Classifications

• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K3/00—Invert sugar; Separation of glucose or fructose from invert sugar
• • 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
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
  • C12N11/12—Cellulose or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

• the present invention relates to a method of fixing an enzyme to a solid support which is insoluble in water. It also relates to a support-enzyme complex and to a method of using such a complex.
• Cellulose has the reactivity of a polyalcohol and can be subjected to oxidation, esterification, etherification, halogenation, etc. reactions.
• the oxycelluloses are coupled to alcohols, amines or proteins by forming esters or amides.
• French patent 2,247,472 (SNPA) describes a method of fixing enzymes on cellulose associated with lignin, but indicates, which seems very surprising, that the cellulose-enzyme association reaction is favored by lignin present in an amount of 5 to 25% by weight, and preferably
• Grains of cereal stalks whose size varies between 0.3 and 10 mm are first delignified on the surface then after washing, treated with thionyl chloride in pyridine, finally brought into contact with an enzyme-buffered solution .
• the present invention relates to a process for obtaining enzymatic derivatives in which the enzyme is fixed by a covalent bond on a support insoluble in water, characterized in that it comprises the following stages:
• the oxidant of step b) is sodium periodate
• the diamine is 1 ethylenededia mine
• the reducing agent is borohydride or sodium cyanoborohydride
• dialdehyde gl utaral dehyde
• the enzyme can be in particular invertase, levan sucrase, lactase or dextranesucrase.
• tender elements containing mainly cellulose and proteins, intended for animal feed.
• GRITS hard elements - called GRITS - containing a large proportion of lignin (greater than 25%), cellulose and xylans. These elements are generally ground and used because of their hardness as fillers or as abrasives.
• the pseudo-spherical particles obtained have dimensions which can vary between a few tens of microns and a few millimeters.
• the invention also relates to an enzymatic derivative in which the enzyme is covalently linked to the support, characterized in that the support consists of the part of the cereal stalks whose lignin content is greater than 25%, the enzyme being invertase, levan sucrase, lactase or dextran.
• Another subject of the invention relates to a process for treating sweet juices using enzymes fixed on the part of the corn cobs whose lignin content is greater than 25%.
• a concentrated sucrose juice is treated by passing over a bed of corn cob particles serving as support for the invertase, with a view to obtaining an inverted syrup.
• sucrose juice is treated by passing over a bed of corn cob particles serving as a support for the levansucrase: a juice containing polyfructose and glucose is obtained.
• Invertase is an enzyme which catalyzes the hydrolysis of sucrose into glucose and fructose, which, according to the invention, has been fixed on supports water insoluble solids, carriers containing both cellulose and lignin.
• the activity of the enzyme preparation is determined by contacting, for 4 minutes at 40 ° C., 10 ml of 0.4 M sucrose solution in 0.1 M acetate buffer, pH 4.5 with 0.1 ml. invertase solution (or immobilized invertase suspension) in the same buffer.
• the reaction is stopped by adding 5 ml of the medium to 0.5 ml of 2N sodium hydroxide solution.
• the content of released reducing sugars is then determined by the dini trosalicylate method (reading of the absorbance at min).
• a calibration is carried out using equimolecular solutions of glucose and fructose in 0.1 M acetate buffer (pH 4.5).
• One unit of enzyme activity (U) is defined as the amount of enzyme that causes the release of 1 g. reducing sugars per minute under the test conditions.
• the specific activity of the invertase preparation (SIGMA Chem. Co is 93.3 U / mg of enzyme.
• the process for treating corn cob particles comprises the following steps:
• Each step is followed by two washes of the stalks by resuspension for 15 min, in 20 ml of the medium used during the previous step - not containing the reactive species - then by three washes by resuspension in the medium used in the next step.
• the optimal oxidation time used is 30 hours.
• a duration of 72 hours was therefore chosen for the amination stage.
• the optimal concentration is therefore 1.25% and the duration of 5 hours.
• the optimal concentration of invertase is therefore 2g 1 -1 and the optimal duration of fixation is 30 hours.
• Table 15 lists the optimal conditions for attaching invertase to corn cobs.
• the immobilization of the invertase was carried out by bringing 100 mg of activated support into contact with 20 ml of invertase solution (SIGMA Chem. Co I 5875) at 2gl _ 1 in 0.1 M acetate buffer, pH 4.5, for 30 hours with rotary stirring at 4 ° C.
• the activity retained was determined by measuring the quantity of reducing sugars (glucose and fructose) released by the action of the enzyme on a 0.4 M sucrose solution at 40 ° C.
• the enzymatic activities obtained with supports treated according to the invention are 40 to 80 times greater than those obtained with the same supports treated according to French patent 2 247 472.
• sucrose 22 g of sucrose hydrolyzed per hour and per g of stems.
• the yield of the immobilization is expressed as the ratio of the activity per mg of immobilized enzyme to the activity per mg of free enzyme.
• Table No. 19 groups together the results concerning the production of glucose and fructose (expressed in g of reducing sugars produced per hour and per g of stalks).
• Immobilized invertase is extremely effective since from the results obtained with the 1 liter reactor for a 68.4% sucrose solution, a productivity of 11 g of reducing sugars per day is obtained at 50 ° C. for the 1 liter volume reactor (the conversion rate being 90%)
• the stability of the catalyst is very good because no significant decrease in activity could be noted after 20 days of continuous operation (with stay at 40 ° C, 50 ° C, 55 ° C, and 60 ° C) for the 1 liter reactor.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

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Cited By (6)

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• 1979
  • 1979-12-21 FR FR7931382A patent/FR2472015A1/fr active Granted
• 1980
  • 1980-12-18 DE DE8181900065T patent/DE3066663D1/de not_active Expired
  • 1980-12-18 EP EP81900065A patent/EP0042395B1/fr not_active Expired
  • 1980-12-18 AT AT81900065T patent/ATE6272T1/de active
  • 1980-12-18 WO PCT/FR1980/000185 patent/WO1981001860A1/fr not_active Ceased
• 1981
  • 1981-08-03 US US06/290,804 patent/US4405715A/en not_active Expired - Fee Related

Patent Citations (2)

Non-Patent Citations (1)

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