WO1983001458A1 - Procede de preparation d'enzymes - Google Patents

Procede de preparation d'enzymes Download PDF

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Publication number
WO1983001458A1
WO1983001458A1 PCT/FI1981/000077 FI8100077W WO8301458A1 WO 1983001458 A1 WO1983001458 A1 WO 1983001458A1 FI 8100077 W FI8100077 W FI 8100077W WO 8301458 A1 WO8301458 A1 WO 8301458A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
enzymes
cell mass
fermentation
fermenter
Prior art date
Application number
PCT/FI1981/000077
Other languages
English (en)
Inventor
Oy Rintekno
Serlachius Oy G.A.
Original Assignee
Lundell, Ralf
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lundell, Ralf filed Critical Lundell, Ralf
Priority to PCT/FI1981/000077 priority Critical patent/WO1983001458A1/fr
Publication of WO1983001458A1 publication Critical patent/WO1983001458A1/fr

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Classifications

    • 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)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
    • 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
    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • 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
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • 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)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • 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)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)

Definitions

  • the present invention is concerned with a process for the preparation of enzymes by making use of 5 the cell mass of mycelium-forming microfungi belonging to the class Fungi imperfecti, the cell mass being obtained from continuous aerobic fermentation.
  • microfungus-based enzymes of industrial production are enzymes that hydro- lyze polysaccharides and their derivatives, such as, for example, amylases, cellulases, hemicellulases, pectinases, 15 and lactases, as well as proteases which hydrolyse pro ⁇ teins and peptides.
  • the hydrolysing enzymes are formed during the stationary/dying stage following after the growth stage of microfungi.
  • the composition of the growing substratum 20 has an essential effect on the formation of enzymes.
  • microfungi The industrial production of enzymes by means 25 of microfungi is based exclusively on the batch fermen ⁇ tation process. ' A microfungus is first grown in a little fermenter, whereupon it is transplanted, usually via an intermediate fermenter, to the production fermenter, wherein the cell mass is grown to the ultimate quantity. 30. After the growth stage, the cells of microfungi produce enzymes by means of the same raw-materials.
  • the duration of the production fermentations is mostly several days, out of which time an essential part is taken by growing the cell mass.
  • the process is 5 frequently modified so that part of the raw-materials are fed into the fermenter during the batch in order to maximize the quantity of cells in the substratum and,
  • the fermentations are aseptic.
  • the vital func ⁇ tions of the cells are directed at the production of new cell mass.
  • a cell mass rich in pro- teins is obtained from growing solutions containing carbohydrates and/or carbohydrate derivatives.
  • the microfungus does not produce "extra" products of metabolism, such as enzymes, but it produces mainly cell mass out of the raw-materials.
  • Patents 3 * 288,683 and 3,652,400 it is known that the microfungus Paecilomyces varioti produces enzymes in batch cultures.
  • the enzymes produ ⁇ ced by means of the said process, glucoamylase- and pro ⁇ tease, are surprisingly favourable as to their properties.
  • the invention came out when the use of the microfungus Paecilomyces varioti as a producer of extra ⁇ cellular enzymes, especially of hydrolases, was studied.
  • OMPI t WIPO * the nutrient solution of the enzyme fermentation, the formation of the enzymes is accelerated and enzyme concentrations are obtained that considerably exceed the concentrations that can be achieved by means of the batch processes representing the prior art technology.
  • Advan ⁇ tages include a clear increase in the yield of enzymes and an essential facilitation of the after-treatment operations.
  • the process in accordance with the invention is mainly characterized in that the microfungus cell mass obtained from continuous culture is, after the substratum used in the culture has been separated and the cell mass washed, introduced into a separate enzyme fermenter, into which a growing solution or suspension containing inorganic and/or organic substances that induce enzymes have been or are introduced, and that the fermentation of enzymes is hereinafter performed under conditions favourable for the formation of enzymes, preferably so that the temperature of the growing solution or suspension is 20 to 50 C, pH 2.5 to 7.0, and that air is introduced into the aerobic process as a quantity of 10 to 50 per cent from the quantity of the growing solution or suspension per minute, and that growing solution or suspension together with the cell mass and with the remaining growing substratum and with the enzyme product are removed from the fermenter batchwise after the enzyme concentration or concentra ⁇ tions have reached a favourable level.
  • microfungus Paecilomyces varioti belongs to the class Fungi imperfecti.
  • the same class includes most of the microfungi, e.g. the microfungi belonging
  • the substratum of continuous, aerobic culture of microfungi may consist of most various carbohydrates and/or suspensions, such as industrial waste, e.g. spent liquors from wood-processing industry.- In addition to this, other rawmaterials of cells are required, especi- 20 ally inorganic salts.
  • the continuous fermentation of microfungi has an aseptic nature. In the process de ⁇ scribed above the separation of the microfungi from the substratum of the continuous culture is essential, especially if the nutrient solution consists of lignin- 25 containing spent liquors from wood-processing industry.
  • Washing is preferably performed by means of a washing liquid, which may be water, whose temperature 30 and pH do not differ essentially from those used in continuous fermentation.
  • the washing liquid should be preferably sterile, in which case the further enzyme fermentation is protected from contaminating micro ⁇ organisms.
  • the requirement of sterility of the washing 35 liquid. is not unconditional, for there are also other means of protection from contamination.
  • OMPI In the process, usually the entire cell mass that is needed for the production of the enzymes is batched right at the initial stage of the process into the enzyme fermenter. It is, however, possible to add to the cell mass during the enzyme fermentation, either during part of the time or throughout the entire duration of the batch.
  • the batching may also be continued after a partial emptying of the fermenter by adding new cell mass to replace the cell mass that was removed from the fermenter.
  • raw-material for the process it is possible to use inorganic and/or organic compounds that have pro ⁇ perties contributing to the maintenance of the cell mass and to the formation of enzymes.
  • the raw-materials may be batched into the enzyme fermenter as a whole at the beginning of the production batch, or they may be added during the batch. If the fermenter is emptied at the beginning of the batch partly, the removed 'portion of - raw-material may be substituted by a new quantity of ra -material. As a rule, the ra -materials are added as sterilized, but this is not always necessary.
  • raw- materials it is possible to use, e.g., various by-products of agricultural products, such as bran and bran extracts, or various by-products of wood-processing industry and extracts from same.
  • Other organic raw-materials besides those mentioned here may also be used.
  • inorganic compounds are also used.
  • water is used, into which the raw-materials have been dissolved or suspended.
  • the temperature of the growing/ enzyme-formation substratum is maintained at 20 to 50°C and the pH at 2.5 to 7.0.
  • the temperature and/or the pH may be varied during the process, but most usually they are maintained at constant values.
  • the fermentation process is aerobic, and the consumption of air per minute
  • OMPI during the process is about 10 to 50 per cent from the quantity of the fermentation substratum.
  • the fermen ⁇ tation broth is frequently stirred mechanically. It has been noticed that it is preferable to carry out the enzyme fermentation as aseptic, whereby the equipment and the raw-materials are sterilized by means of conventional processes before fermentation and the microfungus cells obtained from continuous fermen- tation are washed with water or with a nutrient solution. In all cases, aseptic conditions are, however, not necessary.
  • the procedure of the process in accordance with the invention is.the following.
  • the starting point of the process is a conti ⁇ nuous culture of a microfungus performed by conventional means, from which the culture substratum that has been used and that contains cell mass of the microfungus is constantly available.
  • the washed cell mass and the raw-materials needed for the formation of enzymes and for maintaining the vital functions of the cells are passed into an enzyme fermenter.
  • the fermentation is carried out as an aerobic batch process, which can be continued by removing enzyme- containing solution and by adding a compensating quantity of raw-materials and/or cell mass.
  • the batch is finished by emptying the fermentor completely when it is consi ⁇ dered that the production capacity of the microfungus has been clearly reduced.
  • the fermentation may also be discontinued earlier if other criteria so require.
  • Microbe mass may also be added to the fermenter during the batch if the production of enzymes can be improved thereby.
  • the raw-materials needed in the process and the cell mass can be transferred into the fermenter at the same time or separately. Air is supplied into the fermentation broth as a quantity of about 10 to 50 per
  • the fermenter broth can be stirred mecha ⁇ nically in order to improve the transfer of heat and material.
  • the temperature, 20 to 50 C, and the pH, 2.5 to 7.0, of the culture solution are either maintained as constant or controlled in accordance with a specified program, or they are allowed to adjust themselves freely during the fermentation process.
  • the microfungus mass and solid raw-material residues are sepa ⁇ rated from the enzyme-containing culture broth and washed, whereupon the solid fermentation residue can be used, e.g., as a protein-containing product for fodder or for techni ⁇ cal purposes.
  • the enzyme-containing solution and the washing solution are treated by conventional means in order to recover the enzyme products.
  • the process is rapid, because the cell-growing step of a conventional batch process is omitted totally. This is why the process permits the use of a non-aseptic culture technique.
  • the process is also suitable for the supply of additional raw-materials, as well as for the supply of additional microfungus, whereby the production capacity can be increased further.
  • the process may be connected to a single-cell protein factory using a microfungus, or it may be based on the cell quantity of a continuous production of micro ⁇ fungus constructed for enzyme production only.
  • an additional advantage is that the waste liquors and the solid residues, such as the cell mass and the solid raw-material, residues, can be returned to the single-cell protein factory, and there ⁇ by the environmental problems are reduced to none.
  • the after- treatment of the fermentation solution is more advan ⁇ tageous than the after-treatments of the enzyme-containing fermentation solutions from the processes representing the prior art technology.
  • This reference example illustrates the produc ⁇ tion of enzyme by means of mycelium-forming microfungi in a way in itself known.
  • composition of the pre-growing substratum in shaking flasks (1.5 1) was:
  • composition of the substratum in the trans ⁇ plantation fermenter (30 1) and in the main fermenter (1000 1) was: - wheat bran 40 g/1 - starch 25 g/1
  • the lower limit of the pH had been adjusted at 4.5.
  • the growing temperature was 37°C until the growth maximum of the microfungus, and 30°C at the enzyme-production stage.
  • the microfungus reached its growth maximum about 24 hours after the beginning, at which time the content of cell mass in the growing broth was about 16 grams per kilogram of suspension.
  • the pH-value became higher than 6, it started being adjusted by means of phosphoric acid.' ** The pH was gradually lowered to 4. " 6.
  • the batch fermentation took a total of 87 hours, i.e. 3-5 days.
  • the activities obtained were: enzyme rate of activity enzyme production
  • the glucoamylase activity was determined by using 2mM p-nitrophenyl- ⁇ -_.-D-glucopyranoside as substrate at 45°C, at pH 4.5, the reaction time being 10 minutes.
  • the quantity of p-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .
  • the 5-glucosidase activity was determined by using 1 mM 4-nitrophenyl- ⁇ -D-glucopyranoside as substrate at 50°C, at pH 4.8, the reaction time being 10 minutes.
  • the quantity of 4-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .
  • OMPI The ⁇ -xylosidase activity was determined by using 2 mM o-nitrophenyl- ⁇ -D-xylopyranoside as substrate at 45°C, at pH 4.5, the reaction time being 10 minutes. The quantity of o-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .
  • the growing solution was after-treated for the recovery of the enzymes. From it the bran residue and the yeast mass were separated. The solution was con ⁇ centrated by ultrafiltration to an almost 30-fold con- centration. As the final result, a yellowish brown, highly active enzyme concentrate was obtained. '
  • an 8-litre laboratory fermenter constructed for continuous, aseptic fermenta ⁇ tion was used for the production of the cell mass of the microfungus Paecilomyces varioti.
  • the enzyme fer- mentations were carried out by means of a 4-litre labo ⁇ ratory fermenter which was provided with a filter (100 mesh) for the filtration and washing of the microfungus cell mass.
  • the spent liquor was, before use, diluted with water to a content of about 8 per cent (w/w) , and inor- ganic salts were added to it, whereupon it was sterilized.
  • the continuous fermentation was carried out under the following conditions:
  • the cell concentra ⁇ tion of the microfungus remained at the level of 13 to 14 g/kg of suspension.
  • the waste liquor suspension was pumped straight into the enzyme, fermenter, whereupon the waste liquor was removed through a filter placed on the bottom of the fermenter.
  • the mycelium was washed with a phosphate solution (KH 2 P0 , 10 g/1, pH 4.5).
  • the washing was performed as 4 batches with a total of 4 litres of solution.
  • the washing solutions had been sterilized and cooled to 30 C before the washing stage.
  • the filtration and washing of the mycelium were performed in the fermentation tests in about 30 minutes.
  • the substrate had been sterilized and cooled to 30°C.
  • the mycelium was introduced into the induction
  • OMPI IPO fermenter after 110 hours had elapsed from the starting of continuous fermentation of Paecilomyces varioti.
  • the mycelium content In the continuous fermenter was 19-4 g/kg of suspension. About 5 kg of the suspension was transplanted. The fungus mass was filtered and washed. The mycelium content at the beginning of the test was estimated as about 15. " 5 g/kg of suspension, aeration 4 1/min.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé de préparation d'enzymes consistant à utiliser la masse de cellules de micromycètes formant du mycélium appartenant à la classe des Fungi imperfecti, la masse de cellules étant obtenue à partir d'une fermentation aérobie continue. Selon l'invention, la masse de cellules de micromycètes obtenue à partir d'une culture en continu est, après séparation de la couche utilisée dans la culture et après lavage de la masse de cellules, introduite dans un appareil à fermentation d'enzymes séparé, dans lequel une solution ou suspension de croissance contenant des substances inorganiques et/ou organiques qui induisent des enzymes ont été ou sont introduites, la fermentation des enzymes étant par la suite effectuée dans des conditions favorables à la formation d'enzymes, de préférence la solution ou suspension de croissance se trouvant à une température comprise entre 20 et 50oC, avec un pH compris entre 2,5 et 7,0, et l'air est introduit dans le procédé aérobie en une quantité égale à 10-50 % de la quantité de la solution ou suspension de croissance par minute, et la solution ou suspension de croissance ainsi que la masse de cellules et la couche de croissance restante et le produit enzymatique sont extraits de l'appareil à fermentation de manière discontinue après que la concentration ou les concentrations enzymatiques ont atteint un niveau favorable.
PCT/FI1981/000077 1981-10-26 1981-10-26 Procede de preparation d'enzymes WO1983001458A1 (fr)

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PCT/FI1981/000077 WO1983001458A1 (fr) 1981-10-26 1981-10-26 Procede de preparation d'enzymes

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PCT/FI1981/000077 WO1983001458A1 (fr) 1981-10-26 1981-10-26 Procede de preparation d'enzymes

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0528612A2 (fr) * 1991-08-13 1993-02-24 Sankyo Company Limited Amylase capable de digérer l'amidon brut
US5604128A (en) * 1991-08-13 1997-02-18 Sankyo Company, Limited Isolated cultures of Pestalotiopsis funerea IFO 5427 and Pestalotiopsis negleta FERM BP-3501

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2936266A (en) * 1955-03-03 1960-05-10 Versuchs U Lehranstalt Fur Spi Production of ergot alkaloids
US3288683A (en) * 1963-11-07 1966-11-29 Taisho Pharmaceutical Co Ltd Production of acid-stable proteolytic and amyloytic enzyme
US3652400A (en) * 1965-03-04 1972-03-28 Taisho Pharmaceutical Ltd Process for the preparation of amylase
US3809614A (en) * 1970-08-14 1974-05-07 K Forss Process for the manufacture of protein-containing substances for fodder,foodstuffs and technical applications
FR2302336A1 (fr) * 1975-02-26 1976-09-24 Baxter Laboratories Inc Procede pour la fabrication d'un melange d'enzymes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2936266A (en) * 1955-03-03 1960-05-10 Versuchs U Lehranstalt Fur Spi Production of ergot alkaloids
US3288683A (en) * 1963-11-07 1966-11-29 Taisho Pharmaceutical Co Ltd Production of acid-stable proteolytic and amyloytic enzyme
US3652400A (en) * 1965-03-04 1972-03-28 Taisho Pharmaceutical Ltd Process for the preparation of amylase
US3809614A (en) * 1970-08-14 1974-05-07 K Forss Process for the manufacture of protein-containing substances for fodder,foodstuffs and technical applications
FR2302336A1 (fr) * 1975-02-26 1976-09-24 Baxter Laboratories Inc Procede pour la fabrication d'un melange d'enzymes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Advances in Applied Microbiology, Vol. 25 (1979), New York, Ed. D. PERLMAN: "Applications of Microbial Enzymes in Food Systems and in Biotechnology", TAYLOR M.J. and RICHARDSON T; P. 8-9 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0528612A2 (fr) * 1991-08-13 1993-02-24 Sankyo Company Limited Amylase capable de digérer l'amidon brut
EP0528612A3 (en) * 1991-08-13 1993-06-23 Sankyo Company Limited Amylase capable of digesting raw starch
US5604128A (en) * 1991-08-13 1997-02-18 Sankyo Company, Limited Isolated cultures of Pestalotiopsis funerea IFO 5427 and Pestalotiopsis negleta FERM BP-3501

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