Difference between revisions of "Part:BBa K4380000"

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	This part contains cellulose binding protein, which can be used as a novel way to imobilize proteins on cellulose. The part was used extensively in Vilnius-Lithuania iGEM 2022 team project "NanoFind".		This part contains cellulose binding protein, which can be used as a novel way to imobilize proteins on cellulose. The part was used extensively in Vilnius-Lithuania iGEM 2022 team project "NanoFind".
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	<partinfo>BBa_K4380000 SequenceAndFeatures</partinfo>		<partinfo>BBa_K4380000 SequenceAndFeatures</partinfo>

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Revision as of 14:52, 1 October 2022

Cellulose Binding domain (CBD)

This part contains cellulose binding protein, which can be used as a novel way to imobilize proteins on cellulose. The part was used extensively in Vilnius-Lithuania iGEM 2022 team project "NanoFind".

Sequence and features

Introduction

Cellulose binding domain (CBD) is a protein that has the ability to robustly bind cellulose. The protein coded from this sequence comes from a thermophilic, anaerobic bacterium Clostridium thermocellum . The part can be succesfully used in safety level 1 laboratory for different, but useful reasons. The CBM3 is overexpressed in Escherichia coli and it is possible to take advantage of its affinity properties to purify recombinant proteins on cellulose fibers, (reducing significantly the costs of purification), to imobilize proteins on a cellulose membrane as well as a a purification tag for antimicrobial peptides. CBM3 proteins can not only bind to crystalline cellulose, but can also interact with smaller affinity (~500 lower) to chitin and xyloglucan.

Biology

Many bacterial and fungal enzymes that hydrolyse insoluble carbohydrates share a familiar structure composed of a catalytic domain linked to carbohydrate-binding module (CBM). Carbohydrate-binding modules (CBMs) are non-catalytic domains that anchor glycoside hydrolases into complex carbohydrates. Clostridium thermocellum produces a multi-enzyme complex of cellulases and hemicellulases, termed the cellulosome, which is organized by the scaffoldin protein CipA.

C. thermocellum cellulosome. C. thermocellum scaffoldin (CipA) contains nine type I cohesins and thus organizes a multienzyme complex that incorporates nine enzymes. The C-terminal type II dockerin of CipA binds specifically to type II cohesin modules found in cell surface proteins. Individ- ual enzymes may also adhere directly to the bacterium cell envelope by binding the single type I cohesins found in OlpA and OlpC.

https://www.researchgate.net/figure/Structure-of-the-novel-type-I-Coh-Doc-complexes-CohOlpA-Doc918-and-CohOlpC-Doc124A-The_fig2_232766086 Binding of the cellulosome to the plant cell wall results from the action of CipA family 3 CBM (CBM3), which presents a high affinity for crystalline cellulose. CBMs that are specific for insoluble cellulose (cellulose binding domain – CBD) represent the predominant category. The CBMs can be grouped into distinctive families on the basis of amino acid sequence similarities. CBM3 is a family of protein modules specific for Gram-positive bacterial families. The proteins comprise of around 150 amino acids. The family of proteins is divided into four subgroups: CBM3a, CBM3b, CBM3c, CBM3d. The major ligand recognised by CBM3as and CBM3bs is crystalline cellulose with an affinity (Kd) of 0,4 uM determined by depletion isotherms. The family 3a (scaffoldin) and 3b (mainly free enzymes) are closely similar in their primary structures and both types bind strongly to crystalline cellulose [2,3]. Members of the family IIIc, fails to bind crystalline cellulose, but serves in a 'helper' capacity by feeding a single incoming cellulose chain into the active site of the neighbouring catalytic module pending hydrolysis [4,5].

Structure

The crystal structure of CBM3 has been solved. It has nine beta- strands, which form a compact domain. It is arranged in two antiparallel beta-sheets. The crystal structure of CBM3 has been solved (see <PDB:1NBC>) [2]. It consists of nine β-strands which form a compact domain that has an overall prismatic shape. It is arranged in two antiparallel β-sheets that stack face-to-face to form a β sandwich with jelly roll topology. Two defined surfaces, located on opposite sides of the molecule, contain conserved polar and aromatic residues which are probably involved in the binding of the CBM to cellulose [2,3]. The first one forms a planar strip whereas the second one forms a shallow groove. The structure of CBM3 from C. Thermocellum revealed a classical beta- jelly roll fold consisting . Strukturam strukturos parametrai ir pan.:

C. thermocellum scaffoldin (CipA) contains nine type I cohesins and thus organizes a multienzyme complex that incorporates nine enzymes. The C-terminal type II dockerin of CipA binds specifically to type II cohesin modules found in cell surface proteins. Individ- ual enzymes may also adhere directly to the bacterium cell envelope by binding the single type I cohesins found in OlpA and OlpC.