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김도만,류수진,박관하,ROBYT, JOHN F. 全南大學校 觸媒硏究所 1998 觸媒硏究 論文集 Vol.20 No.-
Acarbose effectively inhibited the synthesis of dextran, and the inhibition pattern was a noncompetitive type with ?? value of 1.35mM. It also inhibited the disproport-ionation reaction of dextransucrase with isomaltotriose and decreased the efficiency of the maltose acceptor reaction. Increased concentration of dextransucrase or maltose in reaction digests, however, decreased the degree of inhibition by acarbose.
Glucansucrases를 이용한 새로운 올리고당의 합성
백진숙,김도만,이진하,장판식,한남수,JohnF.Robyt 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.2
본 연구에서는 dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM과 dextransucrase constitutive mutants인 B-742CB 및 B-1355C에서 얻은 dextransucrase 및 alternansucrase를 이용하여 maltose, lactose, gentiobios 및 raffinose의 수용체반응을 수행하였다. B-512FMCM의 경우 수용체로서 maltose와 gentiobiose는 설탕에 대한 농도비가 1/1이하일 때 수용체에 α1-6으로 D-glucose가 결합한 일련의 올리고당을 생성하였고, 9/1이상에서는 단 두 가지의 수용체산물로서 maltose의 경우에는 panose와 isomaltosyl maltose를, gentiobiose의 경우에는 6^2-α-D-glucopyranosyl gentiobiose와 isomaltosyl gentiobiose만을 생성하였다. 그러나 lactose는 유일한 산물인 2^1-α-D-glycopyranosyl lactose만을 생성하였다. 효소양의 변화에 대해서 maltose와 gentiobiose의 수용체반응에서 생성된 수용체산물(올리고당)의 수와 양은 효소양이 증가함에 따라 증가하였으나 lactose의 경우에는 효소양의 증가에 대해 수용체산물 양의 변화가 크지않았다. B-742CB dextransucrase와 B-1355C alternansucrase를 사용하면 B-512FMCM dextransucrase에서는 단 한가지만의 수용체산물을 생산하였던 lactose와 raffinose를 수용체로하여 여러 가지의 수용체산물(올리고당)을 생산하였다. Dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM and dextransucrase constitutive mutants B-742CB and B-1355C catalyzed the transfer of glucose from sucrose to other carbohydrates which were present or were added to the reaction digests. When the acceptor was a maltose, gentiobiose, lactose or raffinose, there was produced a series of oligosaccharide acceptor products or single product based on the kinds of enzymes and reaction conditions. To obtain the quantitative information about the yield and the distribution of acceptor products and dextran two experimental parameters were studied: a) the ratio of acceptor to sucrose and b) the amount of enzyme at constant carbohydrate concentration (100 mM). As the amount of enzyme increased, the synthesis of acceptor products (of maltose or gentiobiose) increased, and the formation of dextran decreased. As the ratio of acceptor to sucrose increased, the amount of dextran and the number of acceptor-products decreased and the amount of acceptor-products increased. When maltose or gentiobiose was an acceptor, the glucose from sucrose was transferred to the C-6 hydroxyl group of the nonreducing-end glucose residue of acceptors to give a homologous series of isomaltosyl dextrins. In case of lactose or raffinose, there was produced only one acceptor product from B-512FMCM dextransucrase reaction. In the lactose acceptor reaction, the glucose from sucrose was transferred to the C-2 hydroxyl of the reducing end glucose residue of lactose. To get a series of oligosaccharides from lactose or raffinose acceptor reaction we used B-742CB dextransucrase or B-1355C alternansucrase with 500 mM sucrose in reaction digest.
LEE, SO-YOUNG,LEE, JIN-HA,ROBYT, JOHN F.,SEO, EUN-SEONG,PARK, HYEN-JOUNG,KIM, DOMAN 한국미생물 · 생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.2
Lipomyces starkeyi KSM 22 elaborates an enzyme that has both dextranase and amylase activities in a single protein of 100 kDa. Competition studies, using different amounts of dextran and starch as substrates, gave a competition plot consistent with the hypothesis that the hydrolysis of dextran and starch occurs at two independent active sites, each specific for starch and dextran, respectively.
이진하,김도만,백진숙,박관화,한남수,Robyt, JohnF. 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.2
덱스트란수크라제는 sucrose를 이용하여 덱스트란의 합성을 촉진하는데 sucrose 이외에 다른 탄수화물이 효소 반응기 중에 존재하는 경우에는 sucrose의 glucose기를 이 탄수화물에 전달하는 반응을 촉진하여 새로운 구조의 산물을 생산한다. Leuconostoc mesenteroides B-742CB로 부터 얻은 덱스트란수크라제를 이용하여 입자전분과 호화 전분에 sucrose의 glucose를 수식함으로써 변형을 시도하였다. 사용한 전분의 종류(Small starch 또는 Potato starch)에 따라 그리고 전분의 상태(입자 혹은 호화된 상태)에 따라 이론적 수율의 46-72%(s.d.<±5)를 얻었다. 변형 전분을 α-amylase, isoamylase, pullulanase 그리고 endo-dextranase로 처리하여 변형 전의 전분과 가수분해 상태를 비교한 결과 변형전의 산물에 비해 이들 가수분해효소에 대해 저항성을 보였다. 변형에서 얻어진 산물들의 methylation결과 입자 전분을 이용한 변형 반응의 산물의 변형 정도가 더 컸으며, 변형입자를 SEM으로 관찰하면 불규칙한 크기의 작은 조각의 산물을 확인할 수 있었다. 변형 반응으로 sucrose의 glucose가 전분 glucose의 C3, C4, 그리고 C6 위치의 free-OH group에 수식된 새로운 구조의 변형 산물을 생산하였고 이는 iodine에 의한 염색 정도를 크게 감소시켰으며 용해성을 증가시켰다. Many enzymes catalyze a primary reaction and/or secondary reaction. Dextransucrase usually synthesize dextran from sucrose as a primary reaction. The secondary reaction of dextransucrase is the transfer of glucose from sucrose to carbohydrate acceptors. We have reacted dextransucrase from Leuconostoc mesenteroides B-742CB with sucrose and starches; granule or gelatinized starches, and Small or Potato starches. The yield of modified starch was ranged from 46% to 72%(s.d.<±5%) of theoretical depends on various reaction conditions. Modified products were more resistant against the hydrolysis of α-amylase, isoamylase, pullulanase and endo-dextranase than those of native starch. Based on the reactions from enzyme hydrolysis and methylation followed by acid hydrolysis modification of granule starch was more efficient than the modification of gelatinized starch. After modification of granule starch with dextransucrase, there produced a soluble modified starch. After modification the starch granules were fractionated to small size. The positions of glucose substitution of the modified products were determined by methylation followed by acid hydrolysis and analyzed by TLC. The products were modified by the addition of glucose to the position of C3, C4 and C6 free hydroxyl group of glucose residues in the starch.
Cloning and Sequencing of the α-1->6 Dextransucrase Gene from Leuconostoc mensenteroides B-742CB
Kim, Ho Sang,Kim, Do Man,Ryu, Hwa Ja,John F.Robyt 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.4
A dextransucrase gene (dsrB742) that expresses a dextransucrase to synthesize mostly α-1→6 linked dextran with a low amount (3-5%) of α-1→3 branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1-kb PstI fragments were ligated with pGEM-3Zf(-) and transformed into E. coli DH5α. The recombinant clone (pDSRB742) synthesized dextran on an agar plate containing 2%(w/v) sucrose. The dextran synthesized was hydrolyzed with Penicillium endo-dextranase. The hydrolyzate was composed of glucose, isomaltose, isomaltotriose, and branched pentasaccharide. The nucleotide sequence of dsrB742 showed one open reading frame (ORF) composed of 4,524bp encoding dextransucrase. The deduced amino acid sequence revealed a calculated molecular mass of 168.6kDa. It also showed an activity band of 184kDa on a non-denaturing SDS-PAGE (10%). The amino acid sequence of DSRB742 exhibited a 50% similarity with DSRA from L. mesenteroides B-1299, a 70% similarity with DSRS from L. mesenteroides B-512 (F, FMCM) and a 45-56% similarity with Streptococcal GTFs.