Overexpression, purification, and characterization of <i>Paenibacillus</i> cell surface-expressed chitinase ChiW with two catalytic domains

Itoh, Takafumi; Sugimoto, Ikumi; Hibi, Takao; Suzuki, Fumiko; Matsuo, Koichi; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

doi:10.1080/09168451.2014.891935

Cited by 45 publications

(40 citation statements)

References 48 publications

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“…FPU-7 (Fig 1) were overexpressed in Escherichia coli ( E . coli ) and purified as described previously [12]. The expression vector for the recombinant protein composed of CBM-54 of ChiW (Val198 to Phe449) was prepared by DNA truncation from the ChiW-SLHd expression vector with the KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…Genomic and biochemical analyses of the FPU-7 strain have revealed that the bacterium secretes at least seven chitinases, one of which is a unique high-molecular-mass (150 kDa) chitinase, termed ChiW. This enzyme has three surface-layer homology (SLH) domains (~18 kDa) (Fig 1); it is specifically expressed on the surface of the bacterial cell and degrades chitin [11, 12]. In general, the SLH domains are composed of three repeats of highly conserved sequences and bind noncovalently to glycan backbones of the peptidoglycan of Gram-positive bacteria, whereupon the cell wall is surrounded by the congregated proteins with SLH domains as a cell envelope or surface layer [13].…”

Section: Introductionmentioning

confidence: 99%

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Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

et al. 2016

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The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

et al. 2016

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“…DAU101 [16], B. licheniformis [17], Paenibacillus sp. [18], Sanguibacter antarcticus [19], and Stenotrophomonas maltophilia [4]. However, most reported chitinases belong to endochitinases, while a few exochitinases were found in bacteria such as Thermococcus chitonophagus [7], Microbispora sp.…”

Section: Introductionmentioning

confidence: 99%

“…[16]. Until now, Paenibacillus species have been reported to produce some endochitinases [3,11-14,18,21], but no exochitinase from Paenibacillus species has been found.…”

Section: Introductionmentioning

confidence: 99%

An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine

Yan

Yang

et al. 2014

Biotechnol Biofuels

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BackgroundN-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.ResultsA novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N-acetyl chitobiose [(GlcNAc)2] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.ConclusionsThe excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

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“…(30), Paenibacillus sp. (31)(32)(33)(34)(35)(36), Clostridium sp. (37)(38)(39)(40)(41)(42)(43)(44), Thermoanaerobacterium sp.…”

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confidence: 99%

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Conway¹,

Pierce

et al. 2016

Journal of Biological Chemistry

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The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis encodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptor species genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique to C. kronotskyensis and contains SLH domains. Calkro_0111 was produced recombinantly in Escherichia coli as two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo-and exoglucanase activity on the ␤-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs in C. kronotskyensis, when produced in E. coli, was active on a variety of xylans and ␤-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptor and among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii. As such, the gene encoding Calkro_0402 was inserted into the C. bescii genome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.Many bacteria (1, 2) and archaea (3) produce a two-dimensional, para-crystalline array of protein that covers the outside of the cell, referred to as a surface layer (S-layer).5 In bacteria, the majority of S-layer proteins are non-covalently associated with the bacterial cell surface via specialized domains at their N or C terminus, typically from one of three distinct but analogous domain categories: surface layer homology (SLH) (4, 5), CWB2 (pfam04122) (6), or NCAD (previously SLAP, pfam03217) (7,8). In archaea, S-layer proteins are most often attached to the cell membrane. S-layer proteins can be anchored to the membrane via a C-terminal transmembrane helix domain (3, 9) or covalently attached to cell membrane lipid via an archaeosortase, as shown in Haloferax volcanii (10,11). In most microorganisms, the S-layer is composed entirely of one or two proteins (SLPs), which self-assemble. In addition to these SLPs, some Firmicutes produce a family of proteins that also contain SLH, CWB2, or NCAD domains and, as such, are predicted to be associated with the S-layer (12). SLPs and S-layer associated proteins can also contain domains with specialized functions allowing the S-layer to play a role in adherence and biofilm formation (13-16), biogenesis of the cell envelope (17), cell division (18...

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Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains

Cited by 45 publications

References 48 publications

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

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