18-12-2017 дата публикации
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Номер заявки: 00-16-102070542
Дата заявки: 07-06-2016
[1]The present invention refers to mutations of Kluyveromyces marxianus 36907 provided FMEL1 through xylitol enhanced (KCTC18459P) and a method for preparing polyimide foam are disclosed.
[3]Other activates a alcohol xylitol 5, as well as the use of calorie content of the same effects as sweeteners to form its corresponding acid constituent faces are not sugar substitute, [...], number and, pharmaceuticals and oral care number etc. used has been crushed.
[4]In addition, xylitol to methods for insulin action without interposed, since insulin secretion without affecting blood glucose to characterized by disclosed. Thanks to this feature diabetes patient glucose tie etc. can be medical even used to keep cool.
[5]On the other hand, chemical dehydrogenation the case air is xylitol xylose wheel, as well as the method of xylitol yield is low, a large amount of byproducts generated deformation has been inefficient number point at the door. The, rather than chemical method using biological method to produce xylitol 'high production yield' marks and environmental safety can be referred to as among the best method.
[6]However, most of the existing method through biological method not using bacteria or yeast strains of heat resistance when the xylitol production studies non-heat resistance, optimal temperature rather than the temperature suitable for enzymatic activity at temperatures as low strain growth study was performed 30 provided 37 °C kneading step.
[7]On the other hand, of Kluyveromyces marxianus (KluyveromycesMarxianus) And can be used in different substrate, growth has the added feature of fast heat resistance because, steam etc. from widely used to produce biofuel. A heat-resistant strains of Kluyveromyces marxianus (KluyveromycesMarxianus) Required cooling for low cost, efficient enzyme hydrolysis, minimization of wave 1 saccharifying fermentable and contamination.
[8]Introducing a temporal xylose metabolic pathway of Kluyveromyces marxianus capable of producing xylose via recombinant (KluyveromycesMarxianus) Study result is disclosed but is pivotably, efficient of xylitol production speed dial setup.
[10]Korean registration patent number 10 - 1246780 call (registration date: 2013. 03. 18) Is, newly introduced xylitol production of arabinose metabolic pathway is by using Leuconostoc citreum, arabinose xylose (xylose) (arabinose) on mixing medium, used for cellular metabolism of arabinose to xylitol product arabitol (arabitol) number (xylitol) production method relates to the production of an efficient billion, more particularly L-a arabinose isomerase (araA), L a-ribulokinase (araB), L a-ribulose-a 5 a-phosphate (araD) 4 a-epimerase enzymes in the storehouse (Candida tropicalis) strain for the expression of gene (codon optimization) for optimizing code for efficiently after having performed, each glyceride aldehyde - 3 - phosphate dehydrogenase gene (glyceraldehyde-a 3 a-phosphate dehydrogenase) promoter and selecting a marker cassette including inserting a URA3, Candida enzyme (Candida sp.) by introducing, positive number and crystallization process in the production of xylitol which interfere with the xylitol production method disclosed the number generation arabitol billion. Korean registration patent number 10 - 1194752 call (registration date: 20121. 10. 19) Is, xylose reductase (xylose reductase) is permanent expression (constitutive expression) by using Leuconostoc citreum won the xylitol production in the medium to high yield method relates to producing xylitol acid, more particularly neuro [su gun (Neurospora crassa) grow it buys oxidoreductase in the storehouse of xylose (Candida tropicalis) gene (codon optimization) for optimizing code for the expression of high catalytic activity in the obtained performing optimized xylose reductase; optimized for gene expression, aldehyde - 3 - phosphate dehydrogenase (Candida tropicalis) cloned in the storehouse of glyceride (glyceraldehyde-a 3 a-phosphate dehydrogenase) containing exogenous promoter and terminator portion; and selecting URA3 marker including expression vector, said expression vector xylitol dehydrogenase (xylitol dehydrogenase) is it became the fire bow in the storehouse xylose reductase is permanent expressed xylitol producer microorganisms by introducing the number produced therewith, and said batch culture strains cause carbon through number of glucose billion unloaded xylitol method is disclosed.
[11]In the present invention productivity of xylitol of Kluyveromyces marxianus (KluyveromycesMarxianus)Number [...] strains developing a broadcast receiver.
[12]In addition, the present invention refers to said method has a low number of mass-production of xylitol using Leuconostoc citreum [...] the broadcast receiver.
[14]In the present invention of Kluyveromyces marxianus xylitol enhanced (Kluyveromyces Marxianus) A number (KCTC18459P) 36907 provided FMEL1 [...] substrate.
[15]On the other hand, added xylose medium, of Kluyveromyces marxianus (Kluyveromyces Marxianus) To be inoculated with a culture of 36907 provided FMEL1 (KCTC18459P) characterized by a number of xylitol production method [...] substrate.
[16]On the other hand, in the present invention, said production of xylitol method is preferably of Kluyveromyces marxianus (KluyveromycesMarxianus) A 36907 provided FMEL1 (KCTC18459P) 30 provided 40 °C, about 350 provided 450 rpm by culturing is performed now.
[17]In the present invention is heat resistance of Kluyveromyces marxianus yeast (Kluyveromyces Marxianus) 36907 Strain randomly after improving mutations effectively xylitol expressed from recombinant yeasts produce xylose Pa with him.
[18]Finally selected of Kluyveromyces marxianus (Kluyveromyces Marxianus) In the case of strain 36907 provided FMEL1 (KCTC18459P), it is a wool strain of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 Producing xylitol concentration increased compared to 120%, been increased yield of 39%.
[19]On the other hand, in the present invention for improving productivity of Kluyveromyces marxianus xylitol at high temperature (KluyveromycesMarxianus)When the stirring speed optimize conducts an experiment 36907 provided FMEL1 (KCTC18459P), through 40 °C, 400 rpm conditions has been confirmed that the xylitol productivity increases.
[21]In the present invention through mutations of Kluyveromyces marxianus productivity xylitol (KluyveromycesMarxianus) 36907 Provided FMEL1 (KCTC18459P) strain were producer and director.
[22]In addition, when use in, even relatively high temperature can be mass-production of xylitol.
[24]It is a wool strain of Kluyveromyces marxianus is of Figure 1 A (Kluyveromyces Marxianus) 36907 Xylose to xylitol production strains added result confirming the fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol) Of Figure 1 B is random mutations of Kluyveromyces marxianus through enhanced productivity xylitol (KluyveromycesMarxianus) Strains (KCTC18459P) xylose to xylitol added 36907 provided FMEL1 confirmed result production fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol) Of Kluyveromyces marxianus is of Figure 2 A and B (Kluyveromyces Marxianus) Each 30 °C and 40 °C 36907 glucose fermentation results in supply are disclosed. (■: glucose, ○: O. D. , Δ: ethanol) Of Kluyveromyces marxianus is of Figure 2 C and D (Kluyveromyces Marxianus) In each 30 °C and 40 °C (KCTC18459P) glucose fermentation 36907 provided FMEL1 result supply are disclosed. (■: glucose, ○: O. D. , Δ: ethanol) Of Kluyveromyces marxianus of Figure 3 A is in 40 °C (Kluyveromyces Marxianus) 36907 Xylose to xylitol production strains added result confirming the fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol) Of Kluyveromyces marxianus of Figure 3 B is in 40 °C (Kluyveromyces Marxianus) Strains (KCTC18459P) xylose to xylitol added 36907 provided FMEL1 confirmed result production fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol) Figure 4 30 °C of Kluyveromyces marxianus in (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain (A) each 300, 350 (B), (C) 400, 450 and 500 rpm (D) (E) measured about liquor xylitol production are disclosed. (■: xylose, ◇: xylitol, ●: O. D. , Δ: ethanol) Figure 5 40 °C of Kluyveromyces marxianus in (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain (A) each 300, 350 (B), (C) 400, 450 and 500 rpm (D) (E) measured about liquor xylitol production are disclosed. (■: xylose, ◇: xylitol, ●: O. D. , Δ: ethanol) Of Kluyveromyces marxianus in Figure 6 30 and 40 °C (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain prepared liquor xylitol production results of a comparison each cap and are disclosed.
[25]Of Kluyveromyces marxianus (KluyveromycesMarxianus) And can be used in different substrate, fast growth because steam used to heat resistance etc. from biofuel production. Strains having advantages include cooling heat resistance at low cost, efficient hydrolysis of immobilized enzyme as well, provided are a saccharifying fermentation, etc. minimization of contamination.
[26]The present invention refers to a heat-resistant strains of Kluyveromyces marxianus (Kluyveromyces Marxianus) So that xylitol was expressed from recombinant yeasts having excellent production unit. Of the group consisting of Kluyveromyces marxianus candidate experiment 7 (Kluyveromyces Marxianus) During strains of Kluyveromyces marxianus (Kluyveromyces Marxianus) High yield production of xylitol 36907 and viscoelasticity.
[27]Then, random mutagenesis of Kluyveromyces marxianus through (Kluyveromyces Marxianus) Mutant strain of Kluyveromyces marxianus 36907 xylitol than a circular structure (KluyveromycesMarxianus) To ensure 36907 provided FMEL1 (KCTC18459P) queue. Of Kluyveromyces marxianus (Kluyveromyces Marxianus) It is a wool strain of Kluyveromyces marxianus 36907 provided FMEL1 (KCTC18459P) is (KluyveromycesMarxianus) Than xylitol concentration 36907 120%, production yield 39%, 2 times been increased throughput xylitol.
[28]In the present invention for generating random mutations ethyl methane sulfonate (Ethyl methanesulfonate, EMS) a temperature, the material is treated with DNA alkylation reaction of guanine and thymine bases undergo normal distortion and complementary coupling means, the mote it pushed with alkylated is guanine, guanine allow combining alkylated the mote it pushed respectively characterized by disclosed.
[29]One aspect of EMS mutagenesis after derived, DNA single the first node number is desired, G-a C base pairs to A a-T, T-a A transitioning from a point pebble area along diatomic C-a G base pairs caused other. Thereby changing one bio-sequence listing the boundary is equal to code changes with the 3 amino acid consisting of base eventually. This leads to obtain the failure or new function mutations are disclosed.
[30]On the other hand, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of Kluyveromyces marxianus and 36907 (KluyveromycesMarxianus) 36907 Provided FMEL1 resistant strains (KCTC18459P) strain since, in the present invention preferentially relatively high temperature 45 °C xylitol from xylose fermentation database 120 in his time. However, two of Kluyveromyces marxianus (Kluyveromyces Marxianus) To consume both strain 22 - 29 g/L of xylose, xylitol be drooping yield of 10 - 12 g/L have shown.
[31]Thus, in the present invention two of Kluyveromyces marxianus (Kluyveromyces Marxianus) Heat resistant strains on glucose (glucose) in 80 g/L for 45 °C 30 °C further confirms YP-compatible database method for producing fermentation was added. As a result, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of Kluyveromyces marxianus and 36907 (KluyveromycesMarxianus) Acid when used in both strains (KCTC18459P) 36907 provided FMEL1 won, making sure that the capable of exhibiting excellent heat resistance. Only, 30 °C glucose in production rates and ethanol consumption in an almost similar slightly relative to his character 45 °C. Or more with the result of the present invention two of Kluyveromyces marxianus (Kluyveromyces Marxianus) Strains used for maintaining heat resistance is very low when xylose carbon won ATP biosynthesis were determined.
[32]The, xylitol from xylose culture temperature 45 °C 40 °C 5 °C than the low end of a gate and effectively conducts an experiment, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of Kluyveromyces marxianus and 36907 (Kluyveromyces Marxianus) Of xylitol concentration higher than 45 °C (KCTC18459P) 36907 provided FMEL1 fecundity is appeared. 40 °C 144 result in time fermentation, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of xylitol production is 25 g/L (KCTC18459P) 36907 provided FMEL1 fluorescence, of Kluyveromyces marxianus (Kluyveromyces marxianus) 36907 Min of xylitol production is 14 g/L.
[33]On the other hand, in the present invention for optimizing productivity xylitol at high temperature [...] conducts an experiment but, 40 °C, 400 rpm conditions xylitol significant productivity and nickel.
[35]Hereinafter, in the embodiment of the present invention to more detailed content via a broadcast receiver. But, in the embodiment of the present invention is not limited only to scope of the invention, the technical idea of deformation of equivalent comprises corn.
[37][In the embodiment1:7 IsKluyveromycesGene from (Kluyveromyces Marxianus) Acid Xylitol Excellent productivity K. Marxianus Strain selection]
[39]The xylitol production in recent experiments of Kluyveromyces marxianus is used for 7 (Kluyveromyces marxianus)Strain was used in.
[40]Of Kluyveromyces marxianus having a total of 7 (KluyveromycesMarxianus)Strain (124224, 46537, 200963, 36907, 26548, CBS 1555, and CBS 607) for culturing the, each strain YP medium added with 20 g/L of glucose (10 g/L yeast extract, 20 g/L Bacto peptone) to him as about 200 rpm and 30 °C.
[41]Then, to the surface of cultured cells as well as in number twice for sterilising.
[43] (2) Xylitol Excellent productivity KluyveromycesGene from (Kluyveromyces marxianus) Strain selection
[44]Said cultured in Kluyveromyces marxianus (Kluyveromyces Marxianus)In order to identify through a xylitol strains, each strain in a medium added with 80 g/L of xylose YP 200 rpm, 30 °C him as during about 4. After 4, measures have shown to table 1 for each strain xylitol production capacity.
[45]The measurement of the xylitol Rezex Roa provided oRGANIC aCID H + (8%) Column (Phenomenex Inc. , Torrance, CA) column and ' 0. 005 N concentration of H2SO 4' 0 °C using, 0. Performance liquid chromatography (HPLC 1200 Series, Agilent Technologies, Santa Clara, CA) rate 6 ml/min when the analyte, xylose, also have a common measuring and analyzing method such as ethanol metabolites of him.
[46]| Strain name | Consumed Xylose (G/L) | Xylitol (g/L) | Xylitol yield(G/g) | Ethanol(G/L) |
| 124224 | 66. 86 | 6. 68 | 0. 01 | 0. 69 |
| 46537 | 75. 46 | 0 | 0 | 1. 91 |
| 200963 | 39. 05 | 2. 82 | 0. 07 | 2. 43 |
| 36907 | 27. 85 | 13. 27 | 0. 48 | 1. 03 |
| 26548 | 25. 32 | 9. 79 | 0. 39 | 0. 68 |
| CBS 1555 | 72. 73 | 0 | 0 | 0 |
| CBS 607 | 59. 11 | 0 | 0 | 0. 72 |
[48]Measurement results, of Kluyveromyces marxianus (KluyveromycesMarxianus)46537 Of Kluyveromyces marxianus strain (KluyveromycesMarxianus)CBS 1555 in the case of strain, each 75. 46 G/L and 72. 73 G/L xylose consumption while exhibit high, are totally xylitol production nor gone to, ethanol production are also 0 g/L and 1. 91 G/L was to waste.
[49]While, of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 In the case of strain, the xylose consumption 27. Was 85 g/L, xylitol production is 13. As shown in to 27 g/L, 0. 48 G/g high xylitol yield bix, ethanol production is 1. 03 G/L as signal peptides with low.
[50]On the other hand, of Kluyveromyces marxianus (KluyveromycesMarxianus)26548 In the case of strain, 9. 79 G/L when the temperature of the storehouse, of Kluyveromyces marxianus remaining (KluyveromycesMarxianus)124224, 200963 Strains and CBS 607 xylitol or not, his throughput the waste.
[51]Or more and classifies the result of induced by solar heat, of Kluyveromyces marxianus of 7 (Kluyveromyces Marxianus)In the case of strain during most, but high xylose consumption, insignificantly xylitol to have, in addition is not suitable for the production of xylitol from xylose produced ethanol also be administered parenterally.
[52]However, of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 In the case of strain, yield the highest xylitol production and xylitol is shown, suitable for the production of xylitol from xylose strains were predetermined position.
[53]The, finally of Kluyveromyces marxianus (Kluyveromyces Marxianus)36907 Facia expressed from recombinant yeasts, as well as to in in the embodiment 2.
[55][In the embodiment 2: KluyveromycesGene from (KluyveromycesMarxianus)36907 Mutant strain of screening]
[56]In said number of Kluyveromyces marxianus produced therewith (Kluyveromyces Marxianus)36907 Strain in a medium added with 20 g/L 12 hours the molecular pathway YP obtained strains are cultured in the cell culture. First, a mutant form of the strain obtained in order to identify an optimal time for inducing 1. 0 M EMS of each 10 minutes, 20 minutes, 30 minutes and 40 minutes of Kluyveromyces marxianus processing (Kluyveromyces Marxianus)36907 Strain cell death rate were measured.
[57]Measurement results, 1. 0 M 30 minutes when it has processed a of EMS, average 99. 90 To 99. The visibility of 99% cell death rate formate, various of Kluyveromyces marxianus about said random mutation (KluyveromycesMarxianus)36907 Number mutant strain was a high pressure liquid coolant.
[58]Then, each strain of 20 g/L xylose added 580 of Kluyveromyces marxianus YP agar medium of total request the herpesvirus (KluyveromycesMarxianus)36907 Mutant expressed from recombinant yeasts was to separate.
[59]Said 580 of Kluyveromyces marxianus of isolated (Kluyveromyces Marxianus)36907 Mutant strain of xylitol productivity crucial expressed from recombinant yeasts of xylitol was through a function key for each strain.
[60]Said two of Kluyveromyces marxianus isolated from 580 (Kluyveromyces Marxianus)36907 1 Mutant strain. 5 Ml of 80 g/L of xylose added YP medium (24 - well plate) at a temperature of 30 °C in a few 4 using cell cultivator during aeration chamber, after 4, each strain was measured generation xylitol production yield xylitol among high expressed from recombinant yeasts.
[61]Sorting result, xylitol production yield high 10 of Kluyveromyces marxianus of (KluyveromycesMarxianus)36907 A selected mutant strain has been, 250 ml of 80 g/L xylose medium added with 50 ml of each strain to initial absorbance YP flasks (OD600 ) Is 1. 0 So that after inoculation, 30 °C, about 144 hours 100 rpm by using xylitol production yields were measured.
[62]Measurement results, said 10 during strain produced when the two most xylitol expressed from recombinant yeasts, of Kluyveromyces marxianus same (KluyveromycesMarxianus)In the embodiment referred to as well as to 36907 provided FMEL1 (KCTC18459P) and in.
[64][In the embodiment 3: KluyveromycesGene from (KluyveromycesMarxianus)36907 And KluyveromycesGene from (KluyveromycesMarxianus) 36907 Provided FMEL1 (KCTC18459P) strain of xylitol production capacity comparison]
[65]The number of Kluyveromyces marxianus in said experiments in produced therewith (Kluyveromyces Marxianus)36907 Of Kluyveromyces marxianus on (Kluyveromyces Marxianus)36907 Provided FMEL1 strains was a leakage through a xylitol (KCTC18459P).
[66]Said two strain in a medium added with 80 g/L of xylose YP 30 °C, 100 rpm to about 144 hours fermentation production of xylitol was measuring and comparing.
[67]Measurement results, of Kluyveromyces marxianus (KluyveromycesMarxianus) Strains of 78 g/L xylose (KCTC18459P) 36907 provided FMEL1 consume 53 g/L when the temperature of the storehouse, 0. 67 G/g of xylitol raised 0. 36 G/L h reducting the productivity of xylitol and viscoelasticity.
[68]This it is a wool strain of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 Strains 120% compared xylitol concentration, production yield 39%, an article 2 throughput xylitol are disclosed (also 1) result. Of Figure 1 A random mutations of Kluyveromyces marxianus is through enhanced productivity xylitol (KluyveromycesMarxianus) 36907 Xylose to xylitol production strains added result confirming the fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol)
[69]Of Figure 1 B is random mutations of Kluyveromyces marxianus through enhanced productivity xylitol (KluyveromycesMarxianus) Strains (KCTC18459P) xylose to xylitol added 36907 provided FMEL1 confirmed result production fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol)
[70]In addition, advantageously interestingOf Kluyveromyces marxianus (Kluyveromyces Marxianus) 36907 Provided FMEL1 strains (KCTC18459P) throughput ethanol 1. 7 G/L has been delivered, the microorganism of enhanced xylitol production capacity has been maintained even after passaging of at least 10.
[71]The result, selected of Kluyveromyces marxianus (Kluyveromyces Marxianus)(KCTC18459P) strain quite suited for the mass production of xylitol 36907 provided FMEL1 strains result by position of are disclosed.
[73][In the embodiment 4: KluyveromycesGene from (KluyveromycesMarxianus)36907 And KluyveromycesGene from (KluyveromycesMarxianus)(KCTC18459P) identifying 36907 provided FMEL1 heat resistant strain
[74]The experiment of Kluyveromyces marxianus in (Kluyveromyces Marxianus) Of Kluyveromyces marxianus and 36907 (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) heat resistance function key of a strain to him.
[75]A heat-resistant strains of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 Of Kluyveromyces marxianus and (KluyveromycesMarxianus)Method for producing strains (KCTC18459P) xylose 36907 provided FMEL1 YP added high temperature was 45 °C 144 in fermentation time.
[76]Experiment result, both of said two strains of xylitol to consume 10 provided 12g/L 22 provided 29g/L xylose has been confirmed (not shown) representing the result be drooping
[77]The, based on a result of said further confirms the heat resistance to be but two strains, each strain on glucose added 80 g/L in two 45 °C 30 °C YP optimum medium glucose fermentation time was 12 database.
[78]Experiment result, of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 Of Kluyveromyces marxianus strain (KluyveromycesMarxianus)(KCTC18459P) acid when used won 36907 provided FMEL1 strains, were able to confirm that the charge excellent heat resistance. But, when cultured in glucose consumption and production rates and ethanol 30 °C 45 °C character was cultured in a slight elevated compared with the case in an almost similar.
[79]The results of experiments when used to xylose carbon won through the strains, for maintaining very low heat resistance ATP biosynthesis were confirmed (2 also). The, xylitol from xylose culture temperature in order to further reduce the number of passes is been determined.
[80]Of Kluyveromyces marxianus is of Figure 2 A and B (Kluyveromyces Marxianus) Each 30 °C and 40 °C 36907 glucose fermentation results in supply are disclosed. (■: glucose, ○: O. D. , Δ: ethanol)
[81]Of Kluyveromyces marxianus is of Figure 2 C and D (Kluyveromyces Marxianus) In each 30 °C and 40 °C (KCTC18459P) glucose fermentation 36907 provided FMEL1 result supply are disclosed. (■: glucose, ○: O. D. , Δ: ethanol)
[83][In the embodiment5:40 °C Conditions, KluyveromycesGene from (Kluyveromyces Marxianus)36907 And KluyveromycesGene from (KluyveromycesMarxianus)36907 Provided FMEL1 strains (KCTC18459P) Xylitol Production confirmation]
[84]Said experiment result, the present invention of Kluyveromyces marxianus (Kluyveromyces Marxianus)36907 Of Kluyveromyces marxianus strain (Kluyveromyces Marxianus)(KCTC18459P) strains of xylose culture in which 45 °C 36907 provided FMEL1 carbon won significant heat resistance appears to exhibit not, the embodiment 45 °C 5 °C 40 °C conditions than low temperature experiments these strains of his function key production of xylitol.
[85]The same experiment method 40 °C temperature condition in addition to said in the embodiment 4 to xylose fermentation time added YP 144 in a medium by dielectrophoresis.
[86]Experiment result, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of xylitol production is 25 g/L (KCTC18459P) 36907 provided FMEL1 fluorescence, of Kluyveromyces marxianus (KluyveromycesMarxianus)36907 Min of xylitol production is 14 g/L. Of Kluyveromyces marxianus [...] advantageously hereinafter (KluyveromycesMarxianus) Is about 2 times throughput compared 45 °C (KCTC18459P) 36907 provided FMEL1 culture has been extended result.
[87]In addition, of Kluyveromyces marxianus (KluyveromycesMarxianus) Of Kluyveromyces marxianus 36907 provided FMEL1 (KCTC18459P) is (Kluyveromyces Marxianus)36907 Result compared to 76% (3 also) has been increased.
[88]Or more result, of Kluyveromyces marxianus in 40 °C (Kluyveromyces Marxianus)(KCTC18459P) of Kluyveromyces marxianus 36907 provided FMEL1 of xylitol productivity (KluyveromycesMarxianus)36907 Were significantly higher than has been confirmed.
[89]In strains of Kluyveromyces marxianus (Kluyveromyces marxianus) of Figure 3 A is 40 °C 36907 xylose to xylitol production confirming the result added fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol)
[90]Of Kluyveromyces marxianus of Figure 3 B is in 40 °C (Kluyveromyces Marxianus) Strains (KCTC18459P) xylose to xylitol added 36907 provided FMEL1 confirmed result production fermentation in a medium are disclosed. (○: O. D. , ◆: xylitol, ■: xylose, Δ: ethanol)
[92][In the embodiment 6: Xylitol For improving productivity KluyveromycesGene from (Kluyveromyces marxianus) Strains 36907 provided FLEL1 [...] optimization]
[93](1) [...] Identifying optimal conditions
[94]In said in the embodiment of Kluyveromyces marxianus out bits (Kluyveromyces Marxianus)(KCTC18459P) strain fermentation culture productivity of xylitol 36907 provided FMEL1 stirring rate was even.
[95]Of Kluyveromyces marxianus (KluyveromycesMarxianus) Strains of 80 g/L xylose (KCTC18459P) added YP 36907 provided FMEL1 is cultured in medium, 300, 350, 400, 450 and 500 rpm respectively about 30 °C and 40 °C 4 strains when the xylitol production by culturing cap during each comparison, table 3 to 4 and 4 to 5 shown to also to culture result.
[96]30 °C the culture conditions result in 300, 350, 400, 450 and 500 rpm| Rpm | Optical Density(A600Nm) | ConsumedXylose (g/L) | Xylitol (g/L) | Yield (g/g) | Productivity(G/Lh) |
| 300 | 12 | 49 | 23 | 0. 46 | 0. 23 |
| 350 | 37 | 68 | 35 | 0. 44 | 0. 29 |
| 400 | 31 | 77 | 41 | 0. 53 | 0. 57 |
| 450 | 55 | 78 | 26 | 0. 33 | 0. 44 |
| 500 | 55 | 71 | 9 | 0. 12 | 0. 14 |
[98]The culture conditions result in 300, 350, 400, 450 and 500 rpm 40 °C| Rpm | Optical Density (A 600 nm) | Consumed Xylose (g/L) | Xylitol (G/L) | Yield (g/g) | Productivity (G/Lh) |
| 300 | 5. 62 | 42 | 24 | 0. 57 | 0. 25 |
| 350 | 10. 40 | 60 | 36 | 0. 59 | 0. 49 |
| 400 | 18. 60 | 68 | 43 | 0. 64 | 0. 90 |
| 450 | 36. 00 | 77 | 31 | 0. 42 | 0. 46 |
| 500 | 36. 04 | 75 | 17 | 0. 20 | 0. 36 |
[100]Experiment result, 40 °C, 400 rpm of Kluyveromyces marxianus cell culture conditions (KluyveromycesMarxianus)36907 Provided FMEL1 (KCTC18459P) throughput 43 g/L of a strain to xylitol, 0 is made of graphite. 90 (G/L, h) body as, compared to other stirring speed conditions the highest has been confirmed.
[101]The, 40 °C conditions optimized for high yield of xylitol at high temperatures, of about 400 rpm has been confirmed (also 4 and 5).
[102]Figure 4 30 °C of Kluyveromyces marxianus in (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain (A) each 300, 350 (B), (C) 400, 450 and 500 rpm (D) (E) measured about liquor xylitol production are disclosed. (■: xylose, ◇: xylitol, ●: O. D. , Δ: ethanol)
[103]Figure 5 40 °C of Kluyveromyces marxianus in (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain (A) each 300, 350 (B), (C) 400, 450 and 500 rpm (D) (E) measured about liquor xylitol production are disclosed. (■: xylose, ◇: xylitol, ●: O. D. , Δ: ethanol)
[105](2) Said [...] As the biomass productivity of xylitol compared using optimized conditions cap incubator
[106]The in the embodiment at each of Kluyveromyces marxianus [...] in optimum conditions based on said confirmed (KluyveromycesMarxianus) Fermenting strains in xylitol was a leakage productivity 36907 provided FLEL1 viable culture flasks.
[107]'40 °C, 400 rpm' on '30 °C, 400 rpm' optimally of Kluyveromyces marxianus (KluyveromycesMarxianus)(KCTC18459P) strain cultured in YP added 80 g/L of xylose 36907 provided FMEL1 facia, cap culture time in the case of 144, when 72 each of fermentation time, 60 him as time.
[108]Experiment result, optimum conditions of fermentation (40 °C, 400 rpm) result of increasing amount of xylitol in productivity as compared to about 6 culture flasks to 1:1 by capable of doubling (6 also). Of Kluyveromyces marxianus in Figure 6 30 and 40 °C (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P) strain prepared liquor xylitol production results of a comparison each cap and are disclosed.
[110]<bio-deposit id="biod-00001" num="0001"><depositary>Korean bio-technology Institute</depositary><bio-accno>KCTC18459P</bio-accno><date>20160405</date></bio-deposit>
[1]The present invention relates to Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) with improved xylitol production activity through mutation and a production method of xylitol using the same, wherein xylitol can be mass-produced at a relatively high temperature.
Of Kluyveromyces marxianus xylitol enhanced (Kluyveromyces Marxianus) 36907 Provided FMEL1 (KCTC18459P).
Xylose medium added, of Kluyveromyces marxianus (Kluyveromyces Marxianus) To be inoculated with a culture of 36907 provided FMEL1 (KCTC18459P) method characterized by production of xylitol.
According to Claim 2, said culture can, 30 provided 40 °C, optimally performing 350 provided 450 rpm which produces xylitol production method.
