scholarly journals Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12

2000 ◽  
Vol 182 (11) ◽  
pp. 3045-3054 ◽  
Author(s):  
Masayuki Hashimoto ◽  
Takahisa Ikegami ◽  
Shizuka Seino ◽  
Nobuhumi Ohuchi ◽  
Harumi Fukada ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Expression System ◽  
Binding Activity ◽  
Binding Domain ◽  
Bacillus Circulans ◽  
Titration Calorimetry ◽  
Affinity Column ◽  
Chitin Binding Domain ◽  
Wide Range ◽  
Hydrolysis Of

ABSTRACT Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBDChiA1 was produced in Escherichia coliusing a pET expression system and purified by chitin affinity column chromatography. Purified ChBDChiA1 specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBDChiA1 was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBDChiA1 to regenerated chitin. These observations suggest that ChBDChiA1recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBDChiA1 exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBDChiA1 is mediated mainly by hydrophobic interactions. Hydrolysis of β-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of β-chitin microcrystals but greatly enhances the efficiency of degradation.

Involvement of Gln679, in addition to Trp687, in chitin-binding activity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

2013 ◽  
Vol 154 (2) ◽  
pp. 185-193 ◽  
Author(s):  
M. Hara ◽  
H. Sugimoto ◽  
M. Uemura ◽  
K.-i. Akagi ◽  
K. Suzuki ◽  
...  
Keyword(s):  
Binding Activity ◽  
Binding Domain ◽  
Bacillus Circulans ◽  
Chitin Binding Domain

A novel chitin‐binding mode of the chitin‐binding domain of chitinase A1 from Bacillus circulans WL ‐12 revealed by solid‐state NMR

FEBS Letters ◽  
2018 ◽  
Vol 592 (18) ◽  
pp. 3173-3182 ◽  
Author(s):  
Hiroki Tanaka ◽  
Hideo Akutsu ◽  
Izumi Yabuta ◽  
Masashi Hara ◽  
Hayuki Sugimoto ◽  
...  
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Binding Mode ◽  
Binding Domain ◽  
Bacillus Circulans ◽  
Chitin Binding Domain

scholarly journals Immobilization of Cells with Surface-Displayed Chitin-Binding Domain

2006 ◽  
Vol 72 (1) ◽  
pp. 927-931 ◽  
Author(s):  
Jen-You Wang ◽  
Yun-Peng Chao
Keyword(s):  
Escherichia Coli ◽  
Gene Fusion ◽  
Cell Immobilization ◽  
Binding Domain ◽  
Engineering Applications ◽  
Chitin Binding Domain ◽  
Wide Range ◽  
Frame Fusion

ABSTRACT To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to lpp and ompA was constructed and expressed in Escherichia coli. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37°C). These results illustrate the promising use of this approach for engineering applications.

scholarly journals Structure of a consensus chitin-binding domain revealed by solution NMR

2020 ◽  
Author(s):  
Dario Heymann ◽  
Harini Mohanram ◽  
Akshita Kumar ◽  
Chandra S. Verma ◽  
Julien Lescar ◽  
...  
Keyword(s):  
Binding Proteins ◽  
3D Structure ◽  
Binding Domain ◽  
Biomimetic Synthesis ◽  
Binding Motif ◽  
Carbohydrate Binding ◽  
Dosidicus Gigas ◽  
Chitin Binding Domain ◽  
Carbohydrate Degradation ◽  
Wide Range

ABSTRACTCarbohydrate-binding proteins (CBPs) are a versatile group of proteins found in almost every organism on earth. CBPs are involved in enzymatic carbohydrate degradation and also serve as templating scaffolds in the exoskeleton of crustaceans and insects. One specific chitin-binding motif found across a wide range of arthropods’ exoskeletons is the “extended Rebers and Riddiford” consensus (R&R). However, how the R&R motif binds chitin is unclear. Here, we report the 3D structure and molecular level interactions of a chitin-binding domain (CBD-γ) located in a CBP from the beak of the jumbo squid Dosidicus gigas. This CBP is one of four chitin-binding proteins identified in the beak mouthpart of D. gigas and is believed to interact with chitin to form a scaffold network that is infiltrated with a second set of structural proteins during beak maturation. We used solution state NMR spectroscopy to elucidate the molecular interactions between CBD-γ and the soluble chitin derivative pentaacetyl-chitopentaose (PCP) and find that folding of this domain is triggered upon its interaction with PCP. To our knowledge, this is the first experimental 3D structure of a CBP containing the R&R consensus motif, which can be used as a template to understand in more details the role of the R&R motif found in a wide range of CBP-chitin complexes. The present structure also provides molecular information for biomimetic synthesis of graded biomaterials using aqueous-based chemistry and biopolymers.

scholarly journals Fusion of Chitin-Binding Domain From Chitinolyticbacter meiyuanensis SYBC-H1 to the Leaf-Branch Compost Cutinase for Enhanced PET Hydrolysis

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Xue ◽  
Yinping Chen ◽  
Huan Rong ◽  
Ren Wei ◽  
Zhongli Cui ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Hydrolysis ◽  
Polyethylene Terephthalate ◽  
Binding Domain ◽  
Plastic Pollution ◽  
Pet Waste ◽  
Chitin Binding Domain ◽  
C Terminus ◽  
Measurable Activity ◽  
Hydrolysis Of

Polyethylene terephthalate (PET) is a mass-produced petroleum-based non-biodegradable plastic that contributes to the global plastic pollution. Recently, biocatalytic degradation has emerged as a viable recycling approach for PET waste, especially with thermophilic polyester hydrolases such as a cutinase (LCC) isolated from a leaf-branch compost metagenome and its variants. To improve the enzymatic PET hydrolysis performance, we fused a chitin-binding domain (ChBD) from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the previously reported LCCICCG variant, demonstrating higher adsorption to PET substrates and, as a result, improved degradation performance by up to 19.6% compared to with its precursor enzyme without the binding module. For compare hydrolysis with different binding module, the catalytic activity of LCCICCG-ChBD, LCCICCG-CBM, LCCICCG-PBM and LCCICCG-HFB4 were further investigated with PET substrates of various crystallinity and it showed measurable activity on high crystalline PET with 40% crystallinity. These results indicated that fusing a polymer-binding module to LCCICCG is a promising method stimulating the enzymatic hydrolysis of PET.

scholarly journals Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

2004 ◽  
Vol 78 (16) ◽  
pp. 8630-8640 ◽  
Author(s):  
Richard L. Kingston ◽  
Walter A. Baase ◽  
Leslie S. Gay
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Measles Virus ◽  
Expression System ◽  
Spectroscopic Studies ◽  
Mumps Virus ◽  
Binding Domain ◽  
Titration Calorimetry ◽  
N Protein ◽  
P Protein

ABSTRACT We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly α-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (Kd ) was determined to be 13 μM at 20°C and 35 μM at 37°C. Our data are consistent with a model in which an α-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

Limited Proteolysis of Vitronectin by Plasmin Destroys Heparin Binding Activity

1991 ◽  
Vol 66 (03) ◽  
pp. 310-314 ◽  
Author(s):  
David C Sane ◽  
Tammy L Moser ◽  
Charles S Greenberg
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activator Inhibitor ◽  
Binding Activity ◽  
Binding Domain ◽  
Heparin Binding ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

SummaryVitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN.Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

scholarly journals In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1906
Author(s):  
Mona Atabakhshi-Kashi ◽  
Mónica Carril ◽  
Hossein Mahdavi ◽  
Wolfgang J. Parak ◽  
Carolina Carrillo-Carrion ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Hydrophobic Interactions ◽  
Intrinsic Properties ◽  
Wide Range ◽  
Self Assembled ◽  
Uptake Studies ◽  
Targeting Ability ◽  
Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

scholarly journals A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Omid Oftadeh ◽  
Pierre Salvy ◽  
Maria Masid ◽  
Maxime Curvat ◽  
Ljubisa Miskovic ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Biomass Composition ◽  
Industrial Biotechnology ◽  
Overflow Metabolism ◽  
Cellular Expression ◽  
A Genome ◽  
Wide Range ◽  
Eukaryotic Organisms ◽  
Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

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