• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1882-1893
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• 2019

β-Glucosidases and β-xylosidases are two categories of enzymes that could cleave out non-reducing, terminal β-D-glucosyl and β-D-xylosyl residues with release of D-glucose and D-xylose, respectively. In this paper, two functional β-glucosidase Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75℃ and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a K_i value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13-fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the βglucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75℃, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

• Journal of Life Science
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• 제9권1호
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• pp.26-34
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• 1999

A thermostable pullulanase has been isolated and purified from Thermus caldophilus GK-24 to a homogeneity by gel-filtration and ion-exchange chromatography. The specific activity of the purified enzyme was 431-fold increase from the crude culture broth with a recovery of 11.4%. The purified enzyme showed $M_{r}$ of 65 kDa on denaturated and natural conditions. The pI of the enzyme was 6.1 and Schiff staining was negative, suggesting that the enzyme is not a glycoprotein. The enzyme was most active at pH 5.5. The activity was maximal at $75^{\cire}C$ and stable up to $95^{\cire}C$ for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at $4^{\cire}C$ for 24hr. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was simulated by $Mn^{2+}$ ion, }$Ni^{2+}$, $Ca^{2+}$, $Co^{2+}$ ions. The enzyme hydrolyzed the ${\alpha}$-1,6-linkages of amylopectin, glycogens, ${\alpha}$, ${\beta}$-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose and the activity was inhibited by $\alpha$, $\beta$, or $\gamma$-cyclodextrins. The $NH_{2}$-terminal amino acid sequence [(Ala-Pro-Gln-(Asp of Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] was compared with known sequences of various sources and that was compared with known sequences of various sources and that was different from those of bacterial and plant enzymes, suggesting that the enzymes are structurally different.

• BMB Reports
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• 제49권6호
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• pp.349-354
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• 2016

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.57-63
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• 2006

We have cloned a xylanase gene (xynA) from Streptomyces thermocyaneoviolaceus. The deduced amino acid sequences of the XynA, including the active site sequences of glycosyl hydrolase family 10, showed high sequence homology with several xylanases assigned in this category. The XynA was overexpressed under an IPTG inducible T7 promoter control in E. coli BLR(DE3). The overproduced enzymes were excreted into culture supernatants and periplasmic space. The purified XynA had an apparent molecular mass of near 54 kDa, which corresponds to the molecular mass calculated from its gene. The optimum pH and temperature of the purified XynA were determined to be 5.0 and $65^{\circ}C$, respectively. The XynA retained over $90\%$ its activity after the heat treatment at $65^{\circ}C$ for 30 min. The XynA was highly efficient in producing xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) from xylan.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.513-517
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• 2010

A thermostable trehalose synthase (TtTSase) from Thermus thermophilus HJ6 was immobilized on chitosan activated with glutaraldehyde. The yield of immobilization was evaluated as 39.68%. The optimum pH of the immobilized enzyme was similar to that of the free enzyme. However, the optimal temperature ranges were shifted by about $4^{\circ}C$ owing to better thermal stability after immobilization. The half-life of heat inactivation for free and immobilized enzymes was 5.7 and 6.3 days at $70^{\circ}C$, respectively, thus showing a lager thermostability of the immobilized enzyme. When tested in batch reaction, the immobilized enzyme retained its relative activity of 53% after 30 reuses of reaction within 12 days, and still retained 82% of its initial activity even after 150 days at $4^{\circ}C$. A packed-bed bioreactor with immobilized enzyme showed a maximum yield of 56% trehalose from 100 mM maltose in a continuous recycling system (bed volume: 10 ml) under conditions of pH 7.0 and $70^{\circ}C$.

• 미생물학회지
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• 제52권3호
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• pp.344-351
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• 2016

대한민국 동해안 울진 앞 바닷물로부터 50-C로 명명한 해양 미생물을 분리하였다. 50-C 균주는 그람-음성, 호기성 세균이며, 노란색 집락을 형성하고, 극성편모를 갖는 박테리아이다. 이 균주는 $20-50^{\circ}C$, pH 5.5-8.5 범위에서 자라며, 비교적 고온인 $40-50^{\circ}C$, pH 6.5-7.5, 2% (w/v) NaCl에서 최적 성장을 보인다. 16S rRNA 유전자 서열 분석결과 50C-3 균주는 Ruegeria 속에 속하는 R. intermedia CC-GIMAT-$2^T$, R. lacuscaerulensis ITI-$1157^T$의 16S rRNA 유전자 서열과 각각 99.4%, 96.98% 상동성을 보였다. 그러나 50C-3 균주는 운동성, 탄소이용능력, 효소생산능력 등의 생리학적 특성에서 두 균주와는 명확히 다른 특성을 보였다. 50C-3 균주의 DNA G+C content는 66.7 mol%이고, 주요한 respiratory quinone은 ubiquinone-10 (Q-10)이었다. 이와 같은 형태학적, 생리학적, 유전학적 특성을 비교하여, 50C-3 균주는 R. intermedia CC-GIMAT-$2^T$와 같은 종에 속하는 새로운 변종으로 판단되며 Ruegeria sp. 50C-3으로 명명하였다(KCTC23890 =DSM25519). 50C-3 균주는 cellulase, agarase 활성은 없었지만, alkaline phosphatase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase를 생산하였고 이들 모두 $50^{\circ}C$ 에서도 활성이 좋은 내열성 효소일 것으로 판단되었다. 특히, ${\beta}$-galactosidase의 경우 $37^{\circ}C$에서 보다 $50^{\circ}C$에서의 활성이 1.9배 증가하여 산업적으로 활용성이 클 것으로 예상된다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.46-53
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• 2001

A commensal thermophile, Symbiobacterium toebii, isolated from hay compost (toebii) in Korea commensally interacted with a thermophilic Geobacillus toebii sp. nov., which was a new species within the genus Geobacillus on the basis of the phenotypic traits and molecular systematic data. S. toebii required the crude extracts and/or culture supernatant of the Geobacillus toebii for axenic growth and could grow on the temperature between 45 and $70^{\circ}C$ (optimum: $60^{\circ}C$; 2.4 h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was $65 mol\%$, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that Symbiobacterium toebii was closely related with solely reported Symbiobacterium thermophilum. The presence of the commensal thermophile 16S rDNA and accumulation of indole in all the enriched cultures indicate that Symbiobacterium toebii is widely distributed in the various soils. The genome of S. toebii constituted a circular chromosome of 3,280,275 base pairs and there was not an extra-chromosomal element (ECE). It contained about 4,107 predicted coding sequences. Of these protein coding genes, about $45.6\%$ was encoded well-known proteins and annotated the functional assignment of 1,874 open reading frames (ORFs), and the rest predicted to have unknown functions. The genes encoding thermostable tyrosine phenol-lyase and tryptophan indole-lyase were cloned from the genomic DNA of S. toebii and the enzymatic production of L-tyrosine and L-tryptophan was carried out with two thermostable enzymes overexpressed in recombinant E. coli.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.504-511
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• 2016

락툴로오스는 기존에 화학적인 이성화법을 통해 생산해왔던 기능성 당으로서 프로바이오틱스나 장내균총 개선을 위한 의약품으로 활용되어 왔다. 최근 락툴로오스 화학전환법의 단점인 촉매제거와 부산물제거 에너지손실등의 문제를 해결할 수 있는 생물촉매를 이용한 락툴로오스 전환법이 대두되었다. 본연구에서는 유당의 낮은 용해도와 락툴로오스의 효율적전환을 위해 최적의 효소를 선별하여 무작위 돌연변이법으로 유전자를 개량하여 열내성이 $75^{\circ}C$까지 증진되고 활성이 1.3배 향상된 효소를 선별하였다. 이 효소를 정제하여 사용하는 대신 본 연구에서는 과량 발현시킨 대장균을 Ca-alginate로 고정화하여 $70^{\circ}C$에서 200 g/l의 유당과 회분식으로 반응시켜 43%의 전환 수율을 확인하였다. 반복회분식 실험에서 고정화된 담체는 비교적 안정적이었으며 4회 반복반응 후에도 80% 이상의 활성을 유지하고 있었다. 산업적인 방법을 개발하기 위해 고정화 담체를 이용한 반응기의 운전 최적화와 담체의 안정화를 증진시키는 추가적인 연구가 필요하지만, 본 연구에서는 열내성 특성을 이용하여 정제된 효소가 아닌 효소를 발현하는 세포자체를 고정화 시킴으로써 경제성있는 생산에 대한 방법론을 제시하였다.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1210-1215
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• 2006

The properties related to the temperature and oxygen stability of the cytoplasmic hydrogenases from the fermentative strict anaerobic bacterium, Clostridium butyricum NCIB 9576 (Cl. butyricum), and purple sulfur phototrophic bacterium, Thiocapsa roseopersicina NCIB 8347 (T. roseopersicina), were compared. The optimum temperatures for the growth of Cl. butyricum and T. roseopersicina were 37$^{\circ}C$ and 25$^{\circ}C$, respectively, whereas those for the H$_2$ evolution of the cytoplasmic hydrogenases prepared from Cl. butyricum (C-H$_2$ase) and T. roseopersicina (T-H$_2$ase) were 45$^{\circ}C$ and 65$^{\circ}C$, respectively. The T-H$_2$ase was more thermostable than the C-H$_2$ase and retained its full activity for 5 h at 50$^{\circ}C$ under anaerobic conditions and 90% of its activity at 60$^{\circ}C$, whereas the C-H$_2$ase lost its activity drastically at 50$^{\circ}C$. The optimum pHs for H$_2$ oxidation of the C-H$_2$ase and T-H$_2$ase were 9.0 and 7.5, respectively. Both enzymes showed a maximum H$_2$ evolution activity at pH 7.0. Under aerobic conditions, 80% of the T-H$_2$ase activity was retained for 10 h at 30$^{\circ}C$, and 50% of the activity remained after 6 days under the same experimental conditions. However, the C-H$_2$ase was labile to oxygen and lost its activity immediately on exposure to air. Therefore, these properties of the T-H$_2$ase are expected to be advantageous for application in in vitro biological H$_2$ production systems.

• Applied Biological Chemistry
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• 제41권1호
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• pp.18-22
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• 1998

Bacillus licheniformis를 화학적 돌연변이를 시켜 내열성 ${\alpha}-amylase$ 고생산성 변이주 SK-5를 얻었다. 변이주는 모균에 비하여 약 50배 정도의 ${\alpha}-amylase$를 생산하였으며, 그 모양이 가늘고 길이가 길어졌고, 성장속도가 감소되었다. 이 효소의 유전적 변화를 분석하기 위하여 변이주 SK-5로부터 ${\alpha}-amylase$ 유전자 염기배열을 결정한 결과 구조유전자의 염기배열은 동일하였으나 promoter 지역에서 일부 변이가 일어난 것이 확인되어 이것이 부분적으로 효소생산성 증가에 영향을 미칠 것으로 여겨진다. SK-5의 ${\alpha}-amylase$ 생산성이 높기 때문에 이의 배양상층액으로부터 열처리와 황산암모늄 침전 후 한 단계의 hydroxyapatite 컬럼을 사용하여 순수하게 정제된 ${\alpha}-amylase$를 얻을 수 있었다. 변이에 따른 세포외 단백질분해효소의 영향을 검증하기 위하여 SK-5 배양액을 시간별로 준비하여 Western blot으로 분석한 결과 변이주에서 분비되는 ${\alpha}-amylase$의 구조에 변화가 없음을 확인하였다.