Crystal structure and substrate-binding mode of a novel pectate lyase from alkaliphilic Bacillus sp. N16-5

Zheng, Yingying; Huang, Chun‐Hsiang; Liu, Wenting; Ko, Tzu‐Ping; Xue, Yanfen; Zhou, Cheng; Guo, Rey‐Ting; Ma, Yanhe

doi:10.1016/j.bbrc.2012.02.148

Cited by 35 publications

(48 citation statements)

References 28 publications

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“…Zheng et al . [25] found that in crystals of different Pels of the PL1 family, the numbers of Ca 2+ ions and the corresponding binding residues differed from each other. This suggests that the variety of the numbers of Ca 2+ -binding residues may be responsible for different modes of the calcium dependence of Pels.…”

Section: Discussionmentioning

confidence: 99%

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Wang

Zhou

et al. 2014

BMC Biotechnol

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BackgroundBiotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli.ResultsA Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber.ConclusionsThis work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production.

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Section: Discussionmentioning

confidence: 99%

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Wang

Zhou

et al. 2014

BMC Biotechnol

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“…RN1 (GenBank accession number: BAG12908) (Sukhumsiirchart et al 2009), Bsp165PelA from Bacillus sp. N16-5 (GenBank accession number: ACY38198) (Zheng et al 2012), pectate lyase C from Erwinia chrysanthemi (GenBank accession number: P11073) (Tardy et al 1997), pectate lyase from Thermotoga maritima MSB8 (GenBank accession number: AAD35518) (Kluskens et al 2003), and pectate lyase 47 from Bacillus sp. TS-47 (GenBank accession number: BAB40336) (Takao et al 2001), with optimal reaction temperatures from 60 to 90°C.…”

Section: Sequence Alignmentmentioning

confidence: 99%

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming

Liang

Gui

Zhou

et al. 2014

Appl Microbiol Biotechnol

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Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

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“…Inside the internal of β-helix, an asparagine ladder formed by three or four aspargine residues was often observed in some other structures [27,30,31]. The asparagine ladder is characteristic of parallel β-helix structure, but is not consistently seen in all proteins with this fold.…”

Section: Resultsmentioning

confidence: 99%

The substrate/product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485

Huang

Sun

et al. 2012

Biochemical Journal

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Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48–2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel β-helix (residues 1–75 and 201–638) and the flanking region (residues 76–200) folds into a β-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.

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Crystal structure and substrate-binding mode of a novel pectate lyase from alkaliphilic Bacillus sp. N16-5

Cited by 35 publications

References 28 publications

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming

The substrate/product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485

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