Title of Invention	"A PROCESS FOR THE PREPARATION OF CELLULASE FREE XYLANASE"
Abstract	A process for the preparation of cellulase free xylanase A process for the preparation of cellulase free xylanase by growing Tricellula sp. having ATCC No. 62179 in a conventional fermentation medium having a pH ranging between 6.0 to 8.0, containing assimil able carbon 1 to 5 wt% w/v and assimil able nitrogen source and other conventional micronutrients at a temperature ranging between 25 to 35°C for a period of atleast 24 hrs, separating the mycelia from supernatant by conventional methods concentrating the supernatant by conventional methods to obtain the desired cellulase free xylanase.

Title of Invention

"A PROCESS FOR THE PREPARATION OF CELLULASE FREE XYLANASE"

Abstract

A process for the preparation of cellulase free xylanase A process for the preparation of cellulase free xylanase by growing Tricellula sp. having ATCC No. 62179 in a conventional fermentation medium having a pH ranging between 6.0 to 8.0, containing assimil able carbon 1 to 5 wt% w/v and assimil able nitrogen source and other conventional micronutrients at a temperature ranging between 25 to 35°C for a period of atleast 24 hrs, separating the mycelia from supernatant by conventional methods concentrating the supernatant by conventional methods to obtain the desired cellulase free xylanase.

Full Text	This invention relates to a process for the preparation of cellulase free lanase. More particularly it relates to the preparation of the said enzyme using microbial source, specifically Tricellula inaequalis. Still more particularly it relates to the preparation of the said enzyme which is alkali stable and most active at neutral pH. Xylan is one of the major polysaccharides found in the hemicellulosic fraction of plant tissues. It occurs in the biosphere to the extent of 30% of the potential renewable materials. Xylan degrading enzymes of microbial origin act synergistically in nature to depolymerize xylanolytic materials into xylooligosaccharides including various monosaccharides and use it as the carbon sources. The ultimate major product of xylan hydrolysis is D-xylose and it can serve as the feedstock for the production of ethanol using Candida shehatae or Pachysolen tannophilus. Xylose can be used for the production of xylitol. It is an anticariogenic low calory sweetener. Hence, xylitol can find applications in the chewing gum production and as a sweetner for diabetic patients. Current biotechnological approaches to pulp-modification include biological bleaching where hemicellulases are used in the bleaching process. This method replaces hazardous chemical treatments such as chlorine and chlorine dioxide in facilitating lignin extraction. The application of cellulase free xylanases that are alkali-stable, thermostable and active at neutral pH might enhance both the technical and economic feasibilities of the production of dissolving pulp [Prade, R.A., Biotechnol. Genetic. Engg. Rev., (1955) 13, 101-131] In the prior arts Chainia sp. (NCL 82-5-1) was isolated from sand samples' obtained from Rajasthan and identified its capability to produce cellulase free xylanase [Srinivasan et at. (1984) Biotechnol. Lett. 16: 715-718]. Later on many actinomycete strains from American Type Culture Collection were reported to produce xylanase activity with/without cellulase activity. A xylanase component of Streptomyces roseiscleoticus (Chainia rosei) showed similar properties. Our invention demonstrate that the alkali stable, extracellular cellulase free xylanase can be produced using untreated cheap agricultural residues. This needs the appropriate inorganic and /or organic (or combination of both) nitrogen sources and ajDpropriaJ^ The main drawback of the earlier process using different strains is that no fungal source is reported to produce extracellular xylanase by using untreated cheap agricultural residues. Using the process of the present invention production of the cellulase free xylanase from Tricellula inaequalis utilizing cheap and easily available waste materials like agricultural or forest residues including bagasse pith and corn cob powder, is possible. The inventors of the present invention have screened many fungal sources for preparation of extracellular xylanazse. It was found that a fungal source Tricellula inaequallis for the preparation of the desired enzyme. The said culture has got an Accession number of ATCC 62179 (Catalogue of ATTC 17th edition, 1987). The same culture is mentioned in the Catalogue of the Culture Culture of the Commonwealth Mycological Institute with an Accession number CMI 51976 as well as in the catalogue of National Collection of Industrial Microorganisms (National Chemical Laboratory, Pune 411 008, India) with an Accession number NCIM 985. The main object of the present invention is therefore to provide a process for the preparation of cellulase free xylanase using the Tricellula sp. Particularly inaequallis sp. Another object of the present invention is to provide a process which utilizes easily available waste materials like agricultural or forest residues including bagasses pith and corn cob powder. Accordingly the present invention provides a process for the preparation of cellulase free xylanase which comprises growing Tricellula sp. having ATCC No. 62179 (GMI No Ti1976 onH NCIM No. 08G)Hn a conventional fermentation medium having a pH ranging between 6.0 to 8.0, containing assimilable carbon and assimilable nitrogen source and other conventional micronutrients at a temperature ranging between 25 to 35°C for a period of atleast 24 hrs, separating the mycelia from supernatant by conventional methods concentrating the supernatant by conventional methods to obtain the desired cellulase free xylanase. In an embodiment of the present invention the assimilable carbon source used is agricultural residue selected from the group consisting of wheat bran, corn cob, bagasse pith and coconut fibre preferably corn cob. In an another embodiment of the presentipvention the concentration of the carbon sourpe In yet another embodiment of the present invention the nitrogen source used is selected from the group consisting of diammonium hydrogen phosphate, sodium nitrate, ammonium nitrate, urea, corn steep liquor and soya milk. n a feature of the presenllinyentisath^ typical medium con and 0.1% wheat bran and yeast extract In another feature of the present invention the fungus can be maintained on potato dextrose agar (PDA, dextrose 0.5%) slants. In yet another feature of the present invention the xylanazse production could be done in a synthetic medium (hereafter mentioned as M-1) containng following ingredients: The composition of the M-1 is as follows (g/liter): Polyoxythylenesorbitan Monooleate Composition of the trace elements is as follows (mg/liter): Cellulose-123 (Schleicher and Schull Co., FRG) 10 g/liter is supplied as a carbon source. In yet another feature of the invention the quantity of the cellulase free xylanase production can be controlled by using the nitrogen source as described above in the medium. The production of the cellulase free enzyme is also dependent upon the use of the surfactants like Tween-80 and Tween-40 (Polyoxythylenesorbitan Monopalmitate) (0.00 - 0.75%). The present invention is illustrated in the examples below which should not however be construed to limit the scope of the invention. EXAMPLE 1 Tricellula inaegualis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was adjusted to 8.0 at 30°C. The fungus can be maintained on Potato dextrose agar (PDA) but its dextrose content was never exceeded above 0.5%. For the preparation of pre-inoculum the mycelium/spores from the PDA slants were used to inoculate a pre-inoculum medium containing 1% corn cob and 0.1% yeast extract. After 16 to 36 hours of the incubating Tricellula sp. in this medium on a rotary shaker it was transferred to the experimental flasks (I liter capacity) containing 150 ml of the synthetic medium. The fermentation was continued for 2-05 days on a rotary shaker at 28 °C. EXAMPLE 2 Tricellula \naequalis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was adjusted to 7.5. The inoculum, thus developed for 30h was used to produce extracellular cellulase free xylanase. It contained 3.5-5.5 international units of extracellular cellulase free xylanase in the experimental flasks. The 10% of this inoculum was mixed in M-1 medium for the production of said enzyme. In this example the 1% cellulose was replaced by bagasse pith, or coconut fibre or, wheat bran. The amount for surfactant like Tween-80 or Tween-40 in the media can be 0.075%. Under these experimental conditions, minimum 2.5-5.0 units of enzyme was produced with 36 hrs of fermentation. EXAMPLE 3 This example indicates the effect of addition of surfactants in the fermentation medium on the production of xylanase. Tricellula \naequalis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was adjusted to 8.0 at 30° C. The fungus can be maintained on Potato dextrose agar (PDA) but its dextrose content was never exceeded above 0.5%. For the preparation of pre-inoculum the mycelium/spores from the PDA slants were used to inoculate a pre-inoculum medium containing 1% corn cob and 0.1% yeast extract. The pre-inoculum medium prepared was devoid of any surfactants. Under such fermentation conditions Tricellula inaequalisi produces xylanase activity that is totally devoid of cellulase activity. EXAMPLE 4 This example describes the stability of cellulase free xylanase enzyme in a wide range of pH 3-9 at 37 C. The enzyme samples were incubated in the respective buffer solutions for 4 hours at 50° C. All the samples retained atleast 50% of the original activity. The major advantages of this inventions are as follows: 1. Tricellula inaequalis, a fungal source is producing the extracellular cellulase free xylanase only under the specific fermentation conditions. Particularly, it is possible to monitor the production of the enzyme by incorporating the appropriate carbon source in the medium. 2. For the production of the desired enzyme cheap and waste agricultural sources can be used. This contributes to the pollution abatement. 3. The physicochemical properties of the desired enzyme can be suitable for the development of a process of biopulping. We claim: 1. A process for the preparation of cellulase free xylanase which comprises growing Tricellula sp. having ATCC No. 62179 in a conventional fermentation medium having a pH ranging between 6.0 to 8.0, containing assimilable carbon 1 to 5 wt% w/v and assimilable nitrogen source and other conventional micronutrients such as herein described at a temperature ranging between 25 to 35°C for a period of atleast 24 hrs, separating the mycelia from supernatant by conventional methods concentrating the supernatant by conventional methods to obtain the desired cellulase free xylanase. 2. A process as claimed in claim I wherein the assimilable carbon source used is agricultural residue selected from the group consisting of wheat bran, corn cob, bagasse pith and coconut fibre preferably corn cob. 3. A process as claimed in claims I to 2 wherein the nitrogen source used is selected from the group consisting of diammonium hydrogen phosphate, sodium nitrate, ammonium nitrate, urea, corn steep liquor and soya milk. 4. A process for the preparation of cellulase free xylanase as substantially described hereinbefore with reference to examples.

Full Text

This invention relates to a process for the preparation of cellulase free lanase. More particularly it relates to the preparation of the said enzyme using microbial source, specifically Tricellula inaequalis. Still more particularly it relates to the preparation of the said enzyme which is alkali stable and most active at neutral pH.
Xylan is one of the major polysaccharides found in the hemicellulosic fraction of plant tissues. It occurs in the biosphere to the extent of 30% of the potential renewable materials. Xylan degrading enzymes of microbial origin act synergistically in nature to depolymerize xylanolytic materials into xylooligosaccharides including various monosaccharides and use it as the carbon sources. The ultimate major product of xylan hydrolysis is D-xylose and it can serve as the feedstock for the production of ethanol using Candida shehatae or Pachysolen tannophilus. Xylose can be used for the production of xylitol. It is an anticariogenic low calory sweetener. Hence, xylitol can find applications in the chewing gum production and as a sweetner for diabetic patients.
Current biotechnological approaches to pulp-modification include biological bleaching where hemicellulases are used in the bleaching process. This method replaces hazardous chemical treatments such as chlorine and chlorine dioxide in facilitating lignin extraction. The application of cellulase free xylanases that are alkali-stable, thermostable and active at neutral pH might enhance both the
technical and economic feasibilities of the production of dissolving pulp [Prade, R.A., Biotechnol. Genetic. Engg. Rev., (1955) 13, 101-131]

In the prior arts Chainia sp. (NCL 82-5-1) was isolated from sand samples' obtained from Rajasthan and identified its capability to produce cellulase free xylanase [Srinivasan et at. (1984) Biotechnol. Lett. 16: 715-718]. Later on many actinomycete strains from American Type Culture Collection were reported to produce xylanase activity with/without cellulase activity. A xylanase component of Streptomyces roseiscleoticus (Chainia rosei) showed similar properties. Our invention demonstrate that the alkali stable, extracellular cellulase free xylanase can be produced using untreated cheap agricultural residues. This needs the appropriate inorganic and /or organic (or combination of both) nitrogen sources and ajDpropriaJ^
The main drawback of the earlier process using different strains is that no fungal source is reported to produce extracellular xylanase by using untreated cheap agricultural residues. Using the process of the present invention production of the cellulase free xylanase from Tricellula inaequalis utilizing cheap and easily available waste materials like agricultural or forest residues including bagasse pith and corn cob powder, is possible. The inventors of the present invention have screened many fungal sources for preparation of extracellular xylanazse. It was found that a fungal source Tricellula inaequallis for the preparation of the desired enzyme. The said culture has got an Accession number of ATCC 62179 (Catalogue of ATTC 17th edition, 1987). The same culture is mentioned in the Catalogue of the Culture Culture of the Commonwealth Mycological Institute with an Accession number CMI 51976 as well as in the catalogue of National Collection of Industrial Microorganisms (National Chemical Laboratory, Pune 411 008, India) with an Accession number NCIM 985.

The main object of the present invention is therefore to provide a process for the preparation of cellulase free xylanase using the Tricellula sp. Particularly inaequallis sp.
Another object of the present invention is to provide a process which utilizes easily available waste materials like agricultural or forest residues including bagasses pith and corn cob powder.
Accordingly the present invention provides a process for the preparation of cellulase free xylanase which comprises growing Tricellula sp. having ATCC No. 62179 (GMI No Ti1976 onH NCIM No. 08G)Hn a conventional fermentation medium having a pH ranging between 6.0 to 8.0, containing assimilable carbon and assimilable nitrogen source and other conventional micronutrients at a temperature ranging between 25 to 35°C for a period of atleast 24 hrs, separating the mycelia from supernatant by conventional methods concentrating the supernatant by conventional methods to obtain the desired cellulase free xylanase.
In an embodiment of the present invention the assimilable carbon source used is agricultural residue selected from the group consisting of wheat bran, corn cob, bagasse pith and coconut fibre preferably corn cob.
In an another embodiment of the presentipvention the concentration of the carbon sourpe In yet another embodiment of the present invention the nitrogen source used is selected from the group consisting of diammonium hydrogen phosphate, sodium nitrate, ammonium nitrate, urea, corn steep liquor and soya milk.
n a feature of the presenllinyentisath^ typical medium con and 0.1% wheat bran and yeast extract
In another feature of the present invention the fungus can be maintained on potato dextrose agar (PDA, dextrose 0.5%) slants. In yet another feature of the present invention the xylanazse production could be done in a synthetic medium (hereafter mentioned as M-1) containng following ingredients:
The composition of the M-1 is as follows (g/liter):

Polyoxythylenesorbitan Monooleate
Composition of the trace elements is as follows (mg/liter):

Cellulose-123 (Schleicher and Schull Co., FRG) 10 g/liter is supplied as a carbon source.
In yet another feature of the invention the quantity of the cellulase free xylanase production can be controlled by using the nitrogen source as described above in the medium.
The production of the cellulase free enzyme is also dependent upon the use of the surfactants like Tween-80 and Tween-40 (Polyoxythylenesorbitan Monopalmitate) (0.00 - 0.75%).
The present invention is illustrated in the examples below which should not however be construed to limit the scope of the invention.
EXAMPLE 1
Tricellula inaegualis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was adjusted to 8.0 at 30°C. The fungus can be maintained on Potato dextrose agar (PDA) but its dextrose content was never exceeded above 0.5%. For the preparation of pre-inoculum the mycelium/spores from the PDA slants were used to inoculate a pre-inoculum medium containing 1% corn cob and 0.1% yeast extract. After 16 to 36 hours of the incubating Tricellula sp. in this medium on a rotary shaker it was transferred to the experimental flasks (I liter capacity) containing 150 ml of the synthetic medium. The fermentation was continued for 2-05 days on a rotary shaker at 28 °C.
EXAMPLE 2
Tricellula \naequalis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was
adjusted to 7.5. The inoculum, thus developed for 30h was used to produce extracellular cellulase free xylanase. It contained 3.5-5.5 international units of extracellular cellulase free xylanase in the experimental flasks. The 10% of this inoculum was mixed in M-1 medium for the production of said enzyme. In this example the 1% cellulose was replaced by bagasse pith, or coconut fibre or, wheat bran. The amount for surfactant like Tween-80 or Tween-40 in the media can be 0.075%. Under these experimental conditions, minimum 2.5-5.0 units of enzyme was produced with 36 hrs of fermentation.
EXAMPLE 3
This example indicates the effect of addition of surfactants in the fermentation medium on the production of xylanase. Tricellula \naequalis was maintained on media that contains 1% and 0.1% wheat bran and yeast extract, respectively. The pH of the medium was adjusted to 8.0 at 30° C. The fungus can be maintained on Potato dextrose agar (PDA) but its dextrose content was never exceeded above 0.5%. For the preparation of pre-inoculum the mycelium/spores from the PDA slants were used to inoculate a pre-inoculum medium containing 1% corn cob and 0.1% yeast extract. The pre-inoculum medium prepared was devoid of any surfactants. Under such fermentation conditions Tricellula inaequalisi produces xylanase activity that is totally devoid of cellulase activity.
EXAMPLE 4
This example describes the stability of cellulase free xylanase enzyme in a wide range of pH 3-9 at 37 C. The enzyme samples were incubated in the
respective buffer solutions for 4 hours at 50° C. All the samples retained atleast 50% of the original activity.
The major advantages of this inventions are as follows:
1. Tricellula inaequalis, a fungal source is producing the extracellular
cellulase free xylanase only under the specific fermentation conditions.
Particularly, it is possible to monitor the production of the enzyme by
incorporating the appropriate carbon source in the medium.
2. For the production of the desired enzyme cheap and waste agricultural
sources can be used. This contributes to the pollution abatement.
3. The physicochemical properties of the desired enzyme can be suitable
for the development of a process of biopulping.

We claim:
1. A process for the preparation of cellulase free xylanase which comprises
growing Tricellula sp. having ATCC No. 62179 in a conventional
fermentation medium having a pH ranging between 6.0 to 8.0, containing
assimilable carbon 1 to 5 wt% w/v and assimilable nitrogen source and
other conventional micronutrients such as herein described at a
temperature ranging between 25 to 35°C for a period of atleast 24 hrs,
separating the mycelia from supernatant by conventional methods
concentrating the supernatant by conventional methods to obtain the
desired cellulase free xylanase.
2. A process as claimed in claim I wherein the assimilable carbon source
used is agricultural residue selected from the group consisting of wheat
bran, corn cob, bagasse pith and coconut fibre preferably corn cob.
3. A process as claimed in claims I to 2 wherein the nitrogen source used is
selected from the group consisting of diammonium hydrogen phosphate,
sodium nitrate, ammonium nitrate, urea, corn steep liquor and soya milk.
4. A process for the preparation of cellulase free xylanase as substantially
described hereinbefore with reference to examples.

Documents:

0225-del-2001-abstract.pdf

0225-del-2001-claims.pdf

0225-del-2001-correspondence-others.pdf

0225-del-2001-correspondence-po.pdf

0225-del-2001-correspondence.pdf

0225-del-2001-description (complete).pdf

0225-del-2001-form-1.pdf

0225-del-2001-form-19.pdf

0225-del-2001-form-2.pdf

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Patent Number

218343

Indian Patent Application Number

225/DEL/2001

PG Journal Number

22/2008

Publication Date

30-May-2008

Grant Date

31-Mar-2008

Date of Filing

28-Feb-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ANIL LACHKE	NATIONA CHEMICAL LABORATORY, PUNE - 411008, MAHARASHTRA, INDIA.
2	CHINNATHAMBI SATHIVEL	NATIONA CHEMICAL LABORATORY, PUNE - 411008, MAHARASHTRA, INDIA.

PCT International Classification Number

C12N9/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA