Korean Journal of Microbiology (2017) Vol. 53, No. 3, pp. 208-215 pissn 0440-2413 DOI https://doi.org/10.7845/kjm.2017.7062 eissn 2383-9902 Copyright c 2017, The Microbiological Society of Korea Article Streptococcus pyogenes 유래 cyclomaltodextrinase 유전자의발현및효소특성 장명운 1 강혜정 2 정창구 3 오규원 1 이은희 1 손병삼 1 김태집 1 * 1 충북대학교대학원축산 원예 식품공학부식품공학전공, 2 충청북도농업기술원친환경연구과, 3 ( 주 ) 에이피테크놀로지 Functional expression and enzymatic characterization of cyclomaltodextrinase from Streptococcus pyogenes Myoung-Uoon Jang 1, Hye-Jeong Kang 2, Chang-Ku Jeong 3, Gyo Won Oh 1, Eun-Hee Lee 1, Byung Sam Son 1, and Tae-Jip Kim 1 * 1 Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju 28644, Republic of Korea 2 Chungcheongbuk-do Agricultural Research and Extesion Services, Cheongju 28130, Republic of Korea 3 Advanced Protein Technologies Co., Suwon 16229, Republic of Korea (Received August 23, 2017; Revised September 19, 2017; Accepted September 20, 2017) A cyclomaltodextrinase (SPCD) gene was cloned from Streptococcus pyogenes ATCC 700294. Its open reading frame consists of 567 amino acids (66.8 kda), which shows less than 37% of amino acid sequence identity with the other CDasefamily enzymes. The homo-dimeric SPCD with C-terminal six-histidines was expressed and purified from Escherichia coli. It showed the highest activity at ph 7.5 and 45 C, respectively. SPCD has the broad substrate specificities against β-cyclodextrin, starch, and maltotriose to produce mainly maltose, whereas it hydrolyzes pullulan to panose. It can also catalyze the hydrolysis of acarbose to glucose and acarviosineglucose. Interestingly, it showed much higher activity on β -cyclodextrin and acarbose than that on starch, pullulan, or maltotriose, which makes SPCD distinguished from common CDase-family enzymes. Although SPCD has significantly high acarbose-hydrolyzing activity, it showed negligible transglycosylation activity. Keywords: Streptococcus pyogenes, cyclomaltodextrinase (CDase), enzymatic characterization, gene expression These authors contributed equally to this work. *For correspondence. E-mail: tjkim@cbnu.ac.kr; Tel.: +82-43-261-3354; Fax: +82-43-271-4412 Glycoside Hydrolase (GH) family 13에속하는 cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (MAase; EC 3.2.1.133), neopullulanase (NPase; EC 3.2.1.135) 는서로다른명칭을가지지만, 일반적으로 cyclodextrinase 계열의효소로분류된다 (Park et al., 2000; Lee et al., 2002; Park, 2006). CDase 계열효소는 Clostridium (Podkovyrov and Zeikus, 1992), Bacillus (Cha et al., 1998; Kang et al., 2009), Thermus (Kim et al., 1999), Paenibacillus (Kaulpiboon and Pongsawasdi, 2004), Lactobacillus (Oh et al., 2005), Lactococcus (Jang et al., 2013), Listeria (Jang et al., 2016) 속등의다양한미생물로부터연구되었다. 이들효소는 glucose로구성된탄수화물중합체의 α-(1,4)-결합부위를가수분해하여주로 maltose를생성하며, 특이적인당전이활성을나타내어 α-(1,6)- 또는 α-(1,3)- 결합의다양한당전이산물을생성하는것으로알려졌다. 따라서자연계에존재하는각종수용체를이용한새로운탄수화물소재의생산에이들효소를활용하는다양한연구가진행되었다 (Park, 2006). 이들 CDase 계열효소는일반적인 α-amylase 계열효소에비해다양한미생물유전체로부터발견되었으나, 아직이들효소
Streptococcus cyclomaltodextrinase 의효소특성 209 의세포내역할에대해서명확히밝혀지지않았다. Klebsiella oxytoca의경우, cyclodextrin glucanotransferase에의해생성된 cyclodextrin이세포내로운반된후, 다시 CDase 계열효소에의해저분자화되는탄수화물대사경로가보고되었다 (Feederle et al., 1996; Fiedler et al., 1996). 또한 Bacillus subtilis 세포내에서 MAase와 pullulanase의작용에의한 glycogen 대사과정이밝혀졌다 (Shim et al., 2009). 최근에 Listeria 속미생물로부터 maltose 또는 maltodextrin 이용관련유전자클러스터와미생물생리학적특성이연구되었다 (Gopal et al., 2010; Jang et al., 2016). 지금까지진행된 CDase 계열효소에대한연구는주로 Bacillus 속미생물유래효소및유전자에집중되었으나, 최근각종미생물의유전체정보가급속히축적되면서보다다양한미생물로부터서로다른 CDase 계열효소유전자를발굴하고이들의효소특성을비교하여신규효소를개발하려는노력이진행되고있다. 본연구에서는미생물유전체데이터베이스분석을통해 Streptococcus pyogenes ATCC 700294 균주로부터 cyclomaltodextrinase (SPCD) 로예상되는유전자를클로닝및발현하였으며, 기질특이성, 가수분해와당전이활성등다양한효소특성을확인하였다. 또한다른 CDase 계열효소와의유전적 효소적특성을비교함으로써산업적으로유용한신규효소개발을위한정보를제공하고자하였다. 재료및방법 미생물및유전자 Streptococcus pyogenes M1 GAS (ATCC 700294) 의염색체 DNA는한국생명공학연구원에서제공받았으며, 대장균내유전자의항시발현은 phceii/ndei (BioLeaders Co.) 를변형시킨 phcxhd (Kang et al., 2009) 벡터를사용하였다. 유전자클로닝및발현은 Escherichia coli MC1061 균주를사용하였다. 시약및재료일반시약, 효소기질및미생물배지는 Sigma-Aldrich와 Duchefa Biochemie에서구입하여사용하였다. 각종제한효소, DNA polymerase, DNA ligase 등유전자조작용효소류는 Roche Applied Science에서구입하였으며, PCR 및 sequencing 프라이머는 Bioneer에서합성하여사용하였다. SPCD 유전자의클로닝 Streptococcus pyogenes의염색체 DNA를주형으로사용하였으며, SPCD-N (5 -TTTTCATATGAATGTTGCCGGACTC- 3 ) 및 SPCD-C (5 -TTTTCTCGAGGTTACTAAACACAA TATATCC-3 ) PCR 프라이머로 SPCD 유전자를증폭하였다. Taq DNA polymerase (Roche) 와 C1000 thermal cycler (Bio- Rad Laboratories Inc.) 를사용하여 94 o C에서 1분, 54 o C에서 30초, 72 o C에서 1분 30초로 30회반복하고, 72 o C에서 5분간추가로증폭하였다. 증폭된유전자는제한효소 Nde I과 Xho I으로절단하고, 항시발현벡터인 phcxhd에삽입하여 phcxspcd를제조하였다. SPCD 유전자의발현및정제 phcxspcd가형질전환된재조합 E. coli를 LBA (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, 100 mg/ml ampicillin) 액체배지에접종하고 37 o C에서 12시간배양하였다. 원심분리로균체를회수한후, VCX750 ultrasonicator (Sonics & Materials Inc.) 로파쇄하고, AKTA Prime TM (GE Healthcare) 과 HisTrap-FF column (GE Healthcare) 을이용하여정제하였다. 효소단백질정량및분자량측정재조합단백질의정제도는 Mini-protean II (Bio-Rad) 를이용한 12% SDS-PAGE 분석을통해확인하였다. 전기영동으로분리한단백질을 Coomassie blue로염색한후, 단백질표준시료 (Sigma-Aldrich) 와비교하여 monomer의분자량을결정하였다. 단백질의분자량과 4차구조분석을위해 gel permeation chromatography (GPC; Superdex-200 column, 10 x 300 mm, GE Healthcare) 를이용하였으며, 0.5 ml/min 유속의 50 mm sodium phosphate buffer (ph 7.0) 를용매로사용하였다. 효소단백질의농도측정은 BCA TM protein assay kit (Pierce Biotechnology Inc.) 를이용하였다. SPCD 의효소활성측정 SPCD의활성을측정하기위해 1% 의 β-cd, soluble starch, pullulan 기질을각각 50 mm sodium phosphate buffer (ph 7.5) 에녹인후, 적당량의효소를첨가하여최종 100 μl로반응하였다. 37 o C에서 10분간반응하여생성된 maltose의양을 dinitrosalicylic acid (DNS) 법으로측정하였다 (Miller, 1959). Maltotriose와 acarbose 분해활성의경우, 생성된 glucose의양을 AceChem Glucose kit (YD Diagnostics Co.) 로측정하였다. Korean Journal of Microbiology, Vol. 53, No. 3
210 Jang et al. SPCD의효소활성 1 unit는 1분당 1 μmol의 maltose 또는 glucose를생성하는효소의양으로정의하였다. SPCD 의가수분해산물분석최적조건에서 1% 기질과효소를 12시간동안반응한후, 가수분해산물을 thin layer chromatography (TLC) 로분석하였다. Silica gel 60F 254 TLC plate (Merck) 에 1 μl의시료를 spotting하고, isopropanol:ethylacetate: 물 (3:1:1) 의전개용매를사용하였다. TLC plate를발색시약 (3 g N-(1-naphthyl)- ethylene-diamine, 50 ml H 2 SO 4, 950 ml methanol) 에담근후, 건조하고 110 o C에서 10분간발색하였다. SPCD 의당전이산물분석 5% acarbose (Carbosynth) 공여체와 10% α-methyl glucopyranoside (Sigma-Aldrich) 수용체를이용하여최적조건에서 24시간반응하였다. 생성된당전이산물은 CarboPac PA1 컬럼 (0.4 x 25 cm, Thermo Fisher Scientific) 과 electrochemical detector (ED40, Thermo Fisher Scientific) 를사용하여 high performance anion exchange chromatography (HPAEC; ICS- 3000, Thermo Fisher Scientific) 로분석하였다. 이동상 A (150 mm NaOH) 에 1.0 ml/min의유속으로이동상 B (600 mm sodium acetate) 를분당 1% 씩증가시키면서분석하였다. 결과및고찰 SPCD 유전자의탐색세포내에서발현되는 CDase 계열의효소는직쇄형 maltooligosaccharides 또는환형구조의 cyclomaltodextrin (CD) 을 maltose 단위로가수분해하며, 이를통해 α-glucosidase에의한당화과정의효율을높이는것으로알려졌다 (Park et al., 2000; Park, 2006). 최근유전체데이터베이스분석연구를통해이들 CDase 계열효소유전자가자연계의다양한미생물유전체내에널리존재함을확인할수있었다. 또한다양한돌연변이연구를통해 Bacillus subtilis 세포내의탄수화물대사과정에서 maltogenic amylase와 debranching enzyme의생리적기능및역할이보고되었다 (Shim et al., 2009). 본연구에서는 B. subtilis MAase 등기존에알려진 CDase 계열효소유전자의서열을기초로 BLASTP (Altschul et al., 1990) 검색을통해 Streptococcus pyogenes ATCC 700294 유전체 (GenBank accession No. NC_002737.2, Ferretti et al., 2001) 로부터 CDase 로예상되는유전자 (NP_269429.1) 를발굴하였다. 이유전자는기존데이터베이스상에서 α-glycosidase로예상되었으며, 추가분석을통해이유전자가 maltose 또는 maltodextrin 이용유전자의클러스터내에위치하는것을확인하였다. 이러한유전자클러스터구조는지금까지 B. subtilis 168 (Shim et al., 2009), Listeria monocytogenes EGD-e (Gopal et al., 2010) 및 Listeria innocua ATCC 33090 (Jang et al., 2016) 의유전체에서보고된결과와유사하였다. 또한 SPCD의아미노산서열을 SignalP 프로그램 (Petersen et al., 2011) 으로분석한결과, 특이적인 signal peptide 서열이발견되지않아세포내의 cytoplasm 영역에서발현되어 maltodextrin 대사경로에관여할것으로예상하였다. SPCD 의 1 차구조비교 전분등탄수화물중합체를주요기질로하는 (β/α) 8 -barrel 구조의가수분해효소는아미노산서열상동성을기준으로대부분 GH 13 계열로분류되며, 넓은의미에서 CDase 또한이러한계열의효소에속한다 (Henrissat and Bairoch, 1996; Lee et al., 2002). 아미노산서열의상관관계를분석한결과, SPCD는 Thermus MAase (Kim et al., 1999) 와 36.8% 의가장높은상동성을보였으나, Bacillus halodurans (BHCD; Kang et al., 2009), Listeria innocua (LICD; Jang et al., 2016), 그리고유산균인 Lactococcus lactis (LLCD; Jang et al., 2013) 유래 CDase와대부분 35% 내외의상동성을나타냈다 (Table 1). 일반적인세균유래 CDase 계열효소간의상동성이 50% 이상으로높은수준임을감안할때, SPCD와기존효소들의상동성은비교적낮았다. 그러나, SPCD는일반적인 α-amylase와달리 CDase 계열효소에서만특이적으로발견되는특수한 N-말단구조와상동부위 I~IV를가지며, 특히상동부위 II, III, IV 내에위치하는주요활성아미노산잔기인 Asp326, Glu355, Asp422를공유 Table 1. Amino acid sequence identities among various CDase-family enzymes Enzyme a Amino acid sequence identity (%) ThMA BHCD LICD LLCD SPCD 36.8 36.4 34.5 34.5 ThMA 56.0 50.3 46.5 BHCD 50.9 46.6 LICD 47.6 a SPCD, Streptococcus pyogenes CDase in this study; ThMA, Thermus MAase (Kim et al., 2001); BHCD, Bacillus halodurans CDase (Kang et al., 2009); LLCD, Listeria innocua CDase (Jang et al., 2016); LLCD, Lactobacillus lactis CDase (Jang et al., 2013) 미생물학회지제 53 권제 3 호
Streptococcus cyclomaltodextrinase 의효소특성 211 하는것을확인하였다 (Park et al., 2000). 한편상동부위 II와 III의일부아미노산잔기가기존의 CDase 계열효소와상이하므로이로인한효소특성의차이가발생할수있을것으로예상하였다 (Fig. 1). 결론적으로 SPCD는기존의 CDase 계열효소들과 35% 미만의낮은서열상동성을보이지만, 전체적인 1 차구조및상동부위서열의유사성등을고려할때, 신규성이있는 CDase 계열효소의일종일것으로판단하고유전자클로닝, 발현및효소특성연구를진행하였다. SPCD 유전자의클로닝및발현 SPCD의구조유전자 (NP_269429.1) 는 1,701개의염기서열로이루어지며, 총 567개의아미노산잔기를암호화하고있 Fig. 1. Comparison of conserved amino acid sequences among CDasefamily enzymes. ThMA, Thermus MAase; BHCD, Bacillus halodurans CDase; LICD, Listeria innocua CDase; LLCD, Lactococcus lactis CDase; LGMA, Lactobacillus gasseri MAase; SPCD, Streptococcus pyogenes CDase. Catalytic amino acid residues are indicated with closed circles, and non-consensus residues are shown in black boxes. 다. 목적유전자를 SPCD-N 및 SPCD-C 프라이머를이용하여 PCR로증폭하여약 1.7 kb의 SPCD 유전자단편을얻었으며, 이를제한효소 Nde I과 Xho I으로처리한후, phcxhd 발현벡터에삽입하여 phcxspcd를제조하였다. phcxspcd가형질전환된 E. coli를배양하여카복시말단에 6개의 histidine 잔기가결합된형태의재조합 SPCD를얻었다. Ni-NTA 크로마토그래피를통해정제된 SPCD 효소의크기를 SDS-PAGE로분석한결과, 염기서열로부터예상한바와같이약 67 kda의단백질이성공적으로발현및정제되었음을확인하였다 (Fig. 2). SPCD 의복합체구조아미노산서열로부터계산된 SPCD monomer의분자량은 66,820 Da이었으며, GPC 분석을통해결정된재조합 SPCD 의분자량은 147,120 Da이었다. 따라서 SPCD는수용액내에서 homo-dimer 형태로존재할것으로예상하였다 (Fig. 3). 일부호알칼리성세균인 Bacillus sp. I-5 (Lee et al., 2002) 와 B. clarkii 7364 (Nakagawa et al., 2008) 유래 CDase의경우, 6개의 dimer가모여 dodecamer 구조를형성한다고알려졌으나, 일반적인 CDase 계열효소들은주로 homo-dimer 형태로존재하는것으로보고되었다 (Park et al., 2000). 이들효소의아미노말단에는약 100 130개의아미노산으로구성된고유의 N-domain이공통으로존재하여복합체구조의형성에중요한역할을하며, 이과정에서형성된좁고깊은형태의기질결합부위구조가효소의기질특이성및안정성에영향을미치 Fig. 2. Gene expression and purification of SPCD from E. coli. SDS- PAGE analysis showed the expression level and the purity of SPCD from recombinant E. coli. Lane M, protein molecular weight marker; lane CE, cell extract from E. coli harboring phcxspcd; lane PE, SPCD purified by Ni-NTA chromatography. Fig. 3. Determination of quaternary structure of SPCD. Molecular mass of SPCD was estimated by Superdex-200 gel permeation chromatography. The purified SPCD was drawn as a solid line and the molecular weight markers (a dashed line) were used as the mixture of six proteins: a, thyroglobulin (669 kda); b, apoferritin (443 kda); c, α-amylase (200 kda); d, alcohol dehydrogenase (150 kda); e, bovine serum albumin (66 kda); f, carbonic anhydrase (29 kda). Korean Journal of Microbiology, Vol. 53, No. 3
212 Jang et al. 는것으로알려졌다 (Kim et al., 2001; Hondoh et al., 2003; Lee et al., 2002). SPCD도 CDase 계열효소특유의 N-domain을가지며, 이를통해안정적인 homo-dimer 구조를형성하는것으로예상하였다. (A) SPCD 의효소특성재조합 SPCD는 β-cd를기질로할때, 45 o C에서최대활성을나타내었으나, 35 o C와 55 o C에서각각최적온도대비 29.9% 와 15.4% 의낮은활성을보였으며 (Fig. 4A), 이는 SPCD 의낮은열안정성에기인하는것으로예상한다. 한편 50 mm sodium phosphate buffer (ph 7.5) 에서가장높은 β-cd 가수분해활성을보였으나, ph 7.0 및 ph 8.0에서최적 ph 대비 55.0% 및 45.2% 의낮은상대활성을나타내었다 (Fig. 4B). 반응 ph에대한효소활성은 ph 7.0 9.0까지의중성및염기성영역에서인정하였으나, ph 6.0에서 71.2% 로감소하였고 ph 5.0에서는 47.9% 까지감소하였다 ( 자료미제시 ). 대부분의중온성미생물유래 CDase 계열효소가 40 60 o C, ph 5.5 8.0의최적반응조건을가지며 (Park et al., 2000), SPCD 또한유사한최적조건을나타내었으나, 상대적으로온도및 ph에따라효소활성이크게감소하는점에서차이를보였다. (B) Fig. 4. Effects of reaction temperature (A) and ph (B) on β-cd-hydrolyzing activity of SPCD. Optimal temperature was determined by measuring enzymatic activities at various temperatures in 50 mm sodium phosphate buffer (ph 7.5). For ph omtimum, enzymatic activities were measured at various ph and 45 C. SPCD 의기질특이성및가수분해특성 CDase 계열의효소는 β-cd, pullulan, starch, maltooligosaccharides 뿐만아니라 acarbose 등다양한탄수화물기질을가수분해할수있다. 다양한기질에대한 SPCD의효소활성을측정하고, 기존에연구된 CDase 계열효소들과상호비교하였다 (Table 2). 효소유전자분석에서예상한바와같이, SPCD는 β-cd에대해가장높은활성을나타내는 CDase 계열효소의일반적인특징을보였으나, 그활성이 starch 가수분해활성대비 660.1배로매우높아 4.7 82.5배수준인다른 CDase 효소들에비해큰차이를보였다. 반면 pullulan에대한가수분해활성이매우낮아서장시간반응후에활성측정또는분해산물분석이가능한수준이었다. 각기질로부터생성된반응산물을 TLC로분석하여 SPCD 의기질분해특성을확인하였다 (Fig. 5). SPCD는 β-cd의환형구조를 endo-형으로절단하여 7개의 glucose로구성된직쇄 Table 2. Multi-substrate specificity among various CDase-family enzymes Enzyme β-cd (C) Pullulan (P) Specific activity (U/mg) a Starch (S) Maltotriose (M) Acarbose (A) Activity ratio C/S P/S M/S M/A SPCD 336.4 ± 2.9 0.03 ± 0.01 0.5 ± 0.0 0.6 ± 0.0 6.5 ± 0.2 660.1 0.1 1.2 0.1 ThMA 65.2 ± 0.9 5.0 ± 0.1 4.4 ± 0.1 48.9 ± 0.3 27.3 ± 0.3 14.8 1.1 11.1 1.8 BHCD 52.9 ± 0.3 22.8 ± 0.5 11.2 ± 0.1 17.9 ± 0.1 3.0 ± 0.1 4.7 2.0 1.6 6.0 LICD 56.3 ± 0.7 13.0 ± 0.2 3.0 ± 0.0 77.9 ± 0.5 1.6 ± 0.0 18.8 4.3 26.0 48.7 LLCD 16.5 ± 0.9 0.8 ± 0.0 0.2 ± 0.0 8.5 ± 0.1 0.1 ± 0.0 82.5 4.0 42.5 85.0 a Each hydrolyzing activity on β-cd, pullulan, or starch was determined by DNS reducing sugar assay, whereas the activity on matotriose or acarbose was measured by GOD-POD method. 미생물학회지제 53 권제 3 호
Streptococcus cyclomaltodextrinase 의효소특성 213 Fig. 5. TLC analysis of hydrolysis patterns of SPCD on various substrates. SPCD was reacted with 1% of each substrate: C, β-cd; P, pullulan; S, soluble starch; G3, maltotriose; A, acarbose; AG, acarviosine-glucose; M, maltooligosaccharide standards, and the reaction products with (+) or without (-) SPCD. 형의 maltoheptaose로전환하며, 후속분해과정을통해최종산물로 maltose, 부산물로 glucose를생성하였다. Pullulan 및 starch에대한활성은상대적으로매우낮지만, 12시간반응을통해얻어진주요산물은예상과같이각각 maltose와 panose 였으며, 일부 glucose를부산물로생성하였다. Maltotriose의분해산물은최종적으로 glucose와 maltose였으며, maltose는더이상분해되지않고축적되지만, maltotetraose 이상의소당류기질에비해상대적으로느린분해속도를나타냈다. 결론적으로 SPCD는 α-(1,4)-결합을분해하여주로 maltose를생성하는전형적인 CDase 계열의효소임을알수있었으며, pullulan 및 starch와같은고분자중합체기질보다 β-cd 또는 maltotetraose 이상의 oligosaccharides 기질에대해선호도가높은효소특성을가진다. 특히 maltotriose와비교할때 acarbose에대한분해활성이매우높은효소이며, 3개의 glucose로구성된 maltotriose에대한가수분해활성이 β-cd와 acarbose에비해상대적으로낮았다. 일반적인 endo-형의 α-amylase는짧은길이의 oligosaccharides 기질에대해상대적으로낮은활성을보이는점을감안할때 (Park et al., 2000), SPCD 또한 maltotriose 이하의저분자기질에잘작용하지못하는 endo-형의 CDase 로판단하였다. SPCD 의당전이특성 CDase 계열효소의일종인 ThMA, BHCD, LICD 등의당전 Fig. 6. HPAEC analysis of acarbose transfer products. SPCD was reacted with 5% acarbose (donor) and 10% α-methyl glucopyranoside (acceptor) at 30 C for 24 h. α-mg, α-methyl glucopyranoside; G1, glucose; AG, acarviosine-glucose; A, acarbose; TP6, α-(1,6)-transfer product. 이반응에서반응초기에빠르게생성된 α-(1,4)-결합의전이생성물은생성과동시에급속히재분해되지만, 천천히생성된 α- (1,6)- 또는 α-(1,3)-결합의전이생성물은다시분해되지않아최종생성물로축적된다 (Kim et al., 1999). SPCD의당전이활성을확인하고자, 공여체로 acarbose, 수용체로 α-methyl glucopyranoside (α-mg) 를이용한당전이반응을수행하고, 반응산물을 HPAEC로분석하였다. 당전이반응으로부터일부 α- (1,6)-결합전이생성물이얻어졌으나, 생성량이무시할정도로매우적었으며, α-(1,3)-결합전이생성물은관찰되지않았다 (Fig. 6). 따라서 SPCD는상대적으로높은 acarbose 가수분해활성에비해당전이활성은매우낮은특성의효소로생각된다. SPCD의경우, 다른 CDase 계열효소와아미노산상동성이 35% 내외로낮은편이며, 특히상동부위 II (GWRLDVANE) 의 alanine-asparagine 아미노산잔기가 serine-aspartate로치환되어있고, 또한활성부위아미노산잔기인상동부위 III (EIWH) 의 glutamate와인접한 isoleucine 잔기가 asparagine 으로치환되어있는등 1차구조상에서충분히차별화될수있다 (Fig. 1). 특히, 상동부위 II는 ThMA의가수분해및당전이활성조절에중요한역할을하는것으로알려져있으며 (Kim et al., 1999), 또한 acarbose의가수분해특성에도영향을주는것으로보고되었다 (Oh et al., 2008). 따라서 SPCD 특유의가수분해특성이나낮은당전이활성의이유를설명하기위해이러한아미노산잔기에대한단백질공학적연구를추가로진행중이며, CDase 계열효소의기질특이성및당전이활성에관여하는핵심아미노산잔기연구의모델효소로활용가능할것이다. Korean Journal of Microbiology, Vol. 53, No. 3
214 Jang et al. 적요 Streptococcus pyogenes ATCC 700294 유전체로부터 cyclomaltodextrinase (SPCD) 로예상되는유전자를발견하였다. SPCD는총 567개의아미노산으로이루어진 66.8 kda의효소이며, 기존에알려진 CDase 계열효소들과 37% 미만의아미노산서열상동성을가진다. 본연구에서는 SPCD 유전자를클로닝하였으며, 대장균내에서카복시말단에 6개의 histidine 잔기가결합된 dimer 형태로발현및정제되었다. SPCD는 ph 7.5, 45 o C의반응조건에서최대의활성을나타내었으며, β- cyclodextrin, starch, maltotriose를기질로반응하여 maltose 를주산물로생성하였다. 또한 pullulan을 panose 단위로분해하며, acarbose를 glucose와 acarviosine-glucose로가수분해하는 CDase 계열의효소로확인되었다. 그러나, SPCD는다른효소에비해저분자소당류인 β-cyclodextrin에대한활성이매우높고, starch 및 pullulan과같은고분자기질에대해매우낮은활성을보였다. 또한 maltotriose 분해활성이매우낮은반면 acarbose에대해상대적으로높은가수분해활성을가지나, 당전이활성은매우낮아다른 CDase 계열효소들과구별된다. 감사의말 이논문은 2014년도충북대학교학술연구지원사업의연구비지원에의하여연구되었음. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403 410. Cha, H.J., Yoon, H.G., Kim, Y.W., Lee, H.S., Kim, J.W., Kweon, K.S., Oh, B.H, and Park, K.H. 1998. Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur. J. Biochem. 253, 251 262. Feederle, R., Pajatsch, M., Kremmer, E., and Bock, A. 1996. Metabolism of cyclodextrins by Klebsiella oxytoca m5a1: purification and characterisation of a cytoplasmically located cyclodextrinase. Arch. Microbiol. 165, 206 212. Ferretti, J.J., McShan, W.M., Ajdic, D., Savic, D.J., Savic, G., Lyon, K., Primeaux, C., Sezate, S., Suvorov, A.N., Kenton, S., et al. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98, 4658 4663. Fiedler, G., Pajatsch, M., and Bock, A. 1996. Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca. J. Mol. Biol. 256, 279 291. Gopal, S., Berg, D., Hagen, N., Schriefer, E.M., Stoll, R., Goebel, W., and Kreft, J. 2010. Maltose and maltodextrin utilization by Listeria monocytogenes depend on an inducible ABC transporter which is repressed by glucose. PLoS One 5, e10349. Henrissat, B. and Bairoch, A. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695 696. Hondoh, H., Kuriki, T., and Matsuura, Y. 2003. Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase. J. Mol. Biol. 326, 177 188. Jang, M.U., Jeong, C.K., Kang, H.J., Kim, M.J., Lee, M.J., Son, B.S., and Kim, T.J. 2016. Gene cluster analysis and fuctional characterization of cyclomaltodextrinase from Listeria innocua. Microbiol. Biotechnol. Lett. 44, 363 369. Jang, M.U., Kang, H.J., Jeong, C.K., Park, J.M., Yi, A.R., Kang, J.H., Lee, S.W., and Kim, T.J. 2013. Enzymatic characterization of Lactococcus lactis subsp. lactis cyclomaltodextrinase expressed in E. coli. Korean J. Microbiol. Biotechnol. 41, 391 397. Kaulpiboon, J. and Pongsawasdi, P. 2004. Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase. J. Biochem. Mol. Biol. 37, 408 415. Kang, H.J., Jeong, C.K., Jang, M.U., Choi, S.H., Kim, M.H., Ahn, J.B., Lee, S.H., Jo, S.J., and Kim, T.J. 2009. Expression of cyclomaltodextrinase gene from Bacillus halodurans C-125 and characterization of its multisubstrate specificity. Food Sci. Biotechnol. 18, 776 781. Kim, T.J., Kim, M.J., Kim, B.C., Kim, J.C., Cheong, T.K., Kim, J.W., and Park, K.H. 1999. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain. Appl. Environ. Microbiol. 65, 1644 1651. Kim, T.J., Nguyen, V.D., Lee, H.S., Kim, M.J., Cho, H.Y., Kim, Y.W., Moon, T.W., Park, C.S., Kim, J.W., Oh, B.H., et al. 2001. Modulation of the multisubstrate specificity of Thermus maltogenic amylase by truncation of the N-terminal domain and by a salt-induced shift of the monomer/dimer equilibrium. Biochemistry 40, 14182 14190. Lee, H.S., Kim, M.S., Cho, H.S., Kim, J.I., Kim, T.J., Choi, J.H., Park, C., Lee, H.S., Oh, B.H., and Park, K.H. 2002. Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other. J. Biol. Chem. 277, 21891 21897. Miller, C.L. 1959. Use of dinitrosalicylic acid reagent for determination reducing sugar. Anal. Chem. 31, 426 428. Nakagawa, Y., Saburi, W., Takada, M., Hatada, Y., and Horikoshi, K. 2008. Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364. Biochim. Biophys. Acta 1784, 2004 2011. Oh, K.W., Kim, M.J., Kim, H.Y., Kim, B.Y., Baik, M.Y., Auh, J.H., and Park, C.S. 2005. Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in 미생물학회지제 53 권제 3 호
Streptococcus cyclomaltodextrinase 의효소특성 215 Escherichia coli. FEMS Microbiol. Lett. 252, 175 181. Oh, S.W., Jang, M.U., Jeong, C.K., Kang, H.J., Park, J.M., and Kim, T.J. 2008. Modulation of hydrolysis and transglycosylation activity of Thermus maltogenic amylase by combinatorial saturation mutagenesis. J. Microbiol. Biotechol. 18, 1401 1407. Park, K.H. 2006. Function and tertiary- and quaternary-structure of cyclodextrin-hydrolyzing enzymes (CDase), a group of multisubstrate specific enzymes belonging to the alpha-amylase family. J. Appl. Glycosci. 53, 35 44. Park, K.H., Kim, T.J., Cheong, T.K., Kim, J.W., Oh, B.H., and Svensson, B. 2000. Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family. Biochim. Biophys. Acta 1478, 165 185. Petersen, T.N., Brunak, S., von Heijne, G., and Nielsen, H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785 786. Podkovyrov, S.M. and Zeikus, J.G. 1992. Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli. J. Bacteriol. 174, 5400 5405. Shim, J.H., Park, J.T., Hong, J.S., Kim, K.W., Kim, M.J., Auh, J.H., Kim, Y.W., Park, C.S., Boos, W., Kim, J.W., et al. 2009. Role of maltogenic amylase and pullulanase in maltodextrin and glycogen metabolism of Bacillus subtilis 168. J. Bacteriol. 191, 4835 4844. Korean Journal of Microbiology, Vol. 53, No. 3