• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.440-446
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• 2014

The carbohydrate-binding module (CBM) is an important domain of most cellulases that plays a key role in the hydrolysis of cellulose. The neutral endoglucanase (EG1) gene was reconstructed. A redesigned endoglucanase, named EG2, was constructed with a CBM containing a linker from Corticium rolfsii (GenBank Accession No. D49448). The redesigned EG genes were expressed in Escherichia coli, and their characteristics are discussed. Results showed that the degradation of cellulose by EG2 was about double that by EG1. The specific activities of EG1 and EG2 were tested under optimal conditions, and EG2 had higher activity ($169.1{\pm}2.74$ U/mg) toward CMC-Na than did EG1 ($84.0{\pm}1.98$) in the process of cellulose degradation. The optimal pH and temperature, pH stability, and heat stability of EG1 and EG2 were similar. Results indicated that the CBM plays an essential role in the hydrolysis of cellulose. We can improve EG's catalytic power by adding the CBM from Corticium rolfsii.

• Journal of the Korean Wood Science and Technology
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• 제34권3호
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• pp.56-63
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• 2006

셀룰로오스의 가수분해 기작을 구명하기 위하여 Phanerochaete chrysosporium으로부터 74A (PcGHF74A) 유전자를 클로닝한 결과 2162 bp의 염기서열에 해당하는 721개의 아미노산을 가지고 있으며, 다른 사상균에서 유래한 GHF74와 70~77%의 상동성을 나타냈다. Phanerochaete chrysosporium GHF74B (PcGHF74B)는 family 1에 속하는 Cellulose Binding Module (CBM)을 가지고 있으며 셀룰로오스 배양계에서 다양한 전사산물이 존재하였다. PcGHF74B 전사산물에서 나타난 splice variants를 조사하기 위해서 annotation data와 sequence data로부터 primer를 설계하여 RT-PCR분석을 수행하였으며 그 결과 다양한 배양조건에서 splice variants가 존재함을 확인하였다. 첫 번째는 annotation data와 다르게 11번째 intron을 포함하고 있어 full length로 추정되어지는 것으로 2562 bp에 stop codon이 존재했으며, 두 번째는 7번째 exon 1187 bp에 stop codon을 가지고 있으며 12개의 exon으로 구성되어 있다. 세 번째는 10개의 exon과 9개의 intron을 포함하고 있으며 7번째 exon에 stop codon이 존재했다. Splice variants로서 intron에 나타난 stop codon으로 인해 활성단백질의 합성이 일어나지 않을 것이며 비활성 단백질을 생성하거나 원래의 GHF74의 기능이 아닌 다른 새로운 기능을 갖는 단백질을 생성할 수 있을 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• 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.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• 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.