Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA′), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

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Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Applied and Environmental Microbiology ◽

10.1128/aem.67.10.4678-4684.2001 ◽

2001 ◽

Vol 67 (10) ◽

pp. 4678-4684 ◽

Cited By ~ 32

Author(s):

Henrik Wernérus ◽

Janne Lehtiö ◽

Tuula Teeri ◽

Per-Åke Nygren ◽

Stefan Ståhl

Keyword(s):

Metal Binding ◽

Binding Capacity ◽

Surface Display ◽

Surface Proteins ◽

Chimeric Proteins ◽

Phage Display Technology ◽

Binding Domains ◽

Combinatorial Protein Engineering ◽

Display Technology ◽

Cellulose Binding

ABSTRACT Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reeseicellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains

Biochemical Journal ◽

10.1042/bj3300041 ◽

1998 ◽

Vol 330 (1) ◽

pp. 41-45 ◽

Cited By ~ 36

Author(s):

Claude DUPONT ◽

Martin ROBERGE ◽

François SHARECK ◽

Rolf MOROSOLI ◽

Dieter KLUEPFEL

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Streptomyces Lividans ◽

Cellulose Binding Domain ◽

Binding Domains ◽

New Family ◽

Cellulose Binding ◽

Sequence Similarities

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

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