A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag

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.

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

The Journal of Biochemistry ◽

10.1093/jb/mvt043 ◽

2013 ◽

Vol 154 (2) ◽

pp. 185-193 ◽

Cited By ~ 8

Author(s):

M. Hara ◽

H. Sugimoto ◽

M. Uemura ◽

K.-i. Akagi ◽

K. Suzuki ◽

...

Keyword(s):

Binding Activity ◽

Binding Domain ◽

Bacillus Circulans ◽

Chitin Binding Domain

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Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.2862-2869.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 2862-2869 ◽

Cited By ~ 24

Author(s):

Paul A. Colussi ◽

Charles A. Specht ◽

Christopher H. Taron

Keyword(s):

Catalytic Domain ◽

Kluyveromyces Lactis ◽

Killer Toxin ◽

Binding Domain ◽

Affinity Tag ◽

Α Subunit ◽

Chitin Binding Domain ◽

Amino Terminal ◽

Rich Domain ◽

Carboxy Terminal

ABSTRACT Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin α-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.

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Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan

Analytical Biochemistry ◽

10.1016/j.ab.2003.12.029 ◽

2004 ◽

Vol 327 (2) ◽

pp. 278-283 ◽

Cited By ~ 14

Author(s):

Michael P Bernard ◽

Donghui Cao ◽

Rebecca V Myers ◽

William R Moyle

Keyword(s):

Binding Domain ◽

Chitin Binding Domain

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Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PLoS ONE ◽

10.1371/journal.pone.0140953 ◽

2015 ◽

Vol 10 (10) ◽

pp. e0140953 ◽

Cited By ~ 2

Author(s):

Wen-Jie Wang ◽

Wang Cheng ◽

Ming Luo ◽

Qingyu Yan ◽

Hong-Mei Yu ◽

...

Keyword(s):

Binding Domain ◽

Chitin Binding Domain ◽

Peptidoglycan Recognition Protein ◽

Recognition Protein

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Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Applied Microbiology and Biotechnology ◽

10.1007/s00253-008-1772-z ◽

2009 ◽

Vol 82 (3) ◽

pp. 445-451 ◽

Cited By ~ 25

Author(s):

Chung-Jen Chiang ◽

Jen-You Wang ◽

Po-Ting Chen ◽

Yun-Peng Chao

Keyword(s):

Binding Domain ◽

Chitin Binding Domain

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CLONING AND EXPRESSION OF CHITINASE 19 FAMILIE FROM D.CAPENSIS

BIOTECHNOLOGY: STATE OF THE ART AND PERSPECTIVES ◽

10.37747/2312-640x-2020-18-288-290 ◽

2020 ◽

pp. 288-290

Author(s):

A. Chulkin ◽

A. Rozhkova ◽

A. Sinitsyn A.

Keyword(s):

Binding Domain ◽

Full Length ◽

Cloning And Expression ◽

Chitin Binding Domain ◽

Biochemical Characteristics ◽

Family 19 Chitinase ◽

Different Strains

Sequence of family 19 chitinase was cloned from Drosera capensis. Implemented expression in different strains of E.coli and developed a refolding method. Describes the primary biochemical characteristics of the full-length chitinase and its shape without chitin-binding domain.

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