KR820000907B1 - Process for preparation of glucose oxidaze by microorganism - Google Patents

Abstract

A glucose oxidase was produced by fermentation of Penicillium verrculosum NO. 114 (KFCC NO. 10023). Thus, the Penicillium verrculosum NO. 114 was aerobically cultured at 30 ≰C for 4 days in a medium (H 6.0) contg. glucose 5, NaNO3 0.7, KH2PO4 0.1, MgSO4 0.01 and beef extract 0.2%, and then the broth was centrifuged to obtain glucose oxidase with activity of 500-800 units/ml.

Description

Method of preparing glucose oxidase

1 is Chapek agar plate medium of Penicillium Verruculosum No. 114, KFCC NO. 10023.

2 is a wort agar plate medium of Penicillium Verruculosum No. 114, KFCC NO. 10023.

3 is a morphological characteristic of penicillium beruculosum No. 114 (Penicillium Verruculosum No. 114, KFCC NO. 10023) expanded to 600 times.

The present invention provides a new and advanced method for producing glucose oxidaze by culturing penicillium verruculosum No. 114 (KFCC NO.10023) strains in a nutrient medium containing sugars, nitrogen sources and minerals. It relates to a manufacturing method.

Glucose oxidase is an enzyme that catalyzes the production of gluconic acid and hydrogen peroxide by oxidizing glucose. It is used for the production of gluconic acid, the removal of glucose and oxygen, and medically for the diagnosis of glucose content in body fluids such as blood and urine. Used as an enzyme. Industrially, it is widely used in food industry and medicine, such as oxygen removal in canning and beverage production, and glucose removal in storage of dried eggs.

Glucose oxidase is originally known as an enzyme present in animal tissues, but recently, an industrial production method produced by fermentation by microorganisms has been developed and produced industrially. Microorganisms used in the industrial production of glucose oxidase are currently producing a large amount of glucose oxidase by culturing filamentous fungi of Aspergillus genus and penicillium genus, but the yield varies depending on the species. For example, according to US Patent No. 3,701,715 (October 31, 1972), five strains of the genus Aspirugins were cultured in a nutrient medium to produce enzyme yields of 2.4 to 6 units per ml of glucose oxidase.

The present inventors have attempted to search for microorganisms producing high glucose oxidase which is industrially economical for the purpose of industrial production of glucose oxidase. In other words, by taking the soil of each region in Korea, isolates of filamentous fungi of the penicillium, and cultured in synthetic nutrient medium containing glucose as a carbon source, and measuring the activity of glucose oxidase in the medium, the penicillium beru which shows unexpectedly strong enzyme activity Coulosum No. 114 (KFCC No.-10023) was isolated. This strain showed high activity of 15 to 18 units per 1 ml of medium in a glucose-containing synthetic medium. The enzyme activity of this strain is characterized by higher activity than the conventional Aspergillus and Penicillium strains. The production of glucose oxidase by the penicillium strain is a typical advantage because the enzyme is easily eluted out of the cell during the culture, so that the enzyme can be recovered directly from the culture medium without the need of destroying and eluting the cell like the filamentous fungus of the genus Aspergillus. .

The present invention is generally synthesized with penicillium beluchlorium No. 114 (KFCC No. 10023) having the ability to produce glucose oxidase, for example, glucose 5%, sodium nitrate 0.7%, potassium monophosphate 0.1%, Cultured in a medium of 0.05% magnesium sulfate, 0.001% ferric sulfate, and 0.2% (6% pH) of meat extract, accumulate enzymatic activity in the medium, and directly recover the culture medium after the end of the culture to facilitate high yield of glucose oxidase. It relates to a method for preparing glucose oxidase which can be easily recovered.

Determination of the enzyme activity of glucose oxidase using a complex enzymatic assay using peroxidase and ortho-dianisidine is the amount of enzyme that produces 1 micromole of hydrogen peroxide per minute at a temperature of 25 ℃ Enzyme 1 It was made in units.

The bacteriological properties of the penicillium beluclosum No. 114 (KFCC No. 10023) isolated by the inventors are as follows, and this is known as a known method (A Manual of Penicillia, The William and Wilkins Company, 1949, An appraisal). of identification methods for Penicillium species.Mycologia 65,1135 1157,1973).

end. Colony Characteristics: Observed after 10 days incubation at room temperature

1. Chapek agar reputation badge

The surface of colonies is flat and slightly centered, forming a velvety or floccose-funiculose, the color of the mycelia at the center of colonies is green to greenish yellow, and the colonies are yellow to white. Gradually light green. The color of colonies is light yellow to light greenish yellow, and the diameter of colonies is 40-45mm

2. Chapec hancheon deep media (20% per west)

It is a radial colony that forms a mycelial band and gradually becomes greenish yellow by forming light yellow or white mycelium.

3. Mastic agar plate

The colony's surface forms a flocse-like filamentous filament that is more vigorous than a flat, chaffed cloth, as shown in Figure 2. The colony gradually becomes light green and yellow in light green. The color of the colony is gradually white to green. The diameter of colony is 30-40mm

4. Chapek Yeast Extract Agar Plate

Woolly furrow colony with distinct color bands. In the central part of the colony, the surrounding color is light yellow-green, yellow, yellow-green, light pink, and yellow. Production of secretion like water droplets, backside of medium is pink to brown, colony diameter is 40-45mm

I. Morphological features: Observed after 7 days incubation at room temperature (see Figure 3).

1. conidiopore

Derived from Substrical mycellium, the width of substrate mycelium is 2.7μ

2. penicilli

Tablet symmetrical biverticillate

3. Branches

none

4. metulae

6.7-8.5μ long 1.7μ 2-3 reinforcement (2-3 in the verticil)

5. steigmata

8.5μ length 1.5μ 3-5 rotations

6. Conidia

Green Sphere, 1.7-2.7μ Diameter

All. Physiological characteristics: Observed after incubation for 5-7 days in small tank

1. Chapek yeast extract agar medium

Yellowish white colony, diameter 25mm (37 degrees Celsius) of rule

2. Agar medium with 25% glycerin

White colony, colony diameter 4mm (25 ℃)

3. Germination of Chapec Yeast Extract Agar Hazelnut (germination) Not germinated (5 ℃)

As a result of examining the characteristics, behavioral specific and physiological characteristics of colonies according to the identification method of the present invention, the isolate was identified as a strain belonging to the genus Penicillium, and the species was identified as Penicillium berukulosum.

Penicillium beruculium No. 114 (KFCC No. 10023) strain of the present invention was cultured in a medium containing glucose as a main carbon source to induce and produce glucose oxidase as in the experiments of Examples 1,2 and 3. For the preservation of the culture of the strain, either natural medium or synthetic medium can be used. In addition to the carbon source, the medium composition requires magnesium and iron components as sodium nitrate, meat extract, and a small amount of inorganic substances as nitrogen sources.

The following examples will illustrate the invention in more detail, but the scope of the invention is not limited thereto.

Example 1

Penicillium Berukuulosum No.114 (KFCC No.10023) 1 ml of platinum was added to 500 ml of medium (5% glucose, 0.7% sodium nitrate, 0.1% monobasic calcium phosphate, 0.03% magnesium sulfate, 0.001% Ferric sulfate and 0.2% meat extract) were incubated at 30 ° C. for 4 days, followed by incubation in 100 ml of the same medium using 5 ml of the seed stock, followed by incubation at 30 ° C. for 24 hours. The culture was centrifuged to completely remove the cells, and then the culture was used as a coenzyme solution of glucose oxidase.

Enzyme activity was measured in 3 ml of reaction mixture substrate (0.1% phosphate buffer solution, pH 6.0, 2.6 ml 0.1% ortho- dianisidine 0.1 ml, 18% glucose solution 0.3 ml, peroxidase 6 units) and 0.05 ml of crude enzyme solution. Was added to measure the reaction rate per minute of absorbance at 460 nm of spectrophotometer. One unit of enzyme was defined as one unit of an enzyme that produces 1 micromole of hydrogen peroxide at 25 ° C.

The enzyme measurement result was 5-8 units per 1 ml of culture, producing a total of 50-800 units.

Example 2

Penicillium Berulkulsum No. 5 ml of the seed stock solution of 114 (KFCC No. 10023) was inoculated into 100 ml of the medium of Example 1, and then cultured through aeration at 30 ° C., and the enzyme production according to the culture time was measured as follows.

After 24 hours of incubation, the enzyme titer was 550 units per 100ml, and gradually increased as the incubation time increased, reaching a maximum of 1,600 units per 100ml after 72 hours.

The strain culture was centrifuged to remove the cells, and then the culture was used as a coenzyme solution of glucose oxidase.

Example 3

100 ml of seed stock solution of penicillium berukuulosum No. 114 (KFCC No. 10023) was inoculated into 4000 ml of the medium of Example 1, and cultured for about 72 hours in a 5000 ml aeration fermenter under a 30 ° C aeration of 1.5 vvm rotational speed of 350 rpm. Enzyme production amounted to 15 units per ml of culture, totaling 60,000 units.

Example 4

Inoculated in 20,000 ml of the same medium with 500 ml of Penicillium berukuulosum No. 114 (KFCC No. 10023) of Example 3 and aeration for 30,000 ml at 30 ° C., aeration rate of 1.5vvm, and rotation speed of 350 rpm for 75 hours. As a result of cultivation, the enzyme production amounted to 18 units per 1ml of culture, reaching a maximum of 360,000 units.

Claims (1)

Penicillium beruculosum (Penicium verruculosum) No. 114 (KFCC No. 10023) strain cultured in a known medium to prepare a glucose oxidase, characterized in that for producing a glucose oxidase characterized in that the extraction.

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