Fructosyloligosaccharide를 Acceptor 반응의 기질로 사용한 새로운 올리고당의 생합성

이찬용,이충환 한국미생물학회 1999 미생물학회지 Vol.35 No.2

충치 원인균의 일종인 Streptococcus sobrinus ATCC27351 균주를 자외선 조사와 NTG 처리에 의하여 bacitracin 내성변이주를 얻었다. 선발된 변이주들 중 glucosyltransferase 활성이 모균주보다 증가된 변이주 4균주를 선발하였다. 이들의 bacitracin 내성은 모균주 보다 5~48배, glucosyltransferase 활성은 약 2배 증가하였다. 모균주의 glucosyltransferase를 이용하여 maltose acceptor 반응을 하여 \ulcorner 와 image analysis 로 분석하였다. 그 결과 sucrose 와 acceptor를 사용하여 반응을 시킨 결과 모균주와 변이주의 glucose 전이효소 사이에는 acceptor 특이성에 차이가 있었다. 모균주의 효소와는 반응하지 않는 acceptor 인 fructose를 함유하는 1-kestose와 nystose 그리고 turanose를 acceptor 로 하여 변이주 S. sobrinus BR24C 균주의 glucosyltransferase와 반응을 시켰다. 얻어진 acceptor 산물을 정제하여 \sup 1\H, \sup 13\C-NMR 분석을 한 결과, 자연계에서는 발견되지 않는 새로운 구조의 올리고당 \6^{3}$-$\alpha$-D-glucopyranosyl \3^{2}$-O-$\alpha$-D-glucopyranosyl-D-fructose와 \6^{4}$-$\alpha$-D-glucopyranosyl \1^{3}$-$\beta$-D-fructofuranosyl sucrose을 확인하였다. Acceplor reaction of glucosyltr~ansferase of Streptococcus ,SO~~-~IZLIS with f ~ ~ t o ~ y l o l i g o ~ a ~ ~ h a r i d e ~ was studied for the biosynthesis of novel olgisaccharides. First, bacilracin resistant mutants were selected by mutagenesis of Streptococcus sobrimis ATCC27351. Among these mutants 4 strains were selected by resistance to bacitracin and increase of glucosyltransferase. Acceptor reaction of maltose was analyzed by TLC and image analysis. There were differences in the specificity of the acceptor reaclion by Ule glucosylumsferase between mother strain (Streptococcus sobrinus ATCC2735) and bacitracin resistant mutants (Streptococcus sobrinus BR24C, Strepfococcus sobrinus CH-5). Molher strain did ilot show an acceptor reaction with fructosyloligosaccharides such as 1-keqtose and nystose. Acceptor reaction products of turailose and 1-kestose with glucosyltransferase (GW-S) of Streptococcus sobrini~s BR24C were \6^{3}$-$\alpha$-D-glucopyranosyl \3^{2}$-O-$\alpha$-D-fructose (glucose-fructose-glucose) and \6^{4}$-$\alpha$-D-glucopyranosyl \1^{3}$-$\beta$-D-~-h~ctofuranos~~ sucrose (glucose-glucosefructose- fructose). respectively These are novel oligosaccharides which can be produced only by enzymatic reaction.

Streptococcus mutans Ingbritt sucrose-glucan glucosyltransferase특성과 그 활성에 미치는 키틴 유도체들의 효과

주완택 ( Wan Taek Ju ),지명심 ( Myeong Sim Ji ),박노동 ( Ro Dong Park ) 한국응용생명화학회(구 한국농화학회) 2012 Journal of Applied Biological Chemistry (J. Appl. Vol.55 No.3

Sucrose-glucan glucosyltransferase (Gtf) is an importantenzyme involved in the cavity formation process where insolubleglucan is synthesized. In this study, we purified Gtf from Streptcoccusmutans Ingbritt through ammonium sulfate precipitation, SephadexG-150, CM-Sephadex, and DEAE-Sephadex column chromato-graphies. A 13-fold of purification was achieved with a total yieldof 6.3%. The apparent molecular mass of the enzyme was determinedto be 66kDa on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). The optimal pH and temperaturewere established to be 6.0 and 40oC, respectively. The enzymeactivity could be inhibited to 22-59% by 1mM Hg2+, Cu2+ andAl3+, and to 68% by 1mM EDTA. It was also inhibited 40% by2mM xylitol and 35-45% by 0.05% soluble chitosan, glycolchitosan, and glycol chitin. This is the first report to reveal theinhibition effect of chitin derivatives on Gtf activity, which maybe further applicable to develop gargles to overcome cavity.

Cloning and expression of Streptococcus mutans GS-5 glucosyltransferase

김수경,김재곤,백병주,양연미,이경열,박정렬 大韓小兒齒科學會 2008 大韓小兒齒科學會誌 Vol.35 No.1

Dental caries is an infectious disease caused by mutans streptococci, and is a primary etiologic agent of dental caries in humans. The molecular pathogenesis of mutans streptococcal-associated dental caries occurs in three phases. Firstly, S. mutans attaches to tooth surface via a cell surface adhesion termed antigen I/II. In the second phase, the glucosyltransferase(GTFs) synthesize polymers like glucans in the presence of sucrose. In the third phase, the multivalent glucans interacts with glucan binding proteins (GBPs) and they make dental plaque and accumulation of microorganisms. Many studies and clinical trials have indicated that a mucosal immune response to these antigens(Ag I/II, GTFs, GBPs) of S. mutans can influence the pathogenesis of dental caries. So these antigens can be important vaccine candidates for immunologic intervention against dental caries. In this study, we cloned the genes for GTFb, GTFc, GTFd from S. mutans GS-5 and did the nucleotide sequence analysis. And the recombinant proteins of GTFd and N-terminus of GTFd were expressed. Intact GTF which we get from this experiment can be used for antibody production specific for any GTF activity domain through animal experiment. 치아우식은 주로 mutans streptococci에 의해 야기되는 감염성 질환으로서 주 원인균에는 streptococcus mutans가 있다. S. mutans가 치아우식을 유발하는 분자 생물학적 기전은 몇 가지 단계를 포함한다. 먼저 S. mutans는 AgI/II와 같은 세포 표면의 섬유성 단백질을 매개로 치면의 타액성 피막에 일차적으로 부착한다. 두번째 단계에서 자당의 존재하에 glucosyltransferase(GTF)는 glucan과 같은 다당체를 합성하게 된다. 마지막으로 이렇게 합성된 glucan은 glucan binding proteins와 상호작용하여 치면세균막을 형성해서 세균의 군집화를 가능하게 한다. 많은 실험과 임상연구에서 S. mutans의 주요 항원(Ag I/II, GTFs, GBPs)들이 치아우식 병리기전에 영향을 준다고 알려져 왔고, 따라서 이런 항원들이 면역계에 작용하여 치아우식을 막는 백신으로 이용 가능하다. 본 실험은 streptococcus mutans GS-5로부터, GTFb, GTFc, GTFd 유전자를 복제하고 염기서열분석을 하였으며, 이중 GTFd가 먼저 재조합 단백질 생산을 위해 발현 벡터에 클로닝 되었으며, 이로부터 단백질이 발현됨을 확인하였다. 이번 실험에서 얻은 순수 GTF 항원은 동물실험을 통해 특정 GTF 활성부위에 대한 항체 생산에 이용될 수 있을 것이다.

비경구 투여한 Streptococcus mutans 균체 및 Glucosyltransferase에 대한 마우스의 면역항체반응

양규호,정미,정진,장미영,오종석,나희삼,강인철,이현철,Yang, Kyu-Ho,Chung, Mee,Chung, Jin,Chang, Mee-Young,Oh, Jong-Suk,Nah, Hee-Sam,Kang, In-Chol,Lee, Hyun-Chul 대한소아치과학회 2003 大韓小兒齒科學會誌 Vol.30 No.1

치아우식증을 예방하기 위한 백신연구를 위하여 주원인균인 Sterptococcus mutans 균체항원과 이 균에서 분리한 glucosyltransferase (GTF)를 항원으로 하고 이에 면역조절기능이 있는 retinoic acid (RA)를 첨가하여 투여경로와 백신조성이 이들 항원에 대한 면역반응에 미치는 영향을 실험하였다. 균체항원(Ingbritt strain)을 마우스의 피하에 Complete Freund's Adjuvant와 함께 투여하여 생산되는 혈청내 응집항체가는 serotype e (LM-7)와는 강한 교차반응을 보였으나 serotype f (OMZ-175)와는 거의 교차반응을 일으키지 않았다. 면역혈청내 항-GTF 및 항-Ag I/II 항체중 항-GTF IgA는 피하로 투여시 전혀 검출되지 않았으나 이에 RA를 첨가하면 다량의 항체 생산을 관찰하였고 그 정도는 경구투여시의 생산량을 능가하였다. GTF를 alum과 함께 투여하여 생산되는 혈청내 항-GTF 항체중 IgM은 피하로 투여시 상당량이 검출되었고 RA를 첨가하면 그 생산이 증가되었으며 경구로 투여시 대조군에 비하여 약간증가를 보였으나 피하투여시의 그것에는 미치지 못하였다. GTF-특이 IgG는 경구투여시는 전혀 검출되지 아니하였고, 피하투여시에만 현저한 증가를 보였으며, RA첨가는 이에 영향을 미치지 못하였다. 항-GTF IgA는 피하로 투여시 전혀 검출되지 아니하였으나 이에 RA를 첨가하면 증가된 항체생산을 관찰하였고 그 정도는 경구투여시의 생산량을 능가하였다. 이상의 실험성적은 GTF에 대한 항체생산은 투여경로와 항원의 종류에 따라 다양한 반응을 나타내며 RA는 이를 백신에 첨가하면 피하경로를 이용하여 면역하더라도 경구투여와 유사한 IgA-매개 면역반응으로 조절시킬 수 있는 가능성을 나타내었다. Streptococcus mutans is known to be a major causative organism of human dental caries. The development of a vaccine against dental caries involves identification of appropriate antigens of mutans streptococci against which protective immune responses can be mounted, and the selection of a method of immunization that will generate sustained levels of protective antibodies. Antigens receiving most attention include streptococcal surface proteins that are involved in attachment to tooth surfaces and glucosyltransferases (GTF) that synthesize adhesive glucans from sucrose. The induction of antibody responses to orally administered antigens is often difficult due to digestive destruction of antigens and immune tolerance. Here we report the induction of antibody responses to an anti-caries vaccine containing retinoic acid (RA). Subcutaneous immunization with formalin-fixed bacteria or GTF supplemented with RA induced higher serum IgM and IgA responses to GTF compaired to oral adminstration. Antisera induced by Ingbritt strain showed partial cross-reaction with LM-7 strain, but not with OMZ175. These results suggest that subcutaneous immunization with GTF combined with an immunomodulator, RA, may be applied to anti-caries vaccine.

Quantitative comparison of mRNA expression of glucosyltransferase(GTF) between xylitol-resistant(X^(R)) and xylitol-sensitive(X^(s)) mutans streptococci

Kim, Chong-Chul,Lee, Mi-Na,Kim, Young-Jae,Lee, Sung-Hoon 大韓小兒齒科學會 2006 大韓小兒齒科學會誌 Vol.33 No.1

Since the long-term exposure of mutans streptococci to xylitol is known to select for xylitol-resistant(X^(R)) natural mutants, the occurrence and survival of such X^(R) strains were performed in batch culture methods. The aim of the study was to compare the differentiation and quantification of mRNA expression of the gtf genes of X^(R) and X^(S) mutans streptococci. Using a real-time reverse-transcription polymerase chain reaction, the expression of each gtf was determined. In X^(R) strains, the relative levels of transcription of gtfB and gtfC were decreased while that of gtfD was increased, suggesting the presence of independent promoters. It also suggested that mutation related to production of glucosyltransferase occurred under the exposure of xylitol could explain the caries-preventive mechanisms of xylitol. 자일리톨에 장기간 노출된 mutans streptococci는 자일리톨에 내성이 발현되어 자일리톨 내성균주가 생성된다고 알려져 있다. 본 연구의 목적은 mutans streptococci에서 자일리톨 내성균주와 감성균주의 gtf 유전자 발현량을 각각의 유전자별로 정량적으로 분석하고 비교하는 것이다. 실시간 역전사 중합효소연쇄반응법을 이용하여 각각의 gtf 발현을 조사한 결과 gtfD는 증가한 반면. gtfB와 gtfC는 감소하였는데 이는 각 유전자의 독립된 조절기전이 존재함을 보여주는 것이다. 또한 자일리톨에 노출된 mutans streptococci에서의 glucosyltransferase와 연관된 유전자변형이 자일리톨의 치아우식증 예방효과의 작용기전 중 하나임을 알 수 있었다.

The Expression Patterns of AtBSMT1 and AtSAGT1 Encoding a Salicylic Acid (SA) Methyltransferase and a SA Glucosyltransferase, Respectively, in Arabidopsis Plants with Altered Defense Responses

송종태,Yeon Jong Koo,Jong-Beum Park,Yean Joo Seo,Yeon-Jeong Cho,서학수,최양도 한국분자세포생물학회 2009 Molecules and cells Vol.28 No.2

We reported previously that overexpression of a salicylic acid (SA) methyltransferase1 gene from rice (OsBSMT1) or a SA glucosyltransferase1 gene from Arabidopsis thaliana (AtSAGT1) leads to increased susceptibility to Pseudomonas syringae due to reduced SA levels. To further examine their roles in the defense responses, we assayed the transcript levels of AtBSMT1 or AtSAGT1 in plants with altered levels of SA and/or other defense components. These data showed that AtSAGT1 expression is regulated partially by SA, or nonexpressor of pathogenesis related protein1, whereas AtBSMT1 expression was induced in SA-deficient mutant plants. In addition, we produced the transgenic Arabidopsis plants with RNAi-mediated inhibition of AtSAGT1 and isolated a null mutant of AtBSMT1, and then analyzed their phenotypes. A T-DNA insertion mutation in the AtBSMT1 resulted in reduced methyl salicylate (MeSA) levels upon P. syringae infection. However, accumulation of SA and glucosyl SA was similar in both the atbsmt1 and wild-type plants, indicating the presence of another SA methyltransferase or an alternative pathway for MeSA production. The AtSAGT1-RNAi line exhibited no altered phenotypes upon pathogen infection, compared to wild-type plants, suggesting that (an)other SA glucosyltransferase(s) in Arabidopsis plants may be important for the pathogenesis of P. syringae.

Characterization of sterol glucosyltransferase from Salinispora tropica CNB-440: Potential enzyme for the biosynthesis of sitosteryl glucoside

Thuan, N.H.,Yamaguchi, T.,Lee, J.H.,Sohng, J.K. IPC Science and Technology Press ; Elsevier Scienc 2013 Enzyme and microbial technology Vol.52 No.4

A sterol glucosyltransferase-encoded gene was isolated from Salinispora tropica CNB-440, a marine, sediment-dwelling, Gram positive bacterium that produces the potent anticancer compound, salinosporamide A. The full-length gene consists of 1284 nucleotides and encodes 427 amino acids with a calculated mass of 45.65kDa. The gene was then cloned and heterologously expressed in Escherichia coli BL21(DE3). The amino acid sequence shares 39% similarity with the glycosyltransferase from Withania somnifera, which belongs to glycosyltransferase family 1. Enzyme reactions were carried out with the various free sterols (acceptor) and NDP-sugars (donor). The purified protein only showed activity for glucosylation of β-sitosterol with UDP-D-glucose and TDP-D-glucose donors, and optimal activity at pH 7.5 and 37<SUP>o</SUP>C. Among these two donors, UDP-D-glucose was preferred.

단일클론항체를 사용한 타액 내 Streptococcus mutans 검출

이민정,이대우,김미아,이경열,백병주,양연미,김재곤 大韓小兒齒科學會 2015 大韓小兒齒科學會誌 Vol.42 No.1

본 연구의 목적은 Streptococcus mutans의 Ag I/II와 glucosyltransferase에 대해 개발된 단일클론항체를 사용한 타액내 Streptococcus mutans 검출의 효용성을 평가하기 위함이다. 세 군(성인, 미성년자, 그리고 교정치료중인 미성년자)을대상으로 DMFT, Dentocult -SM strip mutans를 이용한 타액내의 colony density (CFU/mL), polymerase chain reaction을이용한 Sreptococcus mutans와 Sreptococcus sobrinus의 존재여부, 그리고 4가지 단일클론항체를 사용한 타액내 Streptococcus mutans 검출 실험을 시행하였다. 그 결과 Streptococcus mutans가 Streptococcus sobrinus보다 치아우식 형성에 더 주요하게 작용한다는 것을 입증하였으며, glucosyltransferases (gtf B, gtf C, gtf D)와 Ag I/II에 대한 단일클론항체들의 Streptococcus mutans를 검출해 내는 능력이 Dentocult -SM strip mutans나 polymerase chain reaction을사용한 검출법보다 뛰어남을 보였다. The purpose of this study was to determine the usefulness of a detection method for Streptococcus mutans insaliva with monoclonal antibodies developed targeting Ag I/II and glucosyltransferases (gtf B, gtf C and gtf D) inStreptococcus mutans. In the three groups tested (adults, minors, and minors under orthodontic treatment), theresults of the DMFT scores, the colony density (CFU/mL) in their saliva was measured using Dentocult -SMstrip mutans, polymerase chain reaction was performed to test whether Streptococcus mutans and Streptococcussobrinus were present, and Streptococcus mutans detecting tests performed in their saliva using four types ofmonoclonal antibody were collected. In conclusion, it was demonstrated that the Streptococcus mutants plays more important role in forming dentalcaries compared to Streptococcus sobrinus, and that the monoclonal antibodies against glucosyltransferases(gtf B, gtf C, gtf D) and Ag I/II of Streptococcus mutans are superior in detecting Streptococcus mutans toDentocult -SM strip mutans or polymerase chain reaction.

재래귤의 성숙시기별 리모노이드 쓴맛 표시자로서 Limonoid UDP-glucosyltransferase 발현 분석

김영미 ( Young Mee Kim ),이도승 ( Do Seung Lee ),전덕현 ( Deok Hyoen Jeon ),송연우 ( Yeon Woo Song ),이동선 ( Dong Sun Lee ),류기종 ( Key Zung Ryu ),조문제 ( Moon Jae Cho ),이동훈 ( Dong Hoon Lee ),김소미 ( So Mi Kim ) 한국식품저장유통학회(구 한국농산물저장유통학회) 2011 한국식품저장유통학회지 Vol.18 No.2

Limonoid UDP-glucosyltransferase (LUGT) is an enzyme that converts limonoids into their corresponding glucosides and ultimately ameliorates limonoid bitterness in Citrus species. In this paper, the LUGT gene was cloned via PCR from 10 Jeju Citrus species. All the deduced glucosyltransferase proteins harbored a highly conserved plant secondary product glucosyltransferase (PSPG) motif within the C terminal region. Phylogenetic analysis based on the amino acid sequence comparison of the LUGT proteins from 10 Citrus species generated three distinct types. The expression patterns of LUGT gene in three representative species from each type were quite different with that of C. unshiu Marc. cv. Miyagawawase(Gungcheon), which his without distinctive juice delayed bitterness. Ourresultssho wth at some Citrus speciessuchas Citrusleiocarpa HORT(Bingul), Citruserythrosa HORT (Dongjunggul), and Citrustachibana TANAKA(Honggul) end emicin Jeju maybe susceptible to intense juice delayed bitterness due to delay inexpression of LUGT.

Sequence and Phylogenetic Analyses of Novel Glucosyltransferase Genes of Mutans Streptococci Isolated from Pig Oral Cavity

Noriko Shinozaki-Kuwahara,Kazuko Takada,Masatomo Hirasawa 한국미생물학회 2008 The journal of microbiology Vol.46 No.2

Nucleotide sequences of water-insoluble glucan-producing glucosyltransferase (gtf) genes of new mutans streptococci isolated from pig oral cavity, Streptococcus orisuis JCM14035, and of Streptococcus criceti HS-6 were determined. The gtf gene of S. orisuis JCM14035 consisted of a 4,401 bp ORF encoding for a 1,466 amino acids, and was revealed to belong to the gtfI group. The percent homology of amino acid sequence of the GTF-I from S. orisuis and S. criceti are 95.0%, however, this score ranges from 77.0% to 78.0% when compared to Streptococcus sobrinus 6715. The deduced N-terminal amino acid sequence was considered responsible for the secretion of GTF-I in S. orisuis JCM14035 and S. criceti HS-6 with high similarity to known GTF proteins from other streptococci. In addition, two other conserved regions, i.e., N-terminal putative catalytic-site and C-terminal glucan binding domain, were also found in GTF-Is of S. orisuis JCM14035 and S. criceti HS-6. Phylogenetic analysis suggested that S. orisuis JCM14035 and S. criceti HS-6, closely related to each other, resemble S. sobrinus and S. downei based on the amino acid sequences of the GTFs