• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.443-448
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• 2008

Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable ${\beta}$-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric ${\beta}$-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1366-1372
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• 2021

Bacterial nanocellulose (BNC) is a biocompatible material with a lot of potential. To make BNC commercially feasible, improvements in its production and surface qualities must be made. Here, we investigated the in situ fermentation and generation of BNC by addition of different cellulosic substrates such as Avicel and carboxymethylcellulose (CMC) and using Komagataeibacter sp. SFCB22-18. The addition of cellulosic substrates improved BNC production by a maximum of about 5 times and slightly modified its structural properties. The morphological and structural properties of BNC were investigated by using Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy and X-ray diffraction. Furthermore, a type-A cellulose-binding protein derived from Clostridium thermocellum, CtCBD3, was used in a novel biological analytic approach to measure the surface crystallinity of the BNC. Because Avicel and CMC may adhere to microfibrils during BNC synthesis or crystallization, cellulose-binding protein could be a useful tool for identifying the crystalline properties of BNC with high sensitivity.

• Journal of Plant Biology
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• v.37 no.2
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• pp.151-158
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• 1994

Aleurone layers of normal and vp1 mutant maize kernels were extracted and centrifuged at 100,000g to yield a cytosol fraction. Binding of [3H]ABA cis, trans (＋)ABA to a soluble macromolecular components present in the cytosol was demonstrated by Sephadex chromatography and non-denaturing PAGE. The binding component was of high molecular weight and seems to be an aggregate of proteins. A rapid DEAE-cellulose filter method for assaying bound [3H]ABA to a soluble protein was adapted. Binding assays were performed with cytosol that had been preheated or incubated with several enzymes, indicating that heat and protease treatments disrupted the binding. This suggested that binding occurred to proteins. Some properties of the ABA binding proteins were described. The [3H]ABA binding were reduced dramatically when unlabeled ABA was added as a competitor, suggesting a specific binding of [3H]ABA. Gel filtration profiles and autoradiogram of [3H]ABA binding showed no difference in the binding components of Vp1 and vp1/vp1 mutant cytosol, indicating that Vp1 protein is not a sole ABA binding protein.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.329-333
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• 2005

This study was designed to investigate the stability of a lipase fused with a cellulose­binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene in Trichoderma hazianum and a lipase gene in Bacillus stearother­mophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the re­action-dependent factors (RDF). RIF includes the reaction conditions such as pH and tempera­ture, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and $50^{\circ}C$ were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal­line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over $70\%$ of the initial activity.

• The Korean Journal of Zoology
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• v.34 no.2
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• pp.196-202
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• 1991

Juvenile honnone binding protein (JHBP) was identified in the last instar larval hemolymph of Lymantria dispar using dextran coated charcoal (DCC) binding assay and gel filtration. The p1 value of JHBP was estimated to be 5.3. JHBP was partially pudfied by polyethylene glycol(PEG) precipitation, DEAE-cellulose ion-exchange chromatography and gel filtration, and was confirmed by DCC binding assay.

• Proceedings of the Korean Nutrition Society Conference
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• 2003.10a
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• pp.90-90
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• 2003

• Membrane Journal
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• v.32 no.5
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• pp.348-356
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• 2022

With the development of the bio industry, membrane chromatography with a high adsorption efficiency is emerging to replace the existing column chromatography used in the downstream processes of pharmaceuticals, food, etc. In this study, through the deacetylation reaction of two commercial cellulose acetate (CA) membranes with different pore sizes, the porous regenerated cellulose (RC) supports for membrane chromatography were obtained to attach the anion exchange ligands. The adsorptive membranes for anion exchange were prepared by attaching an anion exchange ligand ([3-(methacryloylamino) propyl] trimethylammonium chloride) containing quaternary ammonium groups on the RC supports by grafting and UV polymerization. The protein adsorption capacities of the prepared membranes were obtained through both the static binding capacity (SBC) and the dynamic adsorption capacity (DBC) measurement. As a result, the membrane chromatography with the smaller the pore size, the larger the surface area showed the highest protein adsorption capacity. Membrane chromatography which was prepared by using deacetylated commercial CA support with MAPTAC ligand (i.e., RC 0.8 + MAPTAC: 43.69 mg/ml, RC 3.0 + MAPTAC: 36.33 mg/ml) showed a higher adsorption capacity compared to commercial membrane chromatography (28.38 mg/ml).

• BMB Reports
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• v.28 no.5
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• pp.443-450
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• 1995

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.183-183
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• 1994

HIV-1 protcase 를 이용한 in vitro assay system을 개발하기 위하여HIV-1 protease유전자를 Ecoli 발현 벡터를 이용하여 발현시켰다. 가능성있는 Protease유전자의 생간 및 분래를 용이하게 하기위하여 maltose binding protein 의 fusion protein을 이용하였으며 protease 의 autoprocessing을 maltose binding protein 의 polyclonal 항체로 확인하였다, 발현된 protease는 일련의 chromatography 방법 (DEAE, SE cellulose, Superose 12, Mono S) 으로 순수하게 분리되었다. 정제된 protease 는 SDS-PAGE분석으로 단일밴드를 보여주었고, 합성된 undecapeptide를 기질로 하였을때 Km 이 9.8$\mu$M 이었다. 효소 assay 를 위해 기질이 protease 에 의해 절단된 생성물을 HPLC를 사용하여 분석하였다. Protease의 억제제 탐색을 위해 유기합성한 몇개의 기질유사체와 HIV-1 증식을 억제하는 것으로 알려진 천연물의 억제정도를 조사하여 보았다. 이들 test 에 사용한 물질들은 높은 농도에서 protease 의 활성을 저해하는 것으로 보아 좋은 억제제는 아닌것으로 시료되나 본 연구를 통하여 확립된 in vitro assay system 은 추후에도 억제제 탐색을 위하여 계속 활용될 수 있을 것이다.

• Journal of the Korean Wood Science and Technology
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• v.34 no.3
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• pp.56-63
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• 2006

In order to evaluate the mechanism of cellulose hydrolysis, the complementary DNA encoding Glycoside Hydrolase Family (GHF)74 was cloned from Phanerochaete chrysosporium. Depending on the presence of Cellulose Binding Module (CBM), it can be classified as GHF74A or GHF74B. The GHF74A gene from P. chrysosporium (PcGHF74A) consists of 2163 bp encoding a protein of 721 amino acid residues. The PcGHF74A showed homology of 70~77% compared with the GHF74 from other filamentous fungi. The PcGHF74B, which contains CBM and is a member of family 1, was transcribed to various transcripts depending on the nature of carbon sources and their concentration. To study the possible presence of splice variants in GHF74B transcripts in P. chrysospoium, we carried out RT-PCR analysis using primers that designed based on the annotation data and sequenced data. Our result indicated that PcGHF74B was transcribed to several splicing variants in various culture conditions. Especially in the culture of 2% cellulose, three transcript products were observed. First transcript was presumed to be a full length ORF that contained 11th intron with stop codon at position 2562 bp. The second one consisted of 12 exons and 11 introns with stop codon at position 1187 bp with 7th exon. The shortest transcript consisted of 10 exons and 9 introns with stop codon at 910 bp in the 7th exon. These premature stop codon might prevent the synthesis of fully active GHF74 or contribute for the production of protein with distinct function depending on the ambient carbon sources.