Indian Patents. 232755:AN IMPROVED PROCESS FOR THE SINGLE STEP ISOLATION OF ALKALINE PROTEASE FROM A FERMENTATION BROTH

Title of Invention	AN IMPROVED PROCESS FOR THE SINGLE STEP ISOLATION OF ALKALINE PROTEASE FROM A FERMENTATION BROTH
Abstract	A process for the single step isolation of alkaline protease was developed us:::2 commercially available hydrophobic ligands. Higher adsorption was obtained oa butyl and phenyl hydrophobic ligands (94 and 98% respectively). Almost 20 fold purification with 40% yield of alkaline protease was obtained using gradient eiulion in a single slep operation.

Full Text	The present invention relates to an improved process for the single step isolation of alkaline protease from a fermentation broth. More particularly it relates to a process to obtain a highly active alkaline protease in a single step operation from fermentation broth using commercially available hydrophobic ligands. This finds application in food, Pharmaceuticals, detergent and leather processing industries. Alkaline protease is an industrially important enzyme, it is being used in different field such as food, Pharmaceuticals, detergent, leather processing, protein hydrolysis, peptide synthesis etc [Kiatkamjornwong S, siwarungson N, Nganbunsri A. J. Appl. Poly. Sci. 1999, 73,2273 - 2291; Miyazawa T. Amino Acids. 1999, 16 ,191 - 213; Kamini N R, Hemachander C, Mala J G S, Puvanakrishnan R. Current Science. 1999, 77, 80 -86; Matsumoto T, Furuya N, Tateda K, Miyazaki S, Ohno A, Ishii Y, Hirakata Y, Yamaguchi K. J. Medical Microbiol, 1999, 48, 765-770]. However, the reduction in production costs remains a major concern at commercial level. As the number of purification steps increase, the overall yield of the enzyme also decreases, because each step contributes some loss of the enzyme. To avoid this, there is a need to minimize purification steps which will significantly reduced the production costs of the enzyme. Affinity chromatography is one of the ideal methods to obtained highly purified enzyme in single step operation however, a specific adsorbent has to be prepared for each biological material. Its preparation and utilization may pose serious economical problems at industrial level. The hydrophobic interaction, which is general in biological systems, appears to be suitable for the chromatographic purification of biological materials [Shaltiel S.Er-el Z.Proc Natl Acad Sci USA.1973, 70 , 778-781; Kennedy R M Methods Enzymol, 1990, 182, 339 - 352. However, since its invention, it is being used in combination with other methods for the purification of enzymes. So far, only multistep purification methods are being used in the isolation of fungal as well as bacterial alkaline protease from fermentation broth. It was reported that the alkaline protease purified using 6 different steps resulted into 12 fold purification with 3.7 % yield [ Chang, H s., Kwon, T J. sanop Misaengmul Hakhoechi (Korean) . 1998, 26,427 - 434]. It was also reported that the alkaline protease was obtained using 3 different step including affinity chromatography on alpha -casein agarose gives 18.5 fold purification with 39% yield [ Hutadilok Towatana N, Painupong a, Suntinanaler P J.J. Ferment. Bioeng. 1999, 87,5, 581 - 587]. So far, no process is reported which have isolated alkaline protease in a single step operation from fermentation broth using hydrophobic ligands. The main object of the present invention is to provide an improved process for the single step isolation of alkaline protease from a fermentation broth. Yet another object of the present invention is to decrease the production costs of the enzyme by minimizing the purification steps of the enzyme. Still another object of the present invention is to increase the overall yield of the isolation enzyme. Accordingly, the present invention provides an improved process for the single step isolation of alkaline protease from a fermentation broth which comprises, loading a fermentation broth containing alkaline protease preferably from fungus Conidiobolus coronatus on a bed of hydrophobic ligand preferably containing butyl and phenyl , equilibrated with a salt solution preferably ammonium sulphate having concentration ranging from 1.0 M to 2.0 M in a buffer selected from a group consisting of glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane, hydroxidechloride, phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate washing the unadsorbed by the same salt in buffer solution followed by elution at a flow rate ranging between 0.10 to 0.20 ml / min. with decreasing salt gradient from 1.5 to 0 M to obtain the final solution containing the desired product. In one of the embodiment of the present invention, fermentation broth containing alkaline protease used may be obtained from any microbial source, preferably fungus Conidiobolus coronatus. In another embodiment, the hydrophobic ligand used may be selected from commercially available hydrophobic ligands preferably butyl and phenyl. In yet another embodiment of the present invention, the buffer used for equilibration, adsorption, washing, and elution may be selected from the group consisting of glycine - NaOH , borate, Tris ( hydroxy methyl) aminomethane, hydroxidechloride, phosphate and carbonate- cicarbonate, preferably carbonate-bicarbonate. In still another embodiment, the salt used for equilibration, adsorption, washing, and elution may be selected from the group consisting of salt of sodium, potassium, calcium, halides, acetates, phosphates, preferably ammonium sulphate. The process of the present invention is described by following examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner. Example 1 Preparation of the fermentation broth, the fermentation broth was obtained by drawing a fungus Conidiobolus coronatus. As described elsewhere and used as the alkaline protease source without any pretreatment [Sutar II, srinivasan M C, Vartak H G. Biotech Lett. 1991, 13, 191 - 124]. The enzyme activity was estimated using the method described by Kunitz [ Kunitz M.J. Gen Physiol. 1947, 30, 291 - 310]. The reaction mixture ( 2 ml) contained 10 mg of Hammarsten casein (Merck FRG ) in 0.1 M carbonate- bicarbonate buffer, H 9.7 and suitably diluted enzyme. The reaction was terminated after incubation for 10 minutes at 40°C by adding 3 ml of 5% trichloroacetic acid reagent and precipitate Example 2 The 2 ml fermentation broth ( 8.4 mg protein ) was loaded on the different hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 2 M salt. After washing with 5 ml EB containing 2 M ammonium sulphate the desorption was carried out with 5 ml EB containing no salt. The enzyme activity and protein was estimated in collected samples. Table 1 shows the %adsorption obtained on different hydrophobic ligand. Table 1 (Table Removed) Example 3 The 2 ml fermentation broth ( 8.4 mg/ ml protein) was loaded on the butyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 2.0 M salt. After washing with 5 ml EB containing 2 M ammonium sulphate the desorption was carried out with 5 ml EB containing 2.0, 1.5, 1.0 0.5, and 0.0 M salt. The alkaline protease activity and protein was estimated in collected samples. The 7.86 fold purification [ activity in elution ( U/mg) / activity of feed ( U / mg)] of alkaline protease was obtained. Example 4 The 1 ml fermentation broth ( 4.2 mg protein ) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.5 M salt. After washing with 5 ml EB containing 1.5 M ammonium sulphate the desorption was carried out using step-wise elution with 5 ml EB containing 1.5, 1.0 0.5, and 0.0 M salt. The enzyme activity and protein was estimated in collected samples. The 8.3 fold purification [activity in elution ( U/mg) / activity of feed ( U / mg)] of alkaline protease was obtained. Example 5 The 1 ml fermentation broth ( 4.2 mg protein) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.5 M salt. After washing with 5 ml EB containing 1.5 M ammonium sulphate the desorption was carried using gradient elution at the flow rate of 0.15 ml/ min using linearly decreasing salt gradient ( 1.5→0 M) prepared in EB and the alkaline protease activity and protein was estimated in collected samples. The 19.97 fold purification [activity in elution ( U / mg) / activity of feed ( U/ mg)] with 40% yield of alkaline protease was obtained. Example 6 The 1 ml fermentation broth (4.2 mg protein) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.0 M salt. After washing with 5 ml EB containing 1.0 M ammonium sulphate the desorption was carrfied using gradient elution at a flow rate of 0.3 ml/min using linearly decreasing salt gradient (1.0→0M) prepared in EB and the alkaline protease activity and protein was estimated in collected samples. The 20.23 fold purification [activity in elution (U/mg)/ activity of feed (U/mg)] of alkaline protease was obtained. Advantages Important novel feastures and advantages of this invention are as follows : 1. The present invention provides a single step isolation of alkaline protease from a fermentation broth which minimizes the purification steps of the enzyme resulting in the overall reduction of production costs of the enzyme. 2. This process also increases the overall yield of the enzyme significantly. We Claim: 1. An improved process for the single step isolation of alkaline protease from a fermentation broth which comprises, loading a fermentation broth containing alkaline protease preferably from fungus Conidiobolus coronatus on a bed of hydrophobic ligand preferably containing butyl and phenyl , equilibrated with a salt solution preferably ammonium sulphate having concentration ranging from 1.0 M to 2.0 M in a buffer solution selected from a group consisting of glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane, hydroxidechloride, phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate , washing the unadsorbed by the same salt in buffer solution followed by elution at a flow rate ranging between 0.10 to 0.20 ml / min. with decreasing salt gradient from 1.5 to 0 M to obtain the final solution containing the desired product. 2. An improved process as claimed in claim 1, wherein the fermentation broth containing alkaline protease used is obtained from any microbial source, preferably from fungal source. 3. An improved process as claimed in claims 1 to 2 , wherein the hydrophobic ligand used is selected from the group consisting of butyl, hexyl, octyl, decyl and phenyl, hydrophobic ligands, preferably butyl and phenyl. 4. An improved process as claimed in claims 1 to 3, wherein the buffer used for equilibration, adsorption, washing, and elution is selected from the group consisting of glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane, hydroxide- chloride, phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate. 5. An improved process as claimed in claims 1 to 4, wherein the salt used for equilibration, adsorption, washing, and elution is selected from the group consisting of salt of sodium, potassium, calcium, halides, acetates, phosphates and sulphates preferably ammonium. 6. An improved process for the single step isolation of alkaline protease from a fermentation broth substantially as herein described with reference to examples.

Full Text

The present invention relates to an improved process for the single step isolation of alkaline protease from a fermentation broth. More particularly it relates to a process to obtain a highly active alkaline protease in a single step operation from fermentation broth using commercially available hydrophobic ligands. This finds application in food, Pharmaceuticals, detergent and leather processing industries.
Alkaline protease is an industrially important enzyme, it is being used in different field such as food, Pharmaceuticals, detergent, leather processing, protein hydrolysis, peptide synthesis etc [Kiatkamjornwong S, siwarungson N, Nganbunsri A. J. Appl. Poly. Sci. 1999, 73,2273 - 2291; Miyazawa T. Amino Acids. 1999, 16 ,191 - 213; Kamini N R, Hemachander C, Mala J G S, Puvanakrishnan R. Current Science. 1999, 77, 80 -86; Matsumoto T, Furuya N, Tateda K, Miyazaki S, Ohno A, Ishii Y, Hirakata Y, Yamaguchi K. J. Medical Microbiol, 1999, 48, 765-770]. However, the reduction in production costs remains a major concern at commercial level. As the number of purification steps increase, the overall yield of the enzyme also decreases, because each step contributes some loss of the enzyme. To avoid this, there is a need to minimize purification steps which will significantly reduced the production costs of the enzyme. Affinity chromatography is one of the ideal methods to obtained highly purified enzyme in single step operation however, a specific adsorbent has to be prepared for each biological material. Its preparation and utilization may pose serious economical problems at industrial level. The hydrophobic interaction, which is general in biological systems, appears to be suitable for the chromatographic purification of biological materials [Shaltiel S.Er-el Z.Proc Natl Acad Sci USA.1973, 70 , 778-781; Kennedy R M
Methods Enzymol, 1990, 182, 339 - 352. However, since its invention, it is being used in combination with other methods for the purification of enzymes. So far, only multistep purification methods are being used in the isolation of fungal as well as bacterial alkaline protease from fermentation broth. It was reported that the alkaline protease purified using 6 different steps resulted into 12 fold purification with 3.7 % yield [ Chang, H s., Kwon, T J. sanop Misaengmul Hakhoechi (Korean) . 1998, 26,427 - 434]. It was also reported that the alkaline protease was obtained using 3 different step including affinity chromatography on alpha -casein agarose gives 18.5 fold purification with 39% yield [ Hutadilok Towatana N, Painupong a, Suntinanaler P J.J. Ferment. Bioeng. 1999, 87,5, 581 - 587]. So far, no process is reported which have isolated alkaline protease in a single step operation from fermentation broth using hydrophobic ligands.
The main object of the present invention is to provide an improved process for the single step isolation of alkaline protease from a fermentation broth.
Yet another object of the present invention is to decrease the production costs of the enzyme by minimizing the purification steps of the enzyme.
Still another object of the present invention is to increase the overall yield of the isolation enzyme.
Accordingly, the present invention provides an improved process for the single step isolation of alkaline protease from a fermentation broth which comprises, loading a fermentation broth containing alkaline protease preferably from fungus Conidiobolus coronatus on a bed of hydrophobic ligand preferably containing butyl and phenyl , equilibrated with a salt solution preferably ammonium sulphate having concentration ranging from 1.0 M to 2.0 M in a buffer selected from a group consisting of glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane, hydroxidechloride, phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate washing the unadsorbed by the same salt in buffer solution followed by elution at
a flow rate ranging between 0.10 to 0.20 ml / min. with decreasing salt gradient from 1.5 to 0 M
to obtain the final solution containing the desired product.
In one of the embodiment of the present invention, fermentation broth containing alkaline
protease used may be obtained from any microbial source, preferably fungus Conidiobolus
coronatus.
In another embodiment, the hydrophobic ligand used may be selected from commercially
available hydrophobic ligands preferably butyl and phenyl.
In yet another embodiment of the present invention, the buffer used for equilibration, adsorption,
washing, and elution may be selected from the group consisting of glycine - NaOH , borate, Tris
( hydroxy methyl) aminomethane, hydroxidechloride, phosphate and carbonate- cicarbonate,
preferably carbonate-bicarbonate.
In still another embodiment, the salt used for equilibration, adsorption, washing, and elution may
be selected from the group consisting of salt of sodium, potassium, calcium, halides, acetates,
phosphates, preferably ammonium sulphate.
The process of the present invention is described by following examples, which are illustrative
only and should not be construed to limit the scope of the present invention in any manner.
Example 1
Preparation of the fermentation broth, the fermentation broth was obtained by drawing a fungus Conidiobolus coronatus. As described elsewhere and used as the alkaline protease source without any pretreatment [Sutar II, srinivasan M C, Vartak H G. Biotech Lett. 1991, 13, 191 - 124]. The enzyme activity was estimated using the method described by Kunitz [ Kunitz M.J. Gen Physiol. 1947, 30, 291 - 310]. The reaction mixture ( 2 ml) contained 10 mg of Hammarsten casein (Merck FRG ) in 0.1 M carbonate- bicarbonate buffer, H 9.7 and suitably diluted enzyme. The reaction was terminated after incubation for 10 minutes at 40°C by adding 3 ml of 5% trichloroacetic acid reagent and precipitate
Example 2
The 2 ml fermentation broth ( 8.4 mg protein ) was loaded on the different hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 2 M salt. After washing with 5 ml EB containing 2 M ammonium sulphate the desorption was carried out with 5 ml EB containing no salt. The enzyme activity and protein was estimated in collected samples. Table 1 shows the %adsorption obtained on different hydrophobic ligand.
Table 1
(Table Removed)
Example 3
The 2 ml fermentation broth ( 8.4 mg/ ml protein) was loaded on the butyl hydrophobic
ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 2.0 M salt. After washing with 5 ml EB containing 2 M ammonium sulphate the desorption was carried out with 5 ml EB containing 2.0, 1.5, 1.0 0.5, and 0.0 M salt. The alkaline protease activity and protein was estimated in collected samples. The 7.86 fold purification [ activity in elution ( U/mg) / activity of feed ( U / mg)] of alkaline protease was obtained.
Example 4
The 1 ml fermentation broth ( 4.2 mg protein ) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.5 M salt. After washing with 5 ml EB containing 1.5 M ammonium sulphate the desorption was carried out using step-wise elution with 5 ml EB containing 1.5, 1.0 0.5, and 0.0 M salt. The enzyme activity and protein was estimated in collected samples. The 8.3 fold purification [activity in elution ( U/mg) / activity of feed ( U / mg)] of alkaline protease was obtained.
Example 5
The 1 ml fermentation broth ( 4.2 mg protein) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.5 M salt. After washing with 5 ml EB containing 1.5 M ammonium sulphate the desorption was carried using gradient elution at the flow rate of 0.15 ml/ min using linearly decreasing salt gradient ( 1.5→0 M) prepared in EB and the alkaline protease activity and protein was estimated in collected samples. The 19.97 fold purification [activity in elution ( U / mg) / activity of feed ( U/ mg)] with 40% yield of alkaline protease was obtained.
Example 6
The 1 ml fermentation broth (4.2 mg protein) was loaded on the phenyl hydrophobic ligands packed in a column. The equilibration of the column was carried out with 5 ml EB containing 1.0 M salt. After washing with 5 ml EB containing 1.0 M ammonium sulphate the desorption was carrfied using gradient elution at a flow rate of 0.3 ml/min using linearly decreasing salt gradient (1.0→0M) prepared in EB and the alkaline protease activity and protein was estimated in collected samples. The 20.23 fold purification [activity in elution (U/mg)/ activity of feed (U/mg)] of alkaline protease was obtained.
Advantages
Important novel feastures and advantages of this invention are as follows :
1. The present invention provides a single step isolation of alkaline protease
from a fermentation broth which minimizes the purification steps of the enzyme
resulting in the overall reduction of production costs of the enzyme.
2. This process also increases the overall yield of the enzyme significantly.

We Claim:
1. An improved process for the single step isolation of alkaline protease from a
fermentation broth which comprises, loading a fermentation broth containing
alkaline protease preferably from fungus Conidiobolus coronatus on a bed of
hydrophobic ligand preferably containing butyl and phenyl , equilibrated with a
salt solution preferably ammonium sulphate having concentration ranging
from 1.0 M to 2.0 M in a buffer solution selected from a group consisting of
glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane, hydroxidechloride,
phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate ,
washing the unadsorbed by the same salt in buffer solution followed by elution
at a flow rate ranging between 0.10 to 0.20 ml / min. with decreasing salt
gradient from 1.5 to 0 M to obtain the final solution containing the desired
product.
2. An improved process as claimed in claim 1, wherein the fermentation broth
containing alkaline protease used is obtained from any microbial source,
preferably from fungal source.
3. An improved process as claimed in claims 1 to 2 , wherein the hydrophobic
ligand used is selected from the group consisting of butyl, hexyl, octyl, decyl and
phenyl, hydrophobic ligands, preferably butyl and phenyl.
4. An improved process as claimed in claims 1 to 3, wherein the buffer used for
equilibration, adsorption, washing, and elution is selected from the group
consisting of glycine-NaOH, borate, Tris ( hydroxy methyl) aminomethane,
hydroxide- chloride, phosphate and carbonate-bicarbonate, preferably carbonate-bicarbonate.
5. An improved process as claimed in claims 1 to 4, wherein the salt used for
equilibration, adsorption, washing, and elution is selected from the group
consisting of salt of sodium, potassium, calcium, halides, acetates, phosphates
and sulphates preferably ammonium.
6. An improved process for the single step isolation of alkaline protease from a
fermentation broth substantially as herein described with reference to examples.

Documents:

1031-del-2000-abstract.pdf

1031-del-2000-claims.pdf

1031-del-2000-correspondence-others.pdf

1031-del-2000-correspondence-po.pdf

1031-del-2000-description (complete).pdf

1031-del-2000-form-1.pdf

1031-del-2000-form-2.pdf

1031-del-2000-form-3.pdf

1031-del-2000-form-4.pdf

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Patent Number

232755

Indian Patent Application Number

1031/DEL/2000

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

21-Mar-2009

Date of Filing

17-Nov-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	DNYANESHWAR MARUTI THAKAR	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
2	HARSHVARDHAN VISHWANATH ADIKANE	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
3	SANJAY NARAYAN NENE	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA