantially as herein described with reference to examples and drawings accompanying this specification.

Documents:

389-del-2000-abstract.pdf

389-del-2000-claims.pdf

389-del-2000-correspondence-others.pdf

389-del-2000-correspondence-po.pdf

389-del-2000-description (complete).pdf

389-del-2000-drawings.pdf

389-del-2000-form-1.pdf

389-del-2000-form-19.pdf

389-del-2000-form-2.pdf

389-del-2000-form-3.pdf