• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.404-409
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• 2008

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and ${\beta}$-glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1269-1275
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• 2006

The selection of multienzyme complex-producing bacteria under aerobic condition was conducted for improving the degradation of lignocellulosic substances. The criteria for selection were cellulase and xylanase enzyme production, the presence of cellulose-binding domains and/or xylan-binding domains in enzymes to bind to insoluble substances, the adhesion of bacterial cells to insoluble substances, and the production of multiple cellulases and xylanases in a form of a high molecular weight complex. Among the six Bacillus strains, isolated from various sources and deposited in our laboratory, Paenibacillus curdlanolyticus B-6 strain was the best producer of cellulase and xylanase enzymes, which have both cellulose-binding factors (CBFs) and xylan-binding factors (XBFs). Moreover, multiple carboxymethyl cellulases (CMCases) and xylanases were produced by the strain B-6. The zymograms analysis showed at least 9 types of xylanases and 6 types of CMCases associated in a protein band of xylanase and cellulase with high molecular weight. These cells also enabled to adhere to both avicel and insoluble xylan, which were analyzed by scanning electron microscopy. The results indicated that the strain B-6 produced the multienzyme complex, which may be cellulosome or xylanosome. Thus, P. curdlanolyticus B-6 was selected to study the role and interaction between the enzymes and their substrates and the cooperation of multiple enzymes to enhance the hydrolysis due to the complex structure for efficient cellulases and xylanases degradation of insoluble polysaccharides.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1249-1253
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• 2007

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.

• Applied Biological Chemistry
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• 제39권6호
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• pp.494-500
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• 1996

중요한 환경문제로 대두되고 있는 중금속 오염의 대처 방안으로 식물을 이용한 경제적인 정화 방법을 모색하고자 미나리의 중금속 흡수능력을 검정하고 카드늄 결합단백질을 동정하였다. 미나리의 중금속 흡수능력은 미나리를 카드늄 $(Cd^{2+})$, 크롬 $(Cr^{3+})$ 및 납 $(Pb^{2+})$의 농도를 달리한 배양액에서 재배한 후 미나리에 잔류하는 중금속을 정량함으로써 측정되었다. 중금속의 처리 기간은 3일과 7일로 하였고, 생장 저해가 일어나는 농도까지 처리하였다. 카드늄은 16.68 ppm, 크롬은 20 ppm까지 지속적으로 잔류량이 증가하였고 그 농도 이상에서는 생장 저해가 일어나면서 잔류량의 증가율이 둔화되었다. 식물체 부위별 카드늄과 크롬의 잔류량은 뿌리에서 월등하게 높게 나타났고 줄기와 잎의 순으로 낮아졌다. 납의 경우는 카드늄과 크롬에 비하여 잔류량이 가장 높은 뿌리에서 4배 정도 잔류량이 높게 나타났다. 20 ppm의 중금속 용액에서 7일간 재배한 미나리 뿌리에는 건조중량 1 g에 대하여, 카드늄이 6.1 mg, 크롬은 5.2 mg 그리고 납은 23.6 mg이 잔류하고 있었다. 이것은 잔류하고 있는 중금속 중에서 카드늄은 80%, 크롬은 92%그리고 납은 96%이상이 미나리 뿌리에 잔류하고 있는 것이다. 20 ppm카드늄이 함유된 배양액에서 7일간 자란 미나리 뿌리 추출액을 Sephadex G-50과 DEAE-Cellulose 크로마토그래피를 수행하여, polyacrylamide gel 전기영동 상으로 단일 단백질 분리대의 중금속 결합단백질을 정제하였다. 이 단백질의 분자량은 gel filtration 상에서 약 5,000 Da이었고 아미노산의 조성을 보면 산성 아미노산이 27.3%, cystein이 9.9%로서 중금속 결합단백질의 특성을 가지고 있었다. 그러나 그 아미노산 조성은 지금까지 알려진 phytochelatin과는 다른 새로운 중금속 결합단백질로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1732-1740
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• 2021

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1077-1084
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• 2009

A genomic library was constructed to clone a xylanase gene (Mxyn10) from Demequina sp. JK4 isolated from a deep sea. Mxyn10 encoded a 471 residue protein with a calculated molecular mass of 49 kDa. This protein showed the highest sequence identity (70%) with the xylanase from Streptomyces lividans. Mxyn10 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10) and a carbohydrate-binding module (CBM) belonging to family 2. The optimum pH and temperature for enzymatic activity were pH 5.5 and $55^{\circ}C$, respectively. Mxyn10 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 3.5 to 10.0. The protein was not significantly affected by a variety of chemical reagents, including some compounds that usually inhibit the activity of other related enzymes. In addition, Mxyn10 showed activity on cellulose. These properties mark Mxyn10 as a potential enzyme for industrial application and saccharification processes essential for bioethanol production.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1430-1435
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• 2020

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

• 원예과학기술지
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• 제31권6호
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• pp.782-789
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• 2013

당근(Daucus carota L. var. sativa)은 세계적으로 널리 이용되는 작물 중 이며, 영양학적으로도 중요한 작물이다. 하지만, 종자 표피세포에서 생성되는 종자모는 발아를 억제하고 흡수를 저해하여 육묘에 어려움을 야기한다. 이러한 어려움을 타파하기 위해 당근 종자는 기계적인 제모작업을 거쳐 상품화 되고 있다. 이 과정에서 생산상의 여러 가지 단점들이 발생하며, 이를 보완하기 위해 단모종자 당근 품종의 육종이 필요하다. 따라서 본 연구는 단모종자 표현형 CT-ATR 615 OP 666-13개체와 장모종자 표현형 CT-ATR 615 OP 671-9개체 및 단모종자 표현형 CT-SMR 616 OP 659-1 개체와 장모종자 표현형 CT-SMR 616 OP 677-14개체 등 두 조합의 종자 cDNA library를 작성 후 EST 염기서열의 비교분석을 통해 당근 종자모 형질관련 연구에 이용하고자 하였다. 첫째로 EST 염기서열의 BlastX 결과를 바탕으로 각각의 EST를 FunCat 기능별 category로 분류하였다. 그 결과 Metabolism category와 protein folding 및 stabilization, protein binding, C-compound binding category에서 2조합 모두 동일한 유의적인 차이를 확인하였다. 두 번째로 EST 염기서열의 GO data를 바탕으로 seed trichome differentiation 및 cellulose biosynthetic process에 관련된 EST를 선발하였다. 이러한 FunCat category에서의 차이점과 GO data 분석을 통해 확인된 후보 EST 들이 당근 종자모 형성에 많은 영향을 미치는 것으로 생각된다. 마지막으로 분석된 개체 별 EST 염기서열을 바탕으로 33개의 SNP site, 741개의 SSR site를 확인하였다. 확인된 SNP 및 SSR site는 당근 종자모 형성에 관련된 분자마커 개발에 이용할 수 있음은 물론 당근의 여러 형질에 대한 연구에 활용 가능할 것으로 기대된다.