Title of Invention	"METHOD OF PRODUCING ALKALOTHERMOSTABLE XYLANASE FROM BACILLUS PUMIIUS STRAIN MK001 BY SOLID STATE FERMENTATION"
Abstract	This invention relates to a novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising, Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 μ mesh resulting uniform particle size 180-200 n, washing, drying at 50°C, Followed by steaming the bran at 121°C for 30min and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight drying at 50°C, Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KH2 Po4 0.9-1.1 preferably 1.0, Nacl2.2-2.6 preferably 2.5; Mg So4, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 S04, 0.9-1.1 preferably 1.0; Cac12 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Co3, and temperature about 36°C to 39°C, preferably at 37°C, Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg So4, 7H20 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr. Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the solid-state fermentation is carried out up to 120 hours. Extracting the enzyme from fermented wheat bran using 50mM-150mM preferably 100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for 30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g for 10 to 15 min at 4°C.

Title of Invention

"METHOD OF PRODUCING ALKALOTHERMOSTABLE XYLANASE FROM BACILLUS PUMIIUS STRAIN MK001 BY SOLID STATE FERMENTATION"

Abstract

This invention relates to a novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising, Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 μ mesh resulting uniform particle size 180-200 n, washing, drying at 50°C, Followed by steaming the bran at 121°C for 30min and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight drying at 50°C, Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KH2 Po4 0.9-1.1 preferably 1.0, Nacl2.2-2.6 preferably 2.5; Mg So4, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 S04, 0.9-1.1 preferably 1.0; Cac12 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Co3, and temperature about 36°C to 39°C, preferably at 37°C, Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg So4, 7H20 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr. Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the solid-state fermentation is carried out up to 120 hours. Extracting the enzyme from fermented wheat bran using 50mM-150mM preferably 100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for 30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g for 10 to 15 min at 4°C.

Full Text	Method of producing alkalothermostable xylanase from Bacillus pumilus strain MK001 by solid state fermentation. FIELD OF INVENTION This invention relates to the cost effective method for production of xylanase, an enzyme from Bacillus pumilus strain MK001 by solid state fermentation. BACKGROUND OF THE INVENTION The ability to produce xylanase is widespread among bacteria, for example, Anthrobacter, Clostridium, Cellulomonas, Bacillus, Thermononspora, Ruminococcus and Staphylococcus), actinomycetes (Streptomyces, Thermoactinomyces) and yeast (Aureobasidium, Trichosporon). Enzymes are currently produced mostly by submerged fermentation (SmF), a relatively expensive process even when high producing genetically engineered strains are used. An alternative technology for enzyme production is solid state fermentation (SSF). SSF is generally defined as the growth of microorganisms on solid materials in the absence or near absence of free water. The substrate, however must contain enough moisture, which exists in absorbed from within the solid matrix. The major advantages of SSF over SmF are in the operational simplicity, high product concentration and volumetric productivity in a water restricted environment (Pandey, 1999, 2003, Rahardjo et al, 2006). Xylanase are inducible enzymes and attempts have been made to selectively increase the titers of xylanase by microorganisms by addition of xylan-rich substrates to the nutrient medium (fermentation medium) to induce xylanase formation. Purified xylans (birchwood, beech wood and oat spelt) and lignocellulosis residues studied for xylanase production include rice husk, wheat bran, wheat straw sugarcane bagasse, corn cobs, oat husk and corn bran (Saxena et al., 1994, Casimir-Schenkel et al., 1996; Avalos et al., 1996; Saraswat and Bisaria, 1997; Archana and Satyanarayana, 1998; Kuhad el al., 1998 Kuhad et al., 1999; Ganwande and Kamat, 1999; Gomes et al., 2000; Gupta et al., 2001; Taneja et al., 2002; Nascimento et al., 2002; Vega-Estradea, 2002; Taneja et al., 2002; Anthony et al., 2003; Yuan et al., 2005; Nianwe and Kuhad, 2005; Bocchini et al., 2005; Oliveira et al., 2006; Li et al., 2006; Shin and Chen; 2006). The present invention provides a process for producing alkalothermostable xylanase, a promising enzyme for pulp bleaching and production of xylo-oligosaccharides using cost effective wheat bran under solid state fermentation. Xylanase, a repertoire a hydrokytic enzymes, randomly attack internal 0-1, 4 bonds in the polyxylose backbone to yield xylooligosaccharides of various chain lengths as major end products. Xylan ranks second only to cellulose in abundance and comprises up to one third of the total dry weight of higher plants and are structurally diverse heterogeneous molecules with a characteristic backbone consisting on average of 150-200 β-(l,4)-linked D-xylosyl residues to which several substituents are attached. Complete degradation of branched, partially acetylated xylans to xylose and other sugars requires the action of a variety of enzymes among which xylanases hold major importance. The mode of action of xylanases is complex and is realized in conjunction with other (to some extent synergistic) enzymes. The use of xylanses in different bleaching sequences consistently lead to the reduction in consumption of traditionally applied bleaching chemicals. However, the benefits obtained by xylanases are dependent not only on the type of pulp but also on the chemical bleaching sequence used, as well as the final target brightness and environmental goals of the mill. Originally, xylanase were applied in pulp and paper industry with the basic objective to reduce the consumption of chlorine chemicals, especially elemental chlorine. Later, enzymes have been combined with various elemental chlorine free (ECF) and total chlorine free (TCP) bleaching sequences to improve the final brightness of the pulp. Results from laboratory studies and mill trails show about 35-41% reduction in active chlorine, at the chlorination stage for hardwoods and 10-20% for softwoods, whereas savings in total active chlorine were found to be 20-25% for hardwoods and 10-15% for softwoods (Tolan and Canovas, 1992; Skerket et al., 1992; Yang et al., 1992; Werthemann, 1993; Allison et al., 1993; Bajpai et al., 1993, 1994; Clarke et al., 1997; Bajpai, 1997, 1999, 1992b, 1933, 1999; Bim and Franco, 2000; Zhan et al., 2000; Mediros et al., 2002; Teixeira Duarte et al., 2002; Chauhan et al., 2005; Ninawe and Kuhad, 2006; Kapoor et al., 2007; Shatalov and Pereira, 2007). The ability of the xylanase enzyme to reduce bleaching chemical consumption makes it possible to consider significant modifications in the bleaching sequence. It is possible to completely exclude the first chlorination stage in a C/D EOPDEPD and replace it with an enzyme stage (X) to become XEOPDEPD. This has been verified in a mill trail in Spain (Turner et al., 1992). The advantage of this modified sequence is that filtrates from the EOP stage can be recirculated to the recovery system without risk of chloride-initiated corrosion. This approach helps in contributing closure of the water circulation system of the pulp mill and minimizing effluent discharge. A non-chlorine bleaching process (EnZone process) consisting of xylanase treatment (X) in combination with oxygen (O), ozone (Z) and alkaline peroxide (P) bleaching stages has been developed. Hardwood (eucaluptus) kraft pulp delignified via an OXZ sequence had consistently lowered kappa number and higher brightness than OZ bleached pulp. Pulp delignified via an OXZP sequence readily yielded a pulp of brightness 85-0-90.0% (ISO), compared with a brightness of 78.0-84.7% for the OZP pulp. The EnZone-bleached pulp had higher brightness stability, similar tensile index, and slightly lower tear index (Yang et al., 1992), XDEpD was found to be the best alternative to the DEpD sequence as it surpassed the latter in pulp brightness (by 37%) featured a moderate yield loss (9.3%) and provided paper sheets with acceptably smaller breaking length and burst index (20.2 and 13.1% less, respectively). The FDEpD sequence resulted in increase in pulp brightness (8.6%) and higher savings in chlorine consumption (10.9%), however, the yield, the breaking length and burst index of the paper sheets were markedly decreased by 25.2, 24.7 and 41.8%, respectively (Jimenez et al., 1997). Eucalyptus kraft pulp treated with MED 2D supernatant at 10 IU per gram dry weight pulp resulted in a 10.5% reduction in kappa number. XECEDDED-refined bleached pulp resulted in hand sheets with increased brightness compared to the control. Xylanase treatment of pulp release chromophores absorbing at 235 nm (Haarhoff et al., 1999). The use of a novel xylanase A was found beneficial in the elemental chlorine bleaching of oxygen delignified Eucalyptus kraft pulps. The application of the enzyme made it possible to produce fully bleached pulps with higher brightness (89% ISO) and viscosities (above 800cm3/g), without chemical chlorine (XDnEpD and DnEpD sequences) at low chlorine dioxide consumption (Torres et al., 2000). The effects of xylanase and mannanase treatment on the bleachability of oxygen delignified superbatch pulps from different origins have been studied by Surnakki et al., (1997, using peroxide or ozone based sequences. Xylanase pretreatment increased the final brightness of all the pulps more than the mannanase treatment. The combination of the more neutral Trishoderma xylanas, treatment and chelation was more suitable than the acidic Aspergillus kawachii xylanase treatment at pH 2.5 for superbatch pulp TCF bleaching Prof. Eriksson's group 1 as reported the use of an extremely high specific activity xylanase from Orpinomyce sp. strain PC-2 in ECF and TCF bleaching (Shah et al., 2000). An overall brightness gain of 4.3% ISO at the end of an ECF (OXDP) or a TCF (OXAP) bleaching sequence was achieved. A 2.7% ISO brightness gain was achieved at pH 8.0 with the xylanase treatment alone. The same group (Shah et al., 2000) also carried out xylanase treatment from Thermotga maritime of oxygen bleached hardwood kraft pulp at a temperature of 90°C and pH 10 and approximately 25% savings in bleaching chemicals with 90.5% brightness was obtained. The brightness of untreated pulp was only 86% using the same bleaching sequence. Yango mill in Japan has also introduced enzyme bleaching into the hardwood kraft bleaching process (Fukuaga et al., 2000). On-site production of xylanase from Bacillus strain S-2113 was carried out. The principal component culture medium was pulp, which was obtained from the fiber line at the mill. The complete culture broth containing enzyme and bacteria cell was used for enzyme bleaching. The mill operation has been operating successfully for over a year with no adverse effect on pulp quality Yango mill was planning to start up ECF bleaching during the year 2000 and enzyme bleaching as predicted to have an even greater effect. As the TCF market grows, Canadian mills, which are the world's largest exporters of market pulp, have started to investigate chlorine free bleaching with xylanase enzymes European paper makers are now requesting TCF pulps or bleaching pulps with extremely low AOX and/or TOX level in their effluent (Worster, 1993). A combined treatment of chemicals and commercially available enzymes like Cartazyme HS-10, Novozyme 473, VAI Xylanase, Pulpzume HA, pIAFIS have been reported to be more beneficial than chemical treatment alone as these reduce chlorine consumption and the release of total organic chlorine in effluents with enhancement in brightness and reduction in kappa number (Paice et al., 1992; Bajpai et al., 1994; Ragauskas et al., 1994; Kulkarni and Rao, 1996; Wong et al., 1997). Cellulase-free xylandegrading enzyme preparations from Acrophialophora nainiana, Humicola grisea var, thermoidea and two Trichoderma harzianum strains were used as bleaching agnets fro Eucalyptus kraft pulp, prior to a chlorine dioxide and alkaline bleaching sequence. In comparison to the control sequence (performed without xylanase pretreatment), the sequence incorporating enzyme treatment was more effective. Removal of residual lignin was indicated by a reduction in kappa number. Overall, enzyme preparations from T. harzianum were marginally more effective in reducing pulp viscosity and chlorine chemical consumption and improving the brightness of the kraft pulp. However, the highest reduction in pulp viscosity was mediated by the xylanase preparation from A. nainiana (Meqeiros et al., 2002). Damiano et al., (2003) employed two-stage bleaching using a CIO2 chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of kappa number and brightness, respectively 28.5 and 30% less CIO2 was required in comparison to the enzymatically untreated samples. Two extra cellular xylanase (xy) I and xyl and II) produced by the thermo tolerant fungus Aspergillus caespitosus reduced the kappa number and released large amounts of 287 and 465 nm absorbing materials from eucalyptus kraft pulp as compared to control. Xyl II was more effective for bleaching, because it reduced kappa number by 12.6%, only 1.7% with xyl I. This result was probably related with the observed biochemical properties of xyl, II such as higher thermo stability and lower molecular mass, which may allow this enzyme to diffuse more easily into the pulp matrix (Sandrim et al., 2005). Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Cellulose Papel, Limeira, SP, Brazil) showed a reduced of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp (Teixeira Duarte et al., 2003). For the crude enzyme preparation (at charge of 1.5 lU/g dry weight pulp) from H. grisea var. thermoidea the release of chromophores and reducing sugar was observed to be maximal after 3 h of reaction time. In comparison to the control, this release was enhanced by 85, 71, 60, 55 and 78% at 237, 254, 465 and 540 nm, respectively. With A.nainiana enzyme treatment, the highest absorbance values obtained after 4 h and the release of chromophores and reducing sugar was improved by 15, 28, 20, 12 and 40% at 237, 254, 465 and 540 nm, respectively. A defibrillation of the microfibrils was detected or the pulp enzymically treated when compared with the control (Salles et al., 2004). Pre-treatment with enzymes from Penicillium A10 and Aspergillus L22 at a xylanase dosage of 4 lU/g prior to pulping decreased pulp kappa number by 6.29% and 12.07% respectively as compared to the control. High cellulose activity in crude enzymes has a negative influence on pulping. Xylanase pre-bleaching reduced chlorine charge by 20-30%, or increased final brightness by approximately 4-5% ISO, and improved the pup strength properties (Zhap et al., 2006). The effect of enzymatic pretreatment with xylanase preparation (endo-1, 4-p-xylanase ativity; EC 3.2 1.8) on properties and bleachability of organic solvent-based Ethanol-Alkali, Organocell (alkali anthraquinone-methanol) and ASAM (alkali-sulfite-antraquinone-methanol) pulps produced from agro-fibre crop giant reed (Arundo donax L.) has been examined and compared with traditional kraft pulp. The enzyme-assisted removal of xylan-associated lignin fragments and hexenuronic acids caused direct brightening and delignification during the enzymatic stage, while somewhat affecting the pulp strength properties. The xylanase bleach boosting substantially improved subsequent chemical bleaching of organsolv pulps by hydrogen peroxide. The savings in bleaching chemicals with simultaneous increase in brightness and degree of delignification were observed. The enzymatic pre-treatment improved the intrinsic viscosity of bleached organosolv pulps (Shatalov and Pereira, 2007). The substitution of petroleum by renewable fuels produced from biomass is a promising strategy that is rapidly gaining momentum. In 2005, the worldwide production of bioethanol is expected to reach 38.27 million kL but most of it is currently produced from starch or sucrose. The development of technologies for production of ethanol from more widely available lignocellulosic feedstork's would be crucial in efforts to expand bioethanol utilization (Sun and Cheg, 2002; Saha, 2003: Chandel etal., 2006; 2007). Xylitol is a five carbon alcohol sugar that has been recognized as being a "functional food" because of its significant medical and nutritional applications (Yilikari, 1979; Hydrogen et al., 1982; Makinen, 1989, 1995). Xylitol finds increased usage as a sweetener in foods due to its higher sweetening power. It is 2.5 times as sweet as mannitol and has 2-times higher sweetening power than D-sorbitol. Xylitol does not need insulin for its digestion. Therefore it may be used clinically as a sugar substitute for diabetic patients or of glucose -6-phosphote dehydrogenase deficient population (Emodi, 1978). In addition to being a good anticarcinogenic sweetner, xylitol is not utilized by microorganisms, therefore products with xylitol are usually safe from microbial attack. Thus xylitol could be used for protection against dental carries. Xylitol can also be used to prevent some pathology such as, acute otitis (Uhari et al., 1998), experimental osteoporosis (Mattila et al., 1998) and cystic fibrosis (Zabner et al., 2000) and may be also be of value in enhancing the innate antimicrobial defense at the airway surface (Zabner et al., 2000). At present, xylitol for commercial uses is produced through chemical reductin of xylose derived from hemicelluosic hydrolyzates of wood pulp or other xylose rich materials. The chemical route is costlier and not environmentally friendly. Selective microorganisms have the ability to produce xylitol-using xylose through reduction process. However use of pure xylose again challenges the economic viability of the process. This lignocellulose could be used as low cost alternative raw material to produce xylose and eventually for microbial fermentation of xylitol. The key technology required for the successful biological conversion of hemicellulosic biomass to ethanol/xylitol requires: (1) delignification of hemicellulose material rich in xylan; (2) depolymerization of carbohydrate polymers (cellulose and hemicellulose) by xylanases and hemicellulases to produce free sugars, and; (3) fermentation of mixed hexose and pentose sugars mainly by yeats (Pichia stipitis, Candida shehatae and Zymomonas mobilis), to produce ethano/xylitol (Shapack et al., 1987; Screenath and Jeffries 2000; Chandel et al., 2006; Mohagheghi et al., 2006; Davis et al., 2006). A 32.0% xylobiose and 12.0% xylotriose were obtained from chemically (2.0% NaOH) pretreated corncobs powder after 12 h of incubation with Edugragit S-100 immobilized xylanase from S. olivaceovirdis E-86 (Ai et al., 2005). Similarly, cellulose-free xylanase produced by Psedomomonas sp. WLUN024 has been reported to produce high quality xylo-alogosacchride from xylan (Xu et al., 2005). Commercial xylanase preparation Chearzyme, which contains the glycoside hydrolase family 10 endo -1, 4-p-D-xylanase from A.aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX) Rantanen et al., 2007). Hydrolysis of birchwood xylan by free and HP-20 immobilized xylanase from Bacillus pumilus strain MK001 produced i xylogbiose and xylotriose within 1 h of reaction time. Lon-term incubation resulted in steady increase in xylose content. After 10 h of incubation, immobilized enzyme released 40.0% xylobiose and 18.0% xylotriose, on the other hand, free enzyme released 35.0% xylobiose and 14.0% xylotriose from total sugar (9.8 mg/ml immobilized enzyme; 9.3 i mg/ml free enzyme) released by the reaction products (Kapoor and Kuhad, 2007). A 3 h xylanase pretreatment of pulp followed by single-stage peroxide bleaching yielded pulp with a brightness of > 70 ISO units for the production of paper handsheets in which the physical strength of the fibers had been preserved was reported by Garg et al., (1996). Kappa number was reduced by xylanase treatment with an without subsequent alkali extraction, and the pulp brightness after CEDED bleaching at 4% chlorine charge was boosted without significant reducts in fiber strength or interfiber bending. Scanning electron microscopy revealed marked disruption and separation of pulp fibers even at low (5 lU/g dry weight pulp) xylanse doses, and a 30-35% saving in the chlorine charge achieved to obtain pulp brightness comparable to controls could be achieved (Garg et al., 1998). Enzymatic treatment of bagasse pulp carried out using 1.2 IU of enzyme per gram of pulp at 50°C and pH 8 for 2 h resulted in a 4 unit decrease in the kappa number. Similar treatment of the pulp at pH 7 and 9 indicated that the Sam-x xylanase was effective in reducing the kappa number of the pulp over a wide pH ragne (Shah et al., 1999). Pretreatment of pulp with xylanase and its subsequent treatment with 6% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor upto 10% and 17%, respectively (Gupta et al., 2000). Xylanase dose of 3.5 U/g moisture free pulps exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50°C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 45% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (% ISO) by 20% and improved the pulp properties such as tensile strength and burs factor by up to 60% and 8%, respectively (Beg et al., 2000). The xylanase dose of 1.0 lU/g dry weight pulp gave optimum bleach boosting of kraft pulp at pH 8.0 and temperature 55° C for 3 h reaction time (Taneja et al., 2002). Xylanase from Bacillus coagulans has been rested on three non-woody pulps as a prebleaching agent. The effects of crude enzyme on wheat straw, rice straw and jute pulps at two different initial pH of pulp have been studied and a maximum brightness gair of 5.1 points has been achieved with rice straw pulp at an initial pH value of 8. In the case of wheat straw and rice straw pulps, maximum brightness gain has been obtained at the higher pH values (chauhan et al., 2005). The xylanase dose of 10 lU/g moisture free pulp exhibited maximum bleach boosting of soda pulp (pH 9.5-10.0) optionally at 65°C C after 2 n of reaction time. Pre-treatment of pulp with xylanase and its subsequent treatment with 6% hydrochlorite reduced the kappa number by 8.7% enhanced the brightness index by 3.56% and improved other paper properties such as tear index and burst index. The enzymatically-prebleached pulp when treated with 10% reduced level of hypochlorite (5.4%) gave comparable brightness of resultant hand sheets to the fully bleached pulp (6% hypochlorite) (Hinawe and Kuhad 2006). Enzymatic prebleaching of kraft pulp showed 20% reduction in kappa number of the pulp without much change in viscosity. Enzymatic treatment reduced the amount of chloride by 29% without any decrease in brightness. The viscosity of xylanase treated pulp was 4.0 p, whereas the viscosity of the pulp treated exlusivley with chlorine was 4.1 p (Khandeparkar and Bhosle 2007). Recently, soda and different grades of waste pulp fibers (used for making 3-layered duplex sheets-Top layer, TL; Protective layer, PL; Bottom layer, BL) when pretreated with either xylanase (40.0 IU g-1) or laccase mediator system (LMS) (up to 200.0 U g-1) alone and in combination (one after the other) (XLMS) exhibited an increase in release of reducing sugars up to 8810. % soda pulp; up to 736.6% (TL), up to 2157.7% (PL) and up to 198.0 % (BL) waste pulp]. Reduction in kappa number [ up to 17.6% soda pulp; up to 14.0% (TL), up to 25.3 % (PL) and up to 10.9 % (BL), waste pulp], improvement in brightness [ up to 20.4% soda pulp; up to 23.6 % (TL), up to 8.6 % (PL) and up to 5.0 % (BL), waste pulp] as compared to the respective controls. The usage of XLMS along with 15 % reduced level of hypochlorite at CEHHXLMS?EHHXLMS bleaching stage reduced kappa number [5.5 % soda pulp; 11.4 % (TL), 7.9% (PL), waste pulp] and improved brightness [1.0% soda pulp; 0.9% (TL), 1.4 % (PL) waste pulp] as compared to the controls. Scanning electron microscopic (SEM) studies revealed development of cracks, flakes, pores and peeling off the fibers in the enzymes treated pulp samples (Kapoor et al., 2007). Most of the research on bleaching with hemicellulase enzymes has so far focused on softwoods and hardwoods. However, research efforts in this area on nonwoody plants are scanty even though pulp production from nonwoody plants and agricultural residues is significant. Nevertheless, application of xylanases in prebleaching of bamboo kraft pulp, jute, maize, cotton stalks and sarkanda straws has been reported (Bajpai and Bajpai, 1996; Bajpai 2004). The effectiveness of various commercial cellulose less xylanases for prebleaching of bamboo kraft pulp has been reported by Bajpai and Bajpai (1996). Xylanases are primarily being used for the removal of lignin-carbohydrate complex (LCC) that are, otherwise, generated in the kraft (pulping) process and act as physical barrier to the entry of bleaching chemicals (Paice et al., 1992; Vikari et al., 1994; Kuhad et al., 1997; Bajpai 1999; Beg et al., 2001a; Bajapi 2004; Kapoor et al., 2007). Other significant benefits of xylanase treatment include higher brightness ceilings, reduction in the amounts of bleaching chemicals needed to achieve higher brightness and decrease in amounts of organochlorine compounds in the bleach plant effluents (Ragauskas et al., 1994; Bajpai, 1999; Shatalo and Pereira, 2007). However, the use of xylanases for pulp bleaching in the paper industry has been slowed down by the lack of large scale availability of enzymes active and stable at pH values above 8 and temperature around 60°C, which are the prevalent conditions in many bleaching processes (Garg et al., 1998; Beg et al., 2000c; Dhillon et al., 2000: Beg et al., 2001a; Techaoun et al., 2003; Bajapi, 2004; Polizeli et al., 2005; Khandeparker and Bhosle, 2007). Several species of fungi and bacteria have been extensively studied for their xylanase producing abilities. Some of these organisms, although mesophilic produce high levels of xylanases active under slightly acidic (pH 4.0-6.0) or near neutral (6.5-7.5) conditions and temperatures (50-60°C). Due to the high temperature and pH. A pre-requisite, in the pulp and paper industry, is the use of celluase-free xylanases that ensure minimal damage to pulp fibers (cellulose) and also help generating rayon grade or superior quality dissolving pulps. However, majority of the xylanases known exhibit concomitant cellulose activities, especially when these are produced using agricultural residues (lignocelluloses) as substrate for cultivation although usage of agricultural residues economizes the enzyme production process. Therefore it is highly desirable to have alkalostable and thermostable xylanases freem from cellulose or with negligible cellulose activity. The low production amount of xylanase from microorganism is another factor limiting the commercial viability of xylanases. The subject invention highlights production of xylanase with negligible cellulase, active and stable over a broad range of pH (4.5-9.0) and temperature (50-70°C) by B. pumilus strain MK001. The organism is capable of utilizing wheat bran (agricultural by-product) supplemented in a low cost nutrient medium under controlled cultivation conditions to produce hyper-xylanase levels that makes the process of xylanase production cost-effective and commercially viable. OBJECTS OF THE INVENTION The object of the invention is to develop a solid state fermentation (SSF) process for production of cellulase free xylanase. Other object is to develop economically viable solid state fermentation process. Further object is to produce xylanase, which is active and stable over a broad range of pH and temperature. Another object is to use cheap abundantly available and non toxic agricultural by product (wheat bran) to produce xylanase. STATEMENT OF INVENTION This invention relates to a novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising, Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 \i mesh resulting uniform particle size 180-200 n, washing, drying at 50°C, Followed by steaming the bran at 121°C for SOmin and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight drying at 50°C, Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KH2 Po4 0.9-1.1 preferably 1.0, Nacl2.2-2.6 preferably 2.5; Mg So4, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 804, 0.9-1.1 preferably 1.0; Cacl2 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Cos, and temperature about 36°C to 39°C, preferably at 37°C, Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KHa Po4 0.1 Mg 804, 7H2O 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr. Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the solid-state fermentation is carried out up to 120 hours. Extracting the enzyme from fermented wheat bran using 50mM-150mM preferably 100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for 30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g for 10 to 15 min at 4°C. DETAILED DESCRIPTION OF THE INVENTION The present invention was aimed at developing economically viable solid state fermentation (SSF) process for production of cellulose-free xylanase. The process uses cheap, abundantly available and non-toxic agricultural byproduct (what bran) to produce xylanase to the tune of 1,30,000.0 lU/g dry weight bran of xylanase. The present invention is a process for producing xylanase by cultivating a xylanase-producing bacterium idnetiifed as Bacillus pumilus train MK001, in a nutrient medium containing carbon and nitrogen sources with selective salts. Microorganisms of the genus Bacillus is effective as producers of xylanolytic enzymes especially xylanases. It has been found that a simple pre-treatment of the wheat bran have causes an increase in xylanase production by wheat bran while at the same time increasing inductor activity. The pre-treated wheat bran used in the subject process functions as both carbon source for the organism and inducer for xylanase production. The pre-treatment removes the substantial portion of the starch associated with the native wheat bran. The wheat bran is sieved (200n mesh size) and wheat bran resulting there from is autoclaved (121°C, 30 min). This is followed by alakaline pretreatment with 0.2-0.4 N preferably 0.3 N NaOH the time for alkaline pretreatment is from 20-28 hr. preferably (24 h, room temperature, 28°C). The bacterium when grown on pretreated wheat bran (in enamel trays) gives rise to a significant change in the enzymes spectrum by enahcning xylanolytic activity by several percent/fold as compared to when cultivated on untreated wheat bran. 1) In the subject invention, pre-treatment of wheat bran comprises of a) Removal of starch particle's: The crude wheat bran was sieved through a mesh (200 \i) resulting in wheat bran particles of almost same uniform size (180-200 n) and then washed with distilled water (three times). The bran was then oven-dried overnight at 50°C so that the moisture content was completely removed. b) Steam treatment followed by alkaline pretreatment: 700.0 g wheat bran was steamed at 121°C for 30 min and thereafter moistened with 3500.0 ml of 0.3 N NaOH at room temperature in the sealed plastic bag for 24 h. Thereafter, wheat bran was washed with distilled water (three times) and then oven-dried overnight at 50°C so that the moisture content was completely removed. 2) The pre-treated wheat bran was supplemented with salts such as (g/1); KH2PO4, 1.0; NaCl 2.5; MgSO4, 7H2O, 0.1; (NH4), SO4, 1.0; CaCl2, 0.1 and folic acid (0.28% w/w). 3) The pH value for the bacterial cultivation conveniently lies in the range of about 8.5 to about 9.2, preferably at about 9.0. The adjustment of the pH value in the culture medium is conveniently effected with 10.0% (w/v) Na2CO3. 4) The cultivation temperature is conveniently about 36°C to about 39°C. Preferably about 37°C. 5) The fermentation apparatus used can be a conventional enamel tray for which precautions have been taken to exclude foreign infections. Microorganism shall be grown in enamel trays (78x51x8 cm3) containing 700.0 g wheat bran moistened with mineral salt sodium (composition described above) in 1:5 ratio of solid substrate to moisture. 6) The inoculation of the enamel tray is conveniently effected in the following two stages (i)-(ii). (i) The seed culture was prepared by streaking B. pumilus strain MK001 stock culture on a petri plate containing xylan-Horikoshi (Ikura and Horikoshi, 1987) agar (pH 8.0) containing (% w/w birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2PO4 0.1 MgSO4, 7H2O 0.01) and incubating at 37°C for 24 h. Thereafter, 100 ml i. Erlenmeyer flask containing 20.0 ml of Horikoshi medium containing (% w/v glucose 0.5, peptone 0.5, yeast extract 0.5, KH2PO4 0.1 MgSO4, 7H2 loopful of 24 h old .culture D 0.01) (pH 8.0) was inoculated with a of B. pumilus strain MK001 (grown on Horkoshi agar) and incubated at 37°C under shaking conditions (200 rpm) for 6 h. (ii) The enamel tray containing 700.0 g wheat bran is inoculated with the culture medium produced in stage (i), such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently about 12.50 (v/w) to about 12.00, preferably about 12.50. The solid state fermentation was carried out up to 120 hours. The Centration range of nutrients added in solid state media is as follows (g/1): KH2 PO4 0.9-1.1, Nacl 2.2-2.6, Mg So4, 7H2O 0.09-0.11; (NH4)2 SO4 0.9-1.1, Cacb 0.09-1.1, folic acid 0.28-0.05% W/W. enzyme extraction: The separation with the xylanase from the fermented medium under SSF in accordance with the invention and its concentration can be performed according to methods known per se. basic requirements for this are low media temperature (about 5°C to about 15°C.) and neutral pH values (about 5.5 to about 6.0). The procedure conveniently involves: Adding 3500.0 ml of 100.0 mM citrate-phosphate buffer (pH 6.0) to fermented wheat bran (700.0 g) and vortexi (200-250 rpm, 30 min, 37°C). The enzyme can be collected in the supernatant after centrifugation at 10,000x for 10 min at 4°C. The enzyme can be further concentrated using ultrafiltratior, (50.0 KDa). The enzyme activities in the fermentation medium were as follows: (Table Removed) The main field of application for the subject enzyme complex is its utilization as a biobleaching agent in pulp and paper industry. Xylanases are being used primarily for the removal of lignin-carbohydrate complex (LCC) that are generated in the kraft process and act as physical barriers to the entry of bleaching chemicals (Paicet al., 1992, Beg et al., 2000). Enzyme activity determination The activity of the xylanase was determined by incubating the appropriately dilutea enzyme solution with xylan suspension (birch wood xylan from sigma chemicals, MO, USA) in 100.0 mM citrate-phosphate buffer (pH 6.0) at 60°C for 10 minutes. One unit (U) of xylanase was defined as the amount of enzyme required to release 1 ^mol of xylose per milliliter per minute under the assay conditions. The composition in global enzyme markets is intensive which is likely to have a significant effect on Indian market also. To meet the challenges, there is need to develop process for enzyme production which is not only'cost effective but also eco-friendly. The present xylanase could be -used as a prebleaching agent to enhance lignin removal and brightness in the processed pulps by subsequent chemical methods, in pulp and paper industry. The enzyme is well suited for bleaching the pulp and shall reduce the cost of bleaching process and eventually environment pollution. Another application of present xylanase is in production of xylo-oligosaccharides (xylobios, xylotriose, xylotetrose etc) which could be utilized selectively by the beneficial intestinal microllora viz. Bijdiobacteria and are thus expected to be used as a valuable food activities. Moreover xylanase are also useful in improving animal feed and cost-effective conversion of lignocellulose waste material to pentose sugars, which could be fermented to ethanol as biofuel. It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims: Table 1. Comparative account of bleaching conditions and improvement in pulp properties aftei xylanase treatment. (Table Removed) We Claim 1. A novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising i) Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 μ, mesh resulting uniform particle size 180-200 n, washing, drying at 50°C ii) Followed by steaming the bran at 121°C for 30min and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight drying at 50°C iii) Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KHa Po4 0.9-1.1 preferably 1.0, Nacl 2.2-2.6 preferably 2.5; Mg 804, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 So4, 0.9-1.1 preferably 1.0; Cacb 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Cos, and temperature about 36°C to 39°C, preferably at 37°C iv) Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H20 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg So4, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr. v) Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the solid-state fermentation is carried out up to 120 hours. vi) Extracting the enzyme from fermented wheat bran using 50mM-150mM preferably 100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for 30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g for 10 to 15 min at 4°C. 2. The novel method for production of cellulase free xylanase, an enzyme form Bacillus pumilus strain MK001 from wheat bran by solid state fermentation as claimed in claim 1, wherein the enamel tray containing wheat bran is inoculated with the culture medium such that the ratio by volume of the culture medium to the nutrient medium is 12.50 (W/W) to 12.0 preferably 12.50 and the solid state fermentation is carried out upto 120 hours. 3. The novel method for production of cellulase free xylanase, an enzyme form Bacillus pumilus strain MK001 from wheat bran by solid state fermentation as claimed in claim 1, wherein the Microorganism is grown in enamel trays 78x51x8cm3 containing wheat bran moistened with mineral salt in 1:5 ratio of solid substrate to moisture. 4. The novel method for production of cellulase free xylanase, an enzyme form Bacillus pumilus strain MK001 from wheat bran by solid state fermentation as claimed in claim 1, wherein the xylanase is separated from the fermented medium under solid state fermentation at low media temperature of 5°C to 15°C and neutral pH values of 5.5 to 6.0 5. The novel method for production of cellulase free xylanase, an enzyme form Bacillus pumilus strain MK001 from wheat bran by solid state fermentation as claimed in claim 1, wherein the said nutrient containing wheat bran modified as inducer and supplemented with nominal salts and folic acid.

Full Text

Method of producing alkalothermostable xylanase from Bacillus pumilus strain MK001 by solid state fermentation.
FIELD OF INVENTION
This invention relates to the cost effective method for production of xylanase, an enzyme from Bacillus pumilus strain MK001 by solid state fermentation.
BACKGROUND OF THE INVENTION
The ability to produce xylanase is widespread among bacteria, for example, Anthrobacter, Clostridium, Cellulomonas, Bacillus, Thermononspora, Ruminococcus and Staphylococcus), actinomycetes (Streptomyces, Thermoactinomyces) and yeast (Aureobasidium, Trichosporon).
Enzymes are currently produced mostly by submerged fermentation (SmF), a relatively expensive process even when high producing genetically engineered strains are used. An alternative technology for
enzyme production is solid state fermentation (SSF). SSF is generally defined as the growth of microorganisms on solid materials in the absence or near absence of free water. The substrate, however must contain enough moisture, which exists in absorbed from within the solid matrix. The major advantages of SSF over SmF are in the operational simplicity, high product concentration and volumetric productivity in a water restricted environment (Pandey, 1999, 2003, Rahardjo et al, 2006).

Xylanase are inducible enzymes and attempts have been made to selectively increase the titers of xylanase by microorganisms by addition of xylan-rich substrates to the nutrient medium (fermentation medium) to induce xylanase formation. Purified xylans (birchwood, beech wood and oat spelt) and lignocellulosis residues studied for xylanase production include rice husk, wheat bran, wheat straw sugarcane bagasse, corn cobs, oat husk and corn bran (Saxena et al., 1994, Casimir-Schenkel et al., 1996; Avalos et al., 1996; Saraswat and Bisaria, 1997; Archana and Satyanarayana, 1998; Kuhad el al., 1998 Kuhad et al., 1999; Ganwande and Kamat, 1999; Gomes et al., 2000; Gupta et al., 2001; Taneja et al., 2002; Nascimento et al., 2002; Vega-Estradea, 2002; Taneja et al., 2002; Anthony et al., 2003; Yuan et al., 2005; Nianwe and Kuhad, 2005; Bocchini et al., 2005; Oliveira et al., 2006; Li et al., 2006; Shin and Chen; 2006).
The present invention provides a process for producing alkalothermostable xylanase, a promising enzyme for pulp bleaching and production of xylo-oligosaccharides using cost effective wheat bran under solid state fermentation.
Xylanase, a repertoire a hydrokytic enzymes, randomly attack internal 0-1, 4 bonds in the polyxylose backbone to yield xylooligosaccharides of various chain lengths as major end products. Xylan ranks second only to cellulose in abundance and comprises up to one third of the total dry weight of higher plants and are structurally diverse heterogeneous

molecules with a characteristic backbone consisting on average of 150-200 β-(l,4)-linked D-xylosyl residues to which several substituents are attached. Complete degradation of branched, partially acetylated xylans to xylose and other sugars requires the action of a variety of enzymes among which xylanases hold major importance. The mode of action of xylanases is complex and is realized in conjunction with other (to some extent synergistic) enzymes.
The use of xylanses in different bleaching sequences consistently lead to the reduction in consumption of traditionally applied bleaching chemicals. However, the benefits obtained by xylanases are dependent not only on the type of pulp but also on the chemical bleaching sequence used, as well as the final target brightness and environmental goals of the mill.
Originally, xylanase were applied in pulp and paper industry with the basic objective to reduce the consumption of chlorine chemicals, especially elemental chlorine. Later, enzymes have been combined with various elemental chlorine free (ECF) and total chlorine free (TCP) bleaching sequences to improve the final brightness of the pulp. Results from laboratory studies and mill trails show about 35-41% reduction in active chlorine, at the chlorination stage for hardwoods and 10-20% for softwoods, whereas savings in total active chlorine were found to be 20-25% for hardwoods and 10-15% for softwoods (Tolan and Canovas, 1992; Skerket et al., 1992; Yang et al., 1992; Werthemann, 1993; Allison et al.,

1993; Bajpai et al., 1993, 1994; Clarke et al., 1997; Bajpai, 1997, 1999, 1992b, 1933, 1999; Bim and Franco, 2000; Zhan et al., 2000; Mediros et al., 2002; Teixeira Duarte et al., 2002; Chauhan et al., 2005; Ninawe and Kuhad, 2006; Kapoor et al., 2007; Shatalov and Pereira, 2007).
The ability of the xylanase enzyme to reduce bleaching chemical consumption makes it possible to consider significant modifications in the bleaching sequence. It is possible to completely exclude the first chlorination stage in a C/D EOPDEPD and replace it with an enzyme stage (X) to become XEOPDEPD. This has been verified in a mill trail in Spain (Turner et al., 1992). The advantage of this modified sequence is that filtrates from the EOP stage can be recirculated to the recovery system without risk of chloride-initiated corrosion. This approach helps in contributing closure of the water circulation system of the pulp mill and minimizing effluent discharge. A non-chlorine bleaching process (EnZone process) consisting of xylanase treatment (X) in combination with oxygen (O), ozone (Z) and alkaline peroxide (P) bleaching stages has been developed. Hardwood (eucaluptus) kraft pulp delignified via an OXZ sequence had consistently lowered kappa number and higher brightness than OZ bleached pulp.
Pulp delignified via an OXZP sequence readily yielded a pulp of brightness 85-0-90.0% (ISO), compared with a brightness of 78.0-84.7% for the OZP pulp. The EnZone-bleached pulp had higher brightness stability, similar tensile index, and slightly lower tear index (Yang et al.,

1992), XDEpD was found to be the best alternative to the DEpD sequence as it surpassed the latter in pulp brightness (by 37%) featured a moderate yield loss (9.3%) and provided paper sheets with acceptably smaller breaking length and burst index (20.2 and 13.1% less, respectively). The FDEpD sequence resulted in increase in pulp brightness (8.6%) and higher savings in chlorine consumption (10.9%), however, the yield, the breaking length and burst index of the paper sheets were markedly decreased by 25.2, 24.7 and 41.8%, respectively (Jimenez et al., 1997). Eucalyptus kraft pulp treated with MED 2D supernatant at 10 IU per gram dry weight pulp resulted in a 10.5% reduction in kappa number. XECEDDED-refined bleached pulp resulted in hand sheets with increased brightness compared to the control. Xylanase treatment of pulp release chromophores absorbing at 235 nm (Haarhoff et al., 1999). The use of a novel xylanase A was found beneficial in the elemental chlorine bleaching of oxygen delignified Eucalyptus kraft pulps. The application of the enzyme made it possible to produce fully bleached pulps with higher brightness (89% ISO) and viscosities (above 800cm3/g), without chemical chlorine (XDnEpD and DnEpD sequences) at low chlorine dioxide consumption (Torres et al., 2000).
The effects of xylanase and mannanase treatment on the bleachability of oxygen delignified superbatch pulps from different origins have been studied by Surnakki et al., (1997, using peroxide or ozone based sequences. Xylanase pretreatment increased the final brightness of all the pulps more than the mannanase treatment. The combination of the

more neutral Trishoderma xylanas, treatment and chelation was
more suitable than the acidic Aspergillus kawachii xylanase treatment at pH 2.5 for superbatch pulp TCF bleaching Prof. Eriksson's group 1 as reported the use of an extremely high specific activity xylanase from Orpinomyce sp. strain PC-2 in ECF and TCF bleaching (Shah et al., 2000). An overall brightness gain of 4.3% ISO at the end of an ECF (OXDP) or a TCF (OXAP) bleaching sequence was achieved. A 2.7% ISO brightness gain was achieved at pH 8.0 with the xylanase treatment alone. The same group (Shah et al., 2000) also carried out xylanase treatment from Thermotga maritime of oxygen bleached hardwood kraft pulp at a temperature of 90°C and pH 10 and approximately 25% savings in bleaching chemicals with 90.5% brightness was obtained. The brightness of untreated pulp was only 86% using the same bleaching sequence.
Yango mill in Japan has also introduced enzyme bleaching into the hardwood kraft bleaching process (Fukuaga et al., 2000). On-site production of xylanase from Bacillus strain S-2113 was carried out. The principal component culture medium was pulp, which was obtained from the fiber line at the mill. The complete culture broth containing enzyme and bacteria cell was used for enzyme bleaching. The mill operation has been operating successfully for over a year with no adverse effect on pulp quality Yango mill was planning to start up ECF bleaching during the year 2000 and enzyme bleaching as predicted to have an even greater effect.

As the TCF market grows, Canadian mills, which are the world's largest exporters of market pulp, have started to investigate chlorine free bleaching with xylanase enzymes European paper makers are now requesting TCF pulps or bleaching pulps with extremely low AOX and/or TOX level in their effluent (Worster, 1993). A combined treatment of chemicals and commercially available enzymes like Cartazyme HS-10, Novozyme 473, VAI Xylanase, Pulpzume HA, pIAFIS have been reported to be more beneficial than chemical treatment alone as these reduce chlorine consumption and the release of total organic chlorine in effluents with enhancement in brightness and reduction in kappa number (Paice et al., 1992; Bajpai et al., 1994; Ragauskas et al., 1994; Kulkarni and Rao, 1996; Wong et al., 1997).
Cellulase-free xylandegrading enzyme preparations from Acrophialophora nainiana, Humicola grisea var, thermoidea and two Trichoderma harzianum strains were used as bleaching agnets fro Eucalyptus kraft pulp, prior to a chlorine dioxide and alkaline bleaching sequence. In comparison to the control sequence (performed without xylanase pretreatment), the sequence incorporating enzyme treatment was more effective. Removal of residual lignin was indicated by a reduction in kappa number. Overall, enzyme preparations from T. harzianum were marginally more effective in reducing pulp viscosity and chlorine chemical consumption and improving the brightness of the kraft pulp. However, the highest reduction in pulp viscosity was mediated by the xylanase preparation from A. nainiana (Meqeiros et al., 2002).

Damiano et al., (2003) employed two-stage bleaching using a CIO2 chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of kappa number and brightness, respectively 28.5 and 30% less CIO2 was required in comparison to the enzymatically untreated samples. Two extra cellular xylanase (xy) I and xyl and II) produced by the thermo tolerant fungus Aspergillus caespitosus reduced the kappa number and released large amounts of 287 and 465 nm absorbing materials from eucalyptus kraft pulp as compared to control. Xyl II was more effective for bleaching, because it reduced kappa number by 12.6%, only 1.7% with xyl I. This result was probably related with the observed biochemical properties of xyl, II such as higher thermo stability and lower molecular mass, which may allow this enzyme to diffuse more easily into the pulp matrix (Sandrim et al., 2005).
Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Cellulose Papel, Limeira, SP, Brazil) showed a reduced of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp (Teixeira Duarte et al., 2003).
For the crude enzyme preparation (at charge of 1.5 lU/g dry weight pulp) from H. grisea var. thermoidea the release of chromophores and reducing sugar was observed to be maximal after 3 h of reaction time. In comparison to the control, this release was enhanced by 85, 71, 60, 55 and 78% at 237, 254, 465 and 540 nm, respectively. With A.nainiana enzyme treatment, the highest absorbance values obtained after 4 h and the release of chromophores and reducing sugar was improved by 15, 28,

20, 12 and 40% at 237, 254, 465 and 540 nm, respectively. A defibrillation of the microfibrils was detected or the pulp enzymically treated when compared with the control (Salles et al., 2004).
Pre-treatment with enzymes from Penicillium A10 and Aspergillus L22 at a xylanase dosage of 4 lU/g prior to pulping decreased pulp kappa number by 6.29% and 12.07% respectively as compared to the control. High cellulose activity in crude enzymes has a negative influence on pulping. Xylanase pre-bleaching reduced chlorine charge by 20-30%, or increased final brightness by approximately 4-5% ISO, and improved the pup strength properties (Zhap et al., 2006). The effect of enzymatic pretreatment with xylanase preparation (endo-1, 4-p-xylanase ativity; EC 3.2 1.8) on properties and bleachability of organic solvent-based Ethanol-Alkali, Organocell (alkali anthraquinone-methanol) and ASAM (alkali-sulfite-antraquinone-methanol) pulps produced from agro-fibre crop giant reed (Arundo donax L.) has been examined and compared with traditional kraft pulp. The enzyme-assisted removal of xylan-associated lignin fragments and hexenuronic acids caused direct brightening and delignification during the enzymatic stage, while somewhat affecting the pulp strength properties. The xylanase bleach boosting substantially improved subsequent chemical bleaching of organsolv pulps by hydrogen peroxide. The savings in bleaching chemicals with simultaneous increase in brightness and degree of delignification were observed. The enzymatic pre-treatment improved the intrinsic viscosity of bleached organosolv pulps (Shatalov and Pereira, 2007).
The substitution of petroleum by renewable fuels produced from biomass is a promising strategy that is rapidly gaining momentum. In 2005, the worldwide production of bioethanol is expected to reach 38.27 million kL but most of it is currently produced from starch or sucrose.

The development of technologies for production of ethanol from more widely available lignocellulosic feedstork's would be crucial in efforts to expand bioethanol utilization (Sun and Cheg, 2002; Saha, 2003: Chandel etal., 2006; 2007).
Xylitol is a five carbon alcohol sugar that has been recognized as being a "functional food" because of its significant medical and nutritional applications (Yilikari, 1979; Hydrogen et al., 1982; Makinen, 1989, 1995). Xylitol finds increased usage as a sweetener in foods due to its higher sweetening power. It is 2.5 times as sweet as mannitol and has 2-times higher sweetening power than D-sorbitol. Xylitol does not need insulin for its digestion. Therefore it may be used clinically as a sugar substitute for diabetic patients or of glucose -6-phosphote dehydrogenase deficient population (Emodi, 1978). In addition to being a good anticarcinogenic sweetner, xylitol is not utilized by microorganisms, therefore products with xylitol are usually safe from microbial attack.
Thus xylitol could be used for protection against dental carries. Xylitol can also be used to prevent some pathology such as, acute otitis (Uhari et al., 1998), experimental osteoporosis (Mattila et al., 1998) and cystic fibrosis (Zabner et al., 2000) and may be also be of value in enhancing the innate antimicrobial defense at the airway surface (Zabner et al., 2000).
At present, xylitol for commercial uses is produced through chemical reductin of xylose derived from hemicelluosic hydrolyzates of wood pulp or other xylose rich materials. The chemical route is costlier and not environmentally friendly. Selective microorganisms have the ability to produce xylitol-using xylose through reduction process. However use of pure xylose again challenges the economic viability of the process. This lignocellulose could be used as low cost alternative raw material to produce xylose and eventually for microbial fermentation of xylitol.

The key technology required for the successful biological conversion of hemicellulosic biomass to ethanol/xylitol requires: (1) delignification of hemicellulose material rich in xylan; (2) depolymerization of carbohydrate polymers (cellulose and hemicellulose) by xylanases and hemicellulases to produce free sugars, and; (3) fermentation of mixed hexose and pentose sugars mainly by yeats (Pichia stipitis, Candida shehatae and Zymomonas mobilis), to produce ethano/xylitol (Shapack et al., 1987; Screenath and Jeffries 2000; Chandel et al., 2006; Mohagheghi et al., 2006; Davis et al., 2006).
A 32.0% xylobiose and 12.0% xylotriose were obtained from chemically (2.0% NaOH) pretreated corncobs powder after 12 h of incubation with Edugragit S-100 immobilized xylanase from S. olivaceovirdis E-86 (Ai et al., 2005). Similarly, cellulose-free xylanase produced by Psedomomonas sp. WLUN024 has been reported to produce high quality xylo-alogosacchride from xylan (Xu et al., 2005). Commercial xylanase preparation Chearzyme, which contains the glycoside hydrolase family 10 endo -1, 4-p-D-xylanase from A.aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX) Rantanen et al., 2007). Hydrolysis of birchwood xylan by free and HP-20 immobilized xylanase from Bacillus pumilus strain MK001 produced
i
xylogbiose and xylotriose within 1 h of reaction time. Lon-term incubation resulted in steady increase in xylose content. After 10 h of incubation, immobilized enzyme released 40.0% xylobiose and 18.0% xylotriose, on the other hand, free enzyme released 35.0% xylobiose and 14.0% xylotriose from total sugar (9.8 mg/ml immobilized enzyme; 9.3
i
mg/ml free enzyme) released by the reaction products (Kapoor and Kuhad, 2007).

A 3 h xylanase pretreatment of pulp followed by single-stage peroxide bleaching yielded pulp with a brightness of > 70 ISO units for the production of paper handsheets in which the physical strength of the fibers had been preserved was reported by Garg et al., (1996).
Kappa number was reduced by xylanase treatment with an without subsequent alkali extraction, and the pulp brightness after CEDED bleaching at 4% chlorine charge was boosted without significant reducts in fiber strength or interfiber bending. Scanning electron microscopy revealed marked disruption and separation of pulp fibers even at low (5 lU/g dry weight pulp) xylanse doses, and a 30-35% saving in the chlorine charge achieved to obtain pulp brightness comparable to controls could be achieved (Garg et al., 1998). Enzymatic treatment of bagasse pulp carried out using 1.2 IU of enzyme per gram of pulp at 50°C and pH 8 for 2 h resulted in a 4 unit decrease in the kappa number. Similar treatment of the pulp at pH 7 and 9 indicated that the Sam-x xylanase was effective in reducing the kappa number of the pulp over a wide pH ragne (Shah et al., 1999). Pretreatment of pulp with xylanase and its subsequent treatment with 6% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor upto 10% and 17%, respectively (Gupta et al., 2000). Xylanase dose of 3.5 U/g moisture free pulps exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50°C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 45% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (% ISO) by 20% and improved the pulp properties such as tensile strength and burs factor by up to 60% and 8%, respectively (Beg et al., 2000).

The xylanase dose of 1.0 lU/g dry weight pulp gave optimum bleach boosting of kraft pulp at pH 8.0 and temperature 55° C for 3 h reaction time (Taneja et al., 2002). Xylanase from Bacillus coagulans has been rested on three non-woody pulps as a prebleaching agent. The effects of crude enzyme on wheat straw, rice straw and jute pulps at two different initial pH of pulp have been studied and a maximum brightness gair of 5.1 points has been achieved with rice straw pulp at an initial pH value of 8. In the case of wheat straw and rice straw pulps, maximum brightness gain has been obtained at the higher pH values (chauhan et al., 2005).
The xylanase dose of 10 lU/g moisture free pulp exhibited maximum bleach boosting of soda pulp (pH 9.5-10.0) optionally at 65°C C after 2 n of reaction time. Pre-treatment of pulp with xylanase and its subsequent treatment with 6% hydrochlorite reduced the kappa number by 8.7% enhanced the brightness index by 3.56% and improved other paper properties such as tear index and burst index. The enzymatically-prebleached pulp when treated with 10% reduced level of hypochlorite (5.4%) gave comparable brightness of resultant hand sheets to the fully bleached pulp (6% hypochlorite) (Hinawe and Kuhad 2006). Enzymatic prebleaching of kraft pulp showed 20% reduction in kappa number of the pulp without much change in viscosity. Enzymatic treatment reduced the amount of chloride by 29% without any decrease in brightness. The viscosity of xylanase treated pulp was 4.0 p, whereas the viscosity of the pulp treated exlusivley with chlorine was 4.1 p (Khandeparkar and Bhosle 2007). Recently, soda and different grades of waste pulp fibers (used for making 3-layered duplex sheets-Top layer, TL; Protective layer, PL; Bottom layer, BL) when pretreated with either xylanase (40.0 IU g-1) or laccase mediator system (LMS) (up to 200.0 U g-1) alone and in combination (one after the other) (XLMS) exhibited an increase in release

of reducing sugars up to 8810. % soda pulp; up to 736.6% (TL), up to 2157.7% (PL) and up to 198.0 % (BL) waste pulp]. Reduction in kappa number [ up to 17.6% soda pulp; up to 14.0% (TL), up to 25.3 % (PL) and up to 10.9 % (BL), waste pulp], improvement in brightness [ up to 20.4% soda pulp; up to 23.6 % (TL), up to 8.6 % (PL) and up to 5.0 % (BL), waste pulp] as compared to the respective controls. The usage of XLMS along with 15 % reduced level of hypochlorite at CEHHXLMS?EHHXLMS bleaching stage reduced kappa number [5.5 % soda pulp; 11.4 % (TL), 7.9% (PL), waste pulp] and improved brightness [1.0% soda pulp; 0.9% (TL), 1.4 % (PL) waste pulp] as compared to the controls. Scanning electron microscopic (SEM) studies revealed development of cracks, flakes, pores and peeling off the fibers in the enzymes treated pulp samples (Kapoor et al., 2007).
Most of the research on bleaching with hemicellulase enzymes has so far focused on softwoods and hardwoods. However, research efforts in this area on nonwoody plants are scanty even though pulp production from nonwoody plants and agricultural residues is significant. Nevertheless, application of xylanases in prebleaching of bamboo kraft pulp, jute, maize, cotton stalks and sarkanda straws has been reported (Bajpai and Bajpai, 1996; Bajpai 2004). The effectiveness of various commercial cellulose less xylanases for prebleaching of bamboo kraft pulp has been reported by Bajpai and Bajpai (1996).
Xylanases are primarily being used for the removal of lignin-carbohydrate complex (LCC) that are, otherwise, generated in the kraft (pulping) process and act as physical barrier to the entry of bleaching chemicals (Paice et al., 1992; Vikari et al., 1994; Kuhad et al., 1997; Bajpai 1999; Beg et al., 2001a; Bajapi 2004; Kapoor et al., 2007).

Other significant benefits of xylanase treatment include higher brightness ceilings, reduction in the amounts of bleaching chemicals needed to achieve higher brightness and decrease in amounts of organochlorine compounds in the bleach plant effluents (Ragauskas et al., 1994; Bajpai, 1999; Shatalo and Pereira, 2007). However, the use of xylanases for pulp bleaching in the paper industry has been slowed down by the lack of large scale availability of enzymes active and stable at pH values above 8 and temperature around 60°C, which are the prevalent conditions in many bleaching processes (Garg et al., 1998; Beg et al., 2000c; Dhillon et al., 2000: Beg et al., 2001a; Techaoun et al., 2003; Bajapi, 2004; Polizeli et al., 2005; Khandeparker and Bhosle, 2007).
Several species of fungi and bacteria have been extensively studied for their xylanase producing abilities. Some of these organisms, although mesophilic produce high levels of xylanases active under slightly acidic (pH 4.0-6.0) or near neutral (6.5-7.5) conditions and temperatures (50-60°C). Due to the high temperature and pH. A pre-requisite, in the pulp and paper industry, is the use of celluase-free xylanases that ensure minimal damage to pulp fibers (cellulose) and also help generating rayon grade or superior quality dissolving pulps. However, majority of the xylanases known exhibit concomitant cellulose activities, especially when these are produced using agricultural residues (lignocelluloses) as substrate for cultivation although usage of agricultural residues economizes the enzyme production process. Therefore it is highly desirable to have alkalostable and thermostable xylanases freem from cellulose or with negligible cellulose activity. The low production amount of xylanase from microorganism is another factor limiting the commercial viability of xylanases.

The subject invention highlights production of xylanase with negligible cellulase, active and stable over a broad range of pH (4.5-9.0) and temperature (50-70°C) by B. pumilus strain MK001. The organism is capable of utilizing wheat bran (agricultural by-product) supplemented in a low cost nutrient medium under controlled cultivation conditions to produce hyper-xylanase levels that makes the process of xylanase production cost-effective and commercially viable.
OBJECTS OF THE INVENTION
The object of the invention is to develop a solid state fermentation (SSF) process for production of cellulase free xylanase.
Other object is to develop economically viable solid state fermentation process.
Further object is to produce xylanase, which is active and stable over a broad range of pH and temperature.
Another object is to use cheap abundantly available and non toxic agricultural by product (wheat bran) to produce xylanase.
STATEMENT OF INVENTION
This invention relates to a novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising, Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 \i mesh resulting uniform particle size 180-200 n, washing, drying at 50°C, Followed by steaming the bran at 121°C for SOmin and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight

drying at 50°C, Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KH2 Po4 0.9-1.1 preferably 1.0, Nacl2.2-2.6 preferably 2.5; Mg So4, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 804, 0.9-1.1 preferably 1.0; Cacl2 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Cos, and temperature about 36°C to 39°C, preferably at 37°C, Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KHa Po4 0.1 Mg 804, 7H2O 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr. Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the
solid-state fermentation is carried out up to 120 hours. Extracting the
enzyme from fermented wheat bran using 50mM-150mM preferably
100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for
30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g
for 10 to 15 min at 4°C.
DETAILED DESCRIPTION OF THE INVENTION
The present invention was aimed at developing economically viable solid
state fermentation (SSF) process for production of cellulose-free xylanase.
The process uses cheap, abundantly available and non-toxic agricultural
byproduct (what bran) to produce xylanase to the tune of 1,30,000.0
lU/g dry weight bran of xylanase.

The present invention is a process for producing xylanase by cultivating a xylanase-producing bacterium idnetiifed as Bacillus pumilus train MK001, in a nutrient medium containing carbon and nitrogen sources with selective salts. Microorganisms of the genus Bacillus is effective as producers of xylanolytic enzymes especially xylanases.
It has been found that a simple pre-treatment of the wheat bran have causes an increase in xylanase production by wheat bran while at the same time increasing inductor activity. The pre-treated wheat bran used in the subject process functions as both carbon source for the organism and inducer for xylanase production. The pre-treatment removes the substantial portion of the starch associated with the native wheat bran. The wheat bran is sieved (200n mesh size) and wheat bran resulting there from is autoclaved (121°C, 30 min). This is followed by alakaline pretreatment with 0.2-0.4 N preferably 0.3 N NaOH the time for alkaline pretreatment is from 20-28 hr. preferably (24 h, room temperature, 28°C). The bacterium when grown on pretreated wheat bran (in enamel trays) gives rise to a significant change in the enzymes spectrum by enahcning xylanolytic activity by several percent/fold as compared to when cultivated on untreated wheat bran.
1) In the subject invention, pre-treatment of wheat bran comprises of
a) Removal of starch particle's: The crude wheat bran was sieved
through a mesh (200 \i) resulting in wheat bran particles of almost
same uniform size (180-200 n) and then washed with distilled
water (three times). The bran was then oven-dried overnight at
50°C so that the moisture content was completely removed.
b) Steam treatment followed by alkaline pretreatment:
700.0 g wheat bran was steamed at 121°C for 30 min and thereafter moistened with 3500.0 ml of 0.3 N NaOH at room

temperature in the sealed plastic bag for 24 h. Thereafter, wheat bran was washed with distilled water (three times) and then oven-dried overnight at 50°C so that the moisture content was completely removed.
2) The pre-treated wheat bran was supplemented with salts such as
(g/1); KH2PO4, 1.0; NaCl 2.5; MgSO4, 7H2O, 0.1; (NH4), SO4, 1.0;
CaCl2, 0.1 and folic acid (0.28% w/w).
3) The pH value for the bacterial cultivation conveniently lies in the
range of about 8.5 to about 9.2, preferably at about 9.0. The
adjustment of the pH value in the culture medium is conveniently
effected with 10.0% (w/v) Na2CO3.
4) The cultivation temperature is conveniently about 36°C to about
39°C. Preferably about 37°C.
5) The fermentation apparatus used can be a conventional enamel
tray for which precautions have been taken to exclude foreign
infections. Microorganism shall be grown in enamel trays (78x51x8
cm3) containing 700.0 g wheat bran moistened with mineral salt
sodium (composition described above) in 1:5 ratio of solid
substrate to moisture.
6) The inoculation of the enamel tray is conveniently effected in the
following two stages (i)-(ii).
(i) The seed culture was prepared by streaking B. pumilus strain MK001 stock culture on a petri plate containing xylan-Horikoshi (Ikura and Horikoshi, 1987) agar (pH 8.0) containing (% w/w birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2PO4 0.1 MgSO4, 7H2O 0.01) and incubating at 37°C for 24 h. Thereafter, 100 ml
i.
Erlenmeyer flask containing 20.0 ml of Horikoshi medium containing (% w/v glucose 0.5, peptone 0.5, yeast extract 0.5,

KH2PO4 0.1 MgSO4, 7H2 loopful of 24 h old .culture

D 0.01) (pH 8.0) was inoculated with a of B. pumilus strain MK001 (grown on

Horkoshi agar) and incubated at 37°C under shaking conditions (200 rpm) for 6 h.
(ii) The enamel tray containing 700.0 g wheat bran is inoculated with the culture medium produced in stage (i), such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently about 12.50 (v/w) to about 12.00, preferably about 12.50. The solid state fermentation was carried out up to 120 hours. The Centration range of nutrients added in solid state media is as follows (g/1): KH2 PO4 0.9-1.1, Nacl 2.2-2.6, Mg So4, 7H2O 0.09-0.11; (NH4)2 SO4 0.9-1.1, Cacb 0.09-1.1, folic acid 0.28-0.05% W/W.
enzyme extraction: The separation with the xylanase from the fermented medium under SSF in accordance with the invention and its concentration can be performed according to methods known per se. basic requirements for this are low media temperature (about 5°C to about 15°C.) and neutral pH values (about 5.5 to about 6.0).
The procedure conveniently involves:
Adding 3500.0 ml of 100.0 mM citrate-phosphate buffer (pH 6.0) to fermented wheat bran (700.0 g) and vortexi (200-250 rpm, 30 min, 37°C). The enzyme can be collected in the supernatant after centrifugation at 10,000x for 10 min at 4°C. The enzyme can be further concentrated using ultrafiltratior, (50.0 KDa). The enzyme activities in the fermentation medium were as follows:
(Table Removed)

The main field of application for the subject enzyme complex is its
utilization as a biobleaching agent in pulp and paper industry. Xylanases are being used primarily for the removal of lignin-carbohydrate complex (LCC) that are generated in the kraft process and act as physical barriers to the entry of bleaching chemicals (Paicet al., 1992, Beg et al., 2000). Enzyme activity determination
The activity of the xylanase was determined by incubating the appropriately dilutea enzyme solution with xylan suspension (birch wood xylan from sigma chemicals, MO, USA) in 100.0 mM citrate-phosphate buffer (pH 6.0) at 60°C for 10 minutes. One unit (U) of xylanase was defined as the amount of enzyme required to release 1 ^mol of xylose per milliliter per minute under the assay conditions. The composition in global enzyme markets is intensive which is likely to have a significant effect on Indian market also.
To meet the challenges, there is need to develop process for enzyme production which is not only'cost effective but also eco-friendly. The present xylanase could be -used as a prebleaching agent to enhance lignin removal and brightness in the processed pulps by subsequent chemical methods, in pulp and paper industry. The enzyme is well suited for bleaching the pulp and shall reduce the cost of bleaching process and eventually environment pollution. Another application of present xylanase is in production of xylo-oligosaccharides (xylobios, xylotriose, xylotetrose etc) which could be utilized selectively by the beneficial

intestinal microllora viz. Bijdiobacteria and are thus expected to be used as a valuable food activities. Moreover xylanase are also useful in improving animal feed and cost-effective conversion of lignocellulose waste material to pentose sugars, which could be fermented to ethanol as biofuel.
It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims:
Table 1. Comparative account of bleaching conditions and improvement in pulp properties aftei xylanase treatment.
(Table Removed)

We Claim
1. A novel method for production of cellulase free xylanase, an enzyme from Bacillus pumilus strain MK001 from wheat bran by solid state fermentation comprising
i) Pretreatment of wheat bran to remove substantial portion of starch by sieving crude wheat bran particles through 200 μ, mesh resulting uniform particle size 180-200 n, washing, drying at 50°C
ii) Followed by steaming the bran at 121°C for 30min and then moistening with 3.5 litre of 0.2-0.4 N preferably 0.3 N NaoH at room temperature for 24hr. and finally washing and overnight drying at 50°C
iii) Supplementing the pretreated wheat bran with mineral slat solution containing salts as (g/1) KHa Po4 0.9-1.1 preferably 1.0, Nacl 2.2-2.6 preferably 2.5; Mg 804, 7H2o, 0.9-1.1 preferably 0.1; (NH4) 2 So4, 0.9-1.1 preferably 1.0; Cacb 0.9-1.1 preferably 0.1 and folic acid 0.28-0.50% preferably 0.28% W/W, maintaining the bacterial cultivation pH value between 8.5 to about 9.2, preferably at 9.0 by using 10% W/V Na2 Cos, and temperature about 36°C to 39°C, preferably at 37°C
iv) Preparing seed culture by streaking B. pumilus strain MK 001 stock culture on a petri plate containing xylan-Horikoshi agar pH 8.0 containing % w/v birch wood xylan 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg 804, 7H20 0.01, incubating at 37°C for 24h followed by inoculation with a loopful of 24h old culture of B.pumilus strain MK 001 in a flask containing 20ml of Horikoshi medium containing % W/V glucose 0.5, peptone 0.5, yeast extract 0.5, KH2 Po4 0.1 Mg So4, 7H2O 0.01, pH 8.0 at 37°C under shaking conditions 200rpm for 6hr.
v) Inoculating the enamel tray containing about 700.0 g of wheat bran with the culture medium as described in (iv) such that the ratio by volume of the culture medium to the nutrient medium in the enamel tray is conveniently 12.50 (v/w) to about 12.00, preferably 12.5, wherein the solid-state fermentation is carried out up to 120 hours.
vi) Extracting the enzyme from fermented wheat bran using 50mM-150mM preferably 100.0 m M citrate phosphate buffer pH 6.0 vortexing at 200-250rpm for 30 min at 37°C removing solid constituents by centrifuging at 10, 000 x g for 10 to 15 min at 4°C.

2. The novel method for production of cellulase free xylanase, an
enzyme form Bacillus pumilus strain MK001 from wheat bran by
solid state fermentation as claimed in claim 1, wherein the enamel
tray containing wheat bran is inoculated with the culture medium
such that the ratio by volume of the culture medium to the
nutrient medium is 12.50 (W/W) to 12.0 preferably 12.50 and the
solid state fermentation is carried out upto 120 hours.
3. The novel method for production of cellulase free xylanase, an
enzyme form Bacillus pumilus strain MK001 from wheat bran by
solid state fermentation as claimed in claim 1, wherein the
Microorganism is grown in enamel trays 78x51x8cm3 containing
wheat bran moistened with mineral salt in 1:5 ratio of solid
substrate to moisture.
4. The novel method for production of cellulase free xylanase, an
enzyme form Bacillus pumilus strain MK001 from wheat bran by
solid state fermentation as claimed in claim 1, wherein the
xylanase is separated from the fermented medium under solid
state fermentation at low media temperature of 5°C to 15°C and
neutral pH values of 5.5 to 6.0
5. The novel method for production of cellulase free xylanase, an
enzyme form Bacillus pumilus strain MK001 from wheat bran by
solid state fermentation as claimed in claim 1, wherein the said
nutrient containing wheat bran modified as inducer and
supplemented with nominal salts and folic acid.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=KLF2KsSU8Sk4p6nRgTxvwg==&loc=+mN2fYxnTC4l0fUd8W4CAA==

« Previous Patent

Next Patent »

Patent Number

270321

Indian Patent Application Number

984/DEL/2008

PG Journal Number

51/2015

Publication Date

18-Dec-2015

Grant Date

11-Dec-2015

Date of Filing

16-Apr-2008

Name of Patentee

DEPARTMENT OF BIOTECHNOLOGY

Applicant Address

2,7TH FLOOR, CGO COMPLEX, LODHI ROAD, NEW DELHI-110021

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. RAMESH CHANDER KUHAD	DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF DELHI, SOUTH CAMPUS, BENITO JUAREZ ROAD, NEW DELHI-110021
2	MUKESH KAPOOR	INTERNATIONAL CENTER FOR GENETIC ENGINEERING AND BIOTECHNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067

PCT International Classification Number

C12N1/21; C12N9/24

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA