1. 2010 6 1, 2 4 Ⅰ, Ⅱ 2013,,. Bio-ethanol, 2013 STEAM R&E Stropharia rugosoannulata Bio-ethanol Bio-ethanol 30 2014 ISEF-K. 2014 Cellulose Bio-ethanol., Bio-ethanol,. 2010 10 ABS(Access to genetic resource and Benefit Sharing),,, BT. ABS. - 1 -
6 (white rot fungi) 5, 3 HPLC 2., (true fungi) (fungus-like organisms),,..., cellulose..... PCR RAPD - PCR PCR(Polymerase Chain Reaction, ) DNA DNA DNA DNA. PCR DNA 1~2-2 -
, DNA. PCR Denaturation, Annealing, Extension 3. Denaturation DNA, Annealing DNA primer. DNA Polymerase primer DNA. Ribosomal DNA 3 ribosome (18S, 5.8S, 28S) ITS(Internal Transcribed Spacer) region 1. PCR 18S rdna 5.8S rdna ITS1 5.8S rdna 28S rdna ITS2, 5.8S rdna. ribosomal DNA Fig. 1 18S rdna ITS1, 5.8S rdna, ITS2, 28S rdna. ITS1 ITS4 18S ITS1 5.8S ITS2 28S Fig. 1. rdna - RAPD PCR RAPD (random amplified polymorphic DNA) PCR. PCR primer genome fragment. fragment band. band,. RAPD PCR genome sequencing. - 3 -
- (, 2012) -. Stropharia rusosoannulata (, 1999) glucose, yeast extract, KCl, MgSO₄, FeSO₄. Trichoderma reesei (, 1998). ( : Bio-Logic) cellulose cellulose bio-ethanol biomass(, ) cellulose-biomass bio-ethanol. cellulose 2012 bio-ethanol 1~3%. ethanol, bio-ethanol,.,. - 4 -
(ABS: Access to genetic resources and Benefit Sharing).,, bio-ethanol. bio-ethanol simul fermentation 6,. 1. - 6. - DNA sequencing,. 2. - 6 naphthalene, (3 ) UV-Visible spectrophotometer. -,. 3. - YMG, pre-medium homogenizer - 2 fermentation medium - 1, 2, 4, 6, 8, 10-5 -
4. - 3-1, 2, 4, 6, 8, 10-3 - PDA YMG pre-medium. - PDA YMG pre-medium (, ph, Shaking culture, Light Dark condition). - PCR( Polymerase Chain Reaction). - (5,, ) (spectrophotometer). - cellulase. - (5,, ) yeast (Saccharomyces cerevisiae). - yeast(saccharomyces cerevisiae) HPLC( ). - - 6 -
2014 STEAM R&E ( ) 3 4 5 6 7 8 9 10 11 12 예산계정 계획 집행예산 증감 연구장비및재료비 연구활동비 연구과제추진비 연구수당 " DNA ", -80 Potato Dextrose Agar (PDA) 1. NCBI GenBank rdna ITS 6. Table. 1.. - 7 -
Table. 1. The mushroom species used in this study Scientific name Local name Strain Origin Trametes pubescens 흰융털구름버섯 IUM0142 Korea (Kyonggi-do) Flammulina velutips (wild) 팽이 ( 야생 ) IUM5207 Korea (Incheon) Irpex lacteus 기계층버섯 IUM5379 Korea (Kyonggi-do) Flammulina velutips (cultivated) 팽이 ( 재배종 ) IUM2809 Korea, (Seoul) Perreniporia fraxinea 아까시재목버섯 IUM5422 Korea (Kyonggi-do) Irpex consors 송곳니구름버섯 IUM5384 Korea (Kyouggi-do) -. Potato Dextrose Agar(PDA) 1-2. 2 (clamp connection). Fig. 2. 꺽쇄결합 -. Total DNA plate 5mm cork borer Potato Dextrose Broth (PDB) 100 ml 5 120 rpm 25 10. (freezing dry).. 1.5 ml micro tube 500 mg 500 μl protein lysis buffer (50 mm Tris-HCl (ph 7.5), 50 mm EDTA (ph 8.0), 1% sarkosyl) 65 1. 500 μl P.C.I (Phenol : Chloroform : Isoamylalcohol, 25 : 24 : 1) 30 12,000rpm 4 10. tube 500μl 4 5 12,000 rpm DNA. DNA 500 μl 70% 12,000 rpm, 4 5-8 -
DNA. tube DNA pellet DNA, spectrophotometer 260 nm 280 nm Total DNA DNA 20. -. rdna ITS DNA ITS region White (1990) ITS region primer set ITS1(forward) ITS4(reverse) Table. 2.. Table. 2. Primer design for PCR amplification of ITS region used in this study Primer Direction Sequence (5'-3') Length ITS1 forward TCC GTA GGT GAA CCT GCG C 19mer ITS4 reverse TCC TCC GCT TAT TGA TAT GC 20mer PCR( ) BIONEER Taq polymerase kit tube 20 μl. 14.2 μl 3 ; 2 μl 10X PCR buffer (100 mm Tris-HCl, 1.5 mm MgCl 2, 500 mm KCl, ph 8.3); 1.6 μl dntps (0.2 mm datp, dctp, dgtp, dttp); 0.5 μl (50 pmol/ μl ) ITS1 primer; 0.5 μl (50 pmol/ μl ) ITS4 primer; 0.2 μl Taq DNA polymerase (5U/ μl, Bioneer, Korea); 1 μl DNA (100 μg / μl ). PCR 96 30 denaturation, 55 30 annealing, 72 1 extension 30 cycle. -. Sequencing ( ) PCR PCR product. SOLGENT data Email. sequencing sequence sequence NCBI(National Center for Biotechnology Information) website(http://www.ncbi.nlm.nih.gov) - 9 -
ITS region sequence PCR. -. DNA NCBI ITS region sequence, ITS1 primer binding site(18s rdna partial sequence) ITS1 region, 5.8S rdna, ITS2 region, ITS4 primer binding site(28s rdna partial sequence) 5 20 sequence. ITS 6 NCBI GenBank ITS ( ), ( ) 6 12. sequence text file Clustal X (alignment). sequence PAUP 4.0. (Neighbor-Joining method). bootstrapping 1000. 1000 50%. 2. -. 5 6 PDA (Potato dextrose broth 24.4g, Agar 15g, DW 1,000 ml) 7 25 70%. -. PDB 200ml PDB (Potato dextrose broth 24.4g, DW 1,000ml) cork borer 5mm disc 10 25 shaking incubator 10. - 10 -
Fig. 3. PDB 균사배양 -. PDB + N 250 ml 1% naphthalene PDB (Potato dextrose broth 24.4g, DW 1,000ml) 100 ml (PDB+N), PDB homogenizer PDB+N 25mL 25 10. centrifuge. 4. -. Laccase Laccase Bourbounnais et al.(1995). ABTS 1) 5 mm., 2.4 ml sodium acetate buffer(0.1 M, ph 5.0) 100 ml 30 2 420 nm. (umole) 1 unit. -. Lignin peroxidase Lignin peroxidase(lip) Tien Kirk(1983) veratryl alcohol veratryl aldehyde. 50 mm sodium tartrate buffer (ph 5) 2.2 ml 2 mm veratryl alcohol 40 μl 1) ABTS(2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) - 11 -
240 μl 0.2 mm H O 20 μl 25 310 nm. veratryl alcohol (min) veratryl aldehyde (umole) 1 unit. -. Manganese peroxidase Manganese peroxidase(mnp) Glenn Gold(1985) Mn(II) Mn(III) 0.1M sodium succinate buffer 2.5 ml 0.01% phenol red 0.1 mm MnSO. 2.5 ml 200μl 0.1 mm H O 30 2 5 M NaOH 610 nm. Mn(II) (min) Mn(III) (umole) 1 unit. 3. -. YMG(Yeast extract Malt extract Glucose). Table. 3. autoclave clean bench petri-dish. PDA YMG 25 7. Table. 3. YMG (ph 5.8) (%) yeast extract 0.3 malt extract 0.3 glucose 1 poly peptone 0.3 Agar 1.5 -. pre-medium fermentation medium Table 4 pre-medium autoclave. YMG cork borer 5mm disc 10 25 shaking incubator. 10-12 -
homogenizer. Table. 4 pre-medium Fig. 4. homogenizer 를이용한균사파쇄 (ph 5.8) (%) yeast extract 0.3 malt extract 0.3 glucose 1 peptone 0.3 K HPO 0.1 MgSO 0.01 -. fermentation medium 5 (Table. 6) 2 (1mm, 0.5mm ) 0.5mm. carbon source Table. 5 250ml 100ml. Table. 5 fermentation medium (ph 5.8) (%) yeast extract 0.3 malt extract 0.3 carbon source( ) 2 peptone 0.3 K HPO 0.1 MgSO 0.01-13 -
Table. 6 Poplar sawdust wheat bran Oak sawdust rice bran Typha orientalis -. fermentation medium fermentation medium 40ml 1g 25 shaking incubator. -. 1, 2, 4, 6, 8, 10 1, 2, 4, 6, 8, 10 clean bench syringe needle flask 5ml 20. centrifuge tube 1ml 0, 0.1, 0.25, 0.5, 0.75g/L glucose standard tube 1ml. DNS solution 3ml 95 5 spectrophotometer 540nm. glucose x y standard. standard. Fig. 5. fermentation medium 균사접종 Fig. 6. 배양액추출및시약반응후흡광도측정 4. -. 3 -. 1, 2, 4, 6, 8, 10 3 syringe needle 1ml 1M CrO 5ml 30 spectrophotometer 600nm. - 14 -
standard (y=0.7066x+0.0633, R =0.9999). 3. 1. Table. 7., 6 rdna ITS1, 5.8S, ITS2 Strain IUM 0142 IUM 5207 IUM 5379 IUM 2809 IUM 5422 IUM 5384 Name Trametes pubescns Flammulina velutipes (wild) Irpex lacteus Flammulina velutipes (cultivated) Perreniporia fraxinea Irpex consors Nucleotide distribution Sequence information A C G T G+C(%) A+T(%) ITS1 5.8S ITS2 Length(bp) 173 139 130 187 42.8 57.2 233 159 203 629 144 128 130 204 42.6 57.4 235 159 208 606 132 118 125 208 41.7 58.3 245 158 208 583 145 128 130 203 42.6 57.4 246 159 208 606 127 145 135 162 49.4 50.6 237 158 214 571 136 149 128 159 48.6 51.4 237 158 214 574 Fig. 7. ITS 염기서열분석결과와 NCBI genebank 를통한버섯의계통수분류 rdna Table. 7. rdna (bp). Fig. 7. 9 3-15 -
. 2. Table 8. Mushrooms T.pubescens F.velutipes(W) I.lacteus F.velutipes(C) P.fraxinea I.consors Emzyme concentration (U/mL) Laccase LiP MnP 0.12 0.1 0.09 0.12 0.08 0.09 0.07 0.05 0.04 0.11 0.07 0.09 0.06 0.05 0.05 0.11 0.08 0.04 Fig. 8. 리그닌분해효소활성도 -. Laccase 6 1% 10 laccase(lac). Lac ( ), ( ), (Table 8). Lac 0.12 Uml 1 0.06 Uml 1. -. Lignin peroxidase lignin peroxidase(lip) LiP ( ), - 16 -
, ( ),, (Table. 8). LiP 0.1 Uml 1 0.05 Uml 1. -. Manganese peroxidase manganese peroxidase (MnP) MnP ( ), ( ),,,. (W, C) 0.09 Uml 1 0.04 Uml 1.. 3. -. Table. 9. Poplar 에버섯접종후당발생량변화 (g/l) 1 2 4 6 8 10 흰융털구름버섯 0.1 0.13 0.16 0.19 0.19 0.19 팽이버섯 (w) 0.09 0.18 0.19 0.23 0.23 0.23 기계층버섯 0.09 0.14 0.16 0.19 0.2 0.21 팽이버섯 (c) 0.15 0.25 0.29 0.32 0.33 0.29 아까시재목버섯 0.25 0.84 0.9 0.91 0.88 0.85 송곳니구름버섯 0.21 0.25 0.34 0.34 0.31 0.32 Fig. 9. Poplar 에버섯접종후당발생량변화 popular sawdust. 4~6. 1~4. 6,, (c), (w), - 17 -
,. Table. 10. Poplar sawdust+yeast 에버섯접종후당발생량변화 (g/l) 1 2 4 6 8 10 흰융털구름버섯 0.1 0.13 0.13 0.15 0.12 0.1 팽이버섯 (w) 0.07 0.08 0.13 0.16 0.16 0.14 기계층버섯 0.08 0.1 0.12 0.13 0.1 0.11 팽이버섯 (c) 0.14 0.22 0.25 0.22 0.17 0.17 아까시재목버섯 0.19 0.31 0.35 0.21 0.17 0.15 송곳니구름버섯 0.1 0.19 0.22 0.23 0.21 0.16 Fig. 10. Poplar sawdust+yeast 에버섯접종후당발생량변화 yeast, 4~6. 6 8 yeast ethanol. 4, (c),,, (w),. -. Table. 11. Oak sawdust 에버섯접종후당발생량변화 (g/l) 1 2 4 6 8 10 흰융털구름버섯 0.16 0.22 0.23 0.23 0.31 0.34 팽이버섯 (w) 0.11 0.25 0.32 0.36 0.37 0.37 기계층버섯 0.16 0.23 0.28 0.3 0.32 0.32 팽이버섯 (c) 0.17 0.28 0.31 0.3 0.33 0.33 아까시재목버섯 0.31 0.84 0.85 1.08 1.11 1.08 송곳니구름버섯 0.03 0.55 0.56 0.55 0.59 0.58 Fig. 11. Oak sawdust 에버섯접종후당발생량변화 - 18 -
2 2 0.02.. 4,, (w), (c),,. Table. 12. Oak sawdust+yeast 에버섯접종후당발생량변화 (g/l) 1 2 4 6 8 10 흰융털구름버섯 0.1 0.17 0.17 0.15 0.12 0.07 팽이버섯 (w) 0.1 0.19 0.16 0.12 0.08 0.1 기계층버섯 0.1 0.17 0.14 0.14 0.13 0.14 팽이버섯 (c) 0.14 0.27 0.2 0.11 0.15 0.1 아까시재목버섯 0.1 0.17 0.17 0.13 0.14 0.13 송곳니구름버섯 0.07 0.16 0.1 0.06 0.05 0.07 Fig. 12. Oak sawdust+yeast 에버섯접종후당발생량변화 2. yeast. 2 (c), (w),,,,. 4. -. ethanol Table. 13. Poplar sawdust 에버섯접종후 ethanol 발생량변화 (%) 1 2 4 6 8 10 흰융털구름버섯 0.35 0.48 0.48 0.46 0.45 0.46 팽이버섯 (w) 0.3 0.4 0.38 0.36 0.36 0.34 기계층버섯 0.25 0.32 0.32 0.31 0.34 0.25 팽이버섯 (c) 0.37 0.43 0.43 0.5 0.55 0.48 아까시재목버섯 0.26 0.4 0.36 0.34 0.31 0.31 송곳니구름버섯 0.22 0.42 0.54 0.52 0.55 0.47-19 -
Fig. 13. Poplar sawdust 에버섯접종후 ethanol 발생량변화 Yeast 0.3~0.4%.. Table. 14. Poplar sawdust+yeast 에버섯접종후 ethanol 발생량변화 (%) 1 2 4 6 8 10 흰융털구름버섯 0.4 0.59 0.82 0.9 0.75 0.64 팽이버섯 (w) 0.3 0.88 1.17 0.87 0.81 0.73 기계층버섯 0.43 0.79 0.94 1.08 0.94 0.88 팽이버섯 (c) 0.48 0.8 1.02 1.07 1.01 0.98 아까시재목버섯 0.32 2.18 2.43 2.36 2.29 1.95 송곳니구름버섯 0.52 0.83 1.09 1.02 0.98 0.88 Fig. 14. Poplar sawdust+yeast 에버섯접종후 ethanol 발생량변화 4~6. 6. 4, (w),, (c),,. -. ethanol Table. 15. Oak sawdust 에버섯접종후 ethanol 발생량변화 (%) 1 2 4 6 8 10 흰융털구름버섯 0.23 0.49 0.51 0.54 0.57 0.57 팽이버섯 (w) 0.4 0.59 0.55 0.55 0.55 0.48 기계층버섯 0.38 0.67 0.54 0.55 0.49 0.5 팽이버섯 (c) 0.48 0.43 0.44 0.45 0.55 0.47 아까시재목버섯 0.38 0.53 0.54 0.48 0.5 0.51 송곳니구름버섯 0.35 0.54 0.5 0.5 0.51 0.5-20 -
Fig. 15. Oak sawdust 에버섯접종후 ethanol 발생량변화 popular sawdust yeast ethanol.. Table. 16. Oak sawdust+yeast 에버섯접종후 ethanol 발생량변화 1 2 4 6 8 10 흰융털구름버섯 0.31 0.58 0.98 1.02 0.88 0.83 팽이버섯 (w) 0.49 0.71 1.03 1.05 0.97 0.88 기계층버섯 0.6 0.48 0.76 0.83 0.72 0.71 팽이버섯 (c) 0.64 0.86 0.88 0.95 0.96 0.84 아까시재목버섯 0.93 2.18 2.35 2.43 2.25 2.11 송곳니구름버섯 0.97 1.86 1.96 2.13 2.07 1.99 Fig. 16. Oak sawdust+yeast 에버섯접종후 ethanol 발생량변화 popular sawdust 4~6 6. 6,, (w),, (c),. Oak sawdust Poplar sawdust 6 yeast. Rice bran Wheat bran,, (C),.. - 21 -
2 3 4 0.1%. Bio-ethanol, Bio-ethanol. ABS.,.,,. STEAM,. 4. 2014 (2014.12 ) (2014.12 ) (2015.1 ) (2015.1 ) 2015 The Pathfinder (2015.6) ( )(2016 ~) - 22 -
5. ( ), 2009, HomeBook ( ), 1998, 2 ( ), 2013, http://www.scienceall.com/dictionary/ Detection of Cellulolytic Activity in Ophiostoma and Leptographium species by Chromogenic Reaction( ), 2006 Stropharia rugosoannulata (Studies on the Cellulolytic Enzymes Produced by Stropharia rugosoannulata in Synthetic Medium)( ), 1999 Trichoderma reesei (Effects of Mixed Carbon Sources on the Production of Cellulose by Trichoderma reesei)( ), 1998 Comparison of Dyes for east Detection of Extra cellular Cellulases in Fungi( ), 2007 Roseofomes subflexibilis Cellulase (Studies on the Cultural Characteristics of Cellulase Production by Roseofomes subflexibilis)( ), 2003 Plate screening methods for the detection of polysaccharase-producing microorganisms(h.j.ruijssenaars ), 2000 Degradation of three aromatic dyes by white rot fungi and the production of ligninolytic enzymes(jayasinghe, C. ), 2008 Lignin oxidation by laccase in isozymes from Trametes versicolor(bourbounnais, R ), 1995 Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium(tien, M ), 1983 Utilization of damaged sorghum and rice grains for ethanol production by simultaneous saccharification and fermentation(k. Suresh ), 1999 Productionof bioethanol using lignocellulosic hydrolysate by the white rot fungis Hohenbuehelia sp.zw-16(xiaohui Liang ), 2013 Ethanol production via simultaneous saccharification and fermentation of sodium hydroxide treated corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum(micky Vincent ), 2014-23 -