한수지 47(5), 516-521, 2014 Original Article Kor J Fish Aquat Sci 47(5),516-521,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순 박희연 이레나코네바 1 국립수산과학원식품안전과, 1 태평양수산연구소틴로센터 Biochemical Characterization of α-galactosidase-producing Thermophilic Bacillus coagulans KM-1 Ki Ho Nam*, Mi Soon Jang, Hee Yeon Park and Elena Koneva 1 Food and safety Research Division, National Fisheries Research and Development Institute, Busan 619-705, Korea 1 TINRO Centre, Pacific Scientific Research Fisheries Centre, Shevchenko Alley, Vladivostok 690091, Russia A bacterium producing α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) was isolated. The isolate, KM-1 was identified as Bacillus coagulans based on its 16S rrna sequence, morphology, and biochemical properties. α-galactosidase activity was detected the culture supernatant of B. coagulans KM-1. The bacterium showed the maximum activity for hydrolyzing para-nitrophenyl-α-d-galactopyranoside (pnp-αgal) at ph 6.0 and 50 C. It hydrolyzed oligomeric substrates such as melibiose, raffinose, and stachyose liberating a galactose residue, indicating that the B. coagulans KM-1 α-galactosidase hydrolyzed α-1,6 linkage. The results suggest that the decreased stachyose and raffinose contents in fermented soybean meal are due to the α-galactosidase activity. Key words: Bacillus coagulans KM-1, α-galactosidase, Stachyose, Raffinose, Fermented soybean meal 서론 (fish meal). (Kim et al., 2013)., sucrose, maltose, raffinose, stachyose arabinnan. stachyose raffinose -galactosyl -galactosidase ( -D-galactoside galactohydrolase, EC 3,2,1,22) raffinose stachyose,, (Rackis, 1981) (Reddy et al., 1980; Steggerda et al., 1968). (Yoo et al., 2007). (Li et al., 1991). -galactosidase (Min et al., 2002; Kim and Yoon, 1998; Choi et al., 1998; Kim et al., 2004; Irish et al., 1995) -galactosidase (park., 2011; Kim. et al., 2002; Kum and Yoon., 1998; Choi et al., 1998). stachyose raffinose., -galactosidase,,, galactose galactomannan http://dx.doi.org/10.5657/kfas.2014.0516 Kor J Fish Aquat Sci 47(5) 516-521, October 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/)which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 22 September 2014; Revised 20 October 2014; Accepted 29 October 2014 *Corresponding author: Tel: +82. 51. 720. 2652 Fax: +82. 51. 720. 2669 E-mail address: dennis011@korea.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 516 pissn:0374-8111, eissn:2287-8815
Bacillus coagulans KM-1 의생화학적특성 517 melibiose, raffinose, stachyose galactomannan -1,6 -galactose (Dey and Campillo, 1984). (Engraulis japonica) -galactosidase. 배지및시약 재료및방법 Nutrient broth (NB, Difco, St. Louis, MO, USA) Nutrient Agar (NA, Difco) NA. glycerol 15% (V/V) -75 (IIshin DF8514l, Seoul, Korea). 균주의분리 (raw anchovy, Engraulis Japonica ) 10 g 40 ml 5 10 ml 100 ml NB 5 ml 50, 100 rpm (VISION SCI., VS-8480SR, Daejeon, Korea) 24. 1 ml NA 50 incubator colony. α-galactosidase 의활성측정 1 ml 12,000 g 15 -galactosidase. -galactosidase p-nitrophenyl- -D-galactopyranoside (pnp Gal) (Food Research International, 2009)., 700 L 100 mm potassium phosphate buffer (ph 6.5) 100 L 200 L 9.9 mm pnp Gal 5 50, 2 ml 0.2 M borate buffer (ph9.8) 405 nm -galactosidase. 미생물균주의최적발육조건분석 30-70 12 660 nm (Hitachi U-2900, Tokyo, Japan) ph NB ph 4.0-8.0 50 12 660 nm. 주사전자현미경을이용한균주의관찰 Scanning Electron Microscopy (SEM, S-2400, Hitachi, JAPAN). SEM 0.1 M sodium phosphate buffer (ph 7.2-7.4) 2.5% glutaraldehyde (TAAB Laboratoties Equipment Ltd, Berks, UK) 3 0.1 M sodium phosphate buffer 2 1% osmium tetroxide (Sigma, St Louis, MO, USA) 30. 50-100% 5 100% hexamethyldisilazane. SEM 20 kv 7500. 미생물균주의생화학적특성조사 API- ZYM (BioMèrieux, France) 19 -galactosidase API-50CH (BioMèrieux, France). 16S rrna 유전자염기서열과계통수 (phylogenetic tree) 분석 16S rdna., 16S rdna DNA PCR. DNA Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, USA), DNA sequencer (Applied Biosystems model 3730XL, USA). Ez- Taxon server 2.1. NCBI BLASTN 50 EMBL-EBI ClustalW2 Neighbor Joining (Saitou and Nei, 1987) phylogenetic tree. 난소화성탄수화물분해능측정 (Scalabrini et al., 1998) stachyose raffinose 10 g/l NB 5% (w/v) 50 48. 1 ml 12,000 g 15 250 µl 750 L 0.2 m (membrance filter). Stachyose raffinose APS-2 hypersil column (thermo scientific; 250 4.6 mm, 5 m, 120A)
518 남기호ㆍ장미순ㆍ박희연ㆍ이레나코네바 (column, 40 ;, 75% ACN;, 1.5 ml/min). RI (refractive index detector, Dionex). 미생물균주가생산하는 α-galactosidase 의최적반응온도측정 -galactosidase 50 48 8,000 rpm 20. ammonium sulfate 35-85% 4 12. 13,000 g 20 50 mm Tris-HCl (ph7.0) 4 12. -galactosidase p-nitrophenyl- -D-galactopyranoside (pnp Gal) (Sigma, St. Louis, MO, USA). 700 L 100 mm potassium phosphate buffer (ph 6.5) 200 L 9.9 mm pnp Gal 100 L 5 30-70, 2 ml 200 mm borate buffer (ph 9.8) 405 nm -galactosidase. 1 unit 1 1 mol p-nitrophenol, (specific activity) 1 mg 1 unit. 결과및고찰 미생물균주의분리및 α-galactosidase 활성 NA NB colony 20 -galactosidase. (Table 1) KM-1 0.482 Unit/mL 19 stachyose raffinose. 배양시간에따른 KM-1 균주의발육조건 -galactosidase KM-1 Fig. 1. 45-55 50. ph, ph 5.5-7.0 ph 6.0. ph,, -galactosidase (Kim and Yoon, 1998) ph 7.0-7.5. 주사전자현미경을이용한균주의관찰 Table 1. α-galactosidase activity of microorganisms isolated from raw anchovy O.D. (660 nm) Isolate Activity (U/mL) Isolate Activity (U/mL) KM-1 0.482 KM-11 0.154 KM-2 0.230 KM-12 0.161 KM-3 0.128 KM-13 0.143 KM-4 0.153 KM-14 0.112 KM-5 0.133 KM-15 0.113 KM-6 0.112 KM-16 0.135 KM-7 0.154 KM-17 0.164 KM-8 0.151 KM-18 0.160 KM-9 0.152 KM-19 0.135 KM-10 0.132 KM-20 0.157 0.3 0.2 0.1 0 35 40 45 50 55 60 Temperature ( ) Fig. 1. Effect of temperature on growth of Bacillus coagulans KM-1 after 12 hours cultivation. O.D. (660 nm) 0.3 0.2 0.1 0 4 4.5 5 5.5 6 6.5 7 7.5 8 Fig. 2. Effect of ph on growth of Bacillus coagulans KM-1 after 12 hours cultivation. ph
Bacillus coagulans KM-1 의생화학적특성 519 20 -galactosidase KM-1 KM-1 (Gram staining) (Spore staining), KM-1 (Fig. 3) 0.5-0.7 m, 3.3-4.4 m 2 (long rod). (catalase) (Bacillus sp.). API-ZYM 과 API-50CHL Kit 를이용한균주의특성 KM-1 API-ZYM (BioMèrieux, France) 19 (Table 2) KM-1 alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, phosphatase acid, naphthol-asbi-phosphohydrrolase, -galactosidase, -galactosidase -glucosidase. API-50CH (BioMèrieux, France) 50 (Table 3) glycerol, ribose, D-xylose, galactose, D-glucose, D- fructose, D-manose, N-acetyl glucosamine, maltose, melibiose, trehalose, starch, D-arabitol 5-keto-gluconate. 16S rrna 유전자염기서열분석과계통수 (phylogenetic tree) -galactosidase KM-1 16S rdna B. coagulans 99.797% (3/1478), B. acidiproducens 97.171% (40/1414), B. shackletonii 95.831% Fig. 3. Scanning electron microscopy image of Bacillus coagulans KM-1 ( 7,500). Table 2. Enzyme activities of Bacillus coagulans KM-1 by API- ZYM kit Enzyme Activity Enzyme Activity Control - Phosphatase acid + Phosphatase alcaline + Naphthol-ASBIphosphohydrrolase Esterase (C4) + α-galactosidase + Esterase lipase (C8) + β-galactosidase + Lipase (C14) - β-glucuronidase - Leucine arylamidase + α-glucosidase + Valine arylamidase - β-glucosidase - Cystine arylamidase + N-acetyl-βglucosaminidase - Trypsin - α-mannosidase - α-chymotrypsin - α-fucosidase - +, positive; -, negative. Table 3. Carbohydrate utilization pattern of Bacillus coagulans KM-1 by API-50CH kit Carbohydrate Activity Carbohydrate Activity Control - Esculin - Glycerol + Salicin - Erythritol - Celobiose - D-arabinose - Maltose + L-arabinose - Lactose - Ribose + Melibiose + D-xylose + Sucrose - L-xylose - Trehalose + Adonitol - Inulin - β-methyl-d-xyloside - Melezitose - Galactose + D-raffinose - D-glucose + Starch + D-fructose + Glycogen - D-manose + Xylitol - L-sorbose - β-gentiobiose - Rhamnose - D-turanose - Dulcitol - D-lyxose - Inositol - D-tagatose - Manitol - L-fucose - Sorbitol - D-arabitol + α-methyl-d-manoside - L-arabitol - α-methyl-d-glucoside - Gluconate - N-acetyl glucosamine + 2-keto-gluconate - Amygdalin - 5-keto-gluconate + Arbutin - +, positive; -, negative. +
520 남기호ㆍ장미순ㆍ박희연ㆍ이레나코네바 Fig. 4. Phylogenetic tree of Bacillus coagulans KM-1 based on 16S rdna sequencing. Table 4. Non-digestible carbohydrate resolution of Bacillus coagulans KM-1 Carbohydrate (62/1487). KM-1 B. coagulans KM-1 (Fig. 4) (NCBI) (JX569800, 2012. 9. 4) (KCCM 112989P, 2012. 9. 6). 난소화성탄수화물분해능 Fermentation time (h) 0 24 48 Raffinose 0.75* 0.56 0.39 Stachyose 0.49 0.25 0.16 Saccharose 0 0.23 0.42 galactose 0 0.33 0.49 *mg/100 g B. coagulans KM-1 (Table 4) stachyose raffinose 0.49 mg/100 g, 0.75 mg/100 g 24 0.25 mg/100 g, 0.56 mg/100 g, 48 0.16 mg/100 g, 0.39 mg/100 g. Stachyose raffinose saccharose galactose B. coagulans KM-1 -galactosidase stachyose raffinose. Kim and Yoon (1998) -galactosidase stachyose raffinose -galactosidase. 미생물균주가생산하는 α-galactosidase 의최적반응온도 B. coagulans KM-1 -galactosidase Fig. 5. B. coagulans KM-1 α-galactosidase activity (Unit/ ml) 0.1 0.08 0.06 0.04 0.02 0 30 40 50 60 70 Temperature ( ) Fig. 5. Effect of temperature on α-galactosidase activity of Bacillus coagulans KM-1.
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