WO2003022068A1 - Glutaminase - Google Patents
Glutaminase Download PDFInfo
- Publication number
- WO2003022068A1 WO2003022068A1 PCT/EP2002/010061 EP0210061W WO03022068A1 WO 2003022068 A1 WO2003022068 A1 WO 2003022068A1 EP 0210061 W EP0210061 W EP 0210061W WO 03022068 A1 WO03022068 A1 WO 03022068A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glutaminase
- salt
- activity
- lactobacillus
- thermotolerant
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01002—Glutaminase (3.5.1.2)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
Definitions
- the present invention relates to a new salt- resistant and thermotolerant glutaminase from Lactobacillus and a process for the production of such salt-resistant thermotolerant glutaminase from
- Lactobacillus Lactobacillus .
- Hydrolysed proteins have been known for use as seasonings in the form of soy sauce, which traditionally has been prepared by enzymatic hydrolysis requiring a long period of time for preparation.
- soy sauce protein-containing plant materials are inoculated with Aspergilli and the solid culture is fermented for 2 days to make koji. During this time, enzymes are produced which are able to hydrolyse
- Glutaminase is known as playing an important role in such processes, particularly in the moromi stage, to enzymatically convert glutamine to glutamic acid (or glutamate) by deamination.
- This enzyme is regarded as a key enzyme that controls the savoury and umami taste of such kind of fermented products like soy sauce, for example.
- glutaminase comes from Aspergillus strains inoculated in the koji.
- glutaminase from these moulds are reportedly being inhibited by salt concentrations above 10-15% (w/v) which are the practical conditions for soy sauce production, in the moromi stage. It is thus necessary to use a salt-resistant glutaminase in order to achieve efficient glutamic acid production.
- the main reason for the lower glutamic acid content in the enzymatic degradation method is that glutaminase is significantly inhibited by ' either high salt concentration or high temperature.
- Glutaminase from Candida fa ate Japanese Patent Publication N° 38748/1994
- Cryptococcus albidus Japanese Patent Publication N° 48759/1974
- the relative activity in the presence of 18% salt of both enzymes is as low as about 50 to 60 % with respect to 100% activity in the absence of salt.
- US 6063409 discloses a glutaminase from Cryptoccus nodaensis which exhibits higher salt tolerance and better temperature resistance.
- the object of the present invention is to provide a glutaminase that exhibits salt-resistance and thermo-tolerance and that comes from a micro-organism that has a long history of use in food process and that is therefore food-grade.
- the present invention provides a new salt- resistant and thermotolerant glutaminase from Lactobacillus which has a temperature dependence such that it retains more than 80% of its activity between 45°C and 55°C, a salt dependence such that its activity in the presence of up to 15% NaCl is from 80 to 180 % of that without NaCl and a thermal stability such that its energy of activation is about 80 to 150 kJ.mol “1 , preferably 90 to 120 kJ.mol “1 , more preferably 100 to 110 kJ.mol "1 .
- Figure 1 shows the pH dependence of the glutaminase where the vertical axis define the relative activity (R.A.) expressed as percentage of the maximum activity in the indicated pH range.
- Figure 2 shows the temperature dependence of the glutaminase where the vertical axis define the relative activity (R.A.) expressed as percentage of the maximum activity in the temperature range T(°C) .
- Figure 3 shows the salt dependence of the glutaminase where the vertical axis define the relative activity (R.A.) expressed as percentage of the maximum activity in the NaCl .concentration range in percent w/v(NaCl) .
- Figure 4 exhibits the thermal stability of the glutaminase and shows :
- the present salt-resistant thermotolerant glutaminase may be produced by a process which comprises cultivating a micro-organism belonging to the genus Lactobacillus, preferably Lactobacillus rhamnosus having the ability to produce the salt-resistant thermotolerant glutaminase thereby producing the salt- resistant thermotolerant glutaminase and then recovering it .
- a Lactobacillus rhamnosus strain having the required ability was deposited by way of example under the Budapest Treaty at the Collection National de Cultures de Microorganismes (CNCM) , Institut Pasteur, 28 rue du Dondel Roux, 75724 Paris Cedex 15, France, on April 5, 2000 under the reference CNCM 1-2433.
- CNCM Collection National de Cultures de Microorganismes
- the cultivation process is advantageously carried out in a liquid medium in jar, flask or bioreactor, for example.
- the temperature of the medium may range from 35°C to 55°C, and the cultivation time may be of 6 hours or more .
- the culture medium may include as carbon source, monosaccharides and oligosaccharides such as glucose, maltose, dextrines and other carbon source from vegetal origin such as wheat, corn, wheat gluten or soya for example.
- the culture medium may include as nitrogen source includes peptides, proteins and amino acids coming from from vegetal origin such as wheat, corn, wheat gluten or soya as well, for example.
- the culture medium obtained after cultivation may be' used as such for enzyme activity characterisation but may also be subjected to solid/liquid separation and purification.
- the culture medium may also be added into a protein containing medium for the glutaminase to deaminates the glutamine contained.
- the salt-resistant thermotolerant glutaminase according to the present invention is membrane-bound a further step may be necessary to release the enzyme from the cells in order to further characterise it and/or to increase its activity.
- conventional enzyme extraction methods may be applied. For example, it is possible to use mechanical grinding and disruption, sonication or ultrasonication, use of detergents, lysis of the cells using lysozyme or other kind of method allowing release of the enzyme from the membrane.
- a crude enzyme solution of the salt- resistant thermotolerant glutaminase may be prepared.
- the crude enzyme preparation may be subjected to a suitable combination of chromatographic adsorption-elution techniques using various kind of ion exchangers, for example DEAE cellulose, TEAE cellulose, QAE Sephadex or Hydroxyapatite, gel filtration using Sephadex or Superdex whereby a purified enzyme preparation may be obtained.
- Another aspect of the present invention concerns the use of a mixture containing a salt-resistant thermotolerant glutaminase from Lactobacillus in a process for producing a biohydrolysate.
- a culture medium containing the salt- resistant thermotolerant glutaminase according to the present invention may be added in the step of mixing or in the step of fermentation and/or ageing of a process for producing _ biohydrolysates starting from proteinaceous material such as soy sauce process or wheat gluten hydrolysate.
- Such culture medium may be the cell-containing medium obtained after cultivation of Lactobacillus rhamnosus (CNCM 1-2433) .
- This cell- containing medium or cell-fractions-containing medium may also be concentrated by liquid-solid separation means in order to recover cell-containing residue.
- the medium used may also be a mixture in which the enzyme has been released from the cells by sonication, lysis, or any appropriate means.
- thermally denatured proteinaceous materials (soybeans, wheat gluten, ...) are mixed with thermally denatured starchy materials (rice, wheat, corn, ...) and after the water content of the mixture is adjusted to 30-45%, a koji mould is inoculated into it and cultivated in an usual manner to give solid koji which is then mixed with brine and subjected to fermentation and ageing.
- the step of hydrolysing of a protein-containing material may be performed using either endogenous enzymes from koji or technical added enzymes in the presence of a Lactobacillus providing a glutaminase activity, preferably a Lactobacillus rhamnosus, more preferably Lactojbacillus rhamnosus (CNCM 1-2433) .
- a Lactobacillus providing a glutaminase activity preferably a Lactobacillus rhamnosus, more preferably Lactojbacillus rhamnosus (CNCM 1-2433) .
- An other aspect of the present invention concerns the use of a mixture containing a salt-resistant thermotolerant glutaminase from Lactobacillus in a process for producing a biohydrolysate.
- the biohydrolysate is a soy sauce or wheat gluten hydrolysate.
- the salt-resistant thermotolerant glutaminase according to the present invention may be use at the step of hydrolysing and/or at the step of ageing in the biohydrolysate process production.
- the new salt-resistant thermotolerant glutaminase (purified or cell-bound) according to the present invention may thus be added in a process for producing food and drink containing peptides and/or amino acids as a part or a majority of tasting components.
- protein materials or protein-containing materials are hydrolysed with at least one proteolytic enzyme or a proteolytic enzyme-containing material, such as a koji for example.
- Glutaminase activity is tested by determining the formation of L-glutamate from L-glutamine using a colorimetric method (kit Boehringer Mannheim, n°139 092, Darmstadt, Germany).
- the reaction mixture containing 0.5 ml of 60 mM L-glutamine in 50 mM sodium phosphate buffer pH 7.0 is preincubated at 37°C for 3 min and the reaction is started by adding 0.5 ml of the enzyme containing preparation to be tested. The incubation is carried out at 37°C for 20 to 30 min. The reaction is stopped by cooling the samples on ice. The amount of glutamate produced is subsequently enzymatically determined.
- One unit (U) of glutaminase activity is defined as the amount of enzyme that liberates 1 ⁇ mol of glutamate per min under the above conditions.
- Buffer used pH 4 to 6.5 : 100 mM citrate/ 200 mM sodium phosphate pH 7 and 7.5 : 100 mM sodium phosphate pH 8 and 9 : 100 mM Tris/ HCl
- the optimum pH value for the glutaminase according to the present invention is about pH 7.0.
- the enzyme is less active at lower pH values since around 62 % of the activity is lost at pH about 6.0. On the contrary, the enzyme has greater activity in alkaline than acidic pH because about 74 and 69 % of activity still remains at pH 8 and 9 respectively.
- the temperature dependence of glutaminase activity was determined by incubating the enzyme with substrate for 30 min at different temperatures in the range of 25 to 80°C as shown in Figure 2. Glutaminase activity was determined as described in « glutaminase assays » . The optimal temperature for glutaminase activity is around 50°C and the enzyme retains more than 90% of its activity at about 45°C corresponding to the temperature of the cultivation conditions. It is observed that about 50% of activity still remains at 70°C.
- the salt dependence of glutaminase was determined by adding 0-20% (w/v) NaCl to the reaction medium. Relative activities of the enzyme were calculated and expressed as percentage of the activity observed without salt ( Figure 3) . Glutaminase activity is increased in the presence of NaCl concentrations lower than 10%, in particular 1.8-fold improved at 2.5% of NaCl. Furthermore, in the presence of 15% of salt the enzyme activity is about 90% of that without salt.
- Glutaminase-catalysed deamination of glutamine was tested in 50 mM sodium phosphate buffer, pH 7.0, at 37°C using 12 different concentrations of glutamine ranging from 0.5 to 80 mM. Substrate Inhibition was not significant in the glutamine concentration range used.
- the catalytic behaviour of glutaminase with glutamine can be described by Michaelis-Menten equation (2) .
- the catalytic parameters were calculated by weighted linear regression using Lineweaver-Burk inversion and by nonlinear regression of Michaelis-Menten eqn (2) :
- Km value for the glutaminase of the present invention is from about 4 to 6 mM and Vm is from about 100 to 110 U/l of hydrolysate .
- a culture of Lactobacillus rhamnosus (CNCM 1-2433) containing 5.10 8 cfu/ml is grown in a medium containing 22% (w/w) wheat gluten (Roquette, Lestern, France) , 0.1% alcalase (Novozymes, Bagsvaerd, Denmark), 4% (w/w) Flavorzy e L1000 (Novozymes, Bagsvaerd, Denmark) and 30 mM sodium acetate buffer pH 5.8 for 12h at 45°C. The broth is then cooled at 4°C and immediately subjected to analysis.
- Enzyme preparation After cultivation, the medium is washed 3 times with 50 mM sodium phosphate buffer pH 7.0 and finally reconstituted in the same buffer containing 10% (w/v) sucrose and 1 mM PMSF. Because the glutaminase is membrane-bound, the hydrolysate was subjected to 1 mg/ml lysozyme for lh at 37°C. This solution was stored at 4°C and used for enzyme assays.
- the mixture thus obtained was subjected to a traditional moromi fermentation process and in that way, the salt-resistant thermotolerant glutaminase accumulated in the cell-containing residue of medium-example I deaminates the glutamine present in Moromi during the time required to achieve a satisfactory yield of glutamate and to produce a mature soy sauce .
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Nutrition Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Enzymes And Modification Thereof (AREA)
- Soy Sauces And Products Related Thereto (AREA)
- Seasonings (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE60222567T DE60222567T2 (en) | 2001-09-10 | 2002-09-06 | glutaminase |
EP02777040A EP1427293B1 (en) | 2001-09-10 | 2002-09-06 | Glutaminase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01121530A EP1290951A1 (en) | 2001-09-10 | 2001-09-10 | Glutaminase |
EP01121530.8 | 2001-09-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003022068A1 true WO2003022068A1 (en) | 2003-03-20 |
Family
ID=8178588
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2002/010061 WO2003022068A1 (en) | 2001-09-10 | 2002-09-06 | Glutaminase |
Country Status (8)
Country | Link |
---|---|
EP (2) | EP1290951A1 (en) |
CN (1) | CN1291014C (en) |
AR (1) | AR036505A1 (en) |
AT (1) | ATE373426T1 (en) |
DE (1) | DE60222567T2 (en) |
ES (1) | ES2292818T3 (en) |
PT (1) | PT1427293E (en) |
WO (1) | WO2003022068A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1985698A1 (en) | 2007-04-25 | 2008-10-29 | Nestec S.A. | Peptidases from basidiomycetes |
CN103783482B (en) * | 2014-01-20 | 2015-09-16 | 曲阜师范大学 | A kind of preparation method of peanut sauce |
CN104207089B (en) * | 2014-07-29 | 2017-01-11 | 佛山市海天调味食品股份有限公司 | Fermentation technology for increasing content of glutamic acid |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3912822A (en) * | 1973-03-27 | 1975-10-14 | Kikkoman Shoyu Co Ltd | Process for producing a protein hydrolysate |
JPS548785A (en) * | 1977-06-21 | 1979-01-23 | Kikkoman Corp | Preparation of protein hydrolyzate |
JPS6486873A (en) * | 1987-09-29 | 1989-03-31 | Japan Res & Dev Ass | Thermostable and halotolerant glutaminase |
JPH02261379A (en) * | 1989-03-31 | 1990-10-24 | Daiwa Kasei Kk | Salt-tolerant glutaminase |
US5219597A (en) * | 1991-02-20 | 1993-06-15 | Korea Food Research Institute | Method for producing highly concentrated, lactic-acid fermented product utilizing unground grainy rice and improving qualities thereof by the secondary, enzymatic treatment at fermentation |
JPH1142086A (en) * | 1997-07-25 | 1999-02-16 | Daiwa Kasei Kk | Salt-resistant glutaminase |
US6063409A (en) * | 1998-05-28 | 2000-05-16 | Kikkoman Corporation | Species cryptococcus nodaensis, a process for producing salt-resistant thermostable glutaminase by use of the same, and a process for producing glutamic acid-rich protein hydrolysates |
-
2001
- 2001-09-10 EP EP01121530A patent/EP1290951A1/en not_active Withdrawn
-
2002
- 2002-09-06 WO PCT/EP2002/010061 patent/WO2003022068A1/en active IP Right Grant
- 2002-09-06 AT AT02777040T patent/ATE373426T1/en active
- 2002-09-06 EP EP02777040A patent/EP1427293B1/en not_active Expired - Lifetime
- 2002-09-06 CN CNB028176987A patent/CN1291014C/en not_active Expired - Fee Related
- 2002-09-06 ES ES02777040T patent/ES2292818T3/en not_active Expired - Lifetime
- 2002-09-06 PT PT02777040T patent/PT1427293E/en unknown
- 2002-09-06 DE DE60222567T patent/DE60222567T2/en not_active Expired - Lifetime
- 2002-09-09 AR ARP020103408A patent/AR036505A1/en not_active Application Discontinuation
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3912822A (en) * | 1973-03-27 | 1975-10-14 | Kikkoman Shoyu Co Ltd | Process for producing a protein hydrolysate |
JPS548785A (en) * | 1977-06-21 | 1979-01-23 | Kikkoman Corp | Preparation of protein hydrolyzate |
JPS6486873A (en) * | 1987-09-29 | 1989-03-31 | Japan Res & Dev Ass | Thermostable and halotolerant glutaminase |
JPH02261379A (en) * | 1989-03-31 | 1990-10-24 | Daiwa Kasei Kk | Salt-tolerant glutaminase |
US5219597A (en) * | 1991-02-20 | 1993-06-15 | Korea Food Research Institute | Method for producing highly concentrated, lactic-acid fermented product utilizing unground grainy rice and improving qualities thereof by the secondary, enzymatic treatment at fermentation |
JPH1142086A (en) * | 1997-07-25 | 1999-02-16 | Daiwa Kasei Kk | Salt-resistant glutaminase |
US6063409A (en) * | 1998-05-28 | 2000-05-16 | Kikkoman Corporation | Species cryptococcus nodaensis, a process for producing salt-resistant thermostable glutaminase by use of the same, and a process for producing glutamic acid-rich protein hydrolysates |
Non-Patent Citations (4)
Title |
---|
DATABASE WPI Section Ch Week 197909, Derwent World Patents Index; Class D13, AN 1979-16915B, XP002189607 * |
DATABASE WPI Section Ch Week 198919, Derwent World Patents Index; Class D13, AN 1989-141719, XP002189606 * |
DATABASE WPI Section Ch Week 199049, Derwent World Patents Index; Class B05, AN 1990-364396, XP002189605 * |
DATABASE WPI Section Ch Week 199917, Derwent World Patents Index; Class D13, AN 1999-197819, XP002189604 * |
Also Published As
Publication number | Publication date |
---|---|
CN1553775A (en) | 2004-12-08 |
PT1427293E (en) | 2007-10-11 |
ATE373426T1 (en) | 2007-10-15 |
DE60222567D1 (en) | 2007-10-31 |
AR036505A1 (en) | 2004-09-15 |
ES2292818T3 (en) | 2008-03-16 |
CN1291014C (en) | 2006-12-20 |
EP1427293B1 (en) | 2007-09-19 |
EP1427293A1 (en) | 2004-06-16 |
EP1290951A1 (en) | 2003-03-12 |
DE60222567T2 (en) | 2008-06-19 |
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