Title of Invention	"AN IMPROVED PROCESS FOR THE PREPARATION OF PROTEASE ENZYME USING A NOVEL STRAIN OF SPOROSARCINA"
Abstract	This invention relates to an improved process for the preparation of protease enzyme using a novel strain of Sporosarcina which comprises growing the said novel strain of Sporosarcina in a conventional medium such as herein described, supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for period of 15 to 72 hours at room temperature and at neutral pH, separating the cells of Sporosarcina by conventional methods and recovering the desired protease from supematent by conventional dialysis and precipitation methods as described herein.

Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF PROTEASE ENZYME USING A NOVEL STRAIN OF SPOROSARCINA"

Abstract

This invention relates to an improved process for the preparation of protease enzyme using a novel strain of Sporosarcina which comprises growing the said novel strain of Sporosarcina in a conventional medium such as herein described, supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for period of 15 to 72 hours at room temperature and at neutral pH, separating the cells of Sporosarcina by conventional methods and recovering the desired protease from supematent by conventional dialysis and precipitation methods as described herein.

Full Text	The present invention relates to a process for the preparation of protease enzyme form novel strain of sporosarcina. The protease is useful for Biological curing of Collagen and gelatine. The enzyme so prepared from this process is useful in altering the consistency of gelatine to make it suitable for emulsification purposes to be used as a binder for photosensitive and similar chemicals. The hitherto known method of gelatine liquefication derived from protease obtained from plant sources such as papaya and pineapple is dependent on fruit preservation units for the raw matehal and require an elaborate procedure to extract the enzyme. Although the enzyme preparations obtained by these sources of plant origin are at present being utilised by the Industry, the new process herein being patented offers an alternative method to obtain the enzyme through controlled fermentation technique. In contrast to this, the bacterial strain can be grown in controlled fermenters as an additional facility of the user Industry and can be installed at site in which ever location the Industry is located. The advantage of this method lies in the fact that it is possible to produce the enzyme bearing specific properties by varying the substrate that induces the production of the enzyme. Thus it will be possible to predetermine the quality of the enzyme depending upon its use. The strains described have the following salient characters in the wild state: Sporosarcina RRLJ 1 (Bezbaruah J BioSci. 5:267-278,1983) : Gram positive cocci, with heat resistant bodies, with catalase, reaction to glucose neutral, produces hydrogen sulphide from thiosulphate, citrate not utilised, unable to grow in nitrogen free medium, ureolytic and nitrifying, acetyl methyl carbinol negative, methyl red negative starch hydrolysis negetive, indol produced, neutral in milk, active gelatin liquefier. white spreading profusely growing colonies . Nearest affinity to Proteus Produces gas grom glucose, does not form H2S. Does not ferment lactose . Hydrolyses urea slowly (48 h). Habitat: tea plantation soil. Sprosarcina RRLJ 1 was obtained from an earlier collection at RRL Jorhat (Bezbaruah 1983) was grown for 48h under aerated conditions at pH 6.5 in a nutrient media containing g/L-1 KH2PO4,-3, K2HPO4.-O.2, Mg SO4.-7 H2O,-1, (NH4)2SO4,-5, casein hydrolysate, 10 (dissolved in 30 ml of 1N NaOH ).and sucrose 15 . The extra cellular protease produced by the strain in the culture media was recovered . The strain Sporosarcina RRLJ 1 (Bezbaruah J BioSci. 5:267-278,1983) which has been deposited at RRL Jorhat has the following distinguishing characters: Gram positive cocci, with heat resistant bodies, with catalase, reaction to glucose neutral, produces hydrogen sulphide from thiosulphate, citrate not utilised, unable to grow in nitrogen free medium, ureolytic and nitrifying, acetyl methyl carbinol negetive, methyl red negative starch hydrolysis negetive, indol produced, neutral in milk, active gelatin liquefier. white spreading profusely growing colonies . Nearest affinity to Proteus Produces gas grom glucose, does not form H2S. Does not ferment lactose . Hydrolyses urea slowly (48 h). Habitat: tea plantation soil. Protease recovery through conventional methods compnses of precipitation through addition of ammonium sulphate or zinc sulphate and dialysis of the precipitate slurry in phosphate buffer to remove the excess of salts. In this process the enzyme is recovered from the culture media by suspending the supernatent of the culture media in dialysis bags against phosphate buffer and recovery of the enzyme through precipitation with acetone. The acetone precipitate is redissolved in calcium acetate and reprecipitated with acetone to yield pure crystals. The pure crystals are used to treat gelatine to transform it to the desired consistency. The novelty of the process involves culturing the strain Sporosarcina RRLJ 1 in a fermentor under controlled conditions with aeration at a temperature of 30° C. using specific protease inducing substrates such as casein or gelatine. The organism is allowed to grow for 72 h . Recovery of the enzyme through this method is achieved without bulky processing units such as those which would be required to process protease containing plant material. The waste products which will comprise of the spent bacterial nutrient medium and the biomass can be utilised without further treatment as soil additives for agricultural land. The enzyme can be prepared as and when required simply by cultunng the Sprosarcina RRLJ 1 as and when desired. Furthermore, the preparation produced through bacterial fermentation of carbohydrates will yield high quality protease with predictable properties. The enzyme can also be used to degracte protein to release tyrosine selectively and can be incorporated into detergents and tooth pastes to produce high quality detergents and tooth pastes. The mrcro-organism in question RRLJ 1 was originally isolated from tea plantations and cultured in a medium containing peptone, or tryptone, or urea and potassium phosphate, magnesium sulphate, sucrose or glucose. The object of the present invention is to provide an alternative method of protease production. Yet another objective is produce an enzyme with known predictable properties. The main properties of this invention are: 1. Controlled production of protease with desired properties. 2. Production at need or the entire year round depending upon the demand. 3. No problem of effluent disposal . 4. Energy saving . It is a cold process produced through bacterial fermentation. 5. It allows controlled liquefication of the gelatine, to achieve desired viscosity without prior treatment with acid and reduces the curing time. 6. It can be formulated in a powder form for easy handling and transport. Its mode of action are: The protease from the organism is able to release tyrosine from the proteinaceous substrate within 20 min and is active in a pH range of 6-10 (optimum pH 6.5) and a temperature range of 10- 60° C ( optimum 30° C ). Complete liquefication of gelatine within 4 hrs. is possible through addition of enzyme crystals to gelatine. Treatment of the solid gelatine with sensitive chemicals to be used as surfactants prior to addition of the enzyme and allowing the enzyme to react with the gelatine to achieve the desired viscosity will yield the emulsion with the chemicals to be used for coating the selected surface. Depending upon the nature of the chemicals used the gelatin-chemical emulsion will serve as surfactants to yield different types of material. Accordingly the present invention provides an improved process for the preparation of protease enzyme using a novel strain of Sporosarcina which comprises growing the said novel strain of Sporosarcina in a conventional medium such as herein described , supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for period of 15 to 72 hours at room temperature and at neutral pH , separating the cells of Sporosarcina by conventional methods and recovering the desired protease from supematent by conventional dialysis and precipitation methods as described herein . In an embodiment of this invention the growing of Sporosarcina may be effected at neutral pH , room temperature for 48 hours or more. In another embodiment the protease may be recovered by known precipitation and dialysis methods. In still another embodiment of this invention separation of micro-organisms may be effected by centrifugation /filtration. Example 1:The organism is cultured in a medium containing g/1-1 , KH2 PO4, .0.2, MgSO4.7H2O ,-1, (NH4)2 ,-5, casein hydrolysate , -10 ( dissolved in 30 ml of 1N NaOH ) and sucrose 15 for 72 h . At the end of 72 hours the cells are separated from the culture medium and the supematent is dialysed in phosphate buffer (pH 7 ) for 24 h with 4 changes of phosphate buffer at 6 h intervals. The dialysed sample is precipitated in acetone and the precipitate is used for liquefunging gelatine. Example 2: Protein substrate used was casein. Using 20 µg of crude protein it released 10 µg tyrosine in 20 min. Precipitated crude enzyme recovered from 1 L of 72 h old cultures (total volume 5 ml) can liquefy gelatine in 4 h when introduced at a concentration of 0.1 mi to 5 g slabs of 10% gelatine. Example 3: The organism is cultured in a medium containing g/L' KH2PO4,-3, K2HPO4, -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved in 30 ml of IN NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatent is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. The enzyme precipitated through addition of ammonium sulphate was more active than that precipitated with zinc chloride. Example 4: The organism is cultured in a medium containing g/L-1 KH2PO4,-3, K2HPO4, -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved in 30 ml of IN NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatant is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. The enzyme precipitated with ammonium chioride and recovered again after acclimatisation with 10% gelatine for 12 h, was more potent than the gelatine untreated Addition of gelatine into the culture media enhanced the gelatine liquefying property of the strain. Example 5: The organism is cultured in a medium containing g/L-1 KH2PO4,-3, K2HPO4. -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved In 30 ml of 1N NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatent is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. Using a culture media containing 15g/L-1 of sucrose, it was possible to recover 2.514 g precipitate in fresh weight from 100 ml cultures in 72 h. through zinc chloride precipitation. And 2 g precipitate from ammonium chloride precipitation. We Claim: 1. An improved process for the preparation of protease enzyme using a novel strain of Sporosarcina which comprises growing the said novel strain of Sporosarcina in a conventional medium such as herein described , supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for period of 15 to 72 hours at room temperature and at neutral pH , separating the cells of Sporosarcina by conventional methods and recovering the desired protease from supernatent by conventional dialysis and precipitation methods as described herein . 2. An improved process as claimed in claim 1 wherein the protease is recovered by known precipitation and dialysis methods. 3. An improved process as claimed in claim 1 wherein separation of cell of Sporosarcina may be effected by centrifugation /filtration. 4. An improved process for the preparation of protease enzyme using a novel strain of Sporosarcina substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the preparation of protease enzyme form novel strain of sporosarcina. The protease is useful for Biological curing of Collagen and gelatine. The enzyme so prepared from this process is useful in altering the consistency of gelatine to make it suitable for emulsification purposes to be used as a binder for photosensitive and similar chemicals.
The hitherto known method of gelatine liquefication derived from protease obtained from plant sources such as papaya and pineapple is dependent on fruit preservation units for the raw matehal and require an elaborate procedure to extract the enzyme. Although the enzyme preparations obtained by these sources of plant origin are at present being utilised by the Industry, the new process herein being patented offers an alternative method to obtain the enzyme through controlled fermentation technique. In contrast to this, the bacterial strain can be grown in controlled fermenters as an additional facility of the user Industry and can be installed at site in which ever location the Industry is located. The advantage of this method lies in the fact that it is possible to produce the enzyme bearing specific properties by varying the substrate that induces the production of the enzyme. Thus it will be possible to predetermine the quality of the enzyme depending upon its use.
The strains described have the following salient characters in the wild state:
Sporosarcina RRLJ 1 (Bezbaruah J BioSci. 5:267-278,1983) : Gram positive cocci, with heat resistant bodies, with catalase, reaction to glucose neutral, produces hydrogen sulphide from thiosulphate, citrate not utilised,
unable to grow in nitrogen free medium, ureolytic and nitrifying, acetyl methyl carbinol negative, methyl red negative starch hydrolysis negetive, indol produced, neutral in milk, active gelatin liquefier. white spreading profusely growing colonies . Nearest affinity to Proteus Produces gas grom glucose, does not form H2S. Does not ferment lactose . Hydrolyses urea slowly (48 h). Habitat: tea plantation soil.
Sprosarcina RRLJ 1 was obtained from an earlier collection at RRL Jorhat (Bezbaruah 1983) was grown for 48h under aerated conditions at pH 6.5 in a nutrient media containing g/L-1 KH2PO4,-3, K2HPO4.-O.2, Mg SO4.-7 H2O,-1, (NH4)2SO4,-5, casein hydrolysate, 10 (dissolved in 30 ml of 1N NaOH ).and sucrose 15 . The extra cellular protease produced by the strain in the culture media was recovered . The strain Sporosarcina RRLJ 1 (Bezbaruah J BioSci. 5:267-278,1983) which has been deposited at RRL Jorhat has the following distinguishing characters: Gram positive cocci, with heat resistant bodies, with catalase, reaction to glucose neutral, produces hydrogen sulphide from thiosulphate, citrate not utilised, unable to grow in nitrogen free medium, ureolytic and nitrifying, acetyl methyl carbinol negetive, methyl red negative starch hydrolysis negetive, indol produced, neutral in milk, active gelatin liquefier. white spreading profusely growing colonies . Nearest affinity to Proteus Produces gas grom glucose, does not form H2S. Does not ferment lactose . Hydrolyses urea slowly (48 h). Habitat: tea plantation soil.
Protease recovery through conventional methods compnses of precipitation through addition of ammonium sulphate or zinc sulphate and dialysis
of the precipitate slurry in phosphate buffer to remove the excess of salts. In this process the enzyme is recovered from the culture media by suspending the supernatent of the culture media in dialysis bags against phosphate buffer and recovery of the enzyme through precipitation with acetone. The acetone precipitate is redissolved in calcium acetate and reprecipitated with acetone to yield pure crystals. The pure crystals are used to treat gelatine to transform it to the desired consistency.
The novelty of the process involves culturing the strain Sporosarcina RRLJ 1 in a fermentor under controlled conditions with aeration at a temperature of 30° C. using specific protease inducing substrates such as casein or gelatine. The organism is allowed to grow for 72 h . Recovery of the enzyme through this method is achieved without bulky processing units such as those which would be required to process protease containing plant material. The waste products which will comprise of the spent bacterial nutrient medium and the biomass can be utilised without further treatment as soil additives for agricultural land. The enzyme can be prepared as and when required simply by cultunng the Sprosarcina RRLJ 1 as and when desired. Furthermore, the preparation produced through bacterial fermentation of carbohydrates will yield high quality protease with predictable properties. The enzyme can also be used to degracte protein to release tyrosine selectively and can be incorporated into detergents and tooth pastes to produce high quality detergents and tooth pastes. The mrcro-organism in question RRLJ 1 was originally isolated from tea plantations and cultured in a medium containing peptone, or tryptone, or urea and potassium phosphate, magnesium sulphate, sucrose or glucose.
The object of the present invention is to provide an alternative method of protease production.
Yet another objective is produce an enzyme with known predictable properties.
The main properties of this invention are:
1. Controlled production of protease with desired properties.
2. Production at need or the entire year round depending upon the demand.
3. No problem of effluent disposal .
4. Energy saving . It is a cold process produced through bacterial fermentation.
5. It allows controlled liquefication of the gelatine, to achieve desired viscosity without prior treatment with acid and reduces the curing time.
6. It can be formulated in a powder form for easy handling and transport.
Its mode of action are:
The protease from the organism is able to release tyrosine from the proteinaceous substrate within 20 min and is active in a pH range of 6-10 (optimum pH 6.5) and a temperature range of 10- 60° C ( optimum 30° C ). Complete liquefication of gelatine within 4 hrs. is possible through addition of enzyme crystals to gelatine. Treatment of the solid gelatine with sensitive chemicals to be used as surfactants prior to addition of the enzyme and allowing the enzyme to react with the gelatine to achieve the desired viscosity will yield the emulsion with the chemicals to be used for coating the selected surface.
Depending upon the nature of the chemicals used the gelatin-chemical emulsion will
serve as surfactants to yield different types of material.
Accordingly the present invention provides an improved process for the preparation of
protease enzyme using a novel strain of Sporosarcina which comprises growing the said
novel strain of Sporosarcina in a conventional medium such as herein described ,
supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for
period of 15 to 72 hours at room temperature and at neutral pH , separating the cells of
Sporosarcina by conventional methods and recovering the desired protease from
supematent by conventional dialysis and precipitation methods as described herein .
In an embodiment of this invention the growing of Sporosarcina may be effected at
neutral pH , room temperature for 48 hours or more.
In another embodiment the protease may be recovered by known precipitation and
dialysis methods.
In still another embodiment of this invention separation of micro-organisms may be
effected by centrifugation /filtration.
Example 1:The organism is cultured in a medium containing g/1-1 , KH2 PO4, .0.2,
MgSO4.7H2O ,-1, (NH4)2 ,-5, casein hydrolysate , -10 ( dissolved in 30 ml of 1N
NaOH ) and sucrose 15 for 72 h . At the end of 72 hours the cells are separated from
the culture medium and the supematent is dialysed in phosphate buffer
(pH 7 ) for 24 h with 4 changes of phosphate buffer at 6 h intervals. The
dialysed sample is precipitated in acetone and the precipitate is used for liquefunging gelatine.
Example 2:
Protein substrate used was casein. Using 20 µg of crude protein it released 10 µg tyrosine in 20 min.
Precipitated crude enzyme recovered from 1 L of 72 h old cultures (total volume 5 ml) can liquefy gelatine in 4 h when introduced at a concentration of 0.1 mi to 5 g slabs of 10% gelatine.
Example 3:
The organism is cultured in a medium containing g/L' KH2PO4,-3, K2HPO4, -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved in 30 ml of IN NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatent is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. The enzyme precipitated through addition of ammonium sulphate was more active than that precipitated with zinc chloride.
Example 4:
The organism is cultured in a medium containing g/L-1 KH2PO4,-3, K2HPO4, -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved in 30 ml of IN NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatant is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. The enzyme precipitated with ammonium chioride and recovered again after
acclimatisation with 10% gelatine for 12 h, was more potent than the gelatine untreated
Addition of gelatine into the culture media enhanced the gelatine liquefying property of the strain.
Example 5:
The organism is cultured in a medium containing g/L-1 KH2PO4,-3, K2HPO4. -0.2, Mg SO4.7 H2O, -1, (NH4)2SO4,-5, casein hydrolysate,-10 (dissolved In 30 ml of 1N NaOH ).and sucrose 15 for 72 h. At the end of 72 hrs the cells are seperated from the culture medium and the supernatent is dialysed in phosphate buffer (pH 7) for 24 h with 4 changes of phasphate buffer at 6 h intervals. Using a culture media containing 15g/L-1 of sucrose, it was possible to recover 2.514 g precipitate in fresh weight from 100 ml cultures in 72 h. through zinc chloride precipitation. And 2 g precipitate from ammonium chloride precipitation.

We Claim:
1. An improved process for the preparation of protease enzyme using a novel strain of Sporosarcina which comprises growing the said novel strain of Sporosarcina in a conventional medium such as herein described , supplemented with casein hydrolysate or gelatin hydrolysate or a mixture thereof for period of 15 to 72 hours at room temperature and at neutral pH , separating the cells of Sporosarcina by conventional methods and recovering the desired protease from supernatent by conventional dialysis and precipitation methods as described herein .
2. An improved process as claimed in claim 1 wherein the protease is recovered by known precipitation and dialysis methods.
3. An improved process as claimed in claim 1 wherein separation of cell of Sporosarcina may be effected by centrifugation /filtration.
4. An improved process for the preparation of protease enzyme using a novel strain of Sporosarcina substantially as herein described with reference to the examples.

Documents:

2712-del-1998-abstract.pdf

2712-del-1998-claims cancelled.pdf

2712-del-1998-claims.pdf

2712-del-1998-complete specification(granted).pdf

2712-del-1998-correspondence-others.pdf

2712-del-1998-correspondence-po.pdf

2712-del-1998-description (complete).pdf

2712-del-1998-form-1.pdf

2712-del-1998-form-2.pdf

2712-del-1998-form-3.pdf

2712-del-1998-form-9.pdf

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Patent Number

190788

Indian Patent Application Number

2712/DEL/1998

PG Journal Number

34/2003

Publication Date

23-Aug-2003

Grant Date

16-Mar-2004

Date of Filing

11-Sep-1998

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	HARI PRASANNA DEKA BORUAH	REGIONAL RESEARCH LABORATORY, JORHAT, ASSAM, INDIA.
2	BALAMANI BEZBARUAH	REGIONAL RESEARCH LABORATORY, JORHAT, ASSAM, INDIA.

PCT International Classification Number

C12N 9/48

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA