Title of Invention

"A PROCESS OF PREPARING HIGH YIELDS OF XYLANASE PROTEIN FROM BACILLUS SPECIES"

Abstract

This invention relates to a process of preparing high yields of xylanase protein from Bacillus sp. by isolating pool of micro-organisms from soil, screening for a cellulase free xylanase producer Bacillus sp., growing the said screened Bacillus sp. in a fermentor and adding the second growth media at an exponentially increasing rate to get high cell density and xylanase protein while maintaining a constant specific growth after the said first growth media is depleted of the nutrients by the said Bacillus sp., harvesting the cells after 20-24 h of growth and extracting the xylanase protein present in the supernatant of the Bacillus culture by known techniques.

Full Text

FIELD OF INVENTION This invention relates to a process of preparing high yields of xylanase (a non living substance) from Bacillus species. BACKGROUND Xylan is a major constituent of the hemicellulose in wood fibre the breakdown of which is an essential step in paper manufacture. The use of chlorine and chlorine based chemicals for the pulp bleaching step is slowly being phased out because of their negative environmental impact and is being replaced by an environmentally friendly process utilizing xylanases for the breakdown of xylan. This has created a need for theremostable xylanases preferably those, which are active under alkaline conditions for the paper and pulp industry. While fungal cultures usually give high product concentrations they are not easily amenable to scale-up in fermentors. High viscosity, poor oxygen transfer rates and long inycelia are some of the problems in large scale fermentation. Bacillus strains, which are the other important producer of xylanases are much more easier to handle in fermentors. However typical product concentrations achieved are 50-100 U/mL (measured in u mol of xylose formed per mL per minute). This implies that larger reactors are required to produce the same product amount thereby increasing the product costs. Also product is often contaminated with cellulase, which is undesirable since it degrades the cellulose which is used for paper making. OBJECTS OF THE INVENTION An object of this invention is to propose a low cost process high level production of xylanase from Bacillus species. DESCRIPTION OF INVENTION According to this invention there is provided a process of preparing high yields of xylanase (non-living substance) from Bacillus species comprising: The first growth media consists of: KH2PO4 1.0 g/L K2HPO4 1.0g/L NaCl 1.0 g/L CaCl2 0.1 g/L MgSO4 0.2 g/L NH4NO3 1-0 g/L Yeast extract 2.5 g/L Peptone 2.5 g/L Glucose 5.0 g/L Xylan 5.0 g/L Trace elements5 lx 100x trace elements solution contained: 0.4 g/L CoCl2, 0.1 gTL CuSO4.5H2O, 0.05 g/L H3BO3,1-0 g/L MnCl2, 0.2 g/L Na2MoO4.2H2O, 0.2 g/L ZnSO4.7H2O and 0.1 g/L FeCl3 The second growth media consists of: KH2PO4 1.0 g/L K2HPO4 1.0 g/L NaCl 1.0 g/L CaCl2 0.1 g/L MgSO4 0.2 g/L NH4NO3 20.0 g/L Yeast extract 50.0 g/L Peptone 50.0 g/L Glucose 100.0 g/L' Xylan 50.0 g/L Trace elementss lx s 100x trace elements solution contained: 0.4 g/L CoCl2, 0.1 g/L CuSO4.5H2O, 0.05 g/L H3BO3, 1.0 g/L MnCl2,0.2 g/L Na2MoO4 2H2O,0.2 g/L ZnSO4.7H2O and 0.1 g/L FeCl3 The constant specific growth rate is 0.2 h"1 and the harvesting of cells is carried out after 20 h of growth. The invention wilL now be described with reference to the accompanying drawings and foregoing example. Figure 1 shows the schematic representation of feeding strategy used to add the second growth media to grow the organism. Figure 2 shows the cell density and amount of xylanase f&xticin produced in extracellular fraction of the Bacillus culture during the course of growth. The invention will now be described with reference to the following example: EXAMPLE A xylanase producer Bacillus sp. was isolated from soil and grown in a fennentor at a pH of 7.0 to 7.5, temperature 37 °C and 40% dissolved oxygen tension. Initially the organism was grown in 1L media containing 1.0 g/L KH2PO4,1.0 g/L K2HPO4, 1.0 g/L NaCl, 0.1 g/L CaCl2,0.2 g/L MgSO4,1.0 g/L NH4NO3, 2.5 g/L yeast extract, 2.5 g/L peptone, 5.0 g/L Glucose, 5.0 g/L Xylan, lx trace elements solution1. After ~4 h of growth the media started getting depleted of the nutrients, and then the concentrated media (which was 20x with respect to glucose, yeast extract, peptone and ammonium nitrate; 10x with respect to xylan and lx with respect to remaining components) was fed at the rate of 10 mL/h. The feed rate was increased exponentially by a factor of s 100x trace elements solution contained: 0.4 g/L CoCI2, 0.1 g/L CuSO4.5H2O,0.05 g/L H3BO3, 1.0 g/L MnCl2,0.2 g/L Na2MoO4.2H2O,0.2 g/L ZnSO4.7H2O and 0.1 g/L FeCl3 1.26 every 70 minutes thus doubling the feed rate every 3.5 h in order to keep the specific growth rate of the Bacillus organism at 0.2 h-1 (see fig. 1). The feed rate was not increased after 40 mL/h and cells were harvested after 20 h of growth. The cells were removed by centrifugation and the supernatant was assayed for xylanase by DNS method for u moles of xylose formed using xylan as substrate. This process yielded a cell density of 350 g/L and xyalanse activity of 1300U/mLof the culture (SeeFig.2). The substrate for the xylanase is xylan. The assay was performed in two ways; one with 0.5% xylan in the reaction mixture (which is the standard concentration used by other researchers) and the other with 1.0% xylan in the reaction mixture (to ensure sufficient availability of the substrate for the protein). It was observed that the xylanase activity in the second case was double of the first. Thus the maximum xylanase protein achieved by the present method is 1300 x 2 = 2600 U/mL if sufficient substrate is available. The concentration of glucose in second growth media is much more than xylan, to prevent the use of xylan as carbon source and thereby ensuring the availability of xylan for xylanase production. RESULTS: The procedure described earlier by other researchers yielded around 50-100 U of xylanase per mL of the culture. The present invention describe a far more efficient process for production of xylanase from Bacillus sp. The yield from the process described above are greater than 1300 U/mL of culture. Thus the amount of the protein has been increased by -15 times which would cut down the fermentation cost. In addition, the protein is extracellular, thus simple filtration can be used to remove the cells. The supernatant which contains the protein and is also free of cellulase, can be directly used in the pulp and paper industry. Thus, this low cost xylanse protein can effectively replace the environmentally harmful chlorine for bleaching purposes. According to the prior art, the effective yield of xylanase protein was 50-100 U/mL using Bacillus cultures. However this process gives a yield of -1300 U/mL of culture (See fig. 2). Thus, the present process improves upon the earlier process yields. Futher, this process reduces the cost of xylanase production. This in turn will help in replacing the earlier environmentally hazardous chlorine based methods of pulp bleaching with the environmentally friendly method using xylanase. WE CLAIM: 1. A process of preparing high yields of xylanase (non-living substance) from Bacillus species comprising: isolating pool of microorganisms from soil and screening for a cellulase-free xylanase producer Bacillus sp. growing the said screened Bacillus sp. in a fermentor at a pH 7.0 to 7.5, temperature 36-38°C and dissolved oxygen concentration 35-45% in the first growth media, as herein defined, adding the second growth media, as herein defined, at an exponentially increasing rate to get high cell density and xylanase while maintaining a constant specific growth rate, after the said first growth media has depleted of the nutrients by the said Bacillus sp., followed by centrifugafion and harvesting the cells after 20-24 h of growth, and extracting the xylanase present in the supernatant of the Bacillus culture by known techniques. 2. A process as claimed in claim 1 wherein the temperature of the fermentor is 37°C and dissolved oxygen concentration is 40%. 3. A process as claimed in claim 1 wherein the first growth media consists of: KH2PO4 1.0 g/L K2HPO4 1.0 g/L NaCl 1.0 g/L CaCl2 0.1 g/L MgSO4 0.2 g/L NH4NO3 1.0 g/L Yeast extract 2.5 g/L Peptone 2.5 g/L Glucose 5.0 g/L Xylan 5.0 g/L Trace elements5 lx s 100x trace dements solution contained: 0.4 g/L CoCl2, 0.1 g/L CuSO4.SH2O, 0.05 g/L H3BO3 1.0 g/L MnCl2, 0.2 g/L NaMoO.2H2O, 0.2 g/L ZnSO4.7H2O and 0.1 g/L FeCl3 4. A process as claimed in claim 1 wherein the second growth media consists of: KH2PO4 1.0 g/L K2HPQ4 1.0 g/L NaCl 1.0 g/L Cad2 0.1 g/L MgSO4 0.2 g/L NH4NO3 20.0 g/L Yeast extract 50.0 g/L Peptone 50.0 g/L Glucose 100,0 g/L Xylan 50.0 g/L Trace elementss lx s 100x trace elements solution contained: 0.4 g/L CoCl2, 0.1 g/L CuSO4.5H2O, 0.05 g/L H3BO3, 1.0 g/L MnCl2,0.2 g/L Na2MoO4.2H2O,0.2 g/L ZnSO4.7H2O and 0.1 g/L FeCl3 5. A process as claimed in claim 1 wherein the constant specific growth rate is 0.2 h-1. 6. A process as claimed in claim 1 wherein harvesting of cells is carried out after 20 h of growth. 7. A process of preparing high yield of xylanase (nonliving substance) from Bacillus sp. substantially as herein described with reference to the accompanying drawings and forgoing example.

Documents:

822-del-2000-abstract.pdf

822-del-2000-claims.pdf

822-del-2000-complete specification (granted).pdf

822-del-2000-correspondence-others.pdf

822-del-2000-correspondence-po.pdf

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

822-del-2000-drawings.pdf

822-del-2000-form-1.pdf

822-del-2000-form-19.pdf

822-del-2000-form-2.pdf

822-del-2000-form-26.pdf

822-del-2000-form-4.pdf

822-del-2000-petition-138.pdf