KR950009832B1 - New microorganim zymomonas mobilis cp4 and producing method of d-glucitol and d-gluconic acid - Google Patents
New microorganim zymomonas mobilis cp4 and producing method of d-glucitol and d-gluconic acid Download PDFInfo
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- KR950009832B1 KR950009832B1 KR1019880016591A KR880016591A KR950009832B1 KR 950009832 B1 KR950009832 B1 KR 950009832B1 KR 1019880016591 A KR1019880016591 A KR 1019880016591A KR 880016591 A KR880016591 A KR 880016591A KR 950009832 B1 KR950009832 B1 KR 950009832B1
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- glucose
- gluconic acid
- mobilis
- fructose
- glucitol
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- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 title claims description 63
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 title claims description 34
- 235000012208 gluconic acid Nutrition 0.000 title claims description 34
- 229950006191 gluconic acid Drugs 0.000 title claims description 34
- 238000000034 method Methods 0.000 title claims description 19
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 title claims description 12
- 241000588902 Zymomonas mobilis Species 0.000 title claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 33
- 239000008103 glucose Substances 0.000 claims description 32
- 239000000174 gluconic acid Substances 0.000 claims description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- 229930091371 Fructose Natural products 0.000 claims description 26
- 239000005715 Fructose Substances 0.000 claims description 26
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 12
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 10
- 102000004316 Oxidoreductases Human genes 0.000 claims description 6
- 108090000854 Oxidoreductases Proteins 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 4
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- 108010051210 beta-Fructofuranosidase Proteins 0.000 claims description 3
- 239000005515 coenzyme Substances 0.000 claims description 3
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 claims description 3
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims description 2
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- BIRSGZKFKXLSJQ-SQOUGZDYSA-N 6-Phospho-D-gluconate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O BIRSGZKFKXLSJQ-SQOUGZDYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
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- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
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- 239000011616 biotin Substances 0.000 description 2
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- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 2
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- 150000005846 sugar alcohols Chemical class 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- IJOJIVNDFQSGAB-SQOUGZDYSA-N 6-O-phosphono-D-glucono-1,5-lactone Chemical compound O[C@H]1[C@H](O)[C@@H](COP(O)(O)=O)OC(=O)[C@@H]1O IJOJIVNDFQSGAB-SQOUGZDYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
내용 없음.No content.
Description
제1도는 본 발명의 지모모나스 모빌리스 SG11을 이용한 글루시톨(D-Glucitol)의 생산과 시간에 따른 프룩토오스의 농도와의 관계를 나타낸 그래프이고,1 is a graph showing the relationship between the production of glutathol (D-Glucitol) and the concentration of fructose over time by using the Simmonosa mobilis SG11 of the present invention,
제2도는 본 발명의 지모모나스 모빌리스 SG11을 이용한 글루콘산(D-Gluconic acid)의 생산과 시간에 따른 글루코오스의 농도와의 관계를 나타낸 그래프이다.Figure 2 is a graph showing the relationship between the production of glucose and the concentration of glucose over time using D-Gluconic acid using the Simmonosa mobilis SG11 of the present invention.
본 발명은 지모모나스 모빌리스(Zymomonas mobilis) CP4로 부터 돌연변이 유발로 개량된 변이주 지모모나스 모빌리스 SG11(수탁번호 : KCTC 8420 P)과 이를 이용한 글루시톨(D-Glucitol) 및 글루콘산(D-Gluconic acid)을 동시에 제조하는 방법에 관한 것이다.The present invention is a mutant strain Zimomonas mobilis SG11 (Accession No .: KCTC 8420 P) improved by mutagenesis from Zymomonas mobilis CP4 (D-Glucitol) and gluconic acid (D- Gluconic acid) is a method for producing at the same time.
본 발명의 목적화합물인 글루시톨은 당뇨병환자를 위한 저칼로리의 식품으로 이용되며, 아스콜빈산 C의 생산에 필요한 중간물질인 솔보오스(L-Sorbose)로 전환될 수 있는 물질로 유용하다.Glucitol, the target compound of the present invention, is used as a low-calorie food for diabetics, and is useful as a substance that can be converted into sorbose (L-Sorbose), an intermediate required for the production of ascorbic acid C.
또한 글루콘산은 식품첨가물이나 그밖에 콘크리트첨가제 또는 가소제로 사용되는데, 특히 칼슘글루콘산(Calcium Gluconic acid)은 높은 용해성을 갖고 있어 구강이나 정맥의 칼슘요법에도 사용되고 있다.Gluconic acid is also used as a food additive or other concrete additives or plasticizers. In particular, calcium gluconic acid has high solubility and is used for calcium therapy in the oral cavity or vein.
종래에 알려져 있는 글루시톨의 생산방법으로는 덱스트로즈시럽을 높은 압력에서 수소첨가시키는 화학적 방법이 있다.Conventionally known methods of producing glutitol include a chemical method of hydrogenating dextrose syrup at high pressure.
그러나, 이 방법은 물을 전기분해하여 수소를 얻어야 하기 때문에 저정할 수 있는 시설이 필요하다. 다음은 이 수소를 이용하여 약 40-75atm의 압력과 140-150℃의 온도에서 수소첨가를 해야하는데, 이 과정에서 인체에 극히 유해한 니켈, 구리 등의 촉매를 사용해야 한다. 이러한 촉매제를 제거하기 위해서 생성물질인 글루시톨은 여러개의 이온교환수지칼럼(Ion exchange column)을 통과시키지만 약 51ppm 이하의 유해한 촉매가 잔류한다(phillips, M.A. British Biochem, Eng. 8(11) : 767-769, 1963 ; Makkee, et, al., Carbohyo Research, 138 : 225-236, 1985).However, this method requires storage facilities because it requires electrolysis of water to obtain hydrogen. Next, this hydrogen must be hydrogenated at a pressure of about 40-75 atm and a temperature of 140-150 ° C. In this process, catalysts such as nickel and copper, which are extremely harmful to the human body, must be used. To remove these catalysts, the resulting product, glutitol, passes through several ion exchange columns, but remains less than about 51 ppm of harmful catalyst (phillips, MA British Biochem, Eng. 8 (11): 767-769, 1963; Makkee, et, al., Carbohyo Research, 138: 225-236, 1985).
또한, 글루콘산의 생산방법으로는 글루코노박터서복시단스(Gluconnobacter Suboxydans)나 아스퍼질러스 나이저(Aspergillus niger)를 이용한 발효법에 의해서 생산되어 왔다.Gluconic acid has been produced by fermentation using Gluconnobacter Suboxydans or Aspergillus niger.
이 과정에서는 효과 클루코오스옥시테이즈(Glucoseoxydase)에 의해서 글루코오스(Glucose)를 글루코노락톤(Gluconolactone)으로 전환시킨다음 계속적인 가수분해에 의하여 수득하는 방법이 소개 되어 있다. 그러나 이 방법은 가수분해를 하기 때문에 경제성 및 생선성이 낮은 단점이 있다.In this process, a method of converting Glucose to Gluconolactone by effect Glucoseoxydase and then obtaining it by continuous hydrolysis is introduced. However, this method has a disadvantage of low economical efficiency and low fishability due to hydrolysis.
이에 본 발명은 미생물학적 방법에 의해 글루시톨과 글루콘산을 동시에 생산할 수 있고, 수율이 각각 80% 이상 생산할 수 있는 우수한 새로운 변이주 인 지모모나스 모빌리스 SG11을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide an excellent new variant of Zimomonas mobilis SG11, which can simultaneously produce glucitol and gluconic acid by a microbiological method, and can produce more than 80% of yield.
또한 본 발명의 다른 목적은 상기 변이주 지모모나스 모빌리스 SG11을 이용하여 글루시톨과 글루콘산을 동시에 제조하는데 있어 고압과 수소생성에 소요되는 경비를 절감할 수 있을 뿐만아니라 인체에 유해한 촉매제(Ni, Cu)를 사용하지 않음으로서 보다 저렴한 비용으로 높은 생산수율을 얻을 수 있는 새로운 방법을 확립하고, 보다 경제적인 정제 및 분리공정을 사용하여 글루시톨 및 글루콘산을 제조하는 방법을 제공하는데 있다.In addition, another object of the present invention is to reduce the cost required for high pressure and hydrogen production in the simultaneous production of glutathol and gluconic acid using the mutant strain of jimonos mobilis SG11, as well as harmful catalyst (Ni, It is to establish a new method to obtain a high production yield at a lower cost by using no Cu), and to provide a method for producing glutitol and gluconic acid using more economical purification and separation processes.
이하, 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 신균주인 지모모나스 모빌리스 SG11(Zymomonas mobilis SG11)은 지모모나스 모빌리스 CP4의 변이주로서, 다음과 같은 방법으로 제조된다.Zymomonas mobilis SG11, a new strain according to the present invention Zymomonas mobilis SG11, is a mutant strain of Zimomonas mobilis CP4, which is prepared by the following method.
우선, 지모모나스 모빌리스 CP4의 포자를 액체배지에서 성장시킨후 대기수(log phase)에 도달했을때 원심분리기로 세포를 분리한다음, 분리한 세포를 1ml의 완충용액(saline phosphate buffer)으로 씻은다음, N.T.G 100㎍/ml의 제1인산칼륨 1.5~2g/l, 황산암모니움 1~1.5g/l, 글루코오스 200g/l, 효소추출물 8~10g/l을 함유한 배지와 혼합한다음, 30℃에서 30~60분간 방치한후 완충용액으로 씻고 제1인산칼륨 1~1.5g/l, 제2인산칼륨 1~1.5g/l, 염산나트륨 0.4~0.6g/l, 황산암모니움 1~1.5g/l, 인산칼시움 1~1.5mg/l, 비오틴 1~1.5mg/l, 글루코오스 1~1.5%(wt/vol), 프록토오스 1.5~2%(wt/vol)등이 포함이된 배지에서 수일간 진탕배양에서 생산성이 높은 우수균주를 분리한다.First, the spores of Zimomonas mobilis CP4 were grown in a liquid medium, and the cells were separated by centrifugation when reaching the log phase. The cells were washed with 1 ml of saline phosphate buffer. Next, mixed with a
글루시톨(D-glucitol)과 글루콘산(D-gluconic acid)을 생산하기 위해서는 여러종류의 반응조를 사용할 수 있는데, 그중 컬럼반응조(Columm reactor), 유동식 베드 반응조(Fluidized bed reactor), 연속교반반응조(Stirred tank reactor), 세포순환방법(Cell recycle system), 반투막성막을 이용한 세포순환방법(Cell recycle system with membrane module)을 사용할 수 있는데, 이러한 반응조들은 추출한 효소나 세포막투과성을 높인 세포자체를 고정화시켜서 사용하고, 이때 고정화시키는데 필요한 물질은 소디움 알지네이트(Na-alginate)나 카파카라지난(K-Carrageenan)과 같은 물질을 사용한다.Several reactors can be used to produce D-glucitol and D-gluconic acid, including column reactor, fluidized bed reactor and continuous stirring reactor. (Stirred tank reactor), cell recycling system (Cell recycle system) and cell recycling method (Cell recycle system with membrane module) can be used, these reactors by immobilizing the enzyme or the cell itself to increase the cell membrane permeability In this case, the material required for immobilization may be a material such as sodium alginate or Na-alginate or K-Carrageenan.
또한, 효소추출물이나 세포자체를 그대로 사용할때는 세포순환방법, 반투막성막을 이용한 세포순환방법의 반응조를 사용할 수 있으나 글루콘산이 생성됨에 따라 pH가 떨어진다. 이를 위하여 pH가 4.5-6.5를 유지하기 위해서 수산화카리움이나 탄산칼슘과 같은 완충용액을 사용한다.In addition, when the enzyme extract or the cell itself is used as it is, a cell circulation method, a cell circulation method using a semipermeable membrane can be used, but the pH decreases as gluconic acid is produced. To do this, a buffer solution such as potassium hydroxide or calcium carbonate is used to maintain the pH of 4.5-6.5.
본 발명자들은 상기 변이주를 지모모나스 모빌리스 SG11(Zymomonas mobilis SG11)로 명명하고, 이를 1988년 12월 6일자로 한국과학기술원에 수탁번호 KCTC 8420P로 기탁하였다.The present inventors named the mutant strain Zymomonas mobilis SG11, and deposited it with the accession number KCTC 8420P at the Korea Advanced Institute of Science and Technology on December 6, 1988.
이하, 본 발명에 따른 지모모나스 모빌리스 SG11은 다음과 같은 특성을 갖는다.Hereinafter, Zimomonas mobilis SG11 according to the present invention has the following characteristics.
지모모나스 모빌리스 SG11은 그람음성이며 글루코오스와 프룩토오스 또는 슈크로스가 함유된 배지에서 월등한 양의 글루시톨을 생성해내는 것을 제외하고는 친균주의 균학적 특성을 대체로 유사하다.Zimomonas mobilis SG11 is gram-negative and generally similar in mycological properties, except that it produces superior amounts of glutitol in medium containing glucose and fructose or sucrose.
[표 1]TABLE 1
형태학적 특성Morphological characteristics
[표 2]TABLE 2
지모모나스 모빌리스의 당분이용도Sugar use of timothy mobilis
- : 이용하지 못함 + : 이용함-: Not available +: Used
[표 3]TABLE 3
생리학적 특성Physiological characteristics
수회에 걸친 기초배양(subculture)에서 빠른 속도의 성장과 아가(Agar) 배지에서 100%의 콜로니(colony) 생성효율을 얻는데 필요한 BM 배지의 조성은 다음 표 4와 같다.The composition of BM medium required for rapid growth in several subcultures and obtaining 100% colony production efficiency in Agar medium is shown in Table 4 below.
[표 4]TABLE 4
BM 배지의 구성요소Components of the BM Badge
상기 배지의 최종 pH는 6이며, 이외에 칼슘판토테네이트 5mg/l, 티아민하이드로클로라이드 1mg/l, 피리독신하이드로클로라이드 1mg/l, 비오틴 1mg/l, 그리고 니코틴산 1mg/l등을 첨가할 수도 있다.The final pH of the medium is 6, in addition to calcium pantothenate 5mg / l, thiamine hydrochloride 1mg / l, pyridoxine hydrochloride 1mg / l, biotin 1mg / l, nicotinic acid 1mg / l and the like may be added.
RM 배지는 상기의 배지에 글루코오스 20g/l, 효모추출물 10g/l, 그리고 KH2PO42g/l를 조합한다. 특히 아데닌이나 시스테인을 요구할 수도 있다.RM medium is a combination of glucose 20g / l, yeast extract 10g / l, and KH 2 PO 4 2g / l in the medium. In particular, they may require adenine or cysteine.
상기 지모모나스 모빌리스 SG11의 글루시톨과 글루콘산의 생산능력은 실시예에 표기되어 있다.The production capacity of Glycitol and Gluconic Acid of the Zimomonas mobilis SG11 is indicated in the Examples.
한편, 본 발명에서 글루시톨과 글루콘산의 동시생산을 위해서 글루코오스와 프룩토오스 또는 슈크로스(Sucrose를 기질로서 지모모나스 모빌리스 SG11이나 지모모나스 모빌리스 SG11로 부터 추출한 효소복합체(enzyme complex)인 글루코오스/프룩토오스 산화환원효소(Glucose/fructose oxidoreductase)의 존재하에 반응시킨다.Meanwhile, in the present invention, glucose and fructose or sucrose (enzyme complex extracted from Zimomonas mobilis SG11 or Zimomonas mobilis SG11 as substrates for the simultaneous production of glutathol and gluconic acid). React in the presence of Glucose / fructose oxidoreductase.
이 효소는 박테리아인 지모모나스 모빌리스속에 속하는 모든 균주에서 생성되나 본 발명에 사용되는 균주, 지모모나스 모빌리스 SG11은 특히 효소활성이 강하다.This enzyme is produced in all strains belonging to the genus bacteria of the genus Simonus mobilis, but the strain used in the present invention, Simonus mobilis SG11 is particularly strong in enzyme activity.
지모모나스속에 속하는 균주들은 대체로 글루코오스나 프룩토오스를 기질로 이용해서 에탄올을 생산하는 균주로 알려져 있으나 글루코오스+프룩토오스혼합물이나 슈크로스를 이용해서 글루코오스나 프룩토오스를 사용했을 때만큼 많은 에탄올을 생산하지 못한다. 그 이유는 얼마의 글루시톨의 부산물로서 생성되기 때문이다. 이 박테리아는 생성된 글루시톨을 다시 이용하지 못한다.Strains belonging to the genus of Simonomos are generally known to produce ethanol by using glucose or fructose as substrates, but as much as ethanol is produced by using glucose or fructose using glucose + fructose mixture or sucrose. Cannot produce The reason is that it is produced as a by-product of some glutitol. The bacterium cannot use the produced glutitol again.
본 발명에 따른 글루시톨과 글루콘산이 동시에 아주 빠른 속도로 생성되는데, 반응기전은 다음과 같다. 즉 프룩토오스가 글루시톨로 환원됨과 동시에 글루코오스는 글루콘산으로 산환된다. 이 반응에 사용되는 글루코오스/프룩토오스 산화환원효소는 NADP라는 조효소(Co-enzyme)와 함께 글루코오스로부터 프룩토오스로 1분자의 수소이온(H+)을 전달하는 반응을 촉진하는 단일 효소이다.Glucitol and gluconic acid according to the present invention are produced at a very high speed at the same time, before the reactor is as follows. In other words, while fructose is reduced to glutatholate, glucose is converted into gluconic acid. The glucose / fructose oxidoreductase used in this reaction is a single enzyme that promotes the reaction of transferring one molecule of hydrogen ions (H + ) from glucose to fructose together with a co-enzyme called NADP.
환원에너지(reduction potential)면에서 반응식을 나타내면 다음과 같다.The reaction equation in terms of reduction potential is as follows.
상기(1) 식과 (2)식에서 산화환원에너지는 각각 E0=-0.272volt와 E0=-0.320volt이다. 여기서 최소프러스반응(the least half-reaction)인 (2)식은 (1)식에 의해서 산화할 것이다. 따라서 (2)식은 (3)식과 같이 산화의 방향으로 바뀌며,In the above formulas (1) and (2), the redox energy is E 0 = -0.272volt and E 0 = -0.320volt, respectively. Where the least half-reaction (2) is oxidized by (1). Therefore, Equation (2) changes in the direction of oxidation as in Equation (3),
E0=+0.320이다. (1)식과 (3)식으로부터 얻어진 전체반응에너지(overall reaction potential, ΔE0)는 +0.048이므로 반응, 즉, 산화와 환원반응은 동가(同價)의 E0가 이루어질때까지 계속되는 것이다.E 0 = + 0.320. Since the overall reaction potential (ΔE 0 ) obtained from equations (1) and (3) is +0.048, the reaction, ie oxidation and reduction, continues until the equivalent E 0 is achieved.
일반적인 지모모나스 배양에서는 생성된 글루콘산이 6-포스포글루코네이트(6-phospho gluconate)로 전환되어 결국은 에탄올이나 다른 부산물을 생성한다. 즉 50g/l의 각각 글루코오스나 프룩토오스의 배지로부터 25g/l의 글루콘산이 처음 5시간내에 생성되었으나 그후 생성된 글루콘산은 감소되어 완전히 다시 사용되어 에탄올의 농도를 높혔다. 생성된 글루콘산이 재사용되는데는 소모성, 용해성의 여인수(Co-factor)인 ADP-ATP, UDP/UTP 그리고 NAD+/NADH 등과 인산의 존재가 필수적이다.In a typical chymomonas culture, the produced gluconic acid is converted to 6-phospho gluconate, which eventually produces ethanol or other byproducts. That is, 25 g / l of gluconic acid was produced in the first 5 hours from 50 g / l of glucose or fructose medium, but the resulting gluconic acid was reduced and completely used again to increase the concentration of ethanol. In order to reuse the produced gluconic acid, the presence of phosphoric acid such as ADP-ATP, UDP / UTP and NAD + / NADH, which are consumable and soluble co-factors, is essential.
이외에도 글루코오스와 프룩토오스의 발효에 있어 주요물질인 Mg++도 존재되어야 한다. 글루콘산은 제v2 이용되나 글루시톨은 배양액에 그대로 존재되었다.In addition, Mg ++ , a major substance in the fermentation of glucose and fructose, should also be present. Gluconic acid was used for v2 but glucitol remained in the culture.
본 발명에 따르면 이 에탄올이나 그밖에 부산물의 생성을 차단하고 글루시톨과 글루콘산의 동시생산을 유도하는 것이다. 즉 글루콘산 재이용에 필수적인 조효소와 여인수들을 제거해서 생성된 글루콘산이 6-포스포글루코노락톤(6-phospho gluconolactone) 이라는 중간 생성물질로 변화해서 에탄올로의 대사를 중단시켰다.According to the present invention, the production of ethanol or other by-products is blocked and the simultaneous production of glutathol and gluconic acid is induced. Gluconic acid, produced by removing the coenzyme and the number of women essential for gluconic acid reuse, turned into an intermediate called 6-phospho gluconolactone, which stopped metabolism to ethanol.
지나모모나스 모빌리스 SG11을 성장시켜 세포막투과성을 높인후 pH 4.5~5.5의 완충용액으로 세척함으로서 클루콘산 재이용에 필수적인 조효소와 여인수를 제거함으로서 글루콘산으로부터 6-포스포글루코네이트의 생성을 억제했으며, 따라서 글루콘산이 글루시톨과 더불어 축적되어 에탄올이나 그 이외에 부산물이 생성되지 않는다.Gnamomonas mobilis SG11 was grown to increase cell membrane permeability, and then washed with buffer solution of pH 4.5 ~ 5.5 to inhibit the production of 6-phosphogluconate from gluconic acid by removing coenzyme and phosphate essential for gluconic acid reuse. Therefore, gluconic acid accumulates with the glucitol, producing no ethanol or other by-products.
세포막투과성을 높이기 위해서는 30℃에서 지모모나스 모빌리스 SG11이 완전히 생장한 정지기에서 온도를 42-45℃로 높여 방치한후 원심분리기로 5, 000-7, 000rpm에서 얻은 세포를 pH 4.5~5.5의 완충용액으로 두차레 세척했다. 이렇게 세척한 세포를 400g/l의 슈크로스용액과 혼합한 결과 주 생산물인 글루시톨과 글루콘산이 각각 100g/l와 95g/l로 생성되었다. 슈크로스를 기질로 사용시 글루코오스와 프룩토오스를 분해시키기 위해 인버테이즈(Invertase) 효소를 이용할 필요성이 없다. 왜냐하면 세포막투과성이 향상된후에도 본 발명의 지모모나스 모빌리스 SG11은 슈크로스 분해력을 가지고 있기 때문이다.In order to increase the cell membrane permeability, the temperature was raised to 42-45 ° C in the stationary phase where the GIMONIS MOBILIS SG11 was completely grown at 30 ° C, and the cells obtained at 5,000-7, 000rpm were centrifuged at pH 4.5-5.5. Wash twice with buffer. The washed cells were mixed with 400 g / l sucrose solution to produce 100 g / l and 95 g / l, respectively. Using sucrose as a substrate there is no need to use an Invertase enzyme to break down glucose and fructose. The reason for this is that even after the cell membrane permeability is improved, the Zimomonas mobilis SG11 of the present invention has sucrose degrading ability.
그러나, 생산성을 높이기 위해서는 인버테이즈라는 효소를 병행해서 사용할 수도 있다. 기질로서 전분을 이용할 수 있으나 분해를 위해서 전처리가 요구된다.However, in order to increase productivity, an enzyme called invertase can be used in parallel. Starch may be used as the substrate but pretreatment is required for degradation.
세포막투과성을 높인 세포내의 글루코오스/프룩토오스 산화환원효소를 이용해서 반응시킬때의 최적 pH는 4.5~6.5이다. 그러나 pH 5.5~6.5에서 가장 높은 기질의 친화력을 보였다. 이외에도 같은 pH 조건하에서 효소추출물을 이용할 수 있으며 세포를 파괴한후 순수한 효소추출물을 원심분리기를 이용하여 얻을 수 있다.The optimal pH for the reaction using glucose / fructose oxidoreductase in cells with enhanced cell membrane permeability is 4.5 to 6.5. However, the affinity of the substrate was highest at pH 5.5 ~ 6.5. In addition, enzyme extracts can be used under the same pH conditions, and pure enzyme extracts can be obtained using a centrifuge after cell destruction.
이하 본 발명을 실시예에 의하여 구체적으로 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
[실시예 1]Example 1
지모모나스 모빌리스 CP4의 포자를 액체배지에서 성장시킨후 대수기(log phase)에 도달했을때 원심분리기로 세포를 분리한다음, 분리한 세포를 1ml의 완충용액(saline phosphate buffer)으로 씻은다음, N.T.G 100㎍/ml, 제1인산칼륨 2g/l, 황산암모니움 1g/l, 글루코오스 20g/l, 효소추출물 10g/l를 함유한 배지와 혼합한다음, 3℃에서 45분간 방치한후 완충용액으로 씻고 제1인산칼륨 1g/l, 제2인산칼륨 1g/l, 염화나트륨 0.5g/l, 황산암모니움 1g/l, 인산칼시움 1mg/l, 비오틴 1mg/l, 글루코오스 1%(wt/vol). 프룩토오스 2%(wt/vol)등의 포함이된 배지에서 2일간 진탕배양하여 생산성이 높은 우수균주를 분리한다.Spores of Zimomonas mobilis CP4 were grown in liquid medium and cells were separated by centrifugation when they reached the log phase. The cells were washed with 1 ml of saline phosphate buffer.
지모모나스 모빌리스 SG11의 배지조성은 다음 표 5와 같다.The medium composition of Zimomonas mobilis SG11 is shown in Table 5 below.
[표 5]TABLE 5
내지조성Composition
상기 배지에서 배양된 세포를 세포막투과성을 높인후 반응시킬때 용액에 동량의 글루코오스와 프룩토오스(100-300g/l)이다. 또한 슈크로스도 사용할 수 있다.When the cells cultured in the medium are reacted after increasing the cell membrane permeability, the same amount of glucose and fructose in the solution (100-300 g / l). Sucrose can also be used.
상기 표 4의 배지에 30℃에서 배양한 세포를 45℃에서 2시간 또는 4시간 열처리한후 pH 6.2 온도 39℃에서 400g/l의 슈크로스와 반응시킨 결과 글루시톨과 글루콘산의 생산성은 다음 표 6과 같다.The cells cultured at 30 ° C. in the medium of Table 4 were heat treated at 45 ° C. for 2 hours or 4 hours, and then reacted with 400 g / l sucrose at a pH 6.2 temperature of 39 ° C., whereby the productivity of glutathol and gluconic acid was Table 6 is as follows.
[표 6]TABLE 6
세포막투과성을 높인 세포와 400g/l의 슈크로스와의 반응결과Reaction result of 400g / l sucrose with cells with enhanced cell membrane permeability
[실시예 2]Example 2
세포막투과성을 높인 지모모나스 모빌리스 SG11에 글루코오스와 프룩토오스를 pH 6.2, 온도 39℃에서 반응시킨 결과는 다음 표 7과 같다. 빠른속도로 글루코오스는 글루콘산을 또 프룩토오스는 글루시톨로 바뀌었다. pH는 2몰의 소디움카보네이트(Na2CO3)로 조절하였다.The results of the reaction of glucose and fructose at pH 6.2 and a temperature of 39 ° C. to Zimomonas mobilis SG11 with increased cell membrane permeability are shown in Table 7 below. Glucose quickly changed to gluconic acid and fructose to glutitol. pH was adjusted with 2 moles of sodium carbonate (Na 2 CO 3 ).
글루시톨과 글루콘산이 생성되지 않을때에는 pH의 변화가 일어나지 않는다. 그 결과는 다음 표 7과 같다.There is no change in pH when no glycitol and gluconic acid are produced. The results are shown in Table 7 below.
[표 7]TABLE 7
pH 6.2에서의 반응결과Reaction result at pH 6.2
상기 표에서 보는 바와같이 에탄올(ethanol)의 생산은 거의 차단되었다.As shown in the table, the production of ethanol was almost blocked.
[실시예 3]Example 3
세포막투과성을 높인 세포를 칼슘알지네이트에 고정화시켰다. 칼슘알지네이트 겔은 기질이나 생성물(글루시톨과 글루콘산)을 잘 투과시키는 장점이 있다. 즉 투과성을 높인 고농도의 세포를 2%(W/V)의 소디움알지네이트와 1%(W/V)의 celite의 혼합체를 세포와의 비율을 1 : 15로 섞은다음 상온에서 40g/l의 칼슘클로라이드(CaCl2) 용액에 약 1mm 굵기의 주사기 바늘로 떨어뜨리면 지름이 약 1.3mm의 겔비드(gel beads)가 만들어진다. 이들을 글루코오스+프룩토오스용액(각 200g/l)과 CSTR에서 반응시켰다. 이때 pH는 KOH로 조절하였으며 겔의 부서짐을 막기위해 교반속도를 줄였다. 반응온도와 pH는 각각 39℃와 6.2이며, 그 결과는 첨부도면 제1도 및 제2도에 나타나 있다. 20시간내에 글루코오스의 프룩토오스가 완전히 소모되었으며, 이들은 거의 글루시톨과 글루톤산 생산에 이용되었음을 알 수 있다.Cells with increased cell membrane permeability were immobilized with calcium alginate. Calcium alginate gels have the advantage of permeating well to substrates and products (glucitol and gluconic acid). In other words, a mixture of 2% (W / V) sodium alginate and 1% (W / V) celite with a high concentration of cells with high permeability was mixed at a ratio of 1: 15, and then 40 g / l calcium chloride at room temperature. Dropping a (CaCl 2 ) solution with a syringe needle about 1 mm thick produces gel beads of about 1.3 mm in diameter. These were reacted with glucose + fructose solution (200 g / l each) in CSTR. At this time, the pH was adjusted with KOH and the stirring speed was reduced to prevent the gel from breaking. The reaction temperature and pH are 39 ° C. and 6.2, respectively, and the results are shown in FIGS. 1 and 2 of the accompanying drawings. Within 20 hours, the fructose of glucose was completely consumed and it was found that they were almost used for the production of glutathol and glutonic acid.
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