WO2017078422A1 - 네이세리아 고노르호에아 유래 탄산무수화효소를 이용한 탄산무수화 활성이 증가된 형질전환체 - Google Patents

네이세리아 고노르호에아 유래 탄산무수화효소를 이용한 탄산무수화 활성이 증가된 형질전환체 Download PDF

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WO2017078422A1
WO2017078422A1 PCT/KR2016/012576 KR2016012576W WO2017078422A1 WO 2017078422 A1 WO2017078422 A1 WO 2017078422A1 KR 2016012576 W KR2016012576 W KR 2016012576W WO 2017078422 A1 WO2017078422 A1 WO 2017078422A1
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protein
transformant
carbonic anhydrase
activity
anhydrase
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French (fr)
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남윤성
정기준
송석영
최재웅
구본일
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한국과학기술원
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Publication of WO2017078422A1 publication Critical patent/WO2017078422A1/ko

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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
    • C12N9/88—Lyases (4.)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium

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  • the present invention is based on the use of Neisseria gonorhoa derived carbonic anhydrase.
  • the present invention relates to a transformant having increased carbonic anhydride activity and a method of manufacturing the same.
  • Materials such as polymer beads, resins, and gold, such as chitosan and alginic acid, are made in the form of hybrids, which are then combined using amine groups of carbonate anhydrase.
  • Carbonic anhydrase has been used a lot of proprietary carbonic anhydrase, but in some cases, microbial or human anhydrous enzyme.
  • the present invention uses Corynebacterium.
  • the present invention is a sequential link between membrane protein-protein linker and carbonic anhydrase.
  • the present invention also relates to a nucleic acid molecule encoding the amino acid sequence.
  • the present invention also relates to a plasmid containing the nucleic acid molecule.
  • the present invention also relates to a transformant except for humans transformed with the plasmid.
  • the present invention also introduces the nucleic acid molecule into host cells prepared with humans.
  • It relates to a method for producing a transformant in which the carbonic anhydrase, which comprises the step of transforming, is expressed on the cell surface.
  • the present invention also relates to a method of converting carbon dioxide to carbonate using a protein comprising an amino acid sequence having a structure in which a membrane protein-protein linker-carbonate anhydrous enzyme is sequentially linked.
  • the carbonic anhydrase expressed on the surface of the cell according to the present invention is also excellent in its hydration activity and is expected to be utilized for efficient fixation of carbon dioxide as an industrially useful whole cell enzyme system due to its high heat stability.
  • 1 is directed to the production of plasmids for the expression of gCA on the surface of Corynebacterium glutamicum.
  • Figure 3 compares the SDS-PAGE results of (a) porinB- (G 4 S) 2 -ngCA and (b) porinB- (G 4 S) 3- "gCA with negative controls and porinB—ngCA. It is.
  • Figure 6 is a measure of the difference in the expression "in gCA confocal scanning images for porinB- (G 4 S) 2 -ngCA .
  • FIG. 7 shows the results of the hydration experiments of porinB- (G 4 S) 2- «gCA and porinB- (G 4 S) 3- " gCA.
  • Figure 9 shows commercially available carbonated anhydrous enzymes (commercial CA) and E. coli.
  • the present invention is a sequential link between membrane protein-protein linker and carbonic anhydrase.
  • the membrane protein is a protein that connects through the extracellular membrane, but is not particularly limited, but porin-based proteins may be used, for example, from the group consisting of porin B, porin C, or porin H. Any one or more selected may be selected and used, for example, the porin B protein may be used as in the embodiment of the present invention.
  • the protein linker may have a structure of Chemical Formula I, although not particularly limited thereto.
  • G glycine and S: serine
  • n is an integer from 1 to 10.
  • the acid anhydrase include, but doejineun particularly limited in the present invention in Nei ceria (N ⁇ in ⁇ sp.) Can be used that is derived from, an example in Nathan ceria and Norte No. ah (Ne / Men 'a go ⁇ / zoeae).
  • amino acid sequence is not limited in the present invention
  • a labeled protein having an amino acid sequence of Formula II may be additionally linked to the C-terminus of the carbonic anhydrase.
  • D aspartic acid
  • Y tyrosine
  • K lysine
  • the present invention relates to a nucleic acid molecule encoding the amino acid sequence.
  • the present invention relates to a plasmid containing the nucleic acid molecule.
  • the present invention also relates to a transformant except for humans transformed with the plasmid.
  • the transformant is not particularly limited as long as it excludes humans, but is preferably microorganisms, and more preferably, microorganisms of the genus Corynebacterium sp. And more preferably Corynebacterium glutamicum).
  • Corynebacterium glutamicum is significantly more stable against fever than E. coli, a representative microbial cell for protein expression.
  • Glutamicum is non-pathogenic and does not form spores.
  • Non-sporulatag Gram-positive bacteria widely used in industrial applications.
  • Glutadium is more genetically stable due to the lack of a recombinant recovery system and a high copy number plasmid. Furthermore, the cell envelope of Corynebacterium glutamicum is the crystallized protein layer S-. It contains a layer and has a very high strength.
  • Corynebacterium glutamicum can be carried out at high concentrations. Corynebacterium glutamicum is thus a stable whole cell.
  • the present invention is directed to a host cell excluding the nucleic acid molecule.
  • the present invention relates to a method for producing a transformant in which carbonic anhydrase is expressed on the surface of a cell.
  • the present invention also relates to carbonate of carbon dioxide using a protein comprising an amino acid sequence having a structure in which a membrane protein-protein linker- carbonate anhydrase is sequentially linked. It's about how to switch.
  • E. coli XLl-Blue as a host for gene cloning and maintenance
  • Anchoring protein genes such as porin B, porin C, and porin H, were synthesized from the Corynebacterium glutamicum genome by PCR (PCR; Each PCR product was cloned into pCES208 plasmid (Jong-Uk et al., 2008) after ablation with BamHl and Xbal restriction enzymes (C1000 Thermal Cycler, BioRad, Hercules).
  • gonorrhoeae Codon optimized codon frequency of the original ngCA gene (GenScript Co., Piscataway, NJ, US A) for high expression of ngCA derived codon optimized carbonic anhydrase gene Synthesize,
  • Amplification was carried out via polymerase chain reaction.
  • the amplified gene was excised using the same restriction enzyme as above and cloned into the prepared plasmid of each product.
  • a flag-tag (SEQ ID NO: DYKDDDDK) was fused to the «gCA enzyme C-terminus.
  • E. coli was at 37 ° C, LB (Luria-Bertani) medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride; BD, Franklin Lakes, NJ. , USA). Manipulation of DNA was carried out in a generally known manner.
  • LB Lia-Bertani
  • a plasmid was prepared and used to include the genes as shown in Table 1 below (FIG. 1). [Table 1]
  • Each plasmid is expressed in host cells by electrophoresis (Gene Pulser, Bio-Rad).
  • Corynebacterium glutamicum was transformed. As a negative control, cells carrying the pCES208 plasmid were used.
  • All transformed cells contained 250 mL flasks of BHI (brain heart infusion);
  • the medium was treated with antibiotics at a concentration of 25 g / mL.
  • Kanamycin was added.
  • gCA expression levels are similar to those of SDS-PAGE.
  • Protein bands were transferred onto a polyvinyl difluoride membrane (Roche, Basel, Switzerland) for 60 minutes at 100 mA using a transblot device (Bio-Rad). Incubate for 1.5 hours with blocking solution (Tris-buffered saline, 24.7 mM Tris, 137 mM NaCl, 2.7 mM KCl, and 0.5% Tween-20®) and 5 wt% skim milk.
  • blocking solution Tris-buffered saline, 24.7 mM Tris, 137 mM NaCl, 2.7 mM KCl, and 0.5% Tween-20®
  • the peptide linker between the anchoring protein and the enzyme prevents steric hindrance and promotes the fixation of ngCA.
  • «gCA and anchoring proteins are bound to porinB by doublets and triplets of Gly 4 Ser (Gly-Gly-Gly-Gly-Ser) in transformed cells, respectively.
  • the cells were washed twice and reprecipitated in PBS.
  • the cells were diluted at 1: 250 ratio and the monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) bound to fluorescein isothiocyanate (FITC) for 1 hour at 40 ° C.
  • the cells were washed once with PBS solution to remove unbound probes.
  • the stained cells were diluted with a concentration of about 10 6 cells / mL and analyzed by FACS (fluorescent activated cell sorter; MoFlo XDP, Beckman Coulter, Inc., Brea, CA, USA) to measure the fluorescence intensity.
  • FACS fluorescent activated cell sorter
  • FITC fluorescein
  • porinB- (G 4 S) 3- ⁇ CA are compared with those of the negative control and porinB- «gCA.
  • concentration of ngCA was determined to be 0.230 mg / mL, which is very high compared to porinC- «g CA.
  • concentration of ngCA was increased to 0.27 mg / mL, which was higher than that of the control group.
  • control group porinB-ngCA measured 0.15 X 10- 'pg / cell on the cell surface, which was 0.23 of porinC- / CA.
  • Ice dioxide was injected with carbon dioxide (Special gas, Daejeon, Korea). The pH of the solution was recorded immediately after injecting 8 mL of carbon dioxide-saturated water. The solution was magnetically stirred at a rate of 750 rpm over the entire reaction.
  • Equation I The activity of the carbonic anhydrase was determined as the time required for the pH to change from 8.0 to its zero. This time data was converted to the carbonic anhydrase activity in units of WA (Wilbur-Anderson) by Equation I below. Can be [91] [Equation I]
  • Example 1 to Example 4 in Table 2 In order to compare the thermal stability of 3 ⁇ 4 acid anhydrase according to the length of the linker, the experimental group of Example 1 to Example 4 in Table 2 was formed, and the protein linker of each example was prepared in the same process as in the above test example. After the preparation, and heat treatment for 6 hours at 60 ° C, the residual enzyme activity was measured.
  • E. coli isolated production carbonate anhydrous enzyme (free wgCA) was used to purify the expression in the cytoplasm of Escherichia coli, a general production tool of synthetic proteins.
  • the free ngCA is thermally stable compared to surface-expressed carbonate anhydrase.
  • a control group was used for comparison.
  • the purified enzyme was diluted with a concentration of 0.01 mg / tnL.
  • PorinB- (G4S) 2-ngCA showed 43.4% of residual activity after 24 hours at 60 0 C, while BCA showed 4.4% and free ngCA showed 7.1%. Although not shown, at 37 oC, about 121.3% of activity was maintained for 30 days, compared with BCA and ngCA, which decreased by 39.3% and 29.7%, respectively.

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Abstract

본 발명은 네이세리아 고노르호에아 유래 탄산무수화 효소를 이용한 탄산무수화 활성이 증가된 형질전환체 및 그 제조방법에 관한 것이다. 본 발명에 의해 세포 표면에 발현된 탄산무수화 효소의 경우 자체의 수화 활성 또한 우수하며, 열에 대한 안정성이 높아 산업적으로 유용한 전체 세포 효소 시스템으로서 이산화탄소의 효율적 고정에 활용될 수 있을 것으로 기대된다.

Description

명세서
발명의명칭 :네이세리아고노르호에아유래탄산무수화효소를 이용한탄산무수화활성이증가된형질전환체 기술분야
[1] 본발명은네이세리아고노르호에아유래탄산무수화효소를이용한
탄산무수화활성이증가된형질전환체및그제조방법에관한것이다.
배경기술
[2] 이산화탄소방출을감소시키는것은전세계적인기후재앙을방지하기위해 필수적인것이며，산업원료로부터방출되는이산화탄소를고정화하는것은 인류의생존을위해본질적으로필요한요소이다.무기탄산염화 (mineral carbonation)방법은다른이산화탄소고정화방법에비해고정효율이좋기 때문에많은관심을받아왔다.그러나,무기물화 (mineralization)반응의경우 매우느린반웅속도를갖는경우가일반적이다.
[3] 생체에서이산화탄소를중탄산염 (bicarbonate)로변화시키는과정은풍부하게 존재한다.생체에서상기반응은탄산무수화효소 (carbonic anhydrase, CA)의 존재로인해통상의조건에서빠르게진행되며,이산화탄소의무기탄산염화 과정에있어서의율속단계 (rate-Kmiting step)에해당한다.이산화탄소의 무기탄산염화과정에서상기탄산무수화효소의고효율성에도불구하고，짧은 반감기로인해실제적인웅용에는제약이많은상황이다.상기효소의안정성을 증가시키기위해다양한고정화방법이도입되었으나,대용량의이산화탄소를 생산하는장비에사용하기에는고비용이소요되는문제점이있다.탄산무수화 효소를여러유무기물질에고정화해안정화하는방식이여러번제시되었다. 키토산,알긴산과같은고분자비드나레진，금등의재료를하이브리드형태로 제작하여를을만들고，거기에탄산무수화효소의아민기를이용해결합시킨다. 탄산무수화효소는소유래탄산무수화효소가많이사용되었으나,경우에따라 미생물의탄산무수화효소나사람의탄산무수화효소가사용되기도했다.
그러나이경우，재료인탄산무수화효소를정제하는과정이매우복잡하고 값비싸,산업적으로정제탄산무수화효소를사용하기란불가능하다.
[4] 따라서，탄산무수화효소를경제적으로활용하기위해세포의표면상에다수를 발현시키는것이강력한방법이될수있다.이러한방법은효소정제의 필요성을감소시키고，효소를보다쉽게다롤수있도록해주는장점이있다. 미생물의전체세포시스템에서,탄산무수화효소는미생물유전체에재조합된 플라스미드를삽입하여줌으로써세포내에위치시키는것이가능하다.만일 탄산무수화효소가세포질 (cytoplasm)상에존재한다면，이산화탄소고정에따른 pH감소에의해세포내부의항상성에영향을미치게된다.또한,세포막은 확산의장벽으로작용하기때문에세포질에서발현되는효소의활성은제한될 수밖에없다.이대신최근대장균또는효모의주변세포질 (periplasm)또는 표면 (surface)에탄산무수화효소를위치시키는방법이제안되었다.그러나，이와 같은방법은탄산무수화효소의열적안정성측면에있어제약사항이존재한다. 탄산무수화효소는 40 °C근처에서는안정하지만， 50 0C로열처리하였을때 효소의활성이급격히감소하는단점이있다.
발명의상세한설명
기술적과제
[5] 상기와같은문제점을해결하기위해서본발명에서는코리네박테리움
글루타미
Figure imgf000003_0001
g/Mtomi' i)세포의표면에다수의네이세리아 고노르호에아 (Ne^^rw gonorrhoeae)유래탄산무수화효소를발현하여 이산화탄소고정화효과및효소의열적안정성을현저히상승시키고자하였다. 과제해결수단
[6] 본발명은막단백질-단백질링커-탄산무수화효소가순차적으로연결된
구조를갖는이산화탄소의탄산염전환을위한아미노산서열을포함하는 단백질에관한것이다.
[7] 또한본발명은상기아미노산서열을코딩하는핵산분자에관한것이다.
[8] 또한본발명은상기핵산분자를포함하는플라스미드에관한것이다.
[9] 또한본발명은상기플라스미드로형질전환된인간을제외한형질전환체에 관한것이다.
[10] 또한본발명은상기핵산분자를인간을제와한숙주세포에도입하여
형질전환하는단계를포함하는탄산무수화효소가세포표면에발현되는 형질전환체의생산방법에관한것이다.
[11] 또한본발명은막단백질-단백질링커-탄산무수화효소가순차적으로연결된 구조를갖는아미노산서열을포함하는단백질을이용한이산화탄소의탄산염 전환방법에관한것이다.
발명의효과
[12] 본발명에의해세포표면에발현된탄산무수화효소의경우자체의수화활성 또한우수하며，열에대한안정성이높아산업적으로유용한전체세포효소 시스템으로서이산화탄소의효율적고정에활용될수있을것으로기대된다. 도면의간단한설명
[13] 도 1은코리네박테리움글루타미쿰표면에서 «gCA의발현을위한플라스미드 제작에관한것이다.
[14] 도 2는 (a)링커의길이 n=2일때와 (b) n=3일때형광을이용한 ngCA의발현 위치분석에관한것이다.
[15] 도 3은 (a) porinB-(G4S)2-ngCA및 (b) porinB-(G4S)3-"gCA의 SDS-PAGE결과를 음성대조군및 porinB— ngCA의결과와비교한것이다.
[16] 도 4는 (a) porinB-(G4S)2-/igCA및 (b) porinB-(G4S)3-ngCA의웨스턴블롯결과를 음성대조군및 porinB-ngCA의결과와비교한것이다.
[17] 도 5는 porinB-(G4S)2-^CA및 porinB-(G4S)3-"gCA의 SDS-PAGE결과로부터 ng
CA의발현량을측정한것이다.
[18] 도 6은 porinB-(G4S)2-ngCA에대해공초점현미경스캔이미지에서 "gCA의 발현차이를측정한것이다.
[19] 도 7은 porinB-(G4S)2-«gCA및 porinB-(G4S)3-"gCA의수화실험을통한
탄산무수화효소활성을측정한결과이다.
[2이 도 8은링커의길이 n= l , 2, 3및 6일때 60 °C에서 6시간열처리수행시
열안정성을측정한결과이다.
[21 ] 도 9는상업적으로판매되는탄산무수화효소 (commercial CA)및대장균
분리생산탄산무수화효소 (free ngCA)에대한표면발현된탄산무수화효소의열 안정성을 (a)표면발현된 porinB-(G4S)2-^CA에대하여 50 °C에서 30일및 (b) 표면발현된 porinB-(G4S)2-rtgCA에대하여 60 °C에서 48시간동안측정한 결과이다.
발명의실시를위한최선의형태
[22] 본발명은막단백질-단백질링커-탄산무수화효소가순차적으로연결된
구조를갖는이산화탄소의탄산염전환을위한아미노산서열을포함하는 단백질에관한것이다.
[23] 본발명에서상기막단백질은세포외막을통해연결통로를이루는단백질인 경우특별히제한되지는않으나포린 (porin)계열단백질을사용할수있으며， 일예로포린 B，포린 C또는포린 H로이루어진군으로부터선택되는어느하나 이상을선택하여사용할수있고，일예로본발명의실시예에서와같이포린 B 단백질을사용할수있다.
[24] 또한본발명에서상기단백질링커는특별히제한되지는않으나하기화학식 I의구조를갖을수있다.
[25] [화학식 I]
[26] (G4S)n
[27] G:글리신 (glycine), S:세린 (serine)이다;
[28] n은 1내지 10의정수이다.
[29] 본발명에서상기탄산무수화효소는특별히제한되지는않으나네이세리아 속 (N^i n^ sp.)에서유래된것을사용할수있으며，일예로네이세리아 고노르호에아 (Ne/Men'a go ^/zoeae)에서유래된것을사용할수있다.
[30] 탄산무수화효소에는여러종류가있으며，인간유래탄산무수화효소가가장 높은활성을지니고있는것으로알려져있다.그러나，인간유래세포는그 크기와복잡성때문에클로닝 (cloning)에제약이따른다.따라서，차선책으로 인간유래탄산무수화효소 Π의활성에필적할수있는네이세리아
고노르호에아 (N ^en'a gonorrhoeae)유래의탄산무수화효소 ( CA)를사용하는 것이바람직하다.이는상기효소의크기가작고단량체의형태를지니고있어 박테리아에서발현하기에장점이있기때문이다.
[31] 또한본발명에서상기아미노산서열은제한되지는않으나탄산무수화효소의 C-말단에화학식 II의아미노산서열을갖는표지단백질이추가로연결될수 있다.
[32] [화학식 II]
[33] DYKDDDDK
[34] D:아스파르트산 (aspartic acid), Y:티로신 (tyrosine), K:리신 (lysine).
[35]
[36] 또다른양태로,본발명은상기아미노산서열을코딩하는핵산분자에관한 것이다ᅳ
[37] 또한본발명은상기핵산분자를포함하는플라스미드에관한것이다.
[38] 또한본발명은상기플라스미드로형질전환된인간을제외한형질전환체에 관한것이다.
[39] 본발명에서상기형질전환체는인간을제외한대상이라면특별히제한되지는 않으나，바람직하게는미생물올대상으로할수있으며，보다바람직하게는 코리네박테리움속 (Corynebacterium sp.)의미생물을대상으로할수있고,더욱 바람직하게는코리네박테리움
Figure imgf000005_0001
glutamicum)을 대상으로할수있다.
[40] 코리네박테리움글루타미쿰은단백질발현을위한대표적인미생물세포인 대장균에비해열에대한안정성이현저히우수하다.코리네박테리움
글루타미쿰은비병원성 (non-pathogenic)이며 ,포자를형성하지
않는 (non-sporulatag)그람 -양성 (Gram-positive)박테리아로，산업적인용도로 널리사용되어왔다.생물학적유전자조작에있어코리네박테리움
글루타미큼은재조합복원시스템의결핍과높은복제개수 (copy number) 플라스미드로인해유전적으로보다안정하다.더욱이,코리네박테리움 글루타미쿰의세포외피 (cell envelope)는결정화된단백질층인 S-layer를 포함하고있어,매우강도가높다.상기와같은안정적특성에의해，
코리네박테리움글루타미쿰을포함하는산업공정은고농도에서진행될수 있다.이에따라，코리네박테리움글루타미쿰은안정적인전체세포
생촉매 (whole-cell biocatalyst)로의활용을위한플랫폼으로활용할수있다.
[41]
[42] 또다른양태로，본발명은상기핵산분자를인간을제외한숙주세포에
도입하여형질전환하는단계를포함하는탄산무수화효소가세포표면에 발현되는형질전환체의생산방법에관한것이다.
[43]
[44] 또한본발명은막단백질-단백질링커-탄산무수화효소가순차적으로연결된 구조를갖는아미노산서열을포함하는단백질을이용한이산화탄소의탄산염 전환방법에관한것이다.
[45]
[46] 이하,실시예를통하여본발명을보다상세히설명한다.그러나이들실시예는 본발명에대한이해를돕기위한것일뿐,어떤의미로든본발명의범위가이들 예로만한정되는것은아니다.
[47]
[48] [실험재료]
[49] 규주및유저자
[5이 유전자클로닝 (gene cloning)및유지를위한숙주로대장균 (E. coli XLl-Blue;
Stratagene Co., Santa Clara,CA, USA)을사용하였으며，코리네박테리움
Figure imgf000006_0001
glutamicum; ATCC 13032)을탄산무수화효소의 발현을위한주숙주로사용하였다.
[51] 앵커링단백질유전자 (anchoring protein gene)인포린 B(porin B),포린 C(porin C)및포린 H(porin H)는각각코리네박테리움글루타미쿰게놈으로부터 증합효소연쇄반응 (PCR; C1000 Thermal Cycler, BioRad, Hercules)을통해 확보하였다.각각의 PCR산물은 BamHl및 Xbal제한효소로절제한후 pCES208 플라스미드 (Jong-Uk et al., 2008)에클로닝 (cloning)되었다.
[52]
[53] 합성타산무수화효소
[54] 코리네박테리움글루타미큼내에서네이세리아고노르호에아 (Ne^en'a
gonorrhoeae)유래의탄산무수화효소 (ngCA)의고발현을위해원래의 ngCA 유전자 (GenScript Co., Piscataway, NJ, US A)의코돈빈도 (codon frequency)를 조사하여,코돈최적화된탄산무수화효소유전자를합성하고,
중합효소연쇄반웅을통해증폭하였다.
[55] 증폭된유전자를상기와동일한제한효소를사용하여절제한후각각의산물올 준비된플라스미드에클로닝하였다.
[56] 유전자발현여부검출이보다잘이루어지도록하기위해 «gCA효소 C-말단에 표지단백질 (flag-tag;서열 DYKDDDDK)를융합시켰다.
[57] 폴라스미드를제작하는동안，대장균은 37 °C, LB(Luria-Bertani)배지 (트립톤 10 g/L,효모추출물 5 g/L,염화나트륨 5 g/L; BD, Franklin Lakes, NJ, USA)에서 배양시켰다. DNA의조작방법은통상적으로널리알려져있는일반적인 방법으로수행하였다.
[58]
[59] [시험예 1]코리네박테리움글루타미쿰표면에서 MgCA의발현
[60] 하기표 1과같이유전자를포함하도록플라스미드를제작하여사용하였다 (도 1). [Table 1]
막단백질ᅳ단백질링커 -탄산무수화효소
Figure imgf000007_0001
[63] 각플라스미드는전기영동 (Gene Pulser, Bio-Rad)을통해숙주세포인
코리네박테리움글루타미쿰에형질전환하였다.음성대조군으로는 pCES208 플라스미드를가지고있는세포를사용하였다.
[64] 모든형질전환된세포는 250 mL플라스크의 BHI(brain heart infusion; BD,
Franklin Lakes, NJ, USA)배지에접종한후 30 °C에서 20 rpm으로흔들어주면서 48시간동안배양하였다.배지에는항생제로 25 g/mL농도의
카나마이신 (kanamycin)을첨가하였다.
[65]
[66] [시험예 2] wgCA의발현분석
[67] SDS-PAGE및웨스터봄^ (western blot)
[68] 세포질，막，분비소포 (secretosome)에서 "gCA의발현레벨은 SDS-PAGE와
웨스턴블로팅 (western blotting)을순차적으로시행하여측정하였다.상기배양한 세포는 4 °C에서 6,000 rpm으로 10분간원심분리 (Sonic, Vibra cell, Newtown, CT, USA)하여수득하였다.수득한세포는 PBS(phosphate-buffered saline; 135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.2)버퍼에서 재침전시키고，전체,수용성,불용성，막단백질분획으로분리하였다. 12% 폴리아크릴아미드겔에서전기영동한후，상기겔은쿠마시브릴리안트 블루 (Coomassie brilliant blue; 50%메탄올， 10%아세트산， 1 g/L Coomassie brilliant blue R-250)에담가 1시간동안염색 (staining)한후，탈색용액 (destaining solution; 10%메탄올및 10%아세트산)에서탈색하였다.
[69] 단백질밴드는트랜스블롯 (transblot)장치 (Bio-Rad)를이용하여 100 mA로 60 분간폴리비닐디플루오라이드막 (polyvinyl difluoride membrane; Roche, Basel, Switzerland)위로이동시켰다.상기막은실온에서 1.5시간동안블로킹 용액 (Tris-buffered saline, 24.7 mM Tris, 137 mM NaCl, 2.7 mM KCl,및 0.5% Tween-20®)과 5 wt%탈지유 (skim milk)와함께배양하였다.상기막은
HRP(horseradish peroxidase)가결합된단일클론항 -FLAG M2
항체 (Sigma-Aldrich)와함께배양하여표지단백질로표지된단백질의면역 검출 (immune-detection)을수행하였다.배양후，각각의막은 TBS-T로 5분간 4회 세척한후 ECL Kit(Amersham ECL Prime Western Blotting Detection Agent, GE Healthcare Bio-science AB)를이용하여단백질을검출하였다.
[70]
[71] 막분획에서발현된 ngCA의정량을위해 SDS-PAGE겔에서단백질의함량을 실버스테이닝키트 (silver staining kit; GE Healthcare)로분석하였다.막단백질 분획에서발현된 ngCA의비율 (%)확정을위해 Quantity One Software(Bio-Rad)를 사용하였다.
[72]
[73] 상기결과,포린 B는코리네박테리움글루타미쿰에서 / CA에대한고정화
능력이있음을나타냈다.또한，앵커링단백질과효소사이의펩티드링커는 입체장해 (steric hindrance)를방지하고, ngCA의고정화가잘일어나도록한다. 플라스미드에추가적인코돈을삽입함으로써, «gCA와앵커링단백질은각각 형질전환된세포에서 Gly4Ser (Gly-Gly-Gly-Gly-Ser)의더블렛 (doublet)및 트리플렛 (triplet)에의해결합되어 porinB-(G4S)2-ngCA및 porinB-(G4S)3-"gCA를 형성하였디-.
[74]
[75] 발혀세포의유세포분석 (ᅳ flow-cvtometric analysis)
[76] 세포를두번세척하고 PBS에재침전시켰다.그리고 1:250비율로희석된상기 세포를 4 0C에서 1시간동안 FITC(fluorescein isothiocyanate)가결합된단일클론 항 -FLAG M2항체 (Sigma-Aldrich)로면역염색 (immunostaining)하였다.상기 세포를 PBS용액으로 1회세척하여결합되지않은탐침들 (probes)을제거하였다. 염색된세포를약 106 cells/mL의농도로희석한후 FACS(fluorescent activated cell sorter; MoFlo XDP, Beckman Coulter, Inc., Brea, CA, USA)로분석하여형광 강도를측정하였다.
[77]
[78] 공초점휘미경스캐 /confocal microscopy scanning)
[79] 배양된세포를세척한후 PBS에재침전시켰다. FITC(fluorescein
isothiocyanate)가결합된단일클론항 -FLAG M2항체 (Sigma-Aldrich, St. Louis, MO, USA)로면역염색 (immunostaining)한후，세포를 4 %(v/v)
포름알데히드 (Sigma-Aldrich)로고정하고형광측정을위한봉입제 (mounting medium; Vector Laboratories, Inc., Burlingame, CA, USA)에재침전시켰다.세포 샘플을공초점현미경 (Leica TCS SP8 Hyvolution (resolution = 140 nm, Leica Microsystems, Ernst-Leitz-Strasse, Wetzlar, Germany))으로관찰하였다.이때여기 파장 (excitation wavelength)은 488 nm아르곤레이저를사용하였고,영상은 552 nm필터로특정한빛만통과시켰다.
[80]
[81] 상기결과, FITC가세포들의세포질에존재하지않았기때문에,형광세포는 세포질이아닌세포의표면에 CA를나타내게됨을알수있었다 (도 2).대조군 중에서 porinB-ngCA가발현된세포또한다른포린패밀리 (porin family)를 발현하는다른세포에비해형광신호강도가강하였으나， G4S링커가삽입된 porinB-(G4S)2- CA또는 porinB-(G4S)3-"gCA단백질이발현된세포의경우 상대적인형광신호의세기가링커가결합되지않은상기대조군들에비해 현저히상승하는것을확인하였으며，음성대조군으로 ngCA단빡질이발현되지 않은세포의경우형광이나타나지않음을확인하였다.
[82]
[83] 또한, porinB-(G4S)3-^CA의 SDS-PAGE와웨스턴블롯결과를음성대조군및 porinB-«gCA의결과와비교한것을도 3내지도 5에도시하였다.대조군인 porinB-ngCA의경우 ngCA의농도는 0.230 mg/mL로측정되었고,이는 porinC-«g CA의경우에비해매우높은값을나타내었다.반면링커를통해연결된실시예 porinB-(G4S)3-ngCA에서는 ngCA의농도가상기대조군보다높은 0.27 mg/mL 정도로증가하였다.또한대조군인 porinB-ngCA의경우세포표면상의 ngCA의 양이 0.15 X 10-' pg/cell로측정되었고,이는 porinC-/ CA의 0.23 x 101 pg/cell에 비해매우낮은값을나타내었다.반면링커를통해연결된실시예 porinB-(G4S)2- ngCA에서는 ngCA의농도가상기대조군보다높은 0.26 x 10"' pg/cell을 나타내는것을확인하였다 (도 5).
[84]
[85] wgCA의발현차이는공초점현미경스캔이미지에서도확인할수있었다 (도 6).상기결과로부터 porinB-(G4S)2-rtgCA에서 "gCA가세포표면에성공적으로 발현되었음을확인하였다.
[86]
[87] [시험예 3]수화 (hydration)실험을통한탄산무수화효소 (CA)활성측정
[88] 탄산무수화효소는이산화탄소를수화시키며，탄산수소이온 (HCOr)은반응 용액의 pH를낮추는역할을한다.따라서,수화반웅이일어나는동안 pH의 변화를감지할수있다. 40 mL유리병을구멍이 있는격막튜브 (rubber speta sleeve)로덮고， 20 mM Tris-HCl (pH 8.44-8.48; Sigma-Aldrich) 12 mL을
주입하였다. pH측정기 (Thermo scientific)의탐침을유리병을덮고있는튜브의 구멍을통해주입하였다.유리병에 400 의탄산무수화효소샘플을
주입하였다.얼음플라스크에이산화탄소 (Special gas，대전,대한민국)를 주입하였다. 8 mL의이산화탄소-포화된물을주입한직후용액의 pH를 기록하였다.상기용액은전체반웅에걸쳐 750 rpm의속도로마그네틱 교반하였다.
[89] 탄산무수화효소의활성은 pH가 8.0에서그 0으로변화하는데필요한시간으로 결정하였다.이시간데이터는아래수학식 I에의해 WA(Wilbur-Anderson)단위의 탄산무수화효소활성도로변환될수있다. [91] [수학식 I]
[92] Activity(Uml- 10.D.- ' )=(t0-t)/t x df/(Sample solution(ml O.D 1)
[93] 상기식에서， to와 t는각각비촉매화된버퍼및탄산무수화효소로활성화된 용액에서 pH가 8.0에서 7.0으로변화하는데필요한시간을나타낸다.또한 df는 실험회차에따른실험적차이를보정하기위한희석인자 (dilution factor)이다.
[94] 양성대조군으로，상업적으로판매되는소유래탄산무수화효소 (88%단백질 2613 U/mg, Sigma, BCA)가사용되었다.
[95]
[96] 상기방법에따라수화실험을통한탄산무수화효소 (CA)활성을측정한
결과를도 7에도시하였다.이로부터 porinB-(G4S)2-"gCA또는 porinB-(G4S)3- CA를발현시킨경우 pH가빠르게낮아지는것을알수있으며，탄산무수화 효소의활성이가장높음을확인하였다.
[97]
[98] [시험예 4]링커의길이에따른탄산무수화효소의열안정성
[99] [Table 2]
Figure imgf000010_0001
[100]
[101] 링커의길이에따른 ¾산무수화효소의열안정성을비교하기위하여 ,상기표 2의실시예 1내지실시예 4의실험군을구성하고，각실시예의단백질링커는 상기시험예에서와동일한과정을통해제조하고， 60 °C에서각각 6시간동안 열처리한후,잔여효소활성을측정하였다.
[102] 또한링커의길이 n=3인경우와 , n=0인대조군에대하여， 60 °C에서각각 2시간 동안열처리한후,잔여효소활성을측정하였다.
[103]
[104] 이를통해열안정성을측정한결과를도 8에도시하였다.
[105] 링커의길이 n이각각 1, 2， 3및 6인경우에대하여잔여효소활성도를측정한 결과,각각 71.4%, 81.7%, 53.6%및 48.5%의평균활성을나타내어모두높은 활성을유지하고있으며,특히 n=2일때 81.7<¾로가장높은활성을나타내어 열에대한안정성이 porinB-(G4S)2-MgCA가실험군중가장우수함을확인할수 있었으며,이후장기적열적안정성은 p0rinB-(G4S)2-«gCA를통해확인하였다. [107] [시험예 5]상업적으로판매되는탄산무수화효소 (commercial CA)및대장균 분리생산탄산무수화효소 (free ngCA)에대한표면발현된탄산무수화효소의열 안정성
[108] 형질전환된세포와배양된세포를이용하여표면발현된 «gCA의열안정성을 평가하였다.양성대조군으로，상업적으로판매되는소유래탄산무수화효소 (88%단백질, 2613 U/mg, Sigma, BCA)가사용되었다.세포펠릿 (pellets)은 4 °C에서 13,000 rpm으로 10분간원심분리 (Eppendorf 5451R)를통해분리한후약 10 OD(optical density)의농도로 PBS에재침전시켰다.샘플들은 50 °C와 60 °C의 전자식건조기 (Corning)에서최대 30일까지배양하였다.각샘플의세포는 6 시간경과할때마다 4 °C의냉장고로이동시킨후각샘플의수화에의한활성을 상기시험예 3과같은방법으로측정하였다.매회 ^정시측정당일아침에 배양액에서수득한세포를실험에사용하였다.
[109] 대장균분리생산탄산무수화효소 (free wgCA)는합성단백질의일반적생산 도구인대장균의세포질에발현된것올정제하여사용하였다.상기 free ngCA는 표면발현된탄산무수화효소대비하여열안정성을비교하기위한대조군 그룹으로사용하였다.정제된효소는 0.01 mg/tnL의농도로희석하였다.
샘풀들은 50 °C와 60 °C와전자식건조기 (Corning, Corning, NY, USA)에서 배양하였다.각샘플의세포는 6시간경과할때마다 4 °C의냉장고로이동시킨 후각샘플의수화에의한활성을상기시험예 3과같은방법으로측정하였다.
[110]
[111] 상기실험결과를도 9에도시하였다.열처리를수행하지않은효소들에
대하여，6시간간격으로열처리를수행하여잔여활성을측정한값을측정한 결과，상기도면에서확인할수있는바와같이 porinB-(G4S)2-«(?CA의경우 50 °C에서 7일경과후에도평균 58.4%의잔여활성을나타내고있음을알수 있으나,대조군인 BCA는 1.7%， free ngCA는 33.1%로활성이급격히
감소하였으며 , porinB-(G4S)2-ngCA의경우 600C에서 24시간경과후에도 43.4%의잔여활성을나타내고있는반면， BCA는 4.4%, free ngCA는 7.1%로 잔여활성이급격히감소한것을알수있었다.도면에는나타내지않았지만， 37 oC에서는 30일동안약 121.3 %의활성이유지되었고，그에비해 BCA와 ngCA는 각각 39.3 %, 29.7 %로감소하는것으로나타났다.
[112] 또한, porinB-(G4S)r«gCA의경우 24시간경과후에도 60°C에서약 60%,
80°C에서약 35%의잔여활성을나타내고있음을알수있으나，대조군인 BCA 및 free ngCA의경우열처리수행직후부터잔여활성이급격히감소하였으며， BCA의경우각각약 15%와약 5%， free ngCA의경우각각약 5%와약 1%의 잔여활성만이남아，열에대한활성의감소가실시예에비해현저히큼을알수 있었다.
[Π3]
[114] 상기결과로부터,본발명에의해세포표면에발현된탄산무수화효소의경우 자체의수화활성또한우수하며，열에대한안정성이높아산업적으로유용한 전체세포효소시스템으로서이산화탄소의효율적고정에활용될수있는것을 확인하여본발명을완성하였다.

Claims

청구범위
[청구항 1] 막단백질-단백질링커 -탄산무수화효소가순차적으로연결된
구조를갖는이산화탄소의탄산염전환을위한아미노산서열을 포함하는단백질.
[청구항 2] 제 1항에있어서，
상기막단백질은포린 (porin)계열단백질인것을특징으로하는 단백질.
[청구항 3] 제 1항에있어서，
상기단백질링커는하기화학식 I의구조를갖는것을특징으로 하는단백질.
[화학식 I]
(G4S)n
G:글리신 (glycine), S:세린 (serine)이다;
n은 1내지 10의정수이다.
[청구항 4] 제 1항에있어서，
상기탄산무수화효소는네이세리아속 ( sseria sp.)에서유래된 것을특징으로하는단백질.
[청구항 5] 제 4항에있어서，
상기탄산무수화효소는네이세리아고노르호에아 (Ne^en'a gonorr/zoeae)에서유래된것을특징으로하는단백질.
[청구항 6] 제 1항에있어서，
상기아미노산서열은탄산무수화효소의 C-말단에화학식 II의 아미노산서열을갖는표지단백질이추가로연결된것을특징으로 하는단백질
[화학식 II]
DYKDDDDK
D:아스파르트산 (aspartic acid), Y:티로신 (tyrosine), K:리신 (lysine).
[청구항 7] 제 1항내지제 6항중어느한항의아미노산서열을코딩하는
핵산분자.
[청구항 8] 제 7항의핵산분자를포함하는플라스미드.
[청구항 9] 제 8항의플라스미드로형질전환된인간을제외한형질전환체.
[청구항 10] 제 9항에있어서,
상기형질전환체는코리네박테리움속 (Corynebacteiium sp.)인 것을특징으로하는형질전환체ᅳ
[청구항 11] 제 10항에있어서，
상기형질전환체는코리네박테리움글루타미 ^(Corynebacterium glutamicum)인것을특징으로하는형질전환체.
[청구항 12] 제 7항의핵산분자를인간을제외한숙주세포에도입하여 형질전환하는단계를포함하는탄산무수화효소가세포표면에 발현되는형질전환체의생산방법.
[청구항 13] 막단백질-단백질링커 _탄산무수화효소가순차적으로연결된 구조를갖는아미노산서열을포함하는단백질을이용한 이산화탄소의탄산염전환방법.
PCT/KR2016/012576 2015-11-03 2016-11-03 네이세리아 고노르호에아 유래 탄산무수화효소를 이용한 탄산무수화 활성이 증가된 형질전환체 WO2017078422A1 (ko)

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