US3862007A - Extraction of chitinase - Google Patents

Extraction of chitinase Download PDF

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US3862007A
US3862007A US341545A US34154573A US3862007A US 3862007 A US3862007 A US 3862007A US 341545 A US341545 A US 341545A US 34154573 A US34154573 A US 34154573A US 3862007 A US3862007 A US 3862007A
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chitinase
chitin
dilute acid
activity
solution
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US341545A
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Wladimir A Smirnoff
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HER MAJESTY IN RIGHT OF CANADA FORESTRY, Minister of
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Canadian Patents and Development Ltd
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Priority to CA193,673A priority patent/CA1008385A/en
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Assigned to HER MAJESTY IN RIGHT OF CANADA AS REPRESENTED BY THE MINISTER OF FORESTRY reassignment HER MAJESTY IN RIGHT OF CANADA AS REPRESENTED BY THE MINISTER OF FORESTRY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: CANADIAN PATENTS AND DEVELOPMENT LIMITED/SOCIETE CANADIAN DES BREVETS ET D'EXPLOITATION LIMITEE, A COMPANY OF CANADA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification

Definitions

  • Chitin is a horny substance analogous to cellulose occuring in nature e.g. in the carapaces of crabs and beetles, skeletal portions of arthropods, molluscs, shrimp and lobster shells, etc. It is also found in lichens, certain fungi and bacteria. It is insoluble in ordinary solvents, dilute acids or alkalies. Complex enzymes known as chitinases attack chitin and break it down, primarily into N-acetyl-glucosamine.
  • Chitinase is being produced from the fermentation of microorganisms such as Streptomyces bacteria and fungi (e.g. Beauveria, Cordyceps This production apparently requires many steps, prolonged times and is difficult to control.
  • the activity of the chitinase preparation sold by chemical suppliers is usually of the order of 1,200 nephelometric units. It is difficult to obtain quantities of chitinase exceeding 100 mg. and the current price is about $200.00 per gram.
  • Chitinase has been found to potentiate or be a valuable adjuvant to bacterial insecticides such as Bacillus thuringiensis preparations (W.A. Smirnoff US. Pat. Application No. 317,535 filed Dec. 22, 1972). For spraying crops and timber stands large amounts of enzyme preparation are required at a reasonable cost.
  • the method is basically comprised of:
  • the entrails particularly gizzards and intestines are obtained from freshly-killed fowl.
  • the chicken (Gallus gallus L.) is a preferred source which is available from many meat packing plants.
  • the entrails in fresh condition are submerged in dilute acid medium to extract the enzyme.
  • the medium may be entirely aqueous or part aqueous-part watersoluble organic solvent e.g. alcohol.
  • the dilute acid may have to be replenished during the entraction to maintain an acidic pH.
  • the medium is buffered to a pH of about 5 to 5.5.
  • Various suitable buffers are known with a mixture of citric acid and dibasic sodium phosphate being used in the Example 2.
  • An antibacterial agent is usually incorporated to avoid any bacterial contamination.
  • colloidal chitin is added to cause preferential adsorption of the enzyme.
  • a colloidal form of chitin is desired to give good dispersion and high surface area to effect efficient adsorption without excessive amounts and times being required.
  • the colloidal form is also desired to enable hydrolysis (in step c) to proceed to substantial completion in reasonable times.
  • One suitable technique for preparing a colloidal form of chitin is described below. Still other techniques could be used.
  • the chitin-chitinase deposit is washed with neutral or dilute acid solution to remove impurities.
  • neutral or dilute acid solution Preferably the same buffered solution as the extractant is used.
  • the salt added in step (d) is normally ammonium sulphate although some other substantially neutral salt of high water-solubility may be useful. This salting-out effect has been used in recovering other enzymes and high molecular weight proteins.
  • the steps of adsorption on colloidal chitin, removing unadsorbed material, hydrolyzing chitin, adding a neutral salt to the clear solution and recovering precipitated chitinase can be repeated a number of times (until the recovered enzyme material has the desired activity).
  • An activity of at least about 850-900 nephelometric units is preferred for insecticidal applications, and is usually obtained after two adsorptions on chitin.
  • the nephelometric analysis for enzyme activity is based on measuring the decrease in turbidity by-nephelometry (or photometry) in a colloidal suspension of chitin on enzymatic lysis.
  • the nephelometric units measure the activity of enzyme/mg which will cause the turbidity due to 0.3 mg. of colloidal chitin/ml to decrease 50 percent after 2 hours incubation at 37.5C, pH 5.3 (Buffer Solution: citric acid 0.1M and dibasic Na phosphate 0.2M).
  • the chitin was dissolved or dispersed in 15 gallons of concentrated hydrochloric acid. This highly viscous suspension was filtered through a fritted glass filter into a container holding gallons of distilled water. On dilution, the chitin precipitated as a colloidal suspension. This suspension is again filtered and diluted to a final pH of 6.4. The colloidal chitin can then be recovered in solid form by vacuum drying.
  • colloidal chitin Other techniques for obtaining the colloidal chitin can be used and this invention is not limited to the method just described.
  • EXAMPLE 1 300 chicken digestive systems (gizzard, gut and coecums) from freshly-killed chickens were placed in 40 liters of aqueous solution buffered at pH 5.2. After several hours the mixture was filtered, centrifuged and the supernatant collected. About 25 gms of dry colloidal chitin was added and mixed with the solution. The solution was centrifuged and the deposit retained. The deposit was washed with dilute acid solution pH 5.2. The washed residue was dispersed in dilute acid medium (about 1 liter) and hydrolysis allowed to proceed until nearly complete hydrolysis of chitin has occurred (substantial removal of turbidity).
  • the solution was centrifuged and then saturated with ammonium sulphate (saturation at 55 percent).
  • the saturated solution was centrifuged for about 10 min. and the deposit recovered.
  • This enzyme material has an activity of about 125 nephelometric units.
  • EXAMPLE 2 Some 60,000 gizzards and intestine (guts) of freshly killed chickens were acquired and placed in 4,000 liters of buffer solution, pH 5.2.
  • the buffered solution contained 0.02M citric acid and 0.02 M Na HPO plus 10 mg. per liter of an antibiotic (Thymol).
  • the solution was filtered through glass-wool and centrifuged at 4,500 r.p.m. for 10 minutes. The supernatant was collected and placed in a second container.
  • This enzyme preparation was returned to liters of buffer solution pH 5.2 with 1.2 kg of colloidal chitin added. Hydrolysis at 37C with weak stirring was carried out until total reduction of the turbidity was obtained. The solution was centrifuged 15 minutes at 4,500 r.p.m., the supernatant was saturated by l 1 kg of (NH 80,, then stirred and centrifuged 10 minutes at 4,500 r.p.m. The deposit was g. of enzyme material with an activity of 850-900 nephelometric units.
  • a method of extracting chitinase from the entrails of domestic fowl comprising:

Abstract

Chitinase activity is extracted from the entrails (gizzards, intestines) of fowl with dilute acid, colloidal chitin added, the deposit recovered, hydrolysis allowed to proceed in dilute acid, insoluble matter removed, the solution substantially saturated with a neutral salt, and precipitated chitinase material recovered. The enzyme extract is a valuable adjuvant to bacterial insecticides.

Description

United States Patent [1 Smirnoff Jan. 21, 1975 EXTRACTION OF CHITINASE [75] Inventor: V Vladimir A. Smirnoff, Quebec,
Quebec, Canada [73] Assignee: Canadian Patents and Development Limited, Ottawa, Province of Ontario, Canada 22 Filed: Mar. 15,1973
21 Appl. No.: 341,545
[52] US. Cl 195/66 R [51] Int. Cl C07g 7/026 [58] Field of Search 195/65, 66 R, 62
[56] References Cited OTHER PUBLICATIONS Methods in Enzymology, Vol. 8, pages 644650,
Nature, Vol. 192, pages 135-136, (1961).
Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firm-Alan A. Thomson 57] ABSTRACT 9 Claims, No Drawings EXTRACTION OF CHITINASE This invention deals with chitinase preparations and their extraction from fowl entrails. A method of extraction and purification is described.
Chitin is a horny substance analogous to cellulose occuring in nature e.g. in the carapaces of crabs and beetles, skeletal portions of arthropods, molluscs, shrimp and lobster shells, etc. It is also found in lichens, certain fungi and bacteria. It is insoluble in ordinary solvents, dilute acids or alkalies. Complex enzymes known as chitinases attack chitin and break it down, primarily into N-acetyl-glucosamine.
Chitinase is being produced from the fermentation of microorganisms such as Streptomyces bacteria and fungi (e.g. Beauveria, Cordyceps This production apparently requires many steps, prolonged times and is difficult to control. The activity of the chitinase preparation sold by chemical suppliers is usually of the order of 1,200 nephelometric units. It is difficult to obtain quantities of chitinase exceeding 100 mg. and the current price is about $200.00 per gram.
It would be desirable to have a more readily available source of chitinase allowing recovery at lower cost and on a large scale. Chitinase has been found to potentiate or be a valuable adjuvant to bacterial insecticides such as Bacillus thuringiensis preparations (W.A. Smirnoff US. Pat. Application No. 317,535 filed Dec. 22, 1972). For spraying crops and timber stands large amounts of enzyme preparation are required at a reasonable cost.
The digestive tracts of certain creatures such as snails, frogs and sparrows possess considerable chitinolytic activity. I have found that fowl, particularly barnfowl or poultry possess considerable recoverable chitinolytic activity in their digestive tract. Chickens or hens in particular have been found to have significant quantities of chitinase activity in their entrails. Poultry processors and meat packing plants have a steady supply of large numbers of chickens and like fowl. Trials with turkey, goose, and duck stomach and gastric tract yielded lesser amounts of enzyme than with chicken.
An extraction method for separating and recovering chitinase activity from the entrails of fowl has been developed. The method is basically comprised of:
a. contacting the entrails with dilute acid medium until chitinolytic activity is extracted;
b. adding colloidal chitin to the extract, collecting the resulting insoluble deposit and washing the deposit;
c. in further dilute acid medium, allowing hydrolysis of the chitin in the deposit to proceed and removing any insoluble residue;
d. substantially saturating the solution with a neutral salt; and
e. recovering the precipitated chitinase material.
The entrails particularly gizzards and intestines are obtained from freshly-killed fowl. The chicken (Gallus gallus L.) is a preferred source which is available from many meat packing plants.
The entrails in fresh condition are submerged in dilute acid medium to extract the enzyme. The medium may be entirely aqueous or part aqueous-part watersoluble organic solvent e.g. alcohol. The dilute acid may have to be replenished during the entraction to maintain an acidic pH. Preferably the medium is buffered to a pH of about 5 to 5.5. Various suitable buffers are known with a mixture of citric acid and dibasic sodium phosphate being used in the Example 2. An antibacterial agent is usually incorporated to avoid any bacterial contamination.
After separation of the extracting medium from solids, colloidal chitin is added to cause preferential adsorption of the enzyme. A colloidal form of chitin is desired to give good dispersion and high surface area to effect efficient adsorption without excessive amounts and times being required. The colloidal form is also desired to enable hydrolysis (in step c) to proceed to substantial completion in reasonable times. One suitable technique for preparing a colloidal form of chitin is described below. Still other techniques could be used.
Technical or crude chitin can be obtained as a by-' product where chitin-containing natural foods are being processed e.g. from fisheries processing crustacea.
The chitin-chitinase deposit is washed with neutral or dilute acid solution to remove impurities. Preferably the same buffered solution as the extractant is used.
The salt added in step (d) is normally ammonium sulphate although some other substantially neutral salt of high water-solubility may be useful. This salting-out effect has been used in recovering other enzymes and high molecular weight proteins.
The steps of adsorption on colloidal chitin, removing unadsorbed material, hydrolyzing chitin, adding a neutral salt to the clear solution and recovering precipitated chitinase, can be repeated a number of times (until the recovered enzyme material has the desired activity). An activity of at least about 850-900 nephelometric units is preferred for insecticidal applications, and is usually obtained after two adsorptions on chitin.
The nephelometric analysis for enzyme activity is based on measuring the decrease in turbidity by-nephelometry (or photometry) in a colloidal suspension of chitin on enzymatic lysis. The nephelometric units measure the activity of enzyme/mg which will cause the turbidity due to 0.3 mg. of colloidal chitin/ml to decrease 50 percent after 2 hours incubation at 37.5C, pH 5.3 (Buffer Solution: citric acid 0.1M and dibasic Na phosphate 0.2M).
The following Examples are illustrative.
Preparation of Colloidal Chitin A technical or crude chitin was treated as follows. Ten kg. crude chitin was soaked for 24 hours in 15 gallons of dilute hydrochloric acid (concentrated hydrochloric acid diluted one-tenth) and then for 24 hours in 15 gallons of 10 percent sodium hydroxide solution. This soaking was repeated twice more.
After filtration and water removal (washing with acetone), the chitin was dissolved or dispersed in 15 gallons of concentrated hydrochloric acid. This highly viscous suspension was filtered through a fritted glass filter into a container holding gallons of distilled water. On dilution, the chitin precipitated as a colloidal suspension. This suspension is again filtered and diluted to a final pH of 6.4. The colloidal chitin can then be recovered in solid form by vacuum drying.
Other techniques for obtaining the colloidal chitin can be used and this invention is not limited to the method just described.
EXAMPLE 1 300 chicken digestive systems (gizzard, gut and coecums) from freshly-killed chickens were placed in 40 liters of aqueous solution buffered at pH 5.2. After several hours the mixture was filtered, centrifuged and the supernatant collected. About 25 gms of dry colloidal chitin was added and mixed with the solution. The solution was centrifuged and the deposit retained. The deposit was washed with dilute acid solution pH 5.2. The washed residue was dispersed in dilute acid medium (about 1 liter) and hydrolysis allowed to proceed until nearly complete hydrolysis of chitin has occurred (substantial removal of turbidity). The solution was centrifuged and then saturated with ammonium sulphate (saturation at 55 percent). The saturated solution was centrifuged for about 10 min. and the deposit recovered. This enzyme material has an activity of about 125 nephelometric units. On repeating the adsorptionpurification on colloidal chitin once more about 0.5 g of chitinase material was recovered having an activity of 890 nephelometric units.
EXAMPLE 2 Some 60,000 gizzards and intestine (guts) of freshly killed chickens were acquired and placed in 4,000 liters of buffer solution, pH 5.2. The buffered solution contained 0.02M citric acid and 0.02 M Na HPO plus 10 mg. per liter of an antibiotic (Thymol). The solution was filtered through glass-wool and centrifuged at 4,500 r.p.m. for 10 minutes. The supernatant was collected and placed in a second container.
5 kg of colloidal chitin was added and the suspension shaken for minutes. The suspension was centrifuged at 4,500 r.p.m. for minutes and the deposit was collected. The deposit was then washed in 100 liters of buffer pH 5.2 and centrifuged at 4,500 r.p.m. for 15 minutes. The precipitate was added to 100 liters of buffer solution, pH 5.2, and this suspension maintained at 37C with light stirring until nearly complete hydrolysis of the chitin. This solution was centrifuged for 10 minutes at 4,500 r.p.m. and the supernatant treated with 55 kg of ammonium sulfate (saturation at 55 percent). The saturated solution was centrifuged at 4,500 r.p.m., during 10 minutes. The deposit obtained was composed of chitinase having an activity of 100 to 150 nephelometric units.
This enzyme preparation was returned to liters of buffer solution pH 5.2 with 1.2 kg of colloidal chitin added. Hydrolysis at 37C with weak stirring was carried out until total reduction of the turbidity was obtained. The solution was centrifuged 15 minutes at 4,500 r.p.m., the supernatant was saturated by l 1 kg of (NH 80,, then stirred and centrifuged 10 minutes at 4,500 r.p.m. The deposit was g. of enzyme material with an activity of 850-900 nephelometric units.
Further repetiton of the colloidal chitin adsorptionpurification steps would increase the activity still more. However this activity has been found very effective in insecticidal adjuvant use.
I claim:
1. A method of extracting chitinase from the entrails of domestic fowl comprising:
a. contacting with dilute acid medium until the enzyme is dissolved;
b. adding colloidal chitin to the extract, collecting the resulting deposit, and washing the deposit;
c. allowing hydrolysis of the chitin in the deposit to proceed in dilute acid medium and removing any insoluble residue;
(1. substantially saturating the solution with a neutral salt; and
e. recovering the precipitated chitinase material.
2. The method of claim l wherein the dilute acid has a pH buffered at about 5 to 5.5.
3. The method of claim 1 wherein the recovered chitinase material is redispersed in dilute acid medium, colloidal chitin added and hydrolysis allowed to proceed, the solution separated and substantially saturated with a neutral salt, and the resulting chitinase precipitate recovered.
4. The method of claim 3 wherein the adsorptionpurification is repeated until a chitinase having activity of at least about 850 nephelometric units is obtained.
5. The method of claim 2 wherein the buffered pH is 5.2.
6. The method of claim 1 wherein the fowl is chicken.
7. The method of claim 1 wherein the salt is ammonium sulphate.
8. The method of claim 3 wherein the salt is ammonium sulphate.
9. The method of claim 1 wherein the entrails comprise intestine and gizzard.

Claims (8)

  1. 2. The method of claim 1 wherein the dilute acid has a pH buffered at about 5 to 5.5.
  2. 3. The method of claim 1 wherein the recovered chitinase material is redispersed in dilute acid medium, colloidal chitin added and hydrolysis allowed to proceed, the solution separated and substantially saturated with a neutral salt, and the resulting chitinase precipitate recovered.
  3. 4. The method of claim 3 wherein the adsorption-purification is repeated until a chitinase having activity of At least about 850 nephelometric units is obtained.
  4. 5. The method of claim 2 wherein the buffered pH is 5.2.
  5. 6. The method of claim 1 wherein the fowl is chicken.
  6. 7. The method of claim 1 wherein the salt is ammonium sulphate.
  7. 8. The method of claim 3 wherein the salt is ammonium sulphate.
  8. 9. The method of claim 1 wherein the entrails comprise intestine and gizzard.
US341545A 1973-03-15 1973-03-15 Extraction of chitinase Expired - Lifetime US3862007A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587292A (en) * 1987-05-22 1996-12-24 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Diagnosis of fungal infections with a chitinase
US5811095A (en) * 1993-04-30 1998-09-22 Alternative Methods, Inc. Basal and chitinase broth compositions for enhancing anti-fungal activity of a chemical fungicide and methods for preparing and using same
US6193988B1 (en) 1994-01-13 2001-02-27 Stoner, Ii Richard J. Tuber planting system comprising chitin or chitosan
WO2016083911A1 (en) * 2014-11-28 2016-06-02 Uniwersytet Jagielloński A novel method of chitinase a purification by affinity chromatography

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Methods in Enzymology, Vol. 8, Pages 644-650, (1966) *
Nature, Vol. 192, Pages 135-136, (1961) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587292A (en) * 1987-05-22 1996-12-24 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Diagnosis of fungal infections with a chitinase
US5811095A (en) * 1993-04-30 1998-09-22 Alternative Methods, Inc. Basal and chitinase broth compositions for enhancing anti-fungal activity of a chemical fungicide and methods for preparing and using same
US6193988B1 (en) 1994-01-13 2001-02-27 Stoner, Ii Richard J. Tuber planting system comprising chitin or chitosan
WO2016083911A1 (en) * 2014-11-28 2016-06-02 Uniwersytet Jagielloński A novel method of chitinase a purification by affinity chromatography

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Publication number Publication date
CA1008385A (en) 1977-04-12

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