KR100219733B1 - A process for preparing glucose oxydase using aspergillus niger mutant cells - Google Patents

Abstract

The present invention relates to a method for producing aspergillus niger GOD4, a recombinant strain produced by genetic engineering for mass production of glucose oxidase, and a method for mass production of glucose oxidase using the same. More specifically, the expression vector pGDS3 was prepared by assembling the GPD promoter of Aspergillus neidrans and the alpha amylase signal and terminator of Aspergillus olease to the glucose oxidase gene isolated from Aspergillus niger. The present invention relates to a recombinant Aspergillus Niger GOD4 introduced into Fergus nager and a method of fermenting the same, and producing a large amount of glucose oxidase.

Description

Method for preparing glucose oxidase by Aspergillus niger mutant strain

The present invention relates to a method for producing aspergillus niger GOD4, a recombinant strain produced by genetic engineering for mass production of glucose oxidase, and a method for mass production of glucose oxidase using the same. More specifically, after assembling a promoter or signal derived from another species of Aspergillus into a glucose oxidase gene derived from Aspergillus niger ATCC-9029, the expression plasmid expressed in Aspergillus is expressed. The present invention relates to a transformed recombinant strain for introducing and expressing a large amount of glucose oxidase in an Aspergillus host strain and a method for fermenting the same to produce a large amount of glucose oxidase.

Since glucose oxidase is used to measure glucose concentration in a sample, it is widely used in the manufacture of diagnostic kits used to measure blood glucose levels. It is an antioxidant for long-term storage of packaged foods such as beverages and canned foods, and is used to remove oxygen from containers.

친화력을 갖고 있다.Glucose oxidase is an enzyme involved in the conversion of diglunolactone and hydrogen peroxide using betadiglucose and oxygen, and the by-product hydrogen peroxide is converted to oxygen and water by catalase. This enzyme is made up of

Has affinity.

Glucose oxidase was first extracted by German Muller in 1928 from cell extracts of Aspergillus-Niger and Penicillium globules. The location of glucose oxidase in the microorganism is known to be outside the cell membrane in the case of penicillium and simultaneously inside and outside the cell membrane in the case of Aspergillus niger. Rice (Histochemie 7, 202-210 (1966)) and Van Dizucan (Europ. J. Appl. Microbiol. Biotechnol. 9, 275-283 (1980)) are glucose oxidases in mycel's puloxyzomen. Was isolated and Muller extracted the enzyme from the culture of Aspergillus Niger.

180kDa이며, FAD와 결합되어 있는 당단백질로써 각각 70

80kDa인 2개의 서브유니트로 구성되어 있고 각 유니트는 한 분자의 FAD와 견고하게 결합되어 있으며, K_D값은 1.3

10^-10이다.Aspergillus Niger's glucose oxidase structure has a molecular weight of 150

180 kDa, glycoproteins associated with FAD, 70 each

It consists of two subunits of 80 kDa, each unit is firmly bound to one molecule of FAD, and the K _D value is 1.3

10 ^-10 .

The glucose oxidase gene consists of 1815 nucleotides, which coat 605 amino acids. In addition, there is no intron in the gene, and it has a signal consisting of 22 amino acids at its 5 prime end. This signal causes glucose oxidase to be secreted from Aspergillus niger, which in the process is degraded by signal peptidase. Glucose oxidase activates after signal degradation.

Glucose oxidase production is mainly extracted in fermented Aspergillus or Penicillium (US Pat. No. 2,482,724, US Pat. No. 3,701,715). Genetic engineering method using a recombinant yeast strain made by introducing the Aspergillus Niger's glucose oxidase gene into the yeast expression prasmid (US Pat. No. 5,266,688), Aspergillus putidus glucose oxidase gene Glucose oxidase is produced using a recombinant Aspergillus putidus strain (European Patent No. 0 665 291 A1).

The production of glucose oxidase by conventional Aspergillus or penicillium fermentation is 0.29 Unit / mL (Markwell et al., Appl. Microbiol. Biotechnol. 30, 166-169 (1989)) because it is very small. Efforts have been made to increase production by modifying the production strain or expressing it in heterologous strains for mass production purposes. To do this, they may use mutations or genetic engineering methods. When using yeast as a host strain when improving the strain by genetic manipulation, gene introduction into yeast is more convenient than using mold as a host strain. However, when the target gene is glycoprotein, the glycosylation of the expressed protein is excessive. There is a problem.

In addition, in view of the reality that glucose oxidase is currently produced in Aspergillus, the present invention uses the existing production strain Aspergillus niger as a host strain for mass production. Glucose oxidase expression plasmids were prepared by substituting the native promoter and signal genes with strong promoters and signals derived from other species of Aspergillus using the glucose oxidase gene. These plasmids were transformed into Aspergillus niger to allow large amounts of glucose oxidase genes to be expressed in bacteria to allow a large amount of glucose oxidase to be secreted.

1 is a diagram showing the gene combination of the plasmid pGDS3 of the present invention.

Therefore, the present invention assembles the GPD promoter of Aspergillus neidrans and the alpha amylase signal and terminator of Aspergillus olease in the glucose oxidase gene isolated from Aspergillus ligne to prepare the expression vector pGDS3 and host cell. The present invention relates to a recombinant strain Aspergillus Niger GOD4 introduced into Aspergillus Niger and a method of fermenting the same, and producing a large amount of glucose oxidase. Recombinant strain Aspergillus Niger GOD4 of the present invention was deposited with the Korean microbiological preservation center No. KFCC-10945 in the Korean spawn association on January 10, 1997.

Aspergillus Niger NRRL3 was most preferably used as a host cell, and the transformed strain was selected using the resistance of antibiotic hygromycin B. Recombinant strain was able to confirm the expression level through the expression medium, the amount of the O- dianisidine added in the expression medium according to the concentration of the generated H ₂ O ₂ could be measured quantitatively. Aspergillus niger showed a superior expression effect as the host cell compared to other species Aspergillus neidrans or Aspergillus olease of the genus Aspergillus.

0.5g/l, KH₂PO₄0.1

0.5g/l, MgSO₄·7H₂O 0.1

0.3g/l, CaCO₃2

5

, 박토-펩톤 2

6

, 글루코스 4

10

로 구성된 발효배지임을 특징으로 한다.The present invention also provides a method for producing glucose oxidase by batch culturing the recombinant strain Aspergillus Niger GOD4 in fermentation broth, wherein the preferred fermentation broth is (NH ₄ ) ₂ HPO ₄ 0.1

0.5 g / l, KH ₂ PO ₄ 0.1

0.5 g / l, MgSO _{4 7} H ₂ O 0.1

0.3 g / l, CaCO ₃ 2

5

, Bacto-peptone 2

6

, Glucose 4

10

Characterized in that the fermentation medium consisting of.

Hereinafter, the present invention will be described in detail.

Aspergillus Niger's glucose oxidase gene was isolated from cDNA library from mRNA of Aspergillus Niger ATCC-9029. In order to induce effective expression and secretion to this gene, the expression plasmid pGDS3 was prepared by substitution-synthesizing the alpha amylase signal peptide of GPD Aspergillus olease from Aspergillus nyrands.

To assemble the Aspergillus Nydrance GPD promoter, the plasmid pAN 51-1, which is looking for a GPD promoter, was cleaved using the restriction enzymes EcoRI and NcoI, followed by alpha-amylase signaling and glucose oxy from Aspergillus oraise. It was inserted into plasmid pPp * 4.13 (Nael) -Sal having a multidase 5 frame terminal amino acid sequence. This intermediate plasmid was named p435. The plasmid p or + 3.0 / 2.0 Sal containing the glucose oxidase gene was then cleaved with restriction enzyme SalI and then inserted into p435 digested with SalI. The expression prasmid thus prepared was named pGDS3.

The recombinant expression plasmid vector pGDS3 was introduced and transformed into the host strain Aspergillus Niger ATCC-9029 together with pAN 7-1 which is used together as a selection marker by the Tilburn method. Since pAN 7-1 prasmid used as a selection marker has a hygromycin resistance gene, selection of transformed host strains was performed by selecting clones to be grown in a medium containing hygromycin.

The degree of glucose oxidase production produced by the transformed strain was confirmed by measuring enzyme activity. At this time, as a qualitative measurement method, hydrogen peroxide reduced by peroxidase was measured using o-diinishin, and when quantitatively measured, ABTS, which has high water solubility, was measured instead of o-diinishidine. It was.

설탕, 0.2

NaNO₃, 0.05

KCL, 0.05

Mg-Glycerophosphate, 0.035

K₂SO₄, 0.001

FeSO₄, pH 6.8)에서 온도 30

, 250rpm, 4일간 플라스크 배양하였다. 결과 형질전환된 아스퍼질러스 나이거, 아스퍼질러스 오라이제와 아스퍼질러스 나이드란스의 글루코스 옥시다제의 효소활성이 각각 1.9Unit/mL, 1.4Unit/mL, 0.5Unit/mL로 숙주균주는 아스퍼질러스 나이거가 가장 적합한 것으로 나타났다.In the glucose oxidase activity assay, the best clone was selected and named as GOD4, and its expression amount was 1.2Unit / mL. The host strain was changed to increase glucose oxidase production capacity to increase productivity. The expression plasmid pGDS3 was transferred to Aspergillus neidrans and Aspergillus oase, and each transformant was transferred to Schwak-Dox medium (3).

Sugar, 0.2

NaNO ₃ , 0.05

KCL, 0.05

Mg-Glycerophosphate, 0.035

K ₂ SO ₄ , 0.001

FeSO ₄ , pH 6.8), temperature 30

The flask was incubated for 4 days at 250 rpm. Results The enzyme activity of the transformed Aspergillus niger, Aspergillus oliase and Aspergillus neidrans glucose oxidase were 1.9 Unit / mL, 1.4 Unit / mL and 0.5 Unit / mL, respectively. Sniger was found to be the best.

5 L batch fermentation was performed to optimize glucose oxidase production of the recombinant strains. As a result of enzyme titer measurement in fermentation broth after culture, it was found that the transformed strain was about 150 times higher than the Aspergillus Niger wild type strain.

Hereinafter, the present invention will be described in more detail.

Example 1

[Assembly of recombination vectors]

Aspergillus hydrans GPD (glycerolaldehyde-3'-phosphate-dehydrogenase) promoter, alpha amylase signal of Aspergillus orease and glucose oxidase gene of Aspergillus niger for the development of recombinant oxidase The expression vector pGDS3 was developed using a terminator. The cloning method of the expression vector pGDS3 is as follows.

과 아스퍼질러스 나이드란스의 GPD 프로모터가 있는 pAN 52-1 500

를 절단효소 EcoRI과 NcoI으로 37

에서 절단한 후 T₄DNA-라이가제 1유니트를 이용하여 16시간동안 10

하에서 접합을 시킨다. 재결합된 pAN 52-1의 형질전환을 방지하기 위해 잔유하는 T₄DNA-라이가제를 불활성시키기 위하여 이 라이게이숀 프로브를 15분간, 65

에 가열한 후 20유니트 BamHI으로 절단, 37

하에서 4시간 배양한 후, 이·콜라이 5-α 콤피턴트 셀에 형질전환을 시켰다. 얻어진 클론들을 아가로즈 젤상에서 분석하여 아스퍼질러스 나이거의 GPD 프로모터가 올바르게 삽입된 프라스미드 p435를 얻었다. 이 프라스미드에 글루코스 옥시다제 유전자(pGOD2)을 삽입시키기 위하여 p435 200

과 pGOD2 300

을 XmaIII와 HinDIII로 절단, T₄DNA 라이가제 1유니트를 이용하여 16시간, 10

하에서 접합시킨 후, 이·콜라이 5-α 콤피턴트 셀에 형질전환하였다. 얻어지는 클론들을 아가로즈 젤상에서 분석하여 글루코스 옥시다제 유전자의 N-말단부분이 올바르게 삽입된 프라스미드 p450를 얻었다. 나머지 글루코스 옥시다제 유전자를 삽입시키기 위하여 p435 200

과 p450 400

을 XmaIII로 절단한 후 T₄DNA 라이가제 1유니트를 이용하여 16시간, 10

하에서 접합시켰다. 인설트가 결합되지 않고 재결합된 p450의 형질전환을 방지하기 위해 잔유하는 T₄DNA 라이가제를 불활성시켰다. 이를 위해 반응물을 15분간 65

에 가열하였다. 결합된 프라스미드는 이·콜라이 5-α 콤피턴트 셀에 형질전환시켰다. 형질전환체들로부터 프라스미드 DNA를 추출하여 아가로즈 젤상에서 글루코스 옥시다제 유전자가 올바르게 삽입된 프라스미드를 선별하였다. 이렇게 얻은 발현 프라스미드를 pGDS3라 명명하였다. 발현 프라스미드 pGDS3에는 아스퍼질러스 나이드란스의 GPD 프로모터, 아스퍼질러스 오라이제의 알파 아밀라제 시그날시컨스, 아스퍼질러스 나이거의 글루코스 옥시다제 유전자와 터미네이터가 포함되어 있다.PPp. * 4.13 (Nae) -Sal plasmid 200 containing alpha amylase promoter and signal of Aspergillus orisase and N-terminus of glucose oxidase.

And pAN 52-1 500 with GPD promoter from Aspergillus Nylandse

Cleavage with EcoRI and NcoI 37

After cleavage at 10 hours using a T ₄ DNA-ligase 1 unit for 10 hours

Bonding under This ligation probe was run for 15 minutes, 65 minutes to inactivate the remaining T ₄ DNA-ligase to prevent transformation of the recombined pAN 52-1.

Heated to 20 units and cut into BamHI, 37

After 4 hours of incubation, E. coli 5-α competent cells were transformed. The resulting clones were analyzed on agarose gel to obtain plasmid p435 with the Aspergillus Niger GPD promoter correctly inserted. To insert the glucose oxidase gene (pGOD2) into this plasmid p435 200

And pGOD2 300

Was digested with XmaIII and HinDIII, 16 h using T ₄ DNA ligase 1 unit

After conjugation under E. coli 5-α competent cells were transformed. The resulting clones were analyzed on agarose gel to obtain plasmid p450 with the N-terminus of the glucose oxidase gene correctly inserted. P435 200 to insert the remaining glucose oxidase gene

And p450 400

Was digested with XmaIII and then 16 hours, 10 using T ₄ DNA ligase 1 unit.

Conjugation under. Residual T ₄ DNA ligase was inactivated to prevent transformation of p450 that was unbound and rebound. To do this, react the reaction for 15 minutes.

Heated to. Bound plasmids were transformed into E. coli 5-α competent cells. Plasmid DNA was extracted from the transformants to select the plasmid with the glucose oxidase gene correctly inserted on the agarose gel. The expression plasmid thus obtained was named pGDS3. The expression plasmid pGDS3 includes the GPD promoter of Aspergillus ny drans, the alpha amylase signal of Aspergillus oleise, and the glucose oxidase gene and terminator of Aspergillus niger.

Example 2

[Transition of Recombinant Vectors to Host Cells]

Transfer of the recombinant vector pGDS3 to the host cell Aspergillus Niger NRRL3 was carried out by the Tilburn et al., (1983) method. The selection of the transformants was selected by the selection plasmid pAN 7-1 used in the coat transfer treatment.

, 맥아추출물 2

, 박토펩톤 0.1

)에 아스퍼질러스 나이거 NRRL3 포자를 골고루 분산시킨 후 37

하에 포자가 골고루 형성될때까지2

3일간 배양한다. 10ml 0.01

트윈 80에 잘 섞은 후 포자를 적절한 량의 0.02

(w/v) 카제인하이드로라이세이트(caseinhydrolysate)가 포함된 체팍독스배지에 접종한 후 30

에서 균사가 형성될 때까지 배양한다. 여과지에 여과한 후 10mM 포타슘포스페이트 용액으로 잘 세척하였다. 균사를 건조시킨 후 적절한 량의 포타슘포스페이트 용액과 세포외벽을 파쇄하기 위해서 노보자임과 글루큐로닉다제를 넣은 후 2

3시간 배양하여 프로토프라스트를 형성시켰다. 형성된 프로토프라스트는 솔비톨 그래디언트 원심분리를 이용 분리 수집하였다. 각 10

의 발현 프라스미드 pGDS3와 선별 프라스미드 pAN 7-1를 200

의 프로토프라스트에 섞은 후 60

폴리에틸렌글리콜 6000을 첨가 전이를 유도하였다.3 CM medium (glucose 2

, Malt extract 2

Bactopeptone 0.1

Evenly spread the Aspergillus Niger NRRL3 spores

Until the spores are evenly formed

Incubate for 3 days. 10ml 0.01

After mixing well in the Tween 80, the spores should be 0.02

(w / v) 30 days after inoculation into Chefax dox medium containing caseinhydrolysate

Incubate until hyphae form. The filter paper was filtered and washed well with 10 mM potassium phosphate solution. After drying the hyphae, add appropriate amount of potassium phosphate solution and novozyme and glucuronicase to break the extracellular wall.

Incubate for 3 hours to form a protoplast. The formed protoplasts were separated and collected using sorbitol gradient centrifugation. 10 each

Expression of Plasmid pGDS3 and Selected Plasmid pAN 7-1 200

After mixing in the prototype of 60

Polyethyleneglycol 6000 induced addition transitions.

Transformants were screened using the antibiotic hygromycin B resistance. Hygromycin B is inserted into peptidyl-tRNA in ribosomes to interfere with the translaction of peptidyl-tRNA. Selected plasmid pAN 7-1 contains the hygromycin B phosphotransferase gene, and this enzyme inactivates hygromycin B, so that the expression plasmid pGDS3, which has been transcribed with the selected plasmid, contains hygromycin B. Can be selected through the medium.

Example 3

[Expression of recombinant strain]

, 측정시간 4분, 측정간격 30초의 조건하에서 실시하였다. 선별된 우수 재조합 균주들의 글루코스 옥시다제 발현정도의 비교는 플라스크 배양을 실시하여 실험하였다.Transformed recombinant strains can be determined through the expression medium. Glucose oxidase is involved in the reaction that produces the same amount of H ₂ O ₂ per 1 mole of glucose during oxidation, and the concentration of H ₂ O ₂ produced is reduced to H ₂ O by peroxidase. In the presence of dianisidine, O-dianisidine emits electrons that oxidize to a dark brown color. Using this principle, excellent recombinant strains were selected by comparing the expression level of glucose oxidase produced by the production and secretion of transformed recombinant strains by using the expression medium prepared by adding O-Dianisidine to the appropriate medium in a brown ring size. It was. Selected recombinant strains were analyzed quantitatively by analyzing the activity of glucose oxidase expressed by the strain through the flask culture. For quantitative analysis, Ebithishi (2,2'-Azino-di- [3-ethyl benzthiazolinsulfonic acid) was used instead of O-Dianisidine, and the absorbance measurement was 420 nm and temperature 25

The measurement was carried out under the condition of 4 minutes of measurement time and 30 seconds of measurement interval. Comparison of the degree of glucose oxidase expression of the selected excellent recombinant strains was performed by flask culture.

Example 4

[Production Comparison with Other Strains]

Expressions In other host strains of plasmid pGDS3, attempts have been made in Aspergillus nidrans and Aspergillus orisse in addition to Aspergillus niger. Aspergillus neidrans and Aspergillus orease has a high resistance to hygromycin B in itself, and tried to screen for recombinant strains using uridine oxotropes, another screening system. The transformation method and analysis of the expressed glucose oxidase were used in the method of Example 2 and Example 3. The activity of glucose oxidase of the transformed recombinant strain is as follows.

1.88Unit / mL glucose oxidase activity in Aspergillus Niger NRRL3, a recombinant strain host containing expression plasmid pGDS3, was 1.44Unit / mL in Aspergillus Nydrant G191, AO4.1 in Aspergillus orisase. Was 0.46 Unit / mL. The above results suggest that expression of the expression plasmid pGDS3 occurs best in the native host Aspergillus Niger NRRL3.

Example 5

[Fermentation Experiment of Gene Recombinant Mutant]

0.5g/l, KH₂PO₄0.1

0.5g/l, MgSO₄·7H₂O 0.1

0.3g/l, CaCO₃2

5

, 박토-펩톤 2

6

, 글루코스 4

10

를 사용하였고, 발효조건으로는 교반속도 450500rpm, 공기주입 0.8vvm(l/min), pH 5.1, 온도 30

로 하여 실시하였다. 실험한 결과 발효시간에 따른 글루코스 옥시다제의 활성은 다음과 같다.Aspergillus Niger GOD4, which exhibits the highest glucose oxidase activity among the recombinant strains, was cultured using a 5 L batch fermenter. Fermentation broth (NH ₄ ) ₂ HPO ₄ 0.1

0.5 g / l, KH ₂ PO ₄ 0.1

0.5 g / l, MgSO _{4 7} H ₂ O 0.1

0.3 g / l, CaCO ₃ 2

5

, Bacto-peptone 2

6

, Glucose 4

10

Fermentation conditions, stirring speed 450 500 rpm, air injection 0.8 vvm (l / min), pH 5.1, temperature 30

It was carried out as. As a result of experiment, glucose oxidase activity according to fermentation time is as follows.

Recombinant strain Aspergillus Niger GOD4 showed 47.8Unit / mL glucose oxidase activity after 209 hours of fermentation, which was about 150 times higher than 0.3Unit / mL of Aspergillus Niger wild strain. Able to know.

Using the recombinant recombinant strain Aspergillus Niger GOD4 of the present invention, glucose oxidase production could be prepared by a method of about 150 times improved glucose oxidase production compared to the wild type Aspergillus niger strain.

Claims (1)

The expression vector pGDS3 prepared by assembling the GPD promoter of Aspergillus Nyland's GPD promoter and the alpha amylase signal and terminator of Aspergillus orisse was isolated from the glucose oxidase gene isolated from Aspergillus niger. Recombinant strain Aspergillus Niger GOD4 (KFCC-10945) (NH ₄ ) ₂ HPO ₄ 0.1

0.5 g / l, KH ₂ PO ₄ 0.1

0.5 g / l, MgSO _{4 7} H ₂ O 0.1

0.3 g / l, CaCO ₃ 2

5

, Bacto-peptone 2

6

, Glucose 4

10

Method of producing glucose oxidase by batch culture in a fermentation medium consisting of.

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