• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1547-1556
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• 2009

A novel thermostable $\alpha$-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2,253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, p-nitrophenyl-$\alpha$-D-glucopyranide, and dextrin with an exotype cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at $60^{\circ}C$ and pH 5.5. The half-life was 2 h at $60^{\circ}C$. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce $\alpha$-1,4-linked maltotriose and $\alpha$-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce $\alpha$-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1447-1456
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• 2018

The amylosucrase encoding gene from Deinococcus geothermalis DSM 11300 (DgAS) was codon-optimized and expressed in Escherichia coli. The enzyme was employed for biosynthesis of three different dihydroxybenzene glucosides using sucrose as the source of glucose moiety. The reaction parameters, including temperature, pH, and donor (sucrose) and acceptor substrate concentrations, were optimized to increase the production yield. This study demonstrates the highest ever reported molar yield of hydroquinone glucosides 325.6 mM (88.6 g/l), resorcinol glucosides 130.2 mM (35.4 g/l) and catechol glucosides 284.4 mM (77.4 g/l) when 400 mM hydroquinone, 200 mM resorcinol and 300 mM catechol, respectively, were used as an acceptor substrate. Furthermore, the use of commercially available amyloglucosidase at the end of the transglycosylation reaction minimized the gluco-oligosaccharides, thereby enhancing the target productivity of mono-glucosides. Moreover, the immobilized DgAS on Amicogen LKZ118 beads led to a 278.4 mM (75.8 g/l), 108.8 mM (29.6 g/l) and 211.2 mM (57.5 g/l) final concentration of mono-glycosylated product of hydroquinone, catechol and resorcinol at 35 cycles, respectively, when the same substrate concentration was used as mentioned above. The percent yield of the total glycosides of hydroquinone and catechol varied from 85% to 90% during 35 cycles of reactions in an immobilized system, however, in case of resorcinol the yield was in between 65% to 70%. The immobilized DgAS enhanced the efficiency of the glycosylation reaction and is therefore considered effective for industrial application.

• Applied Biological Chemistry
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• 제48권1호
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• pp.16-22
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• 2005

키토산분해효소(Chitosanases, EC 3.2.1.132)는 당질가수분해효소군의 하나로, 아미노당인 D-glucosamine polymer인 chitosan의 ${\beta}-1,4-glycoside$ 결합을 가수분해하는 효소이며, 세균, 곰팡이, 식물 등에 널리 분포한다. 본 논문에서는 chitosanase의 N-말단 아미노산의 서열과 입체구조에 근거한 family 및 clan 분류, 작용모형, 절단 유형, subclass 분류, 및 family-subclass 상관성을 검토하였다. 아미노산 서열과 입체구조와 기질의 분해패턴 사이에는 깊은 상관이 있음을 확인 제시하였다. 다양한 종 유래 chitosanase의 1차구조의 해명과 진화적 상관 규명, 나아가 보다 정교한 chitosanase의 정의와 다양한 산업적 응용에의 가능성도 검토하였다.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.461-466
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• 1986

본 연구에서는 CGTase 생산성이 높은 균주인 Bacillus sp.를 자연계에서 분리하였고 이 균주의 특성, 분비효소의 특성, 효의 생산조건 및 효소이용 가능성에 대한 기초연구를 실시하였다. 분리균주는 운동성이 있는 내성포자형성 간균이었고 분비효소의 최적작용조건은 pH 6.0, 45$^{\circ}C$이며 pH 6-10 범위에서 안정하였다. 효소생산조건은 탄소원으로 corn starch 1%, 질소원으로서 corn steep liquor 5%, urea 0.1%, ammonium sulfate 0.25%를 동시에 첨가하는 것이 가장 양호하였으며 본 생산균을 30$\ell$ jar fermentor로 3$0^{\circ}C$, 200rpm, 0.6vvm에서 60시간 정도 배양하였을 때 최대의 효소 생산력을 나타내었다. 또한 본 효소를 이용하여 stevioside를 acceptor로 하고 전분가수분해물을 donor로 하여 당전이반응을 실시한 결과 상당한 전이효과가 나타났으므로 기타 전이제품 및 cyclodextrin 생산에 응용이 가능할 것으로 기대된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권3호
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• pp.174-180
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• 2000

Cyclodextrin glucanotransferase (CGTasa) from Thermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of amount ratio of the adsorbed enzyme to intial amount in the solution. Immobilixation of CGTase shifted the optimum temperature for the enzyme to peoduce transglycosylated xylitol from 7$0^{\circ}C$ to 9$0^{\circ}C$ and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuoncly produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10%(w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10%(w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10%(w/v) xylitor as the glycosyl acceptor, 20mL/h of medium fiow rate, and 6$0^{\circ}C$. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g.L-1.h-1, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1916-1924
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• 2017

In this study, we synthesized a glycosylated derivative of caffeic acid phenethyl ester (CAPE) using the amylosucrase from Deinococcus geothermalis with sucrose as a substrate and examined its solubility, chemical stability, and anti-inflammatory activity. Nuclear magnetic resonance spectroscopy showed that the resulting glycosylated CAPE (G-CAPE) was the new compound caffeic acid phenethyl ester-4-O-${\alpha}-{\small{D}}$-glucopyranoside. G-CAPE was 770 times more soluble than CAPE and highly stable in Dulbecco's modified Eagle's medium and buffered solutions, as estimated by its half-life. The glycosylation of CAPE did not significantly affect its anti-inflammatory activity, which was assessed by examining lipopolysaccharide-induced nitric oxide production and using a nuclear factor erythroid 2-related factor 2 reporter assay. Furthermore, a cellular uptake experiment using high-performance liquid chromatography analysis of the cell-free extracts of RAW 264.7 cells demonstrated that G-CAPE was gradually converted to CAPE within the cells. These results demonstrate that the glycosylation of CAPE increases its bioavailability by helping to protect this vital molecule from chemical or enzymatic oxidation, indicating that G-CAPE is a promising candidate for prodrug therapy.

• KSBB Journal
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• 제19권2호
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• pp.164-167
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• 2004

효소인 싸이클로덱스트린글루카노트란스퍼라제 (CGTase)는 효소의 활성도에 칼슘이 관련된 분자내 당 전이반응에 의해 녹말과 그에 관련된 $\alpha$-1,4-glucan의 기질을 싸이클로덱스트린으로 생성시키는 산업적으로 가장 많이 응용되는 효소 중에 하나이다. 수용성 녹말을 기질로 하여 CLEC화한 Bacillus macerans $\alpha$-CGTase 효소를 극한의 반응 조건인 계면활성제나 유기 용매가 혼합된 반응조건에서 실험한 결과, 이들 조건이 싸이클로덱스트린의 생산을 증가시키는 영향을 초래하였고 특히, 계면활성제인 SDS와 $\beta$-OG는 전체 싸이클로덱스트린의 생성을 증가시켰고 이 중에서 SDS와 Lubrol PX는 알파싸이클로덱스트린의 생성의 특이성의 결과를, 반면에 Triton x-100과 Tween 80은 알파싸이클로덱스트린의 생성을 억제하는 결과를 보였다. 유기용매인 DMSO, formamide, MPD, ethylene glycol 또한 사이클로 덱스트린의 전체 수율과 특이성에 영향을 미치는 효력을 보였다.

• Food Science and Biotechnology
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• 제18권3호
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• pp.776-781
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• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1698-1704
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• 2012

Resveratrol, or its glycoside form piceid, is a dietary antioxidant polyphenolic compound, found in grapes and red wine that has been shown to have protective effects against cardiovascular disease. However, very low water solubility of the compound may limit its application in the food and pharmaceutical industries. The amylosucrase (AMAS) of Alteromonas macleodii Deep ecotype was expressed in Escherichia coli and showed high glycosyltransferase activity to produce the glucosyl piceid when piceid was used as an acceptor. The conversion yield of piceid glucoside was 35.2%. Biotransformation using culture of the E. coli harboring the amas gene increased the yield up to 70.8%. The transfer product was purified by reverse phase chromatography and recycling preparative HPLC, and the molecular structure of the piceid glucoside was determined using NMR spectroscopy. The piceid glucoside was identified as glucosyl-${\alpha}$-($1{\rightarrow}4$)-piceid. The solubility of glucosyl piceid was 5.26 and 1.14 times higher than those of resveratrol and piceid, respectively. It is anticipated that dietary intake of this compound is more effective by enhancing the bioavailability of resveratrol in the human body because of its hydrophilic properties in the intestinal fluid.

• 한국미생물·생명공학회지
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• 제44권3호
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• pp.363-369
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• 2016

Listeria innocua ATCC 33090 유전체로부터 maltose/maltodextrin 이용과 관련한 유전자 클러스터를 발견하였으며, 그로부터 cyclomaltodextrinase (LICD)로 예상되는 유전자를 클로닝하고, 대장균 내에서 발현하였다. LICD는 총 591개의 아미노산으로 이루어진 68.6 kDa 크기의 효소이며, 일반적인 CDase 계열 효소들과 39−58%의 아미노산 서열 상동성을 나타내었다. 재조합 LICD는 37℃, pH 7.0의 조건에서 최대 활성을 나타내었으며, cyclodextrin, starch, maltotriose에 작용하여 주로 maltose를 생성하였다. 또한 pullulan을 분해하여 panose를, 그리고 acarbose를 분해하여 glucose와 acarviosine-glucose를 생성하는 전형적인 CDase 계열 효소임을 확인하였다. 그러나, starch 및 pullulan과 같은 고분자기질 대비 cyclodextrin 및 maltotriose의 저분자 소당류에 대해 상대적으로 높은 활성을 나타내며, acarbose 분해 활성이 매우 낮아 다른 효소들과 차별성을 가진다. 또한 LICD는 acarbose 공여체를 가수분해하여 수용체에 전이하는 당전이 활성을 보였다.